Engineered adenylate kinase variants

WO2026222220A1PCT designated stage Publication Date: 2026-10-22CODEXIS INC
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Patent Information

Application Number
PCT/US2026/023887
Authority / Receiving Office
WO · WO
Patent Type
Applications
Current Assignee / Owner
Priority Date
2025-05-20
Filing Date
2026-04-15
Publication Date
2026-10-22

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Abstract

The present disclosure provides engineered adenylate kinases, and recombinant polynucleotides encoding the engineered adenylate kinases. The present disclosure further provides uses of the engineered adenylate kinases for converting NMP to NDP.
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Description

Docket No. 5244.286WO3 ENGINEERED ADENYLATE KINASE VARIANTSCROSS-REFERENCE

[0001] This application claims priority to U.S. Provisional Application No. 63 / 809,198 filed on May 20, 2025 and U.S. Provisional Application No. 63 / 789,728, filed on April 16, 2025 which are incorporated herein by reference in their entirety.REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

[0002] The Sequence Listing concurrently submitted herewith as file name CX10-286WO3.xml, created on April 15, 2026, with a file size of 1,818,624 bytes, is part of the specification and is hereby incorporated by reference herein in its entirety.TECHNICAL FIELD

[0003] The present disclosure relates to engineered adenylate kinase variants, polynucleotides encodingthe engineered adenylate kinase variants, and uses of the engineered enzymes.BACKGROUND

[0004] Adenylate kinase (AK or AdyK) is an ATP:AMP phosphotransferase, an enzyme that belongs to the nucleoside monophosphate (NMPs) kinase family, and mediates the interconversion of adenine nucleotides (ATP + AMP <-> 2 ADP). The enzyme is ubiquitous in nature, being present in archaea, bacteria, and as well eukarya, with the exception of sulfur bacteria, and is a critical enzyme in energy metabolism and equilibrating adenine nucleotides in vivo. Although ATP is the substrate for most AKs, the human AK isoenzyme 3 uses GTP instead (GTP:AMP phosphotransferase).

[0005] Adenylate kinase can be used as an ATP regenerating system in presence of excess ADP to convert two ADP molecules, one as a phosphate acceptor and the other as a phosphate donor, to produce ATP + AMP. Adenylate kinase is also adaptable for use in enzyme cascades for the conversion of AMP to ADP as well as other NDPs. However, adenylate kinases can display bias in substrate specificity and have different activities towards certain NDP substrates.SUMMARY

[0006] The present disclosure provides engineered adenylate kinase enzymes, polynucleotides encoding the engineered adenylate kinase enzymes, and method of using the adenylate kinase enzymes. In some embodiments, the adenylate kinases have been engineered to have an improved property as compared to a reference adenylate kinase.

[0007] In one aspect, the present disclosure provides an engineered adenylate kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a referenceDocket No. 5244.286WO3 sequence corresponding to amino acid residues 12-231 of an even-numbered SEQ ID NO. of SEQ ID NOs: 16-360 and 372-1018, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 16-360 and 372-1018, wherein the amino acid sequence comprises one or more substitutions relative to a reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, 144, 252, 402, 446, 710, or 808, or relative to a reference sequence corresponding to SEQ ID NO: 16, 144, 252, 402, 446, 710, or 808.

[0008] In some embodiments, the engineered adenylate kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, 144, 252, 402, 446, 710, or 808, or to a reference sequence corresponding to SEQ ID NO: 16, 144, 252, 402, 446, 710, or 808, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 16, 144, 252, 402, 446, 710, or 808.

[0009] In some embodiments, the engineered adenylate kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or to the reference sequence corresponding to SEQ ID NO: 16, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0010] In some embodiments, the engineered adenylate kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0011] In some embodiments, the engineered adenylate kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-231 of an even-numbered SEQ ID NO. of SEQ ID NOs: 18-360 and 372-1018, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 18-360 andDocket No. 5244.286WO3 372-1018, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0012] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution at amino acid position 4, 14, 15, 16, 17, 18, 31, 33, 35, 37, 38, 40, 46, 47, 52, 53, 54, 55, 56, 59, 61, 62, 66, 68, 69, 74, 80, 81, 82, 83, 84, 87, 89, 91, 92, 93, 95, 96, 104, 105, 109, 111, 112, 115, 117, 121, 122, 125, 126, 132, 133, 135, 136, 139, 142, 143, 146, 148, 149, 154, 157, 159, 162, 163, 166, 168, 169, 170, 178, 179, 180, 182, 183, 184, 187, 192, 194, 199, 202, 203, 204, 205, 207, 212, 214, 215, 217, 218, 219, 220, 222, 223, 225, 227, 228, and 229, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0013] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a at least a substitution or amino acid residue 4N, 14P, 15A / H / T, 16C / G / M / T, 171, 18V, 31F / V / W, 33L, 35C / E, 37M, 37E / H / Q / R, 38D, 40T, 46E, 47L, 52L, 53D / M / S / T / V, 54G / R / V, 55Q, 55E / G / N / R / S, 56A / E, 59G, 61G, 62H, 66D, 68V, 69V, 74H, SOS, 81P, 82L, 83R, 84A / T, 87G / R / S, 89A / D / F / G / L / P / Q / S / V / W, 91L, 92S, 93E, 93G / L / V, 95W, 96L, 104L / R, 104Q, 105L / Q / S, 109A, 111N, 112C / T, 115C / H / N, 117A / E / G / R / T / V, 121V, 122S, 125L, 126C / I, 132R, 133W, 135E / N, 136L / R, 139A / H, 142V, 1431, 146A / G / H, 148Q, 149A / L / M / Q, 154E, 157R, 159V, 162D / M / V, 163E / R, 166E, 168C, 169E, 170K / M, 178A / F / G / I / L / M / N / R / T / V / Y, 179A, 180A / P / Q, 182L, 183L, 184P / Q / S, 187D / R, 192L / M / V, 194L, 199F, 202E / G / N, 203Q, 204D, 205Q, 2051, 205C / L, 2071 / N, 212A, 214K / P, 215V, 217A / E, 218L / V, 219L / T, 220G, 222A, 223D / M / V, 225H / T, 225A / Q, 227S, 228L / S, and 229G, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0014] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or amino acid residue H4N, A14P, Y15A / H / T, L16C / G / M / T, V17I, L18V, R31F / V / W, I33L, A35C / E, H37M, T37E / H / Q / R, G38D, P40T, D46E, I47L, V52L, A53D / M / S / T / V, K54G / R / V, E55Q, Q55E / G / N / R / S, N56A / E, V59G, S61G, S62H, W66D, L68V, K69V, P74H, P80S, V81P, V82L, L83R, R84A / T, K87G / R / S, K89A / D / F / G / L / P / Q / S / V / W, C91L, E92S, G93E, K93G / L / V, F95W, I96L, Q104L / R, R104Q, R105L / Q / S, Y109A, D111N, S112C / T, E115C / H / N, Q117A / E / G / R / T / V, L121V, T122S, V125L, L126C / I, D132R, E133W, V135E / N, V136L / R, L139A / H, R142V, V143I, K146A / G / H, G148Q, R149A / L / M / Q, I154E, P157R, K159V, E162D / M / V, L163E / R, D166E, G168C, V169E, S170K / M, K178A / F / G / I / L / M / N / R / T / V / Y, E179A, E180A / P / Q, V182L, R183L, M184P / Q / S, K187D / R, K192L / M / V, Q194L, Y199F, K202E / G / N, K203Q, G204D, I205Q, L205I, Q205C / L, A207I / N, S212A, E214K / P, 1215V, P217A / E, I218L / V, V219L / T, A220G, V222A, L223D / M / V, A225H / T, I225A / Q, G227S, V228L / S, and I229G, or combinations thereof,Docket No. 5244.286WO3 wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0015] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution at amino acid position 37, 93, 125, or 205, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ. ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0016] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or amino acid residue 37H / M, 93G, 125L, or 205Q, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0017] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or amino acid residue T37H / M, K93G, V125L, or I205Q, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0018] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution at amino acid position 4, 47, 95, or 96; or substitution 14P, 15A / T, 16C / G / T, 37E / H / M / Q, 52L, 53T / V, 54V, 55N / R, 56A, 69V, 74H, 89D / F / G / S / W, 93L, 115C / H / N, 117A / E / T / V, 125L, 1261, 132R, 133W, 135E / N, 146A / G, 157R, 166E, 168C, 170M, 178A / F / G / I / L / M / N / R / T / V / Y, 180A / Q, 187D / R, 203Q, 205C / Q, 214K, 217E, or 220G; or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.|0019| In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution 4N, 14P, 15A / H / T, 16C / G / M / T, 171, 18V, 31F / V / W, 33L, 35C / E, 37M, 37E / H / Q / R, 38D, 40T, 46E, 47L, 52L, 53D / M / S / T / V, 54G / R / V, 55Q, 55E / G / N / R / S, 56A / E, 59G, 61G, 62H, 66D, 68V, 69V, 74H, SOS, 81P, 82L, 83R, 84A / T, 87G / R / S, 89A / D / F / G / L / P / Q / S / V / W, 91L, 92S, 93E, 93G / L / V, 95W, 96L, 104L / R, 104Q, 105L / Q / S, 109A, 111N, 112C / T, 115C / H / N, 117A / E / G / R / T / V, 121V, 122S, 125L, 126C / I, 132R, 133W, 135E / N, 136L / R, 139A / H, 142V, 1431, 146A / G / H, 148Q, 149A / L / M / Q, 154E, 157R, 159V, 162D / M / V, 163E / R, 166E, 168C, 169E, 170K / M, 178A / F / G / I / L / M / N / R / T / V / Y, 179A, 180A / P / Q, 182L, 183L, 184P / Q / S, 187D / R, 192L / M / V, 194L, 199F, 202E / G / N, 203Q, 204D, 205Q, 2051, 205C / L, 2071 / N, 212A, 214K / P, 215V, 217A / E, 218L / V, 219L / T, 220G, 222A, 223D / M / V, 225H / T, 225A / Q, 227S, 228L / S, and 229G, or combinations thereof, wherein the amino acid positions are relative to theDocket No. 5244.286WO3 reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0020] In some aspects, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution H4N, A14P, Y15A / H / T, L16C / G / M / T, V17I, L18V, R31F / V / W, I33L, A35C / E, H37M, T37E / H / Q / R, G38D, P40T, D46E, I47L, V52L, A53D / M / S / T / V, K54G / R / V, E55Q, Q55E / G / N / R / S, N56A / E, V59G, S61G, S62H, W66D, L68V, K69V, P74H, P80S, V81P, V82L, L83R, R84A / T, K87G / R / S, K89A / D / F / G / L / P / Q / S / V / W, C91L, E92S, G93E, K93G / L / V, F95W, I96L, Q104L / R, R104Q, R105L / Q / S, Y109A, D111N, S112C / T, E115C / H / N, Q117A / E / G / R / T / V, L121V, T122S, V125L, L126C / I, D132R, E133W, V135E / N, V136L / R, L139A / H, R142V, V143I, K146A / G / H, G148Q, R149A / L / M / Q, I154E, P157R, K159V, E162D / M / V, L163E / R, D166E, G168C, V169E, S170K / M, K178A / F / G / I / L / M / N / R / T / V / Y, E179A, E180A / P / Q, V182L, R183L, M184P / Q / S, K187D / R, K192L / M / V, Q194L, Y199F, K202E / G / N, K203Q, G204D, I205Q, L205I, Q205C / L, A207I / N, S212A, E214K / P, 1215V, P217A / E, I218L / V, V219L / T, A220G, V222A, L223D / M / V, A225H / T, I225A / Q, G227S, V228L / S, and I229G, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0021] In some embodiments, where the amino acid sequence of the engineered adenylate kinase comprises at least a substitution at amino acid position 4, 47, 95, or 96; or substitution 14P, 15A / T, 16C / G / T, 37E / H / M / Q, 52L, 53T / V, 54V, 55N / R, 56A, 69V, 74H, 89D / F / G / S / W, 93L, 115C / H / N, 117A / E / T / V, 125L, 1261, 132R, 133W, 135E / N, 146A / G, 157R, 166E, 168C, 170M, 178A / F / G / I / L / M / N / R / T / V / Y, 180A / Q, 187D / R, 203Q, 205C / Q, 214K, 217E, or 220G; or combinations thereof, the amino acid sequence can further comprise a substitution at amino acid position 11, 13, 14, 15, 16, 18, 20, 21, 22, 26, 27, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 97, 98, 99, 100, 101, 102, 104, 105, 108, 109, 110, 111, 112, 113, 115, 116, 117, 118, 119, 120, 122, 123, 124, 125, 126, 127, 128, 129, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 146, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 161, 162, 163, 166, 168, 169, 170, 172, 173, 175, 178, 179, 180, 181, 182, 183, 184, 186, 187, 188, 190, 191, 192, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, or 231, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0022] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set at amino acid position(s) 37, 47, 52, 53, 54, 55, 56, 89, 92, 96,Docket No. 5244.286WO3 115, 125, 132, 135, 146, 168, 179, 180, 187, 220, 15, 40, 91, 93, 95, 117, 122, 166, 178, 203, 37 / 53 / 74 / 93 / 205, 37 / 74 / 93, 37 / 74 / 93 / 125, 37 / 74 / 205, 37 / 93 / 125 / 205, 37 / 125, 37 / 166, 47 / 55 / 126 / 178, 47 / 89 / 126 / 178, 47 / 89 / 146 / 178, 47 / 117 / 126 / 178, 47 / 178, 53 / 74 / 93 / 166, 53 / 74 / 125, 53 / 93 / 125 / 133 / 166 / 205, 55 / 126 / 178, 74, 74 / 93 / 133, 74 / 125, 74 / 205, 89 / 117 / 178, 89 / 126, 89 / 146 / 178, 89 / 178, 117 / 126, 117 / 126 / 178, 117 / 178, 126 / 146 / 178, 126 / 178, 133, 146 / 178, 205, 37 / 53, 37 / 53 / 74 / 125 / 205, 47 / 54 / 178, 47 / 146 / 178, 53 / 74 / 93, 53 / 93 / 125 / 166, 53 / 133, 54 / 117 / 126 / 146, 89 / 117 / 146 / 178, 89 / 126 / 178, 93 / 125, or 166 / 205, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0023] In some embodiments, the amino acid sequence of the engineered adenylate kinase further comprises at least a substitution at amino acid position 11, 13, 14, 15, 16, 18, 20, 21, 22, 26, 27, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 97, 98, 99, 100, 101, 102, 104, 105, 108, 109, 110, 111, 112, 113, 115, 116, 117, 118, 119, 120, 122, 123, 124, 125, 126, 127, 128, 129, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 146, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 161, 162, 163, 166, 168, 169, 170, 172, 173, 175, 178, 179, 180, 181, 182, 183, 184, 186, 187, 188, 190, 191, 192, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, or 231, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0024] In some embodiments, the amino acid sequence of the engineered adenylate kinase further comprises at least a substitution or amino acid residue 11D, 13G / S, 14C / E, 15F, 16M, 18C / E / L, 20A / P / R / T / V, 21L / N / R / S / T / V, 22A / S, 26L, 27C / S, 29Q / R, 30G / L / S / Y, 31R, 32F / I / L / N, 34A / E / S / V, 35A / E / F / L / S, 36A / E / I / L / M / Q / R / S / V / Y, 37F / G / I / U / R / Y, 38C / F / L / R, 39A / M / Q / T, 40C / F / L / S / T, 42T, 43A / C / G / H / N, 44S, 46E / F / L / M / Y, 48F / L, 50A / C / L / N / Q / R / S / V, 51F / G / K / L / M / R / T, 52A / H, 53A / M / S, 54G / H / I / M / Q / R / T, 55G / L / Q / S, 56E / Q / T, 57H / L / P / S, 58C / G / Y, 59E / G / H / M / P / R / T / V / W / Y, 60A / C / P / S / W, 61A / E / N / P / S / V, 62A / E / G / I / L / S, 63S, 64R, 65A / D / F / K / P / T, 66A / C / D / E / F / G / H / N / P / Q / R / S / T / V / W, 67R, 68A / D / E / G / I / L / Q / R / S / T / V / W / Y, 69F / L / Q / R / Y, 70R, 71T, 731 / R, 74C / G / Q / S, 75L, 76G / L / P, 77A / I / M / Q / S, 78C / G / N / T, 79A / K / L / P / W, 80A / D / G / L / N / P / R / S / V / W, 81 F / l / L / Q / S, 82 l / L / P / Q / T, 83G / H / L / S / T, 84A / E / H / M / S, 87A / E / I / K / L / M / R / V / Y, 88N / R / T, 89A / H / I / L / P / Q / T / V, 90C / E / F / K / S / T / V, 91A / F / G / L / S / V, 92L / S, 93A / E / G / P / S / T / V / Y, 94A / C / F / M / Q / S / T / V / Y, 971, 98A / G / Q, 99A / C, 100F / S / V, 101-, 102A / C / N, 104A / F / H / I / L / Q / R / S / W, 105G / K / L / M / R / S, 108M / R / S / V, 109E / Y, 110C / F, 111E / P / R, 112A / C / E / K / M / N / Q / R / T, 113E / W / Y, 115G / K / R, 116A / E / F / L, 117L / N / S, 118A / G / L / R / S, 119F / K / L / P / R / S, 120G / K / L / R / S / T, 122A / H / S, 123S, 124V, 1251, 126A / E / S / V, 1271 / L / P / S, 128C / E / I / K / N / R / S, 1291 / L / P / S,Docket No. 5244.286WO3 131A / G / V, 132G / K / L / T / V, 133A / E / F / L / Q / S / V / W, 134L, 135 l / K / M / P / R / S, 136A / I / L / S / V / Y, 137L, 138C / I / M / V, 139A / H / L / R, 140G, 141G / V, 142F / L / M / W, 143A / C / G / P / R / S / T / V, 146D / H / N / R / V, 148F / H / M / P / Q / R / S / T, 149L, 150C / G / L / P / S / V, 151A / F / R / Y, 152F / H, 153C / K / S / V, 154Q / R, 155A / F / T / W, 156A / C / N / T / V, 157P / V, 161G / L, 162G, 163G / Q / S, 166C / F / L / P / S, 168G / L / N / Q / S, 169A / D / I / Y, 170A / G / H / P / R / S, 172A / H / M / S / T, 173F / K / R / S / T / V, 175F / L / S, 178E / G / N / R, 179A / C / G / I / L / P / V, 180G / H / P, 181C / I / V, 182A / G / I / L / Q / S, 183A / L / Q, 1841 / K / M / N / R / S / T / V, 186L, 187G / Y, 188G / L, 190A / C / E / G / H / N / Q / R, 191D / K, 192A / H / I / W, 194F / L / R / V / Y, 195G / I / M / R, 1961, 197A / L / Q / V, 198G, 199R, 200A, 201A / F / K / L / S, 202E / G / M / S, 203A / E / L / R, 204R / S, 205L / P, 206W, 207A / L / T, 208E, 210V, 211A, 212C / H / I / L / M / N / P / Q / R / S / W, 213L / P / Q / S, 214A / E / L7M / P / R / T / W, 215A / P / V, 216A / D / E / G / H / L / M / N / P / R / S / T / V, 217E / G / H / P / T, 218A / I / L, 219A / F / G / L / P / S / T / W, 220E / V, 221D, 2221 / T, 223T, 224G / S / T, 225A / L / Q / R / T / V, 226K / L / P / R / S / T, 227P, 228D / F / G / I / L / P / Q / R / S / T / V, 229C / I, 230A / M / P / Q, or 231A / Q / R / T, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0025] In some aspects, the amino acid sequence of the engineered adenylate kinase further comprises at least a substitution or amino acid residue G11D, M13G / S, A14C / E, Y15F, L16M, F18C / E / L, G20A / P / R / T / V, P21L / N / R / S / T / V, P22A / S, K26L, G27C / S, Y29Q / R, A30G / L / S / Y, K31R, I32F / I / L / N, Q34A / E / S / V, E35A / F / L / S, K36A / E / I / L / M / Q / R / S / V / Y, T37F / G / I / L / R / Y, G38C / F / L / R, I39A / M / Q / T, P40C / F / L / S / T, I42T, S43A / C / G / H / N, T44S, D46E / F / L / M / Y, F48L, D50A / C / L / N / Q / R / S / V, I51F / G / K / L / M / R / T, V52A / H, K53A / M / S, K54G / H / I / M / Q / R / T, E55G / L / Q / S, N56E / Q / T, D57H / L / P / S, E58C / G / Y, L59E / G / H / M / P / R / T / V / W / Y, G60A / C / P / S / W, K61A / E / N / P / S / V, K62A / E / G / I / L / S, I63S, K64R, E65A / D / F / K / P / T, I66A / C / D / E / F / G / H / N / P / Q / R / S / T / V / W, M67R, E68A / D / G / I / L / Q / R / S / T / V / W / Y, K69F / L / Q / R / Y, G70R, E71T, V73I / R, P74C / G / Q / S, D75L, E76G / L / P, L77A / I / M / Q / S, V78C / G / N / T, N79A / K / L / P / W, E80A / D / G / L / N / P / R / S / V / W, V81F / I / L / Q / S, V82I / L / P / Q / T, K83G / H / L / S / T, R84A / E / H / M / S, S87A / E / I / K / L / M / R / V / Y, E88N / R / T, K89A / H / I / L / P / Q / T / V, D90C / E / F / K / S / T / V, C91A / F / G / L / S / V, E92L / S, K93A / E / G / P / S / T / V / Y, G94A / C / F / M / Q / S / T / V / Y, L97I, D98A / G / Q, G99A / C, Y100F / S / V, P101-, R102A / C / N, V104A / F / H / I / L / Q / R / S / W, A105G / K / L / M / R / S, E108M / R / S / V, F109E / Y, L110C / F, D111E / P / R, S112A / C / E / K / M / N / Q / R / T, F113E / W / Y, E115G / K / R, S116A / E / F / L, Q117L / N / S, N118A / G / L / R / S, K119F / K / L / P / R / S, Q120G / K / L / R / S / T, T122A / H / S, A123S, A124V, V125I, L126A / E / S / V, F127I / L / P / S, D128C / E / I / K / N / R / S, V129I / L / P / S, E131A / G / V, D132G / K / L / T / V, V133A / E / F / L / Q / S / W, V134L, V135I / K / M / P / R / S, Q136A / I / L / S / V / Y, R137L, L138C / I / M / V, T139A / H / L / R, S140G, R141G / V, R142F / L / M / W, I143A / C / G / P / R / S / T / V, K146D / H / N / R / V, G148F / H / M / P / Q / R / S / T, R149L, I150C / G / L / P / S / V, Y151A / F / R, N152F / H, M153C / K / S / V, I154Q / R, S155A / F / T / W, L156A / C / N / T / V, P157V, D161G / L, E162G, L163G / Q / S, D166C / F / L / P / S, K168G / L / N / Q / S, V169A / D / I / Y, K170A / G / H / P / R / S, V172A / H / M / S / T, Q173F / K / R / S / T / V, D175F / L / S, K178E / G / N / R, E179A / C / G / I / L / P / V, E180G / H / P,Docket No. 5244.286WO3 T181C / I / V, V182A / G / I / L / Q / S, R183A / L / Q, H 1841 / K / M / N / R / S / T / V, Y186L, K187G / Y, V188G / L, L190A / C / E / G / H / N / Q / R, E191D / K, K192A / H / I / W, Q194F / L / R / V / Y, P195G / I / M / R, V196I, 1197A / L / Q / V, D198G, Y199R, Y200A, G201A / F / K / L / S, K202E / G / M / S, K203A / E / L / R, G204R / S, I205L / P, L206W, K207A / L / T, R208E, D210V, G211A, T212C / H / I / L / M / N / P / Q / R / S / W, I213L / P / Q / S, G214A / E / L / M / P / R / T / W, I215A / P / V, D216A / D / E / G / H / L / M / N / P / R / S / T / V, N217E / G / H / P / T, V218A / I / L, V219A / F / G / L / P / S / T / W, A220E / V, E221D, V222I / T, L223T, K224G / S / T, I225A / L / Q / R / T / V, I226K / L / P / R / S / T, G227P, W228D / F / G / I / L / P / Q / R / S / T / V, S229C / I, D230A / M / P / Q, or K231A / Q / R / T, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEO. ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0026] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set at amino acid position(s) 37, 47, 52, 53, 54, 55, 56, 89, 92, 96, 115, 125, 132, 135, 146, 168, 179, 180, 187, 220, 15, 40, 91, 93, 95, 117, 122, 166, 178, 203, 37 / 53 / 74 / 93 / 205, 37 / 74 / 93, 37 / 74 / 93 / 125, 37 / 74 / 205, 37 / 93 / 125 / 205, 37 / 125, 37 / 166, 47 / 55 / 126 / 178, 47 / 89 / 126 / 178, 47 / 89 / 146 / 178, 47 / 117 / 126 / 178, 47 / 178, 53 / 74 / 93 / 166, 53 / 74 / 125, 53 / 93 / 125 / 133 / 166 / 205, 55 / 126 / 178, 74, 74 / 93 / 133, 74 / 125, 74 / 205, 89 / 117 / 178, 89 / 126, 89 / 146 / 178, 89 / 178, 117 / 126, 117 / 126 / 178, 117 / 178, 126 / 146 / 178, 126 / 178, 133, 146 / 178, 205, 37 / 53, 37 / 53 / 74 / 125 / 205, 47 / 54 / 178, 47 / 146 / 178, 53 / 74 / 93, 53 / 93 / 125 / 166, 53 / 133, 54 / 117 / 126 / 146, 89 / 117 / 146 / 178, 89 / 126 / 178, 93 / 125, or 166 / 205, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ. ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0027] In some embodiments, the amino acid sequence of the engineered adenylate kinase further comprises at least a substitution or substitution set 37E, 37Q, 47L, 52L, 53S, 54V, 55S, 56A, 56E, 89D, 89F, 89S, 89W, 92S, 96L, 115H, 125L, 132R, 135E, 135N, 146A, 168C, 179A, 180A, 187R, 220G, 15A, 40T, 53T, 53V, 54G, 54R, 55G, 55N, 55R, 89A, 89G, 89L, 89P, 89 89V, 91L, 93G, 93L, 93V, 95W, 115N, 117A, 117E, 117V, 122S, 166E, 178A, 178F, 1781, 178M, 178N, 178R, 178T, 178V, 178Y, 180P, 203 37H, 37H / 74H / 93G / 125L, 37H / 74H / 205a 37H / 93G / 125L / 205a 37H / 125L, 37R / 53M / 74H / 93G / 205a 37R / 74H / 93G, 37R / 166E, 47L / 55S / 126I / 178G, 47L / 89G / 126I / 178G, 47L / 89G / 146G / 178L, 47L / 117T / 126I / 178G, 47L / 178G, 53M / 74H / 93G / 166E, 53M / 74H / 125L, 53M / 93G / 125L / 133W / 166E / 205a 55S / 126I / 178L, 74H, 74H / 93G / 133W, 74H / 125L, 74H / 205a 89G / 117T / 178G, 89G / 126I, 89G / 146G / 178G, 89G / 178L, 117T, 117T / 1261, 117T / 126I / 178G, 117T / 126I / 178L, 117T / 178L, 126I / 146G / 178G, 126I / 178G, 133W, 146G, 146G / 178G, 178G, 205a 37H / 53M, 37R, 37R / 53M, 37R / 53M / 74H / 125L / 205Q, 47L / 54R / 178G, 47L / 146G / 178G, 47L / 178L, 53M, 53M / 74H / 93G, 53M / 93G / 125L / 166E, 53M / 133W, 54R / 117T / 126I / 146G, 74H, 89G / 117T / 146G / 178L, 89G / 126I / 178G, 93G / 125L, 166E / 205a or 178L, wherein the amino acid positions are relative to theDocket No. 5244.286WO3 reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0028] In some embodiments, the amino acid sequence of the engineered adenylate kinase further comprises at least a substitution or substitution setT37E, T37Q, I47L, V52L, A53S, K54V, Q55S, N56A, N56E, K89D, K89F, K89S, K89W, E92S, I96L, E115H, V125L, D132R, V135E, V135N, K146A, G168C, E179A, E180A, K187R, A220G, Y15A, P40T, A53T, A53V, K54G, K54R, Q55G, Q55N, Q55R, K89A, K89G, K89L, K89P, K89Q, K89V, C91L, K93G, K93L, K93V, F95W, E115N, Q117A, Q117E, Q117V, T122S, D166E, K178A, K178F, K178I, K178M, K178N, K178R, K178T, K178V, K178Y, E180P, K203Q, T37H, T37H / P74H / K93G / V125L, T37H / P74H / I205Q, T37H / K93G / V125L / I205Q, T37H / V125L, T37R / A53M / P74H / K93G / I205Q, T37R / P74H / K93G, T37R / D166E, I47L / Q55S / L126I / K178G, I47L / K89G / L126I / K178G, I47L / K89G / K146G / K178L, I47L / Q117T / L126I / K178G, I47L / K178G, A53M / P74H / K93G / D166E, A53M / P74H / V125L, A53M / K93G / V125L / E133W / D166E / I205Q, Q55S / L126I / K178L, P74H, P74H / K93G / E133W, P74H / V125L, P74H / I205Q, K89G / Q117T / K178G, K89G / L126I, K89G / K146G / K178G, K89G / K178L, Q117T, Q117T / L126I, Q117T / L126I / K178G, Q117T / L126I / K178L, Q117T / K178L, L126I / K146G / K178G, L126I / K178G, E133W, K146G, K146G / K178G, K178G, I205Q, T37H / A53M, T37R, T37R / A53M, T37R / A53M / P74H / V125L / I205Q, I47L / K54R / K178G, I47L / K146G / K178G, I47L / K178L, A53M, A53M / P74H / K93G, A53M / K93G / V125L / D166E, A53M / E133W, K54R / Q117T / L126I / K146G, K89G / Q117T / K146G / K178L, K89G / L126I / K178G, K93G / V125L, D166E / I205Q, or K178L, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0029] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least one substitution set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2 or 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0030] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set of an engineered adenylate kinase variant set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2 or 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0031] In some embodiments, the engineered adenylate kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference amino acid sequenceDocket No. 5244.286WO3 comprising a substitution or substitution set of an engineered adenylate kinase variant set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2 or 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0032] In some embodiments, the engineered adenylate kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0033] In some embodiments, the engineered adenylate kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12-231 of an even-numbered SEQ ID NO. of SEQ ID NOs: 18-360 and 372-1018, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 18-360 and 372-1018.

[0034] In some embodiments, the engineered adenylate kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0035] In some embodiments, the engineered adenylate kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-231 of an even-numbered SEQ ID NO. of SEQ ID NOs: 18-360 and 372-1018, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 18-360 and 372-1018, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0036] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution at amino acid position 4, 14, 15, 16, 17, 18, 31, 33, 35, 37, 38, 40, 46, 47, 52, 53, 54, 55, 56, 59, 61, 62, 66, 68, 69, 74, 80, 81, 82, 83, 84, 87, 89, 91, 92, 93, 95, 96, 104, 105, 109, 111,Docket No. 5244.286WO3 112, 115, 117, 121, 122, 125, 126, 132, 133, 135, 136, 139, 142, 143, 146, 148, 149, 154, 157, 159, 162, 163, 166, 168, 169, 170, 178, 179, 180, 182, 183, 184, 187, 192, 194, 199, 202, 203, 204, 205, 207, 212, 214, 215, 217, 218, 219, 220, 222, 223, 225, 227 , 228, and 229, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ. ID NO: 144, 252, 402, 446, 710, or 808.

[0037] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or amino acid residue 4N, 14P, 15A / H / T, 16C / G / M / T, 171, 18V, 31F / V / W, 33L, 35C / E, 37M, 37E / H / Q / R, 38D, 40T, 46E, 47L, 52L, 53D / M / S / T / V, 54G / R / V, 55Q, 55E / G / N / R / S, 56A / E, 59G, 61G, 62H, 66D, 68V, 69V, 74H, 80S, 81P, 82L, 83R, 84A / T, 87G / R / S, 89A / D / F / G / L / P / Q / S / V / W, 91L, 92S, 93E, 93G / L / V, 95W, 96L, 104L / R, 104Q, 105L / Q / S, 109A, 111N, 112C / T, 115C / H / N, 117A / E / G / R / T / V, 121V, 122S, 125L, 126C / I, 132R, 133W, 135E / N, 136L / R, 139A / H, 142V, 1431, 146A / G / H, 148Q, 149A / L / M / Q, 154E, 157R, 159V, 162D / M / V, 163E / R, 166E, 168C, 169E, 170K / M, 178A / F / G / I / L / M / N / R / T / V / Y, 179A, 180A / P / Q, 182L, 183L, 184P / Q / S, 187D / R, 192L / M / V, 194L, 199F, 202E / G / N, 203Q, 204D, 205Q, 2051, 205C / L, 2071 / N, 212A, 214K / P, 215V, 217A / E, 218L / V, 219L / T, 220G, 222A, 223D / M / V, 225H / T, 225A / Q, 227S, 228L / S, and 229G, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0038] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution at amino acid position 37, 93, 125, or 205, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808. In some aspects, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or amino acid residue 37H / M, 93G, 125L, or 205Q, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0039] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution at amino acid position 4, 47, 95, or 96; or substitution 14P, 15A / T, 16C / G / T, 37E / H / M / Q, 52L, 53T / V, 54V, 55N / R, 56A, 69V, 74H, 89D / F / G / S / W, 93L, 115C / H / N, 117A / E / T / V, 125L, 1261, 132R, 133W, 135E / N, 146A / G, 157R, 166E, 168C, 170M, 178A / F / G / I / L / M / N / R / T / V / Y, 180A / Q, 187D / R, 203Q, 205C / Q, 214K, 217E, or 220G; or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ IDDocket No. 5244.286WO3 NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0040] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution at amino acid position 4, 47, 95, or 96; or substitution 4N, 14P, 15A / H / T, 16C / G / M / T, 171, 18V, 31F / V / W, 33L, 35C / E, 37M, 37E / H / Q / R, 38D, 40T, 46E, 47L, 52L, 53D / M / S / T / V, 54G / R / V, 55Q, 55E / G / N / R / S, 56A / E, 59G, 61G, 62H, 66D, 68V, 69V, 74H, SOS, 81P, 82L, 83R, 84A / T, 87G / R / S, 89A / D / F / G / L / P / Q / S / V / W, 91L, 92S, 93E, 93G / L / V, 95W, 96L, 104L / R, 104Q, 105L / Q / S, 109A, 111N, 112C / T, 115C / H / N, 117A / E / G / R / T / V, 121V, 122S, 125L, 126C / I, 132R, 133W, 135E / N, 136L / R, 139A / H, 142V, 1431, 146A / G / H, 148Q, 149A / L / M / Q, 154E, 157R, 159V, 162D / M / V, 163E / R, 166E, 168C, 169E, 170K / M, 178A / F / G / I / L / M / N / R / T / V / Y, 179A, 180A / P / Q, 182L, 183L, 184P / Q / S, 187D / R, 192L / M / V, 194L, 199F, 202E / G / N, 203Q, 204D, 205Q, 2051, 205C / L, 2071 / N, 212A, 214K / P, 215V, 217A / E, 218L / V, 219L / T, 220G, 222A, 223D / M / V, 225H / T, 225A / Q, 227S, 228L / S, or 229G; or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ. ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0041] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution 4N, 14P, 15A / H / T, 16C / G / M / T, 171, 18V, 31F / V / W, 33L, 35C / E, 37M, 37E / H / Q / R, 38D, 40T, 46E, 47L, 52L, 53D / M / S / T / V, 54G / R / V, 55Q, 55E / G / N / R / S, 56A / E, 59G, 61G, 62H, 66D, 68V, 69V, 74H, 80S, 81P, 82L, 83R, 84A / T, 87G / R / S, 89A / D / F / G / L / P / Q / S / V / W, 91L, 92S, 93E, 93G / L / V, 95W, 96L, 104L / R, 104Q, 105L / Q / S, 109A, 111N, 112C / T, 115C / H / N, 117A / E / G / R / T / V, 121V, 122S, 125L, 126C / I, 132R, 133W, 135E / N, 136L / R, 139A / H, 142V, 1431, 146A / G / H, 148Q, 149A / L / M / Q, 154E, 157R, 159V, 162D / M / V, 163E / R, 166E, 168C, 169E, 170K / M, 178A / F / G / I / L / M / N / R / T / V / Y, 179A, 180A / P / Q, 182L, 183L, 184P / Q / S, 187D / R, 192L / M / V, 194L, 199F, 202E / G / N, 203Q, 204D, 205Q, 2051, 205C / L, 2071 / N, 212A, 214K / P, 215V, 217A / E, 218L / V, 219L / T, 220G, 222A, 223D / M / V, 225H / T, 225A / Q, 227S, 228L / S, or 229G, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0042] In some embodiments, the amino acid sequence can further comprise at least a substitution at amino acid position 11, 13, 14, 15, 16, 18, 20, 21, 22, 26, 27, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 97, 98, 99, 100, 101, 102, 104, 105, 108, 109, 110, 111, 112, 113, 115, 116, 117, 118, 119, 120, 122, 123, 124, 125, 126, 127, 128, 129, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 146, 148, 149, 150, 151, 152, 153, 154, 155,Docket No. 5244.286WO3 156, 157, 161, 162, 163, 166, 168, 169, 170, 172, 173, 175, 178, 179, 180, 181, 182, 183, 184, 186, 187, 188, 190, 191, 192, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, or 231, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ. ID NO: 144, 252, 402, 446, 710, or 808.

[0043] In some aspects, the amino acid sequence can further comprise at least a substitution or amino acid residue 11D, 13G / S, 14C / E, 15F, 16M, 18C / E / L, 20A / P / R / T / V, 21L / N / R / S / T / V, 22A / S, 26L, 27C / S, 29Q / R, 30G / L / S / Y, 31R, 32F / I / L / N, 34A / E / S / V, 35A / E / F / L / S, 36A / E / I / L / M / Q / R / S / V / Y, 37F / G / I / L / R / Y, 38C / F / L / R, 39A / M / Q / T, 40C / F / L / S / T, 42T, 43A / C / G / H / N, 44S, 46E / F / L / M / Y, 48F / L, 50A / C / L / N / Q / R / S / V, 51F / G / K / L / M / R / T, 52A / H, 53A / M / S, 54G / H / I / M / Q / R / T, 55G / L / Q / S, 56E / Q / T, 57H / L / P / S, 58C / G / Y, 59E / G / H / M / P / R / T / V / W / Y, 60A / C / P / S / W, 61A / E / N / P / S / V, 62A / E / G / I / L / S, 63S, 64R, 65A / D / F / K / P / T, 66A / C / D / E / F / G / H / N / P / Q / R / S / T / V / W, 67R, 68A / D / E / G / I / L / Q / R / S / T / V / W / Y, 69F / L / Q / R / Y, 70R, 71T, 731 / R, 74C / G / Q / S, 75 L, 76G / L / P, 77A / I / M / Q / S, 78C / G / N / T, 79A / K / L / P / W, 80A / D / G / L / N / P / R / S / V / W, 81F / I / L / Q / S, 821 / L / P / Q / T, 83G / H / L / S / T, 84A / E / H / M / S, 87A / E / I / K / L / M / R / V / Y, 88N / R / T, 89A / H / I / L / P / Q / T / V, 90C / E / F / K / S / T / V, 91A / F / G / L / S / V, 92L / S, 93A / E / G / P / S / T / V / Y, 94A / C / F / M / Q / S / T / V / Y, 971, 98A / G / Q, 99A / C, 100F / S / V, 101-, 102A / C / N, 104A / F / H / I / L / Q / R / S / W, 105G / K / L / M / R / S, 108M / R / S / V, 109E / Y, 110C / F, 111E / P / R, 112A / C / E / K / M / N / Q / R / T, 113E / W / Y, 115G / K / R, 116A / E / F / L, 117L / N / S, 118A / G / L / R / S, 119F / K / L / P / R / S, 120G / K / L / R / S / T, 122A / H / S, 123S, 124V, 1251, 126A / E / S / V, 1271 / L / P / S, 128C / E / I / K / N / R / S, 1291 / L / P / S, 131A / G / V, 132G / K / L / T / V, 133A / E / F / L / Q / S / V / W, 134L, 1351 / K / M / P / R / S, 136A / I / L / S / V / Y, 137L, 138C / I / M / V, 139A / H / L / R, 140G, 141G / V, 142F / L / M / W, 143A / C / G / P / R / S / T / V, 146D / H / N / R / V, 148F / H / M / P / Q / R / S / T, 149L, 150C / G / L / P / S / V, 151A / F / R / Y, 152F / H, 153C / K / S / V, 154Q / R, 155A / F / T / W, 156A / C / N / T / V, 157P / V, 161G / L, 162G, 163G / Q / S, 166C / F / L / P / S, 168G / L / N / Q / S, 169A / D / I / Y, 170A / G / H / P / R / S, 172A / H / M / S / T, 173F / K / R / S / T / V, 175F / L / S, 178E / G / N / R, 179A / C / G / I / L / P / V, 180G / H / P, 181C / I / V, 182A / G / I / L / Q / S, 183A / L / Q, 1841 / K / M / N / R / S / T / V, 186L, 187G / Y, 188G / L, 190A / C / E / G / H / N / Q / R, 191D / K, 192A / H / I / W, 194F / L / R / V / Y, 195G / I / M / R, 1961, 197A / L / Q / V, 198G, 199R, 200A, 201A / F / K / L / S, 202E / G / M / S, 203A / E / L / R, 204R / S, 205 L / P, 206W, 207A / L / T, 208 E, 210V, 211A, 212C / H / I / L / M / N / P / Q / R / S / W, 213L / P / Q / S, 214A / E / L / M / P / R / T / W, 215A / P / V, 216A / D / E / G / H / L / M / N / P / R / S / T / V, 217E / G / H / P / T, 218A / I / L, 219A / F / G / L / P / S / T / W, 220E / V, 221D, 2221 / T, 223T, 224G / S / T, 225A / L / Q / R / T / V, 226K / L / P / R / S / T, 227P, 228D / F / G / I / L / P / Q / R / S / T / V, 229C / I, 230A / M / P / Q, or 231A / Q / R / T, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.Docket No. 5244.286WO3

[0044] In some embodiments, the engineered adenylate kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, or to the reference sequence corresponding to SEQ ID NO: 144, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ. ID NO: 144, or relative to the reference sequence corresponding to SEQ ID NO: 144.

[0045] In some embodiments, the engineered adenylate kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-231 of an even-numbered SEQ ID NO. of SEQ ID NOs: 252-286, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 252-286, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, or relative to the reference sequence corresponding to SEQ ID NO: 144.

[0046] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set at amino acid position(s) 15, 16, 37, 69, 80, 82, 112, 115, 170, 178, 187, 205, or 217, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, or relative to the reference sequence corresponding to SEQ ID NO: 144. In some aspects, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution set, or amino acid residue(s) 15T, 16C, 16G, 16T, 37M, 69V, 80S, 82L, 112C, 112T, 115C, 170M, 178A, 178L, 178V, 187D, 205C, or 217E, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, or relative to the reference sequence corresponding to SEQ ID NO: 144. In some aspects, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution set, or amino acid residue(s) Y15T, L16C, L16G, L16T, H37M, K69V, P80S, V82L, S112C, S112T, E115C, S170M, K178A, K178L, K178V, K187D, Q205C, or P217E, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, or relative to the reference sequence corresponding to SEQ ID NO: 144.

[0047] In some embodiments, the engineered adenylate kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 252, or to the reference sequence corresponding to SEQ ID NO: 252,Docket No. 5244.286WO3 wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 252, or relative to the reference sequence corresponding to SEQ ID NO: 252.

[0048] In some embodiments, the engineered adenylate kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-231 of an even-numbered SEQ ID NO. of SEQ ID NOs: 288-360, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 288-360, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 252, or relative to the reference sequence corresponding to SEQ ID NO: 252.

[0049] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set at amino acid position(s) 4 / 15, 14 / 15 / 93, 14 / 170 / 178, 14 / 214, 15 / 93 / 157 / 178 / 205 / 214, 15 / 115 / 157, 15 / 115 / 170, 15 / 170 / 180, 15 / 214, 93, 93 / 157 / 170 / 214, 93 / 205, 115, 157 / 170, 157 / 205 / 214, 178 / 180 / 214, 180, 214, 14, 14 / 15 / 157 / 170 / 178 / 180 / 214, 14 / 15 / 157 / 205, 14 / 157 / 170 / 178 / 180 / 205, 14 / 205, 15, 15 / 93 / 157 / 214, 15 / 93 / 180, 15 / 93 / 205, 15 / 157, 15 / 157 / 205, 15 / 178, 15 / 205, 93 / 157 / 205, 93 / 157 / 214, 93 / 170 / 205, 93 / 178 / 205, 93 / 180, or 205, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 252, or relative to the reference sequence corresponding to SEQ ID NO: 252.

[0050] In some aspects, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set, or amino acid residue(s) 4N / 15T, 14P / 15T / 93E, 14P / 170M / 178T, 14P / 214P, 15T / 93E / 157R / 178T / 205L / 214K, 15T / 115H / 157R, 15T / 115H / 170M, 15T / 170M / 180Q, 15T / 214P, 93E, 93E / 157R / 170M / 214K, 93E / 205L, 115H, 157R / 170M, 157R / 205L / 214P, 178T / 180Q / 214P, 180Q, 214P, 14P, 14P / 15T / 157R / 170M / 178T / 180Q / 214K, 14P / 15T / 157R / 205L, 14P / 157R / 170M / 178T / 180Q / 205L, 14P / 205L, 15T, 15T / 93E / 157R / 214P, 15T / 93E / 180Q, 15T / 93E / 205L, 15T / 157R, 15T / 157R / 205L, 15T / 178T, 15T / 205L, 93E / 157R / 205L, 93E / 157R / 214K, 93E / 170M / 205L, 93E / 178T / 205L, 93E / 180Q, or 205L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 252, or relative to the reference sequence corresponding to SEQ ID NO: 252.

[0051] In some aspects, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set, or amino acid residue(s) H4N / Y15T, A14P / Y15T / G93E, A14P / S170M / K178T, A14P / E214P, Y15T / G93E / P157R / K178T / Q205L / E214K, Y15T / E115H / P157R, Y15T / E115H / S170M, Y15T / S170M / E180Q, Y15T / E214P, G93E, G93E / P157R / S170M / E214K, G93E / Q205L, E115H, P157R / S170M, P157R / Q205L / E214P, K178T / E180Q / E214P, E180Q, E214P, A14P,Docket No. 5244.286WO3 A14P / Y15T / P157R / S170M / K178T / E180Q / E214K, A14P / Y15T / P157R / Q205L, A14P / P157R / S170M / K178T / E180Q / Q205L, A14P / Q205L, Y15T, Y15T / G93E / P157R / E214P, Y15T / G93E / E180Q, Y15T / G93E / Q205L, Y15T / P157R, Y15T / P157R / Q205L, Y15T / K178T, Y15T / Q205L, G93E / P157R / Q205L, G93E / P157R / E214K, G93E / S170M / Q205L, G93E / K178T / Q205L, G93E / E180Q, or Q205L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 252, or relative to the reference sequence corresponding to SEQ ID NO: 252.

[0052] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set at amino acid position(s) 180 / 205, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 252, or relative to the reference sequence corresponding to SEQ ID NO: 252. In some aspects, the amino acid sequence of the engineered adenylate kinase comprises or further comprises at least a substitution or substitution set 180Q / 205L, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 252, or relative to the reference sequence corresponding to SEQ ID NO: 252. In some aspects, the amino acid sequence of the engineered adenylate kinase further comprises at least a substitution or substitution set E180Q / Q205L wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 252, or relative to the reference sequence corresponding to SEQ ID NO: 252.

[0053] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set at amino acid position(s) 18, 31, 35, 38, 59, 62, 87, 104, 105, 109, 139, 143, 146, 149, 184, 202, 207, 219, 222, 223, 227, 228, and 229, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 402, or relative to the reference sequence corresponding to SEQ ID NO: 402.

[0054] In some embodiments, the amino acid sequence of the engineered adenylate kinase further comprises at least a substitution or substitution set of 18V, 31F, 31V, 31W, 35C, 35E, 38D, 59G, 62H, 87G, 104Q, 105Q, 109A, 139H, 1431, 146H, 149A, 149L, 149Q, 184P, 202E, 202G, 207N, 219L, 219T, 222A, 223V, 227S, 228L, 228S, and 229G, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 402, or relative to the reference sequence corresponding to SEQ ID NO: 402.

[0055] In some embodiments, the amino acid sequence of the engineered adenylate kinase further comprises at least a substitution or substitution set L18V, R31F, R31V, R31W, A35C, A35E, G38D, V59G, S62H, K87G, R104Q R105Q, Y109A, L139H, V143I, K146H, R149A, R149L, R149Q, M184P, K202E, K202G, A207N, V219L, V219T, V222A, L223V, G227S, V228L, V228S, and I229G wherein the amino acidDocket No. 5244.286WO3 positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 402, or relative to the reference sequence corresponding to SEQ ID NO: 402.

[0056] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set at amino acid position(s) 33, 33 / 54, 33 / 54 / 83 / 104 / 109, 33 / 54 / 104, 33 / 54 / 104 / 109, 33 / 54 / 104 / 109 / 207, 33 / 54 / 104 / 109 / 223, 33 / 54 / 104 / 154 / 223, 33 / 54 / 104 / 199 / 223, 33 / 54 / 104 / 223, 33 / 54 / 154, 33 / 54 / 154 / 199, 33 / 54 / 154 / 223, 33 / 54 / 207 / 223, 33 / 54 / 223, 33 / 104, 33 / 104 / 109, 33 / 104 / 109 / 199 / 223, 33 / 104 / 207, 33 / 104 / 223, 33 / 109, 33 / 109 / 199 / 207, 33 / 109 / 199 / 223, 33 / 109 / 202 / 223, 33 / 154, 33 / 154 / 223, 33 / 207, 33 / 223, 54, 54 / 83, 54 / 83 / 223, 54 / 104, 54 / 104 / 109, 54 / 104 / 223, 54 / 154 / 199, 55, 55 / 87, 55 / 87 / 105, 55 / 87 / 105 / 139, 55 / 87 / 105 / 139 / 184, 55 / 87 / 105 / 139 / 225, 55 / 87 / 105 / 184, 55 / 87 / 105 / 184 / 202 / 225, 55 / 87 / 105 / 225, 55 / 87 / 139 / 184, 55 / 87 / 184, 55 / 87 / 184 / 202 / 225, 55 / 87 / 184 / 225, 55 / 87 / 225, 55 / 105, 55 / 105 / 139 / 184, 55 / 105 / 139 / 184 / 225, 55 / 105 / 139 / 225, 55 / 105 / 184, 55 / 105 / 184 / 225, 55 / 105 / 225, 55 / 139, 55 / 139 / 184 / 225, 55 / 139 / 225, 55 / 184, 55 / 184 / 202, 55 / 184 / 202 / 225, 55 / 184 / 225, 55 / 225, 83, 87, 87 / 105, 87 / 105 / 139, 87 / 105 / 182 / 184 / 225, 87 / 105 / 184 / 202, 87 / 105 / 184 / 225, 87 / 139 / 184 / 225, 87 / 139 / 225, 87 / 184, 87 / 184 / 202, 87 / 202 / 204, 87 / 225, 104, 104 / 109, 104 / 109 / 154, 104 / 109 / 154 / 199, 104 / 109 / 199, 104 / 109 / 199 / 207, 104 / 109 / 199 / 207 / 223, 104 / 109 / 207, 104 / 207, 104 / 223, 105, 105 / 111 / 184 / 225, 105 / 139 / 184, 105 / 139 / 184 / 202, 105 / 139 / 184 / 225, 105 / 184, 105 / 184 / 202, 105 / 225, 109 / 199, 139 / 184, 139 / 184 / 225, 139 / 202 / 225, 139 / 225, 154, 154 / 199, 184, 184 / 202, 184 / 225, 199, 199 / 223, 202, 202 / 225, 207, 223, and 225, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 446, or relative to the reference sequence corresponding to SEQ ID NO: 446.

[0057] In some embodiments, the amino acid sequence of the engineered adenylate kinase further comprises at least a substitution or substitution set 33L, 33L / 54G, 33L / 54G / 83R / 104L / 109A, 33L / 54G / 104L, 33L / 54G / 104L / 109A, 33L / 54G / 104L / 109A / 207I, 33L / 54G / 104L / 154E / 223M, 33L / 54G / 104L / 199F / 223M, 33L / 54G / 104R / 109A / 223M, 33L / 54G / 104R / 223M, 33L / 54G / 154E, 33L / 54G / 154E / 199F, 33L / 54G / 154E / 223M, 33L / 54G / 207I / 223D, 33L / 54G / 223D, 33L / 104L / 223M, 33L / 104R, 33L / 104R / 109A, 33L / 104R / 109A / 199F / 223M, 33L / 104R / 207I, 33L / 109A, 33L / 109A / 199F / 207I, 33L / 109A / 199F / 223D, 33L / 109A / 199F / 223M, 33L / 109A / 202N / 223D, 33L / 154E, 33L / 154E / 223M, 33L / 207I, 33L / 223M, 54G, 54G / 83R, 54G / 83R / 223D, 54G / 104L / 223M, 54G / 104R, 54G / 104R / 109A, 54G / 154E / 199F, 55E, 55E / 87S, 55E / 87S / 105L / 225A, 55E / 87S / 105Q / 139A, 55E / 87S / 105Q / 139A / 184S, 55E / 87S / 105Q / 184S, 55E / 87S / 105S, 55E / 87S / 105S / 139A / 184Q, 55E / 87S / 105S / 139A / 225Q, 55E / 87S / 105S / 184Q, 55E / 87S / 105S / 184Q / 202E / 225Q, 55E / 87S / 139A / 184Q, 55E / 87S / 184Q, 55E / 87S / 184Q / 225Q, 55E / 87S / 184S, 55E / 87S / 184S / 202E / 225A, 55E / 87S / 184S / 225Q, 55E / 87S / 225Q, 55E / 105L, 55E / 105Q, 55E / 105Q / 139A / 184Q / 225A, 55E / 105Q / 139A / 225Q, 55E / 105Q / 184S, 55E / 105Q / 225Q, 55E / 105S, 55E / 105S / 139A / 184Q,Docket No. 5244.286WO3 55E / 105S / 139A / 225A, 55E / 105S / 184Q / 225A, 55E / 105S / 184Q / 225Q, 55E / 105S / 225A, 55E / 139A, 55E / 139A / 184Q / 225A, 55E / 139A / 225A, 55E / 184Q, 55E / 184Q / 202E, 55E / 184Q / 202E / 225A, 55E / 184Q / 202E / 225Q, 55E / 184Q / 225A, 55E / 184Q / 225Q, 55E / 184S, 55E / 184S / 202E, 55E / 184S / 202E / 225Q, 55E / 184S / 225Q, 55E / 225A, 55E / 225Q, 83R, 87S, 87S / 105L / 182L / 184S / 225Q, 87S / 105L / 184Q / 202E, 87S / 105L / 184S / 225Q, 87S / 105Q, 87S / 105Q / 139A, 87S / 105S, 87S / 139A / 184Q / 225Q, 87S / 139A / 184S / 225Q, 87S / 139A / 225Q, 87S / 184Q, 87S / 184S, 87S / 184S / 202E, 87S / 202E / 204D, 87S / 225A, 104L, 104L / 109A, 104L / 109A / 154E, 104L / 109A / 199F, 104L / 109A / 199F / 207I / 223M, 104L / 109A / 207I, 104L / 207I, 104R / 109A, 104R / 109A / 154E / 199F, 104R / 109A / 199F / 207I, 104R / 109A / 207I, 104R / 223M, 105L, 105L / 139A / 184Q / 225Q, 105L / 139A / 184S / 202E, 105Q, 105Q / 111N / 184Q / 225Q, 105Q / 139A / 184S / 225Q, 105Q / 225Q, 105S, 105S / 139A / 184Q, 105S / 139A / 184Q / 225A, 105S / 139A / 184Q / 225Q, 105S / 139A / 184S / 225A, 105S / 184Q, 105S / 184Q / 202E, 105S / 225Q, 109A / 199F, 139A / 184Q, 139A / 184S / 225Q, 139A / 202E / 225A, 139A / 225A, 154E, 154E / 199F, 184Q, 184Q / 202E, 184Q / 225A, 184S, 199F, 199F / 223M, 202E, 202E / 225A, 2071, 223D, 223M, 225A, and 225Q, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 446, or relative to the reference sequence corresponding to SEQ. ID NO: 446.

[0058] In some embodiments, the amino acid sequence of the engineered adenylate kinase further comprises at least a substitution or substitution set I33L, I33L / K54G, I33L / K54G / L83R / Q104L / Y109A, I33L / K54G / Q104L, I33L / K54G / Q104L / Y109A, I33L / K54G / Q104L / Y109A / A207I, I33L / K54G / Q104L / I154E / L223M, I33L / K54G / Q104L / Y199F / L223M, I33L / K54G / Q104R / Y109A / L223M, I33L / K54G / Q104R / L223M, I33L / K54G / I154E, I33L / K54G / I154E / Y199F, I33L / K54G / I154E / L223M, I33L / K54G / A207I / L223D, I33L / K54G / L223D, I33L / Q104L / L223M, I33L / Q104R, I33L / Q104R / Y109A, I33L / Q104R / Y109A / Y199F / L223M, I33L / Q104R / A207I, I33L / Y109A, I33L / Y109A / Y199F / A207I, I33L / Y109A / Y199F / L223D, I33L / Y109A / Y199F / L223M, I33L / Y109A / K202N / L223D, I33L / I154E, I33L / I154E / L223M, I33L / A207I, I33L / L223M, K54G, K54G / L83R, K54G / L83R / L223D, K54G / Q104L / L223M, K54G / Q104R, K54G / Q104R / Y109A, K54G / I154E / Y199F, Q55E, Q55E / K87S, Q55E / K87S / R105L / I225A, Q55E / K87S / R105Q / L139A, Q55E / K87S / R105Q / L139A / M184S, Q55E / K87S / R105Q / M184S, Q55E / K87S / R105S, Q55E / K87S / R105S / L139A / M184Q, Q55E / K87S / R105S / L139A / I225Q, Q55E / K87S / R105S / M184Q, Q55E / K87S / R105S / M184Q / K202E / I225Q, Q55E / K87S / L139A / M184Q, Q55E / K87S / M184Q, Q55E / K87S / M184Q / I225Q, Q55E / K87S / M184S, Q55E / K87S / M184S / K202E / I225A, Q55E / K87S / M184S / I225Q, Q55E / K87S / I225Q, Q55E / R105L, Q55E / R105Q, Q55E / R105Q / L139A / M184Q / I225A, Q55E / R105Q / L139A / I225Q, Q55E / R105Q / M184S, Q55E / R105Q / I225Q, Q55E / R105S, Q55E / R105S / L139A / M184Q, Q55E / R105S / L139A / I225A, Q55E / R105S / M184Q / I225A, Q55E / R105S / M184Q / I225Q, Q55E / R105S / I225A, Q55E / L139A, Q55E / L139A / M184Q / I225A, Q55E / L139A / I225A, Q55E / M184Q, Q55E / M184Q / K202E,Docket No. 5244.286WO3 Q55E / M184Q / K202E / I225A, Q55E / M184Q / K202E / I225Q, Q55E / M184Q / I225A, Q55E / M184Q / I225Q, Q55E / M184S, Q55E / M184S / K202E, Q55E / M184S / K202E / I225Q, Q55E / M184S / I225Q, Q55E / I225A, Q.55E / I225C L83R, K87S, K87S / R105L / V182 L / M 184S / I225CL K87S / R105L / M184Q / K202E, K87S / R105L / M184S / I225Q, K87S / R105Q, K87S / R105Q / L139A, K87S / R105S, K87S / L139A / M184Q / I225Q, K87S / L139A / M184S / I225Q, K87S / L139A / I225Q, K87S / M184Q, K87S / M184S, K87S / M184S / K202E, K87S / K202E / G204D, K87S / I225A, Q104L, Q104L / Y109A, Q104L / Y109A / I154E, Q104L / Y109A / Y199F, Q104L / Y109A / Y199F / A207I / L223M, Q104L / Y109A / A207I, Q104L / A207I, Q104R / Y109A, Q104R / Y109A / I154E / Y199F, Q104R / Y109A / Y199F / A207I, Q104R / Y109A / A207I, Q104R / L223M, R105L, R105L / L139A / M184Q / I225Q, R105L / L139A / M184S / K202E, R105Q, R105Q / D111N / M184Q / I225C R105Q / L139A / M184S / I225Q, R105Q / I225Q, R105S, R105S / L139A / M184Q, R105S / L139A / M184Q / I225A, R105S / L139A / M184Q / I225Q, R105S / L139A / M184S / I225A, R105S / M184Q, R105S / M184Q / K202E, R105S / I225Q, Y109A / Y199F, L139A / M184Q, L139A / M184S / I225Q, L139A / K202E / I225A, L139A / I225A, I154E, I154E / Y199F, M184Q, M184Q / K202E, M184Q / I225A, M184S, Y199F, Y199F / L223M, K202E, K202E / I225A, A207I, L223D, L223M, I225A, and l225Qwherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 446, or relative to the reference sequence corresponding to SEQ ID NO: 446.

[0059] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set at amino acid position(s) 15 / 61 / 136 / 212, 16, 16 / 53, 16 / 53 / 68, 16 / 53 / 68 / 84 / 170 / 223, 16 / 53 / 68 / 170 / 192, 16 / 53 / 170, 16 / 53 / 170 / 218 / 223, 16 / 53 / 223, 16 / 68, 16 / 68 / 84 / 170 / 218, 16 / 68 / 170, 16 / 68 / 170 / 192 / 218 / 223, 16 / 68 / 170 / 218, 16 / 68 / 192 / 218 / 223, 16 / 68 / 218, 16 / 68 / 218 / 223, 16 / 68 / 223, 16 / 84, 16 / 84 / 170, 16 / 84 / 170 / 192, 16 / 84 / 170 / 192 / 218, 16 / 170, 16 / 170 / 192, 16 / 170 / 192 / 218, 16 / 170 / 218, 16 / 170 / 218 / 223, 16 / 170 / 223, 16 / 192, 16 / 192 / 223, 16 / 218 / 223, 16 / 223, 16 / 223 / 225, 17, 17 / 61 / 136, 17 / 61 / 136 / 205 / 212, 17 / 61 / 205, 17 / 66 / 136 / 212, 17 / 66 / 205, 17 / 66 / 205 / 217, 17 / 117 / 212, 17 / 136, 17 / 136 / 205, 17 / 205, 17 / 217, 53, 53 / 68, 53 / 68 / 84 / 170 / 218, 53 / 68 / 218, 53 / 84 / 170 / 218, 53 / 84 / 192 / 223, 53 / 170, 53 / 170 / 218, 53 / 170 / 223, 53 / 192, 53 / 223, 61, 61 / 66, 61 / 205, 66, 68, 68 / 84, 68 / 84 / 218, 68 / 170, 68 / 170 / 192 / 218, 68 / 170 / 218, 68 / 170 / 223, 68 / 192, 68 / 218, 84, 84 / 170 / 192 / 223, 84 / 170 / 218, 84 / 192, 84 / 192 / 218 / 223, 84 / 192 / 223, 84 / 218, 136, 136 / 205, 136 / 205 / 212, 136 / 212, 170, 170 / 192, 170 / 192 / 218, 170 / 192 / 218 / 223, 170 / 192 / 223, 170 / 218, 170 / 218 / 223, 183, 192, 192 / 218 / 223, 192 / 223, 205, 212 / 217, 217, 218, 218 / 223, and 223, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ. ID NO: 710, or relative to the reference sequence corresponding to SEQ ID NO: 710.

[0060] In some embodiments, the amino acid sequence of the engineered adenylate kinase further comprises at least a substitution or substitution set 15H / 61G / 136L / 212A, 16M, 16M / 53D, 16M / 53D / 68V, 16M / 53D / 68V / 84A / 170K / 223M, 16M / 53D / 68V / 170K / 192L, 16M / 53D / 170K,Docket No. 5244.286WO3 16M / 53D / 170K / 218L / 223M, 16M / 53D / 223M, 16M / 68V, 16M / 68V / 84A / 170K / 218V, 16M / 68V / 170K, 16M / 68V / 170K / 192L / 218V / 223M, 16M / 68V / 170K / 218V, 16M / 68V / 192L / 218L / 223M, 16M / 68V / 218V, 16M / 68V / 218V / 223M, 16M / 68V / 223M, 16M / 84A / 170K, 16M / 84A / 170K / 192L, 16M / 84A / 170K / 192L / 218V, 16M / 84T, 16M / 170K, 16M / 170K / 192L, 16M / 170K / 192L / 218L, 16M / 170K / 218L, 16M / 170K / 218L / 223M, 16M / 170K / 218V, 16M / 170K / 223M, 16M / 192L, 16M / 192L / 223M, 16M / 192M, 16M / 218V / 223M, 16M / 223M, 16M / 223M / 225T, 171, 17I / 61G / 136L, 17I / 61G / 136R / 205I / 212A, 17I / 61G / 205I, 17I / 66D / 136L / 212A, 17I / 66D / 2051, 17I / 66D / 205I / 217A, 17I / 117R / 212A, 17I / 136L / 2051, 17I / 136R, 171 / 2051, 17I / 217A, 53D, 53D / 68V, 53D / 68V / 84A / 170K / 218V, 53D / 68V / 218L, 53D / 84A / 170K / 218L, 53D / 84A / 192L / 223M, 53D / 170K, 53D / 170K / 218V, 53D / 170K / 223M, 53D / 192L, 53D / 223M, 61G, 61G / 66D, 61G / 205I, 66D, 68V, 68V / 84A, 68V / 84T / 218L, 68V / 170K, 68V / 170K / 192L / 218V, 68V / 170K / 218L, 68V / 170K / 223M, 68V / 192L, 68V / 218V, 84A, 84A / 170K / 218V, 84A / 192L, 84A / 192L / 218L / 223M, 84A / 192L / 223M, 84A / 218V, 84T, 84T / 170K / 192L / 223M, 84T / 218L, 136L, 136R, 136R / 205I, 136R / 205I / 212A, 136R / 212A, 170K, 170K / 192L, 170K / 192L / 218L / 223M, 170K / 192L / 218V, 170K / 192L / 218V / 223M, 170K / 192L / 223M, 170K / 218V, 170K / 218V / 223M, 183L, 192L, 192L / 218L / 223M, 192L / 218V / 223M, 192L / 223M, 2051, 212A / 217A, 217A, 218L, 218L / 223M, 218V, 218V / 223M, and 223M, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 710, or relative to the reference sequence corresponding to SEQ ID NO: 710.

[0061] In some embodiments, the amino acid sequence of the engineered adenylate kinase further comprises at least a substitution or substitution set Y15H / S61G / V136L / S212A, L16M, L16M / A53D, L16M / A53D / L68V, L16M / A53D / L68V / R84A / S170K / L223M, L16M / A53D / L68V / S170K / K192L, L16M / A53D / S170K, L16M / A53D / S170K / I218L / L223M, L16M / A53D / L223M, L16M / L68V, L16M / L68V / R84A / S170K / I218V, L16M / L68V / S170K, L16M / L68V / S170K / K192L / I218V / L223M, L16M / L68V / S170K / I218V, L16M / L68V / K192L / I218L / L223M, L16M / L68V / I218V, L16M / L68V / I218V / L223M, L16M / L68V / L223M, L16M / R84A / S170K, L16M / R84A / S170K / K192L, L16M / R84A / S170K / K192L / I218V, L16M / R84T, L16M / S170K, L16M / S170K / K192L, L16M / S170K / K192L / I218L, L16M / S170K / I218L, L16M / S170K / I218L / L223M, L16M / S170K / I218V, L16M / S170K / L223M, L16M / K192L, L16M / K192L / L223M, L16M / K192M, L16M / I218V / L223M, L16M / L223M, L16M / L223M / A225T, V17I, V17I / S61G / V136L, V17I / S61G / V136R / L205I / S212A, V17I / S61G / L205I, V17I / W66D / V136L / S212A, V17I / W66D / L205I, V17I / W66D / L205I / P217A, V17I / Q117R / S212A, V17I / V136L / L205I, V17I / V136R, V17I / L2051, V17I / P217A, A53D, A53D / L68V, A53D / L68V / R84A / S170K / I218V, A53D / L68V / I218L, A53D / R84A / S170K / I218L, A53D / R84A / K192L / L223M, A53D / S170K, A53D / S170K / I218V, A53D / S170K / L223M, A53D / K192L, A53D / L223M, S61G, S61G / W66D, S61G / L205I, W66D, L68V, L68V / R84A, L68V / R84T / I218L, L68V / S170K, L68V / S170K / K192L / I218V, L68V / S170K / I218L, L68V / S170K / L223M, L68V / K192L, L68V / I218V, R84A,Docket No. 5244.286WO3 R84A / S170K / I218V, R84A / K192L, R84A / K192L / I218L / L223M, R84A / K192L / L223M, R84A / I218V, R84T, R84T / S170K / K192L / L223M, R84T / I218L, V136L, V136R, V136R / L205I, V136R / L205I / S212A, V136R / S212A, S170K, S170K / K192L, S170K / K192 L / 1218L / L223 M, S170K / K192L / I218V, S170K / K192L / I218V / L223M, S170K / K192L / L223M, S170K / I218V, S170K / I218V / L223M, R183L, K192L, K192L / I218L / L223M, K192L / I218V / L223M, K192L / L223M, L205I, S212A / P217A, P217A, I218L, I218L / L223M, 1218V, I218V / L223M, and L223M wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 710, or relative to the reference sequence corresponding to SEQ ID NO: 710.

[0062] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set at amino acid position(s) 46, 55, 81, 87, 117, 121, 126, 142, 148 / 202, 149, 159, 162, 163, 169, 192, 194, 215, and 225, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 808, or relative to the reference sequence corresponding to SEQ ID NO: 808.

[0063] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises the substitution set46E, 55Q, 81P, 87R, 117G, 121V, 126C, 142V, 148Q / 202N, 149M, 159V, 162D, 162M, 162V, 163E, 163R, 169E, 192V, 194L, 215V, and 225H, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 808, or relative to the reference sequence corresponding to SEQ ID NO: 808.

[0064] In some embodiments, the amino acid sequence of the engineered adenylate kinase further comprises at least a substitution or substitution set D46E, E55Q, V81P, K87R, Q117G, L121V, L126C, R142V, G148Q / K202N, R149M, K159V, E162D, E162M, E162V, L163E, L163R, V169E, K192V, Q194L, 1215V, and A225H wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 808, or relative to the reference sequence corresponding to SEQ ID NO: 808.

[0065] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least one substitution set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2 or 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0066] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set of an engineered adenylate kinase variant set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2 or 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, 252, 402, 446,Docket No. 5244.286WO3 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0067] In some embodiments, the engineered adenylate kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference amino acid sequence comprising a substitution or substitution set of an engineered adenylate kinase variant set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2 or 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ. ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0068] In some embodiments, the engineered adenylate kinase comprises an amino acid sequence comprising residues 12-231 of an even-numbered SEQ ID NO. of SEQ ID NOs: 18-360 and 372-1018, or comprises an even-numbered SEQ ID NO. of SEQ ID NOs: 18-360 and 372-1018.

[0069] In some embodiments, the engineered adenylate kinase comprises an amino acid sequence comprising residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or wherein the amino acid sequence comprises SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0070] In some embodiments, the engineered adenylate kinase has adenylate kinase activity and at least one improved property as compared to a reference adenylate kinase. In some embodiments, the engineered adenylate kinase has an improved property selected from i) increased activity on unmodified nucleoside monophosphate (NMP), ii) increased stability, iii) increased thermostability, iv) increased activity on 2'-fluoro modified nucleoside monophosphate, and v) increased activity on 2'-O-methyl modified nucleoside monophosphate, or any combinations of i), ii), iii), iv), and v) as compared to a reference adenylate kinase. In some embodiments, the reference adenylate kinase has an amino acid sequence corresponding to residues 12-231 of SEQ ID NO: 16, 144, 252, 402, 446, 710, or 808, or an amino acid sequence corresponding to SEQ ID NO: 16, 144, 252, 402, 446, 710, or 808. In some embodiments, the reference adenylate kinase has an amino acid sequence corresponding to residues 12-231 of SEQ ID NO: 16, or an amino acid sequence corresponding to SEQ ID NO: 16.

[0071] In some embodiments, the engineered adenylate kinase is purified. In some embodiments, the engineered adenylate kinase is immobilized on a support medium.

[0072] In a further aspect, the present disclosure provides a recombinant polynucleotide comprising a polynucleotide sequence encoding an engineered adenylate kinase described herein.

[0073] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,Docket No. 5244.286WO3 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34-693 of SEQ ID NO: 143, 251, 401, 445, 709, or 807, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 143, 251, 401, 445, 709, or 807, wherein the recombinant polynucleotide encodes an adenylate kinase.

[0074] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34-693 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 17-359 and 371-1017, or to a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO. of SEQ ID NOs: 17-359 and 371-1017, wherein the recombinant polynucleotide encodes an engineered adenylate kinase.

[0075] In some embodiments, the polynucleotide sequence encoding the engineered adenylate kinase has preferred codons or is codon optimized. In some embodiments, the polynucleotide sequence is codon optimized for expression in a bacterial cell, fungal cell, or a mammalian cell.

[0076] In some embodiments, the polynucleotide sequence comprises nucleotide residues 34-693 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 17-359 and 371-1017, or comprises SEQ ID NO: 17-359 and 371-1017.

[0077] In some embodiments, the polynucleotide sequence comprises nucleotide residues 34-693 of SEQ ID NO: 143, 251, 401, 445, 709, or 807, or comprises SEQ ID NO: 143, 251, 401, 445, 709, or 807.

[0078] In another aspect, the present disclosure provides an expression vector comprising a recombinant polynucleotide encoding an engineered adenylate kinase described herein. In some embodiments, the recombinant polynucleotide of the expression vector is operably linked to a control sequence. In some aspects, the control sequences comprises a promoter.

[0079] In a further aspect, the present disclosure also provides a host cell comprising an expression vector comprising a recombinant polynucleotide encoding an engineered adenylate kinase. In some aspects, the host cell is a prokaryotic cell or eukaryotic cell. In some embodiments, the host cell is a bacterial cell, fungal cell, insect cell, or mammalian cell.

[0080] In a further aspect, the host cells are used for producing an engineered adenylate kinase. In some embodiments, a method of producing an engineered adenylate kinase polypeptide comprises culturing a host cell comprising an expression vector described herein under suitable culture conditions such that the engineered adenylate kinase polypeptide is produced. In some embodiments, the method further comprises recovering or isolating the adenylate kinase polypeptide from the cultureDocket No. 5244.286WO3 and / or host cells. In some embodiments, the method further comprises purifying the adenylate kinase polypeptide.

[0081] In a further aspect, the engineered adenylate kinase is provided as a composition. In some embodiments, the composition further comprises a nucleoside monophosphate substrate. In some embodiments, the nucleoside in the composition is a modified nucleoside monophosphate. In some aspects, the modified nucleoside monophosphate has a modified sugar moiety, a modified nucleobase, a modified phosphate or a combination thereof.

[0082] In a further aspect, the engineered adenylate kinase is used for converting a nucleoside monophosphate to the corresponding nucleoside diphosphate. In some embodiments, a method of converting a nucleoside monophosphate (NMP) to the nucleoside diphosphate (NDP), comprises contacting a nucleoside monophosphate with an engineered adenylate kinase described herein in the presence of substrate NTP under suitable reaction conditions to convert the nucleoside monophosphate to the corresponding nucleoside diphosphate.

[0083] In some embodiments of the method, the nucleoside monophosphate comprises a nucleobase selected from adenine, cytosine, guanine, thymine, uracil, xanthine, hypoxanthine, 2,6-diaminopurine, purine, 6,8-diaminopurine, 5-methylcytosine (m5C), 2-th iouridine, pseudouridine, dihydrouridine, inosine, and 7-methylguanosine (m7G).

[0084] In some embodiments, the nucleoside monophosphate has at the 2'-position of the sugar moiety a H or OH.

[0085] In some embodiments, the nucleoside monophosphate is AMP, GMP, UMP, CMP, orTMP, and wherein the nucleoside monophosphate has at the 2'-position of the sugar moiety an OH, thereby by resulting in corresponding product rADP, rGDP, rUDP, rCDP, or rTDP, respectively.

[0086] In some embodiments, the nucleoside monophosphate is AMP, GMP, UMP, CMP, orTMP, and wherein the nucleoside monophosphate has at the 2'-position of the sugar moiety an H, thereby by resulting in corresponding product dADP, dGDP, dUDP, dCDP, or dTDP, respectively.

[0087] In some embodiments of the method, the substrate nucleoside monophosphate is an unmodified nucleoside monophosphate.

[0088] In some embodiments of the method, the substrate nucleoside monophosphate is a modified nucleoside monophosphate. In some embodiments, the modified nucleoside monophosphate comprises a modified sugar moiety, modified nucleobase, or modified phosphate, or any combinations thereof. In some embodiments, the modified nucleoside monophosphate comprises a modified sugar moiety. In some embodiments, the modified sugar moiety is a modified 2'-position of the sugar moiety. In some aspects, the modified 2'-position 2 is a 2'-O-R', wherein in R' is alkyl, aminoalkyl, cyanoalkyl, -Docket No. 5244.286WO3 C(O)-a Ikyl, or 2'-halo. In some aspects, the modified 2'-position is 2'-O-alkyl is 2'-O-CH3, 2'-O-CH2CH3, 2'-O-(2-methoxyethyl), 2'-O-allyl, 2'-0-propargyl, 2'-0-aminoethyl, 2'-0-cyanoethyl, or 2'-O-acetalester. In some aspects, the 2'-position is 2'-halo. In some aspects, the 2'-position is 2'-F, 2'-CL, or 2'-Br.

[0089] In some embodiments of the method, the modified sugar moiety is modified at the 3'-position. In some aspects, the 3'-position of the sugar moiety comprises a blocking group, wherein the blocking group is formate, benzoylformate, acetate, propionate, isobutyrate, aminoxy (-ONH2), O-methyl, O-methoxymethyl, O-methylthiomethyl, O-benzyloxymethyl, O-allyl, 3'-0-(2-nitrobenzyl), O-azidomethyl (O-CH2N3), O-tert-butyldithiomethyl, phosphate, diphosphate, or triphosphate.

[0090] In some embodiments of the methods, the modified nucleoside monophosphate comprises a modified nucleobase. In some aspects, the modified nucleobase is selected from 5-bromo-uracil, 5-iodo-uracil, 6-mCEPh-purine, 6-phenylpyrrolocytidine, N2-alkyl 8-oxoguanosine, difluorotoluene, difluorobenzene, dichlorobenzene, imidazole, and benzimidazole.

[0091] In some embodiments, the modified nucleoside monophosphate comprises a modified phosphate. In some aspects, the modified nucleoside monophosphate comprises a nucleoside 5'-thiophosphate, thereby resulting in product nucleoside 5'-l-thiodiphosphate (NDPaS). In some aspects, product NDPaS is the (Rp)-NDPaS, (Sp)-NDPaS, or a mixture of (Rp)-NDPaS and (Sp)-NDPaS diastereomers. Certain aspects of the methods further comprise the step of separating the (Rp)-NDPaS and (Sp)-NDPaS diastereomers.

[0092] In some embodiments, of the method, the phosphate donor NTP is rATP or dATP. In some aspects of the methods, the phosphate donor NTP has the same nucleotide structure of the nucleoside monophosphate.

[0093] In some embodiments of the methods, the method further comprises regenerating NTP using an NTP regenerating system. In some aspects of the methods, the NTP regenerating system comprises creatine kinase, pyruvate kinase, polyphosphate kinase, and / or acetate-kinase. In some aspects of the methods, the NTP regenerating system is acetate kinase and substrate acetyl phosphate. In some aspects, the NTP regenerating system comprises pyruvate oxidase and substrates pyruvate and inorganic phosphate.

[0094] In some embodiments, the method comprises a catalase.

[0095] In some embodiments, the application provides a modified nucleoside-5'-0-thiotriphosphate comprising a modified nucleobase produced by an engineered adenylate kinase of the application.

[0096] In some embodiments, a diastereomeric mixture of nucleotides produced by an engineered adenylate kinase of the application is provided.Docket No. 5244.286WO3

[0097] In some embodiments an NTP with a phosphate group at the 3' position of the sugar ( NQP) produced by an engineered adenylate kinase is provided. In some aspects, the NQP comprises a modification of the nucleoside, NTP and / or NQP at the 2' position of the sugar. In some aspects, the NQP further comprises a modification of the nucleoside, NTP and / or NQP at the nucleobase comprising a noncanonical nucleobase known to those skilled in the art, a removable tag, a cleavable linker, or a radio, a photo, and / or a chemical sensor; a modification of the nucleoside, NTP and / or NQP at the phosphate chain, optionally comprising an a-phosphothioate linkage or other non-phosphodiester linkage; or a combination of a modification at the nucleobase and a modification at the phosphate chain.DETAILED DESCRIPTION

[0098] The present disclosure provides engineered adenylate kinases for the interconversion of ATP:AMP, and recombinant polynucleotides encoding the engineered adenylate kinases. In some embodiments, the engineered adenylate kinases has adenylate kinase activity, and one or more improved properties as compared to the parent wild-type enzyme, including increased activity on NDPs, including NDPs other than ADP, and increased activity on modified NDPs.Abbreviationsand Definitions

[0099] In reference to the present invention, the technical and scientific terms used in the descriptions herein will have the meanings commonly understood by one of ordinary skill in the art, unless specifically defined otherwise. Accordingly, the following terms are intended to have the following meanings.

[0100] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a polypeptide" includes more than one polypeptide.

[0101] Similarly, "comprise," "comprises," "comprising" "include," "includes," and "including" are interchangeable and not intended to be limiting. Thus, as used herein, the term "comprising" and its cognates are used in their inclusive sense ( / .e., equivalent to the term "including" and its corresponding cognates).

[0102] It is to be further understood that where descriptions of various embodiments use the term "comprising," those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language "consisting essentially of" or "consisting of."

[0103] "About" means an acceptable error for a particular value. In some instances, "about" means within 0.05%, 0.5%, 1.0%, or 2.0%, of a given value range. In some instances, "about" means within 1, 2, 3, or 4 standard deviations of a given value.Docket No. 5244.286WO3

[0104] "EC" number refers to the Enzyme Nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reactions they catalyze.

[0105] "ATCC" refers to the American Type Culture Collection whose biorepository collection includes genes and strains.

[0106] " NCBI" refers to National Center for Biological Information and the sequence databases provided therein.

[0107] "Adenylate kinase," "AdyK," or "AK" refers to an enzyme that catalyzes the interconversion of ATP, ADP, and AMP through transfer of phosphoryl groups. In some embodiments, adenylate kinase also includes enzymes capable of catalyzing the interconversion of NTP, NDP and NMP. In some embodiments, adenylate kinase are enzymes classified in EC 2.7.3.4.

[0108] "Creatine kinase" refers to an enzyme that catalyzes the reversible interconversion of creatine:ATP to creatine phosphate:ADP.

[0109] "Polyphosphate kinase" refers to an enzyme that catalyzes the transfer of phosphate group(s) from high-energy, phosphate-donating molecules, such as polyphosphate (PolyPn), to specific substrates / molecules. Two families of polyphosphate kinases, PPKK1 and PPK2, are known. PPKls preferentially synthesize polyphosphate from NTP and the corresponding reverse reaction, and PPK2s preferentially consume polyphosphate to phosphorylate nucleoside mono- or diphosphates, and the corresponding reverse reactions. In some embodiments, polyphosphate kinase includes enzymes classified in EC 2.7.4.1.

[0110] "Acetate kinase ("AcK") refers to enzymes that are capable of catalyzing the phosphorylation of nucleoside diphosphates or analogues thereof, to nucleoside triphosphates or the corresponding analogues, using acetyl phosphate or another phosphoryl group donor. Acetate kinases as used herein includes naturally occurring, wild-type enzymes or engineered enzymes. In some embodiments, acetate kinases are naturally occurring, wild-type basic metabolic enzymes found primarily in prokaryotes that catalyze the following reaction: acetate + ATP <->acetyl phosphate + ADP. Acetyl phosphate is an intermediate in the formation of acetyl-CoA. In some embodiments, acetate kinases are derived from the naturally occurring, wild-type enzymes.

[0111] "Adenosine kinase," "AdoK," or "Adk," refer to an enzyme that catalyzes the phosphorylation of adenosine (A or ADO) to adenosine-5'-monophosphate (AMP). In some embodiments, adenosine kinase is classified in EC 2.7.1.20. Although the primary substrate is adenosine, adenosine kinases as used herein include enzymes that act on other nucleosides.Docket No. 5244.286WO3

[0112] "Pyruvate oxidase" or "Pox" refers to an enzyme that catalyzes the catalyzes the reaction between pyruvate, inorganic phosphate, and oxygen to generate acetyl phosphate and H2O2. In some embodiments, pyruvate oxidase include enzymes classified in EC 1.2. 3.3.

[0113] " Catalase" refers to an enzyme that converts hydrogen peroxide (H2O2) to H2O and O2. Catalase can be used to remove residual hydrogen peroxide in applications where hydrogen peroxide is present or is a product in a process. In some embodiments, catalases includes enzymes classified in EC 1.11.1.6.

[0114] "Protein," "polypeptide," and "peptide" are used interchangeably herein to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). Included within this definition are D-and L-amino acids, and mixtures of D- and L-amino acids, as well as polymers comprising D- and L-amino acids, and mixtures of D- and L-amino acids.

[0115] " Fusion protein," and "chimeric protein" and "chimera" refer to hybrid proteins created through the joining of two or more polynucleotides that originally encode separate proteins. In some embodiments, fusion proteins are created by recombinant technology (e.g., molecular biology techniques known in the art).

[0116] "Amino acids" are referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by IUPAC-IUB Biochemical Nomenclature Commission.Nucleotides, likewise, may be referred to by their commonly accepted single letter codes. The abbreviations used for the genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartate (Asp or D), cysteine (Cys or C), glutamate (Glu or E), glycine (Gly or G), glutamine (Gin or Q.), histidine (His or H), isoleucine (lie or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Vai or V). When the three-letter abbreviations are used, unless specifically preceded by an "L" or a "D" or clear from the context in which the abbreviation is used, the amino acid may be in either the L- or D-configuration about a-carbon (Ca). For example, whereas "Ala" designates alanine without specifying the configuration about the a-carbon, "D-Ala" and "L-Ala" designate D-alanine and L-alanine, respectively. When the one-letter abbreviations are used, upper case letters designate amino acids in the L-configuration about the a-carbon and lower case letters designate amino acids in the D-configuration about the a-carbon. For example, "A" designates L-alanine and "a" designates D-alanine. When polypeptide sequences are presented as a string of one-letter or three-letter abbreviations (or mixtures thereof), the sequences are presented in the amino (N) to carboxy (C) direction in accordance with common convention.Docket No. 5244.286WO3

[0117] "Polynucleotide" and "nucleic acid' refer to two or more nucleotides that are covalently linked together. The polynucleotide may be wholly comprised of ribonucleotides (i.e., RNA), wholly comprised of 2' deoxyribonucleotides (i.e., DNA), or comprised of mixtures of ribo- and 2' deoxyribonucleotides. While the nucleosides will typically be linked together via standard phosphodiester linkages, the polynucleotides may include one or more non-standard linkages. The polynucleotide may be singlestranded or double-stranded, or may include both single-stranded regions and double-stranded regions. Moreover, while a polynucleotide will typically be composed of the naturally occurring encoding nucleobases (i.e., adenine, guanine, uracil, thymine and cytosine), it may include one or more modified and / or synthetic nucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc. In some embodiments, such modified or synthetic nucleobases are nucleobases encoding amino acid sequences.

[0118] The abbreviations used for the genetically encoding nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless specifically delineated, the abbreviated nucleosides may be either ribonucleosides or 2'-deoxyribonucleosides. The nucleosides may be specified as being either ribonucleosides or 2'-deoxyribonucleosides on an individual basis or on an aggregate basis. When nucleic acid sequences are presented as a string of one-letter abbreviations, the sequences are presented in the 5' to 3' direction in accordance with common convention, and the phosphates are not indicated.

[0119] "Nucleobase" or "base" refers to those naturally occurring and synthetic heterocyclic moieties commonly known to those who utilize nucleic acid or polynucleotide technology to thereby generate polymers that can hybridize to polynucleotides in a sequence-specific manner. Non-limiting examples of nucleobases include, among others, adenine, cytosine, guanine, thymine, uracil, 5-propynyl-uracil, 2-thio-5-propynyl-uracil, 5-methylcytosine, pseudoisocytosine, pseudoisouridine, 2-thiouracil and 2-thiothymine, 2-aminopurine, N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine, N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine) and N8-(7-deaza-8-aza-adenine).

[0120] " Nucleoside" refers to glycosylamines comprising a nucleobase, and a 5-carbon sugar (e.g., ribose, deoxyribose, or arabinose). Non-limiting examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine, and inosine. In contrast, the term "nucleotide" refers to the glycosylamines comprising a nucleobase, a 5-carbon sugar, and one or more phosphate groups, as further described herein. In some embodiments, nucleosides can be phosphorylated by kinases to produce nucleotides.

[0121] "Nucleoside monophosphate" or "NMP" refers to glycosylamines comprising a nucleobase, a 5-carbon sugar (e.g., ribose, deoxyribose, or arabinose), and a phosphate moiety at the 5'-position. In some embodiments herein, "nucleoside monophosphate" is abbreviated as "NMP". Non-limiting examples of nucleoside monophosphates include cytidine monophosphate (CMP), uridineDocket No. 5244.286WO3 monophosphate (UMP), adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), and inosine monophosphate (IMP). In some embodiments, "nucleoside monophosphate" may refer to a non-natural nucleoside monophosphate. The terms "nucleoside" and "nucleotide" may be used interchangeably in some contexts.

[0122] "Nucleoside diphosphate" or "NDP" refers to glycosylamines comprising a nucleobase, a 5-carbon sugar (e.g., ribose, deoxyribose, or arabinose), and a diphosphate (i.e., pyrophosphate) moiety at the 5'-position. In some embodiments herein, "nucleoside diphosphate" is abbreviated as "NDP". Non-limiting examples of nucleoside diphosphates include cytidine diphosphate (CDP), uridine diphosphate (UDP), adenosine diphosphate (ADP), guanosine diphosphate (GDP), thymidine diphosphate (TDP), and inosine diphosphate (IDP). In some embodiments, "nucleoside diphosphate" may refer to a non-natural nucleoside diphosphate. The terms "nucleoside" and "nucleotide" may be used interchangeably in some contexts.

[0123] "Nucleoside triphosphate" or "NTP" refers to glycosylamines comprising a nucleobase, a 5-carbon sugar (e.g., ribose, deoxyribose, or arabinose), and a triphosphate moiety at the 5'-position. In some embodiments herein, "nucleoside triphosphate" is abbreviated as "NTP". Non-limiting examples of nucleoside triphosphates include cytidine triphosphate (CTP), uridine triphosphate (UTP), adenosine triphosphate (ATP), guanosine triphosphate (GTP), thymidine triphosphate (TTP), and inosine triphosphate (ITP). In some embodiments, "nucleoside triphosphate" may refer to a non-natural nucleoside triphosphate. The terms "nucleoside" and "nucleotide" may be used interchangeably in some contexts.

[0124] "Modified" or "non-natural" in context of a nucleoside or nucleotide refers to a nucleoside or nucleotide that has been altered to a non-naturally occurring nucleoside or nucleotide. In some embodiments, modifications to nucleoside or nucleotides include modifications to the nucleobase, sugar moiety, and / or phosphate. In some embodiments, the common modifications of the 2'-position of the sugar residue with fluoro (F) or -O-CH3 is denoted by "fN" and "mN", respectively, where N denotes the nucleobase on the nucleoside or nucleotide. In some embodiments, the presence of a 5'-thiophosphate is denoted by "*N".

[0125] "Locked nucleoside" or "locked nucleotide" refers to nucleoside or nucleotide, respectively, in which the ribose moiety is modified with a bridge connecting the 2' oxygen and 4' carbon (see, e.g., Obika et al., Tetrahedron Letters, 1997, 38(50) :8735- 8738; Orum et al., Current Pharmaceutical Design, 2008, 14(11):1138-1142). In some embodiments, the bridge is a methylene or ethylene bridge. In some embodiments, the ribose moiety of the locked nucleoside or locked nucleotide is in the C3'-endo (beta-D) or C2'-endo (alpha-L) conformation.Docket No. 5244.286WO3

[0126] "Biocatalysis, " "biocatalytic," "biotransformation," and "biosynthesis" refer to the use of enzymes to perform chemical reactions on organic compounds.

[0127] "Wild-type" and "naturally occurring" refer to the form found in nature. For example, a wildtype polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and which has not been intentionally modified by human manipulation.

[0128] "Recombinant," "engineered," "variant," "non-natural," and "non-naturally occurring" when used with reference to a cell, nucleic acid, or polypeptide, refers to a material, or a material corresponding to the natural or native form of the material, which has been modified in a manner that would not otherwise exist in nature. In some embodiments, the cell, nucleic acid or polypeptide is identical a naturally occurring cell, nucleic acid or polypeptide, but is produced or derived from synthetic materials and / or by manipulation using recombinant techniques. Non-limiting examples include, among others, recombinant cells expressing genes that are not found within the native (nonrecombinant) form of the cell or express native genes that are otherwise expressed at a different level.

[0129] "Percent (%) sequence identity" is used herein to refer to comparisons among polynucleotides or polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences. Optimal alignment of sequences for comparison can be conducted by any suitable method, including, but not limited to the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 1981, 2:482), by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 1970, 48:443), by the search for similarity method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA., 1988, 85:2444), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection, as known in the art. Examples of algorithms that areDocket No. 5244.286WO3 suitable for determining percent sequence identity and sequence similarity include, but are not limited to the BLAST and BLAST 2.0 algorithms, which are described by Altschul et al. (See Altschul et al., J. Mol. Biol., 1990, 215:403-410; and Altschul et al., NucL Acids Res., 1977, 25(17):3389-3402, respectively). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as, the neighborhood word score threshold (See, Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (See, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89:10915). Exemplary determination of sequence alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison Wl), using default parameters provided.

[0130] "Substantial identity" refers to a polynucleotide or polypeptide sequence that has at least 80 percent sequence identity, at least 85 percent identity, at least between 89 to 95 percent sequence identity, or more usually, at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 residue positions, frequently over a window of at least 30-50 residues, wherein the percentage of sequence identity is calculated by comparing the reference sequence to a sequence that includes deletions or additions which total 20 percent or less of the reference sequence over the window of comparison. In some specific embodiments applied to polypeptides, the term "substantial identity" means that two polypeptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 89 percent sequence identity, at least 95 percent sequenceDocket No. 5244.286WO3 identity or more (e.g., 99 percent sequence identity). In some embodiments, residue positions that are not identical in sequences being compared differ by conservative amino acid substitutions.

[0131] “ Reference sequence" refers to a defined sequence used as a basis for a sequence and / or activity comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotide or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, at least 100 residues in length or the full length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing sequences of the two polynucleotides or polypeptides over a "comparison window" to identify and compare local regions of sequence similarity. In some embodiments, a "reference sequence" can be based on a primary amino acid sequence, where the reference sequence is a sequence that can have one or more changes in the primary sequence.

[0132] "Comparison window" refers to a conceptual segment of contiguous nucleotide positions or amino acids residues wherein a sequence may be compared to a reference sequence. In some embodiments, the comparison window is at least 15 to 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. In some embodiments, the comparison window can be longer than 15-20 contiguous residues, and includes, optionally 30, 40, 50, 100, or longer windows.

[0133] "Corresponding to," "reference to," and "relative to" when used in the context of the numbering of a given amino acid or polynucleotide sequence refer to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as that of an engineered adenylate kinase, can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although the gaps are present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned.

[0134] "Amino acid difference", "residue difference" and "substitution" refer to a difference in the amino acid residue at a position of a polypeptide sequence relative to the amino acid residue at aDocket No. 5244.286WO3 corresponding position in a reference sequence. The positions of amino acid differences generally are referred to herein as "Xn," where n refers to the corresponding position in the reference sequence upon which the residue difference is based. For example, a "residue difference at position X15 as compared to SEQ ID NO: 16" refers to a difference of the amino acid residue at the polypeptide position corresponding to position 15 of SEQ. ID NO: 16. Thus, if the reference polypeptide of SEQ ID NO: 16 has a tyrosine at position 15, then a "residue difference at position X15 as compared to SEQ ID NO: 16" refers to an amino acid substitution of any residue other than tyrosine at the position of the polypeptide corresponding to position 15 of SEQ ID NO: 16. In most instances herein, the specific amino acid residue difference at a position is indicated as "XnY" where "Xn" specified the corresponding position as described above, and "Y" is the single letter identifier of the amino acid found in the engineered polypeptide (i.e., the different residue than in the reference polypeptide). In some instances (e.g., in the Tables presented in the Examples), the present invention also provides specific amino acid differences denoted by the conventional notation "AnB", where A is the single letter identifier of the residue in the reference sequence, "n" is the number of the residue position in the reference sequence, and B is the single letter identifier of the residue substitution in the sequence of the engineered polypeptide. In some instances, an amino acid residue difference or substitution may be a deletion and may be denoted by a In some instances, a polypeptide of the present invention can include one or more amino acid residue differences relative to a reference sequence, which is indicated by a list of the specified positions where residue differences are present relative to the reference sequence. In some embodiments, the amino acid difference, e.g., a substitution, is denoted by the abbreviation "nB," without the identifier for the residue in the reference sequence. In some embodiments, the phrase "an amino acid residue nB" denotes the presence of the amino residue in the engineered polypeptide, which may or may not be a substitution in context of a reference polypeptide or amino acid sequence.

[0135] In some instances, a polypeptide of the present disclosure can include one or more amino acid residue differences relative to a reference sequence, which is indicated by a list of the specified positions where residue differences are present relative to the reference sequence. In some embodiments, where more than one amino acid can be used in a specific residue position of a polypeptide, the various amino acid residues that can be used are separated by a(e.g., X15A / X15T, X15A / T, or 15A / T).

[0136] "Amino acid substitution set” or "substitution set" refers to a group of amino acid substitutions in a polypeptide sequence, as compared to a reference sequence. A substitution set can have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions. In some embodiments, a substitution set refers to the set of amino acid substitutions that is present in any of the variant adenylate kinasesDocket No. 5244.286WO3 listed in the Tables provided in the Examples. In these substitution sets, the individual substitutions are separated by a semicolon (";"; e.g., T37R;D166E) or slash (" / "; e.g., T37R / D166E or 37R / 166E).

[0137] "Conservative amino acid substitution" refers to a substitution of a residue with a different residue having a similar side chain, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar defined class of amino acids. By way of example and not limitation, in some embodiments, an amino acid with an aliphatic side chain is substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with an hydroxyl side chain is substituted with another amino acid with an hydroxyl side chain (e.g., serine and threonine); an amino acids having aromatic side chains is substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is substituted with another amino acid with a basis side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and / or a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively.

[0138] "Non-conservative substitution" refers to substitution of an amino acid in the polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affects (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine) (b) the charge or hydrophobicity, or (c) the bulk of the side chain. By way of example and not limitation, an exemplary non-conservative substitution can be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.

[0139] "Deletion" refers to modification to the polypeptide by removal of one or more amino acids from the reference polypeptide. Deletions can comprise removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids making up the reference enzyme while retaining enzymatic activity and / or retaining the improved properties of an engineered adenylate kinase enzyme. Deletions can be directed to the internal portions and / or terminal portions of the polypeptide. In various embodiments, the deletion can comprise a continuous segment or can be discontinuous. Deletions are typically indicated by in amino acid sequences.

[0140] "Insertion" refers to modification to the polypeptide by addition of one or more amino acids to the reference polypeptide. Insertions can be in the internal portions of the polypeptide, or to the carboxy or amino terminus. Insertions as used herein include fusion proteins as is known in the art. TheDocket No. 5244.286WO3 insertion can be a contiguous segment of amino acids or separated by one or more of the amino acids in the naturally occurring polypeptide.

[0141] “ Functional fragment" and "biologically active fragment" are used interchangeably herein to refer to a polypeptide that has an amino-terminal and / or carboxy-terminal deletion(s) and / or internal deletions, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is being compared (e.g., a full-length engineered adenylate kinase of the present invention) and that retains substantially all of the activity of the full-length polypeptide.

[0142] "Isolated polypeptide" refers to a polypeptide which is substantially separated from other contaminants that naturally accompany it (e.g., protein, lipids, and polynucleotides). The term embraces polypeptides which have been removed or purified from their naturally occurring environment or expression system (e.g., within a host cell or via in vitro synthesis). The recombinant adenylate kinase polypeptides may be present within a cell, present in the cellular medium, or prepared in various forms, such as lysates or isolated preparations. As such, in some embodiments, the recombinant adenylate kinase polypeptides can be an isolated polypeptide.

[0143] "Substantially pure polypeptide" or "purified protein" refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis it is more abundant than any other individual macromolecular species in the composition), and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight. However, in some embodiments, the composition comprising adenylate kinase comprises adenylate kinase that is less than 50% pure (e.g., about 10%, about 20%, about 30%, about 40%, or about 50%). Generally, a substantially pure adenylate kinase composition comprises about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species by mole or % weight present in the composition. In some embodiments, the object species is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, the isolated recombinant adenylate kinase polypeptides are substantially pure polypeptide compositions.

[0144] "Improved enzyme property" refers to at least one improved property of an enzyme. In some embodiments, the present invention provides engineered adenylate kinase polypeptides that exhibit an improvement in any enzyme property as compared to a reference adenylate kinase polypeptide and / or a wild-type adenylate kinase polypeptide, and / or another engineered adenylate kinase polypeptide. Thus, the level of "improvement" can be determined and compared between various adenylate kinaseDocket No. 5244.286WO3 polypeptides, including wild-type, as well as engineered adenylate kinases. Improved properties include, but are not limited, to such properties as increased protein expression, increased thermoactivity, increased thermostability, increased pH activity, increased stability, increased enzymatic activity, increased substrate specificity or affinity, increased specific activity, increased resistance to substrate or end-product inhibition, increased chemical stability, improved chemoselectivity, improved solvent stability, increased tolerance to acidic pH, increased tolerance to proteolytic activity (i.e., reduced sensitivity to proteolysis), reduced aggregation, increased solubility, and altered temperature profile. In additional embodiments, the term is used in reference to the at least one improved property of adenylate kinase enzymes. In some embodiments, the present invention provides adenylate kinase polypeptides that exhibit an improvement in any enzyme property as compared to a reference adenylate kinase polypeptide and / or a wild-type adenylate kinase polypeptide, and / or another engineered adenylate kinase polypeptide. Thus, the level of "improvement" can be determined and compared between various adenylate kinase polypeptides, including wild-type, as well as engineered adenylate kinases.

[0145] "Increased enzymatic activity" and "enhanced catalytic activity" refer to an improved property of the engineered polypeptides, which can be represented by an increase in specific activity (e.g., product produced / time / weight protein) or an increase in percent conversion of the substrate to the product (e.g., percent conversion of starting amount of substrate to product in a specified time period using a specified amount of enzyme) as compared to the reference enzyme. In some embodiments, the terms refer to an improved property of engineered adenylate kinase polypeptides provided herein, which can be represented by an increase in specific activity (e.g., product produced / time / weight protein) or an increase in percent conversion of the substrate to the product (e.g., percent conversion of starting amount of substrate to product in a specified time period using a specified amount of adenylate kinase) as compared to the reference adenylate kinase enzyme. In some embodiments, the terms are used in reference to improved adenylate kinase enzymes provided herein. Exemplary methods to determine enzyme activity of the engineered adenylate kinases of the present invention are provided in the Examples. Any property relating to enzyme activity may be affected, including the classical enzyme properties of Km, Vmaxor kcat, changes of which can lead to increased enzymatic activity. For example, improvements in enzyme activity can be from about 1.1 fold the enzymatic activity of the corresponding wild-type enzyme, to as much as 2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold or more enzymatic activity than the naturally occurring adenylate kinase or another engineered adenylate kinase from which the adenylate kinase polypeptides were derived.

[0146] "Conversion" refers to the enzymatic conversion (or biotransformation) of a substrate(s) to the corresponding product(s). "Percent conversion" refers to the percent of the substrate that is converted to the product within a period of time under specified conditions. Thus, the "enzymatic activity" orDocket No. 5244.286WO3 "activity" of an adenylate kinase polypeptide can be expressed as "percent conversion" of the substrate to the product in a specific period of time.

[0147] "Stringent hybridization conditions" is used herein to refer to conditions under which nucleic acid hybrids are stable. As known to those of skill in the art, the stability of hybrids is reflected in the melting temperature (Tm) of the hybrids. In general, the stability of a hybrid is a function of ion strength, temperature, G / C content, and the presence of chaotropic agents. The Tmvalues for polynucleotides can be calculated using known methods for predicting melting temperatures (See e.g., Baldino et al., Meth. Enzymol., 1989, 168:761-777; Bolton et aL, Proc. Natl. Acad. Sci. USA., 1962, 48:1390; Bresslauer et al., Proc. Natl. Acad. Sci. USA, 1986, 83:8893-8897; Freier et al., Proc. Natl. Acad. Sci. USA., 1986, 83:9373-9377; Kierzek et al., Biochem., 1986, 25:7840-7846; Rychlik et al., Nucl. Acids Res., 1990, 18:6409-6412 (erratum, Nucl. Acids Res., 1991, 19:698); Sambrook et al., supra); Suggs et aL, 1981, in "Developmental Biology Using Purified Genes," Brown et al. [eds.], pp. 683-693, Academic Press, Cambridge, MA (1981); and Wetmur, Crit. Rev. Biochem. Mol. Biol., 1991, 26:227-259). In some embodiments, the polynucleotide encodes the polypeptide disclosed herein and hybridizes under defined conditions, such as moderately stringent or highly stringent conditions, to the complement of a sequence encoding an engineered adenylate kinase enzyme of the present invention.

[0148] "Hybridization stringency" relates to hybridization conditions, such as washing conditions, in the hybridization of nucleic acids. Generally, hybridization reactions are performed under conditions of lower stringency, followed by washes of varying but higher stringency (see, e.g., Sambrook et aL, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 2001; Ausubel et aL, Current Protocols in Molecular Biology, John Wiley & Sons, 2003). The term "moderately stringent hybridization" refers to conditions that permit target-DNA to bind a complementary nucleic acid that has about 60% identity, preferably about 75% identity, about 85% identity to the target DNA, with greater than about 90% identity to target-polynucleotide. Exemplary moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5x Denhart's solution, 5xSSPE, 0.2% SDS at 42 °C, followed by washing in 0.2xSSPE, 0.2% SDS, at 42 °C. "High stringency hybridization" refers generally to conditions that are about 10 °C or less from the thermal melting temperature Tmas determined under the solution condition for a defined polynucleotide sequence. In some embodiments, a high stringency condition refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 0.018M NaCI at 65 °C (i.e., if a hybrid is not stable in 0.018M NaCI at 65 °C, it will not be stable under high stringency conditions, as contemplated herein). High stringency conditions can be provided, for example, by hybridization in conditions equivalent to 50% formamide, 5x Denhart's solution, SxSSPE, 0.2% SDS at 42 °C, followed by washing in O.lxSSPE, and 0.1% SDS at 65 °C. Another high stringency condition is hybridizing in conditions equivalent to hybridizing in 5X SSC containing 0.1% (w / v) SDS at 65 °C and washing in O.lx SSC containing 0.1% SDS at 65 °C. Other highDocket No. 5244.286WO3 stringency hybridization conditions, as well as moderately stringent conditions, are described in the references cited above.

[0149] "Coding sequence" refers to that portion of a nucleic acid (e.g., a gene) that encodes an amino acid sequence of a protein.

[0150] "Codon optimized" refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate in that most amino acids are represented by several codons, called "synonyms" or "synonymous" codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, the polynucleotides encoding the adenylate kinase enzymes may be codon optimized for optimal production in the host organism selected for expression.

[0151] "Preferred," "optimal," and "high codon usage bias" codons when used alone or in combination refer(s) interchangeably to codons that are used at higher frequency in the protein coding regions than other codons that code for the same amino acid. The preferred codons may be determined in relation to codon usage in a single gene, a set of genes of common function or origin, highly expressed genes, the codon frequency in the aggregate protein coding regions of the whole organism, codon frequency in the aggregate protein coding regions of related organisms, or combinations thereof. Codons whose frequency increases with the level of gene expression are typically optimal codons for expression. A variety of methods are known for determining the codon frequency (e.g., codon usage, relative synonymous codon usage) and codon preference in specific organisms, including multivariate analysis, for example, using cluster analysis or correspondence analysis, and the effective number of codons used in a gene (See e.g., GCG Codon Preference, Genetics Computer Group Wisconsin Package;CodonW, Peden, University of Nottingham; McInerney, Bioinform., 1998, 14:372-73; Stenico et al., Nucl. Acids Res., 1994, 222437-46; and Wright, Gene, 1990, 87:23-29). Codon usage tables are available for many different organisms (See e.g., Wada et aL, Nucl. Acids Res., 1992, 20:2111-2118; Nakamura et aL, Nucl. Acids Res., 2000, 28:292; Duret, et al., supra; Henaut and Danchin, in Escherichia coli and Salmonella, Neidhardt, et al. (eds.), ASM Press, Washington D.C., p. 2047-2066 (1996)). The data source for obtaining codon usage may rely on any available nucleotide sequence capable of coding for a protein. These data sets include nucleic acid sequences actually known to encode expressed proteins (e.g., complete protein coding sequences-CDS), expressed sequence tags (ESTS), or predicted coding regions of genomic sequences (See e.g., Mount, "Bioinformatics: Sequence and Genome Analysis,"Docket No. 5244.286WO3 Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); Uberbacher, Meth. Enzymol., 1996, 266:259-281; and Tiwari et al., Comput. Appl. Biosci., 1997, 13:263-270).

[0152] "Control sequence" refers herein to include all components, which are necessary or advantageous for the expression of a polynucleotide and / or polypeptide of the present application. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, pro-peptide sequence, promoter sequence, signal peptide sequence, initiation sequence and transcription terminator. In some embodiments, the control sequences include a promoter, and transcriptional and translational stop signals.

[0153] "Operably linked" or "operatively linked" is defined herein as a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) at a position relative to a polynucleotide of interest such that the control sequence directs or regulates the expression of the polynucleotide, and where appropriate the encoded polypeptide of interest.

[0154] "Promoter sequence" refers to a nucleic acid sequence that defines and / or initiates expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence contains transcriptional control sequences, which mediate the expression of a polynucleotide of interest. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0155] "Heterologous" refers to the relationship between two or more nucleic acid or protein sequences (e.g., a promoter sequence, signal peptide, terminator sequence, etc.) that are derived from different sources and are not associated in nature.

[0156] "Vector" refers to a polynucleotide construct for introducing a polynucleotide sequence into a cell. In some embodiments, the vector is an expression vector that is operably linked to a suitable control sequence capable of effecting the expression in a suitable host of the polynucleotide of interest and where appropriate the encoded polypeptide.|0157| "Expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.

[0158] " Produces" refers to the production of proteins and / or other compounds by cells. It is intended that the term encompass any step involved in the production of polypeptides including, but not limitedDocket No. 5244.286WO3 to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.

[0159] "Culturing" refers to the growing of a population of microbial cells under any suitable conditions (e.g., using a liquid, gel or solid medium).

[0160] " Host cell" and "host strain" refer to suitable hosts for expression vectors comprising DNA provided herein (e.g., the polynucleotides encoding the adenylate kinase variants). In some embodiments, the host cells are prokaryotic or eukaryotic cells that have been transformed or transfected with vectors constructed using recombinant DNA techniques as known in the art.

[0161] "pH stable" refers to an adenylate kinase polypeptide that maintains similar activity (e.g., more than 60% to 80%) after exposure to high or low pH (e.g., 4.5-6 or 8 to 12) for a period of time (e.g., 0.5-24 hrs) compared to the untreated enzyme.

[0162] "Thermostable" refers to an adenylate kinase polypeptide that maintains similar activity (more than 60% to 80% for example) after exposure to elevated temperatures (e.g., 40-80 °C) for a period of time (e.g., 0.5-24 h) compared to the wild-type enzyme exposed to the same elevated temperature.

[0163] "Solvent stable" refers to an adenylate kinase polypeptide that maintains similar activity (more than e.g., 60% to 80%) after exposure to varying concentrations (e.g., 5-99%) of solvent (ethanol, isopropyl alcohol, dimethylsulfoxide [DMSO], tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl adenylate, methyl tert-butyl ether, etc.) for a period of time (e.g., 0.5-24 h) compared to the wild-type enzyme exposed to the same concentration of the same solvent.

[0164] "Thermo- and solvent stable" refers to an adenylate kinase polypeptide that is both thermostable and solvent stable.

[0165] "Stereoselectivity" refers to the preferential formation in a chemical or enzymatic reaction of one stereoisomer over another. Stereoselectivity can be partial, where the formation of one stereoisomer is favored over the other, or it may be complete where only one stereoisomer is formed. When the stereoisomers are enantiomers, the stereoselectivity is referred to as enantioselectivity, the fraction (typically reported as a percentage) of one enantiomer in the sum of both. It is commonly alternatively reported in the art (typically as a percentage) as the enantiomeric excess ("e.e.") calculated therefrom according to the formula [major enantiomer - minor enantiomer] / [major enantiomer + minor enantiomer]. Where the stereoisomers are diastereoisomers, the stereoselectivity is referred to as diastereoselectivity, the fraction (typically reported as a percentage) of one diastereomer in a mixture of two diastereomers, commonly alternatively reported as the diastereomeric excess ("d.e."). Enantiomeric excess and diastereomeric excess are types of stereomeric excess.Docket No. 5244.286WO3

[0166] "Regioselectivity" and "regioselective reaction" refer to a reaction in which one direction of bond making or breaking occurs preferentially over all other possible directions. Reactions can completely (100%) regioselective if the discrimination is complete, substantially regioselective (at least 75%), or partially regioselective (x%, wherein the percentage is set dependent upon the reaction of interest), if the product of reaction at one site predominates over the product of reaction at other sites.

[0167] "Chemoselectivity" refers to the preferential formation in a chemical or enzymatic reaction of one product over another.

[0168] "Suitable reaction conditions" refers to those conditions in the enzymatic conversion reaction solution (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffers, co-solvents, etc.) under which an adenylate kinase polypeptide of the present invention is capable of converting a substrate to the desired product compound. Some exemplary "suitable reaction conditions" are provided herein.

[0169] "Loading," such as in "compound loading" or "enzyme loading" refers to the concentration or amount of a component in a reaction mixture at the start of the reaction.

[0170] "Substrate" in the context of an enzymatic conversion reaction process refers to the compound or molecule acted on by the engineered enzymes provided herein (e.g., engineered adenylate kinase polypeptides).

[0171] "Product" in the context of an enzymatic conversion process refers to the compound or molecule resulting from the action of an enzymatic polypeptide on a substrate.

[0172] "Increasing" yield of a product (e.g., a nucleoside triphosphate or analogue) from a reaction occurs when a particular component present during the reaction (e.g., an adenylate kinase enzyme) causes more product to be produced, compared with a reaction conducted under the same conditions with the same substrate and other substituents, but in the absence of the component of interest.

[0173] "Substantially free" of a particular enzyme if the amount of that enzyme compared with other enzymes that participate in catalyzing the reaction is less than about 2%, about 1%, or about 0.1% (wt / wt).

[0174] "Fractionating" a liquid (e.g., a culture broth) means applying a separation process (e.g., salt precipitation, column chromatography, size exclusion, and filtration) or a combination of such processes to provide a solution in which a desired protein comprises a greater percentage of total protein in the solution than in the initial liquid product.

[0175] "Alkyl" refers to saturated hydrocarbon groups of from 1 to 18 carbon atoms inclusively, either straight chained or branched, more preferably from 1 to 8 carbon atoms inclusively, and mostDocket No. 5244.286WO3 preferably 1 to 6 carbon atoms inclusively. An alkyl with a specified number of carbon atoms is denoted in parenthesis (e.g., (Ci-C4)alkyl refers to an alkyl of 1 to 4 carbon atoms).

[0176] "Alkenyl" refers to groups of from 2 to 12 carbon atoms inclusively, either straight or branched containing at least one double bond but optionally containing more than one double bond.

[0177] "Alkynyl" refers to groups of from 2 to 12 carbon atoms inclusively, either straight or branched containing at least one triple bond but optionally containing more than one triple bond, and additionally optionally containing one or more double bonded moieties.

[0178] "Heteroalkyl, "heteroalkenyl," and heteroalkynyl," refer to alkyl, alkenyl and alkynyl as defined herein in which one or more of the carbon atoms are each independently replaced with the same or different heteroatoms or heteroatomic groups. Heteroatoms and / or heteroatomic groups which can replace the carbon atoms include, but are not limited to, -O-, -S-, -S-O-, -NR'-, -PH-, -S(O)-, -S(O)2-, -S(O) NR'-, -S(O)2NR'-, and the like, including combinations thereof, where each Ra is independently selected from hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

[0179] "Alkoxy" refers to the group -OR' wherein R' is an alkyl group is as defined above including optionally substituted alkyl groups as also defined herein.

[0180] "Amino" refers to the group -NH,. Substituted amino refers to the group -N HR', NR'R', and NR'R'R', where each R' is independently selected from substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, aryl, heteroaryl, heteroarylalkyl, acyl, alkoxycarbonyl, sulfanyl, sulfinyl, sulfonyl, and the like. Typical amino groups include, but are limited to, dimethylamino, diethylamino, trimethylammonium, triethylammonium, methylysulfonylamino, furanyl-oxy-sulfamino, and the like.

[0181] " Oxo" refers to =0.

[0182] "Oxy" refers to a divalent group -O-, which may have various substituents to form different oxy groups, including ethers and esters.

[0183] "Carboxy" refers to -COOH.

[0184] "Carbonyl" refers to -C(O)-, which may have a variety of substituents to form different carbonyl groups including acids, acid halides, aldehydes, amides, esters, and ketones.

[0185] "Alkyloxycarbonyl" refers to -C(O)OR', where R' is an alkyl group as defined herein, which can be optionally substituted.

[0186] As used herein, "aminocarbonyl" refers to -C(O)NH2. Substituted aminocarbonyl refers to -C(O)NR'R', where the amino group NR'R' is as defined herein.

[0187] "Halogen" and "halo" refer to fluoro, chloro, bromo and iodo.Docket No. 5244.286WO3

[0188] "Hydroxy" refers to -OH.

[0189] "Cyano" refers to -CN.

[0190] "Aryl" refers to an unsaturated aromatic carbocyclic group of from 6 to 12 carbon atoms inclusively having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl). Exemplary aryls include phenyl, pyridyl, naphthyl and the like.

[0191] "Heterocycle," "heterocyclic," and interchangeably "heterocycloalkyl," refer to a saturated or unsaturated group having a single ring or multiple condensed rings, from 2 to 10 carbon ring atoms inclusively and from 1 to 4 hetero ring atoms inclusively selected from nitrogen, sulfur or oxygen within the ring. Such heterocyclic groups can have a single ring (e.g., piperidinyl or tetrahydrofuryl) or multiple condensed rings (e.g., indolinyl, dihydrobenzofuran or quinuclidinyl). Examples of heterocycles include, but are not limited to, furan, thiophene, thiazole, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, pyrrolidine, indoline and the like.

[0192] "Heteroaryl" refers to an aromatic heterocyclic group of from 1 to 10 carbon atoms inclusively and 1 to 4 heteroatoms inclusively selected from oxygen, nitrogen and sulfur within the ring. Such heteroaryl groups can have a single ring (e.g., pyridyl or furyl) or multiple condensed rings (e.g., indolizinyl or benzothienyl).

[0193] "Heteroarylalkyl" refers to an alkyl substituted with a heteroaryl (i.e., heteroaryl-alkyl- groups), preferably having from 1 to 6 carbon atoms inclusively in the alkyl moiety and from 5 to 12 ring atoms inclusively in the heteroaryl moiety. Such heteroarylalkyl groups are exemplified by pyridylmethyl and the like.

[0194] "Heteroarylalkenyl" refers to an alkenyl substituted with a heteroaryl (i.e., heteroaryl-alkenyl-groups), preferably having from 2 to 6 carbon atoms inclusively in the alkenyl moiety and from 5 to 12 ring atoms inclusively in the heteroaryl moiety.

[0195] "Heteroarylalkynyl" refers to an alkynyl substituted with a heteroaryl (i.e., heteroaryl-alkynyl-groups), preferably having from 2 to 6 carbon atoms inclusively in the alkynyl moiety and from 5 to 12 ring atoms inclusively in the heteroaryl moiety.

[0196] "Membered ring" is meant to embrace any cyclic structure. The number preceding the term "membered" denotes the number of skeletal atoms that constitute the ring. Thus, for example, cyclohexyl, pyridine, pyran and thiopyran are 6-membered rings and cyclopentyl, pyrrole, furan, and thiophene are 5-membered rings.Docket No. 5244.286WO3

[0197] "Phosphate" as used herein refers to a functional group comprised of an orthophosphate ion (phosphorous atom covalently linked to four oxygen atoms). The orthophosphate ion is commonly found with one or more hydrogen atoms or organic groups. A phosphate group or chain may be modified, as further described herein.

[0198] "Phosphorylated" as used herein refers to the addition or presence of one of more phosphoryl groups (phosphorous atom covalently linked to the three oxygen atoms).

[0199] "Thiophosphate" refers to an instance where a non-bridging oxygen in a phosphate group of a phosphodiester bond, NMP, NDP, NTP or NQP is replaced with a sulfur. In some embodiments, nucleoside 5'-thiomonophosphate is referred to as NMP-S or NMPaS. In some embodiments, nucleoside-5'-l-thio(diphosphate) and nucleoside-5'-l-thio(triphosphate) are referred to as NDPaS or aS-NDP, and NTPaS or aS-NTP, respectively. In some embodiments, nucleoside-5'-2-thio(diphosphate) and nucleoside-5'-2-thio(triphosphate) are referred to as NDPbS and NTPbS, respectively.

[0200] "Dithiophosphate" refers to an instance where two non-bridging oxygens in a phosphate group of a phosphodiester bond, NMP, NDP, NTP or NQP are replaced with two sulfurs.

[0201] Unless otherwise specified, positions occupied by hydrogen in the foregoing groups can be further substituted with substituents exemplified by, but not limited to, hydroxy, oxo, nitro, methoxy, ethoxy, alkoxy, substituted alkoxy, trifluoromethoxy, haloalkoxy, fluoro, chloro, bromo, iodo, halo, methyl, ethyl, propyl, butyl, alkyl, alkenyl, alkynyl, substituted alkyl, trifluoromethyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, thio, alkylthio, acyl, carboxy, alkoxycarbonyl, carboxamido, substituted carboxamido, alkylsulfonyl, alkylsulfinyl, alkylsulfonylamino, sulfonamido, substituted sulfonamido, cyano, amino, substituted amino, alkylamino, dialkylamino, aminoalkyl, acylamino, amidino, amidoximo, hydroxamoyl, phenyl, aryl, substituted aryl, aryloxy, arylalkyl, arylalkenyl, a rylalkynyl, pyridyl, imidazolyl, heteroaryl, substituted heteroaryl, heteroaryloxy, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, substituted cycloalkyl, cycloalkyloxy, pyrrolidinyl, piperidinyl, morpholino, heterocycle, (heterocycle)oxy, and (heterocycle)alkyl; and preferred heteroatoms are oxygen, nitrogen, and sulfur. It is understood that where open valences exist on these substituents they can be further substituted with alkyl, cycloalkyl, aryl, heteroaryl, and / or heterocycle groups, that where these open valences exist on carbon they can be further substituted by halogen and by oxygen-, nitrogen-, or sulfur-bonded substituents, and where multiple such open valences exist, these groups can be joined to form a ring, either by direct formation of a bond or by formation of bonds to a new heteroatom, preferably oxygen, nitrogen, or sulfur. It is further understood that the above substitutions can be made provided that replacing the hydrogen with the substituent does not introduce unacceptable instability to the molecules of the present invention, and is otherwise chemically reasonable.Docket No. 5244.286WO3

[0202] "Optional" and "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. One of ordinary skill in the art would understand that with respect to any molecule described as containing one or more optional substituents, only sterica lly practical and / or synthetically feasible compounds are meant to be included.

[0203] "Optionally substituted" refers to all subsequent modifiers in a term or series of chemical groups. For example, in the term "optionally substituted arylalkyl, the "alkyl" portion and the "aryl" portion of the molecule may or may not be substituted, and for the series "optionally substituted alkyl, cycloalkyl, aryl and heteroaryl," the alkyl, cycloalkyl, aryl, and heteroaryl groups, independently of the others, may or may not be substituted.Engineered Adenylate Kinase Polypeptides

[0204] In one aspect, the present disclosure provides adenylate kinase enzymes engineered to have improved properties compared to the parent adenylate kinase enzyme. In some embodiments, the engineered adenylate kinase enzymes exhibit, among others, increased activity on nucleoside monophosphate (NMP) substrates, increased thermostability, and / or increased activity on modified NMP substrates. The engineered adenylate kinases are useful in the conversion of nucleoside monophosphate substrates to their corresponding nucleoside diphosphate products, particularly in enzyme cascade systems.

[0205] In some embodiments, an engineered adenylate kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-231 of an even-numbered SEQ ID NO. of SEQ ID NOs: 16-360, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 16-360, wherein the amino acid sequence comprises one or more substitutions relative to a reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, 144, 252, 402, 446, 710, or 808, or relative to a reference sequence corresponding to SEQ ID NO: 16, 144, 252, 402, 446, 710, or 808.

[0206] In some embodiments, the engineered adenylate kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, 144, 252, 402, 446, 710, or 808, or to a reference sequence corresponding to SEQ ID NO: 16, 144, 252, 402, 446, 710, or 808, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 16, 144, 252, 402, 446, 710, or 808.Docket No. 5244.286WO3

[0207] In some embodiments, the engineered adenylate kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or to the reference sequence corresponding to SEQ ID NO: 16, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ. ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0208] In some embodiments, the engineered adenylate kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0209] In some embodiments, the engineered adenylate kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-231 of an even-numbered SEQ ID NO. of SEQ ID NOs: 18-360 and 372-1018, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 18-360 and 372-1018, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0210] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution at amino acid position 4, 14, 15, 16, 17, 18, 31, 33, 35, 37, 38, 40, 46, 47, 52, 53, 54, 55, 56, 59, 61, 62, 66, 68, 69, 74, 80, 81, 82, 83, 84, 87, 89, 91, 92, 93, 95, 96, 104, 105, 109, 111, 112, 115, 117, 121, 122, 125, 126, 132, 133, 135, 136, 139, 142, 143, 146, 148, 149, 154, 157, 159, 162, 163, 166, 168, 169, 170, 178, 179, 180, 182, 183, 184, 187, 192, 194, 199, 202, 203, 204, 205, 207, 212, 214, 215, 217, 218, 219, 220, 222, 223, 225, 227, 228, and 229, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0211] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution, or amino acid residue 4N, 14P, 15A / H / T, 16C / G / M / T, 171, 18V, 31F / V / W, 33L, 35C / E, 37M, 37E / H / Q / R, 38D, 40T, 46E, 47L, 52L, 53D / M / S / T / V, 54G / R / V, 55Q, 55E / G / N / R / S, 56A / E,Docket No. 5244.286WO3 59G, 61G, 62H, 66D, 68V, 69V, 74H, 80S, 81P, 82L, 83R, 84A / T, 87G / R / S, 89A / D / F / G / L / P / Q / S / V / W, 91L, 92S, 93E, 93G / L / V, 95W, 96L, 104L / R, 104Q, 105L / Q / S, 109A, 111N, 112C / T, 115C / H / N, 117A / E / G / R / T / V, 121V, 122S, 125L, 126C / I, 132R, 133W, 135E / N, 136L / R, 139A / H, 142V, 1431, 146A / G / H, 148Q, 149A / L / M / Q, 154E, 157R, 159V, 162D / M / V, 163E / R, 166E, 168C, 169E, 170K / M, 178A / F / G / I / L / M / N / R / T / V / Y, 179A, 180A / P / Q, 182L, 183L, 184P / Q / S, 187D / R, 192L / M / V, 194L, 199F, 202E / G / N, 203Q, 204D, 205Q, 2051, 205C / L, 2071 / N, 212A, 214K / P, 215V, 217A / E, 218L / V, 219L / T, 220G, 222A, 223D / M / V, 225H / T, 225A / Q, 2275, 228L / S, and 229G, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0212] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution H4N, A14P, Y15A / H / T, L16C / G / M / T, V17I, L18V, R31F / V / W, I33L, A35C / E, H37M, T37E / H / Q / R, G38D, P40T, D46E, I47L, V52L, A53D / M / S / T / V, K54G / R / V, E55Q, Q55E / G / N / R / S, N56A / E, V59G, S61G, S62H, W66D, L68V, K69V, P74H, P80S, V81P, V82L, L83R, R84A / T, K87G / R / S, K89A / D / F / G / L / P / Q / S / V / W, C91L, E92S, G93E, K93G / L / V, F95W, I96L, Q104L / R, R104Q, R105L / Q / S, Y109A, D111N, S112C / T, E115C / H / N, Q117A / E / G / R / T / V, L121V, T122S, V125L, L126C / I, D132R, E133W, V135E / N, V136L / R, L139A / H, R142V, V143I, K146A / G / H, G148Q, R149A / L / M / Q, I154E, P157R, K159V, E162D / M / V, L163E / R, D166E, G168C, V169E, S170K / M, K178A / F / G / I / L / M / N / R / T / V / Y, E179A, E180A / P / Q, V182L, R183L, M184P / Q / S, K187D / R, K192L / M / V, Q194L, Y199F, K202E / G / N, K203Q, G204D, I205Q, L205I, Q205C / L, A207I / N, S212A, E214K / P, 1215V, P217A / E, I218L / V, V219L / T, A220G, V222A, L223D / M / V, A225H / T, I225A / Q, G227S, V228L / S, and I229G, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0213] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution at amino acid position 37, 93, 125, or 205, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0214] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or amino acid residue 37H / M, 93G, 125L, or 205Q, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0215] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution at amino acid position 4, 47, 95, or 96; or substitution 14P, 15A / T, 16C / G / T, 37E / H / M / Q, 52L, 53T / V, 54V, 55N / R, 56A, 69V, 74H, 89D / F / G / S / W, 93L, 115C / H / N, 117A / E / T / V, 125L,Docket No. 5244.286WO3 1261, 132R, 133W, 135E / N, 146A / G, 157R, 166E, 168C, 170M, 178A / F / G / I / L / M / N / R / T / V / Y, 180A / Q, 187D / R, 203Q, 205C / Q 214K, 217E, or 220G; or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0216] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution 4N, 14P, 15A / H / T, 16C / G / M / T, 171, 18V, 31F / V / W, 33L, 35C / E, 37M, 37E / H / Q / R, 38D, 40T, 46E, 47L, 52L, 53D / M / S / T / V, 54G / R / V, 55Q, 55E / G / N / R / S, 56A / E, 59G, 61G, 62H, 66D, 68V, 69V, 74H, 80S, 81P, 82L, 83R, 84A / T, 87G / R / S, 89A / D / F / G / L / P / Q / S / V / W, 91L, 92S, 93E, 93G / L / V, 95W, 96L, 104L / R, 104Q, 105L / Q / S, 109A, 111N, 112C / T, 115C / H / N, 117A / E / G / R / T / V, 121V, 122S, 125L, 126C / I, 132R, 133W, 135E / N, 136L / R, 139A / H, 142V, 1431, 146A / G / H, 148Q, 149A / L / M / Q, 154E, 157R, 159V, 162D / M / V, 163E / R, 166E, 168C, 169E, 170K / M, 178A / F / G / I / L / M / N / R / T / V / Y, 179A, 180A / P / Q 182L, 183L, 184P / Q / S, 187D / R, 192L / M / V, 194L, 199F, 202E / G / N, 203Q, 204D, 205Q, 2051, 205C / L, 2071 / N, 212A, 214K / P, 215V, 217A / E, 218L / V, 219L / T, 220G, 222A, 223D / M / V, 225H / T, 225A / Q, 227S, 228L / S, and 229G, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0217] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution H4N, AMP, Y15A / H / T, L16C / G / M / T, V17I, L18V, R31F / V / W, I33L, A35C / E, H37M, T37E / H / Q / R, G38D, P40T, D46E, I47L, V52L, A53D / M / S / T / V, K54G / R / V, E55Q, Q55E / G / N / R / S, N56A / E, V59G, S61G, S62H, W66D, L68V, K69V, P74H, P80S, V81P, V82L, L83R, R84A / T, K87G / R / S, K89A / D / F / G / L / P / Q / S / V / W, C91L, E92S, G93E, K93G / L / V, F95W, I96L, Q104L / R, R104Q, R105L / Q / S, Y109A, D111N, S112C / T, E115C / H / N, Q117A / E / G / R / T / V, L121V, T122S, V125L, L126C / I, D132R, E133W, V135E / N, V136L / R, L139A / H, R142V, V143I, K146A / G / H, G148Q, R149A / L / M / Q, I154E, P157R, K159V, E162D / M / V, L163E / R, D166E, G168C, V169E, S170K / M, K178A / F / G / I / L / M / N / R / T / V / Y, E179A, E180A / P / Q, V182L, R183L, M184P / Q / S, K187D / R, K192L / M / V, Q194L, Y199F, K202E / G / N, K203Q, G204D, I205Q, L205I, Q205C / L, A207I / N, S212A, E214K / P, 1215V, P217A / E, I218L / V, V219L / T, A220G, V222A, L223D / M / V, A225H / T, I225A / Q, G227S, V228L / S, and I229G, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0218] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution set at amino acid positions 37 / 93 / 125 / 205, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16. In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution set, or amino acidDocket No. 5244.286WO3 residues 37H / 93G / 125L / 205Qor 37M / 93G / 125L / 205Q, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16. In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution set T37H / K93G / V125L / I205Q or T37M / K93G / V125L / I205Q, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0219] In some embodiments, where the amino acid sequence comprises at least a substitution at amino acid position 4, 47, 95, or 96; or substitution 14P, 15A / T, 16C / G / T, 37E / H / M / Q, 52L, 53T / V, 54V, 55N / R, 56A, 69V, 74H, 89D / F / G / S / W, 93L, 115C / H / N, 117A / E / T / V, 125L, 1261, 132R, 133W, 135E / N, 146A / G, 157R, 166E, 168C, 170M, 178A / F / G / I / L / M / N / R / T / V / Y, 180A / Q, 187D / R, 203Q, 205C / Q, 214K, 217E, or 220G; or combinations thereof, the amino acid sequence of the engineered adenylate kinase can further comprises at least a substitution at amino acid position 11, 13, 14, 15, 16, 18, 20, 21, 22, 26, 27, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 97, 98, 99, 100, 101, 102, 104, 105, 108, 109, 110, 111, 112, 113, 115, 116, 117, 118, 119, 120, 122, 123, 124, 125, 126, 127, 128, 129, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 146, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 161, 162, 163, 166, 168, 169, 170, 172, 173, 175, 178, 179, 180, 181, 182, 183, 184, 186, 187, 188, 190, 191, 192, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, or 231, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0220] In some embodiments, the amino acid sequence of the engineered adenylate kinase can further comprise at least a substitution or amino acid residue 11D, 13G / S, 14C / E, 15F, 16M, 18C / E / L, 20A / P / R / T / V, 21L / N / R / S / T / V, 22A / S, 26L, 27C / S, 29Q / R, 30G / L / S / Y, 31R, 32F / I / L / N, 34A / E / S / V, 35A / E / F / L / S, 36A / E / I / L / M / Q / R / S / V / Y, 37F / G / I / L / R / Y, 38C / F / L / R, 39A / M / Q / T, 40C / F / L / S / T, 42T, 43A / C / G / H / N, 44S, 46E / F / L / M / Y, 48F / L, 50A / C / L / N / Q / R / S / V, 51F / G / K / L / M / R / T, 52A / H, 53A / M / S, 54G / H / I / M / Q / R / T, 55G / L / Q / S, 56E / Q / T, 57H / L / P / S, 58C / G / Y, 59E / G / H / M / P / R / T / V / W / Y, 60A / C / P / S / W, 61A / E / N / P / S / V, 62A / E / G / I / L / S, 63S, 64R, 65A / D / F / K / P / T, 66A / C / D / E / F / G / H / N / P / Q / R / S / T / V / W, 67R, 68A / D / E / G / I / L / Q / R / S / T / V / W / Y, 69F / L / Q / R / Y, 70R, 71T, 731 / R, 74C / G / Q / S, 75L, 76G / L / P, 77A / I / M / Q / S, 78C / G / N / T, 79A / K / L / P / W, 80A / D / G / L / N / P / R / S / V / W, 81 F / l / L / Q / S, 82 l / L / P / Q / T, 83G / H / L / S / T, 84A / E / H / M / S, 87A / E / I / K / L / M / R / V / Y, 88N / R / T, 89A / H / I / L / P / Q / T / V, 90C / E / F / K / S / T / V, 91A / F / G / L / S / V, 92L / S, 93A / E / G / P / S / T / V / Y, 94A / C / F / M / Q / S / T / V / Y, 971, 98A / G / Q, 99A / C, 100F / S / V, 101-, 102A / C / N, 104A / F / H / I / L / Q / R / S / W, 105G / K / L / M / R / S, 108M / R / S / V, 109E / Y, 110C / F, 111E / P / R,Docket No. 5244.286WO3 112A / C / E / K / M / N / Q / R / T, 113E / W / Y, 115G / K / R, 116A / E / F / L, 117L / N / S, 118A / G / L / R / S, 119F / K / L / P / R / S, 120G / K / L / R / S / T, 122A / H / S, 123S, 124V, 1251, 126A / E / S / V, 1271 / L / P / S, 128C / E / I / K / N / R / S, 1291 / L / P / S, 131A / G / V, 132G / K / L / T / V, 133A / E / F / L / Q / S / V / W, 134L, 135 l / K / M / P / R / S, 136A / I / L / S / V / Y, 137L, 138C / I / M / V, 139A / H / L / R, 140G, 141G / V, 142F / L / M / W, 143A / C / G / P / R / S / T / V, 146D / H / N / R / V, 148F / H / M / P / Q / R / S / T, 149L, 150C / G / L / P / S / V, 151A / F / R / Y, 152F / H, 153C / K / S / V, 154Q / R, 155A / F / T / W, 156A / C / N / T / V, 157P / V, 161G / L, 162G, 163G / Q / S, 166C / F / L / P / S, 168G / L / N / Q / S, 169A / D / I / Y, 170A / G / H / P / R / S, 172A / H / M / S / T, 173F / K / R / S / T / V, 175F / L / S, 178E / G / N / R, 179A / C / G / I / L / P / V, 180G / H / P, 181C / I / V, 182A / G / I / L / Q / S, 183A / L / Q, 1841 / K / M / N / R / S / T / V, 186L, 187G / Y, 188G / L, 190A / C / E / G / H / N / Q / R, 191D / K, 192A / H / I / W, 194F / L / R / V / Y, 195G / I / M / R, 1961, 197A / L / Q / V, 198G, 199R, 200A, 201A / F / K / L / S, 202E / G / M / S, 203A / E / L / R, 204R / S, 205L / P, 206W, 207A / L / T, 208E, 210V, 211A, 212C / H / I / L / M / N / P / Q / R / S / W, 213L / P / Q / S, 214A / E / L / M / P / R / T / W, 215A / P / V, 216A / D / E / G / H / L / M / N / P / R / S / T / V, 217E / G / H / P / T, 218A / I / L, 219A / F / G / L / P / S / T / W, 220E / V, 221D, 2221 / T, 223T, 224G / S / T, 225A / L / Q / R / T / V, 226K / L / P / R / S / T, 227P, 228D / F / G / I / L / P / Q / R / S / T / V, 229C / I, 230A / M / P / Q, or 231A / Q / R / T, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ. ID NO: 16.

[0221] In some embodiments, the amino acid sequence of the engineered adenylate kinase can further comprise at least a substitution or amino acid residue G11D, M13G / S, A14C / E, Y15F, L16M, F18C / E / L, G20A / P / R / T / V, P21L / N / R / S / T / V, P22A / S, K26L, G27C / S, Y29Q / R, A30G / L / S / Y, K31R, 132 F / l / L / N, Q.34A / E / S / V, E35A / F / L / S, K36A / E / I / L / M / Q / R / S / V / Y, T37F / G / I / L / R / Y, G38C / F / L / R, I39A / M / Q / T, P40C / F / L / S / T, I42T, S43A / C / G / H / N, T44S, D46E / F / L / M / Y, F48L, D50A / C / L / N / Q / R / S / V, I51F / G / K / L / M / R / T, V52A / H, K53A / M / S, K54G / H / I / M / Q / R / T, E55G / L / Q / S, N56E / Q / T, D57H / L / P / S, E58C / G / Y, L59E / G / H / M / P / R / T / V / W / Y, G60A / C / P / S / W, K61A / E / N / P / S / V, K62A / E / G / I / L / S, I63S, K64R, E65A / D / F / K / P / T, I66A / C / D / E / F / G / H / N / P / Q / R / S / T / V / W, M67R, E68A / D / G / I / L / Q / R / S / T / V / W / Y, K69F / L / Q / R / Y, G70R, E71T, V73I / R, P74C / G / Q / S, D75L, E76G / L / P, L77A / I / M / Q / S, V78C / G / N / T, N79A / K / L / P / W, E80A / D / G / L / N / P / R / S / V / W, V81 F / l / L / Q / S, V82I / L / P / Q / T, K83G / H / L / S / T, R84A / E / H / M / S, S87A / E / I / K / L / M / R / V / Y, E88N / R / T, K89A / H / I / L / P / Q / T / V, D90C / E / F / K / S / T / V, C91A / F / G / L / S / V, E92L / S, K93A / E / G / P / S / T / V / Y, G94A / C / F / M / Q / S / T / V / Y, L97I, D98A / G / Q, G99A / C, Y100F / S / V, P101-, R102A / C / N, V104A / F / H / I / L / Q / R / S / W, A105G / K / L / M / R / S, E108M / R / S / V, F109E / Y, L110C / F, D111E / P / R, S112A / C / E / K / M / N / Q / R / T, F113E / W / Y, E115G / K / R, S116A / E / F / L, Q117L / N / S, N118A / G / L / R / S, K119F / K / L / P / R / S, Q120G / K / L / R / S / T, T122A / H / S, A123S, A124V, V125I, L126A / E / S / V, F127I / L / P / S, D128C / E / I / K / N / R / S, V129I / L / P / S, E131A / G / V, D132G / K / L / T / V, V133A / E / F / L / Q / S / W, V134L, V135I / K / M / P / R / S, Q136A / I / L / S / V / Y, R137L, L138C / I / M / V, T139A / H / L / R, S140G, R141G / V, R142F / L / M / W, I143A / C / G / P / R / S / T / V, K146D / H / N / R / V, G148F / H / M / P / Q / R / S / T, R149L, I150C / G / L / P / S / V, Y151A / F / R, N152F / H, M153C / K / S / V, I154Q / R, S155A / F / T / W, L156A / C / N / T / V, P157V,Docket No. 5244.286WO3 D161G / L, E162G, L163G / Q / S, D166C / F / L / P / S, K168G / L / N / Q / S, V169A / D / I / Y, K170A / G / H / P / R / S, V172A / H / M / S / T, Q173 F / K / R / S / T / V, D175F / L / S, K178E / G / N / R, E179A / C / G / I / L / P / V , E180G / H / P, T181C / I / V, V182A / G / I / L / Q / S, R183A / L / Q, H184I / K / M / N / R / S / T / V, Y186L, K187G / Y, V188G / L, L190A / C / E / G / H / N / Q / R, E191D / K, K192A / H / I / W, Q194F / L / R / V / Y, P195G / I / M / R, V196I, I197A / L / Q / V, D198G, Y199R, Y200A, G201A / F / K / L / S, K202E / G / M / S, K203A / E / L / R, G204R / S, I205L / P, L206W, K207A / L / T, R208E, D210V, G211A, T212C / H / I / L / M / N / P / Q / R / S / W, I213L / P / Q / S, G214A / E / L / M / P / R / T / W, I215A / P / V, D216A / D / E / G / H / L / M / N / P / R / S / T / V, N217E / G / H / P / T, V218A / I / L, V219A / F / G / L / P / S / T / W, A220E / V, E221D, V222I / T, L223T, K224G / S / T, I225A / L / Q / R / T / V, I226K / L / P / R / S / T, G227P, W228D / F / G / I / L / P / Q / R / S / T / V, S229C / I, D230A / M / P / Q, or K231A / Q / R / T, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0222] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set at amino acid position(s) 37, 47, 52, 53, 54, 55, 56, 89, 92, 96, 115, 125, 132, 135, 146, 168, 179, 180, 187, 220, 15, 40, 91, 93, 95, 117, 122, 166, 178, 203, 37 / 53 / 74 / 93 / 205, 37 / 74 / 93, 37 / 74 / 93 / 125, 37 / 74 / 205, 37 / 93 / 125 / 205, 37 / 125, 37 / 166, 47 / 55 / 126 / 178, 47 / 89 / 126 / 178, 47 / 89 / 146 / 178, 47 / 117 / 126 / 178, 47 / 178, 53 / 74 / 93 / 166, 53 / 74 / 125, 53 / 93 / 125 / 133 / 166 / 205, 55 / 126 / 178, 74, 74 / 93 / 133, 74 / 125, 74 / 205, 89 / 117 / 178, 89 / 126, 89 / 146 / 178, 89 / 178, 117 / 126, 117 / 126 / 178, 117 / 178, 126 / 146 / 178, 126 / 178, 133, 146 / 178, 205, 37 / 53, 37 / 53 / 74 / 125 / 205, 47 / 54 / 178, 47 / 146 / 178, 53 / 74 / 93, 53 / 93 / 125 / 166, 53 / 133, 54 / 117 / 126 / 146, 89 / 117 / 146 / 178, 89 / 126 / 178, 93 / 125, or 166 / 205, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0223] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set, or amino acid residue(s) 37E, 37Q, 47L, 52L, 53S, 54V, 55S, 56A, 56E, 89D, 89F, 89S, 89W, 92S, 96L, 115H, 125L, 132R, 135E, 135N, 146A, 168C, 179A, 180A, 187R, 220G, 15A, 40T, 53T, 53V, 54G, 54R, 55G, 55N, 55R, 89A, 89G, 89L, 89P, 89Q, 89V, 91L, 93G, 93L, 93V, 95W, 115N, 117A, 117E, 117V, 122S, 166E, 178A, 178F, 1781, 178M, 178N, 178R, 178T, 178V, 178Y, 180P, 203Q, 37H, 37H / 74H / 93G / 125L, 37H / 74H / 205Q, 37H / 93G / 125L / 205Q, 37H / 125L, 37R / 53M / 74H / 93G / 205Q, 37R / 74H / 93G, 37R / 166E, 47L / 55S / 126I / 178G, 47L / 89G / 126I / 178G, 47L / 89G / 146G / 178L, 47L / 117T / 126I / 178G, 47L / 178G, 53M / 74H / 93G / 166E, 53M / 74H / 125L, 53M / 93G / 125L / 133W / 166E / 205Q, 55S / 126I / 178L, 74H, 74H / 93G / 133W, 74H / 125L, 74H / 205Q, 89G / 117T / 178G, 89G / 126I, 89G / 146G / 178G, 89G / 178L, 117T, 117T / 1261, 117T / 126I / 178G, 117T / 126I / 178L, 117T / 178L, 126I / 146G / 178G, 126I / 178G, 133W, 146G, 146G / 178G, 178G, 205Q, 37H / 53M, 37R, 37R / 53M, 37R / 53M / 74H / 125L / 205Q, 47L / 54R / 178G, 47L / 146G / 178G, 47L / 178L, 53M,Docket No. 5244.286WO3 53M / 74H / 93G, 53M / 93G / 125L / 166E, 53M / 133W, 54R / 117T / 126I / 146G, 74H, 89G / 117T / 146G / 178L, 89G / 126I / 178G, 93G / 125L, 166E / 205Q, or 178L, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ. ID NO: 16.

[0224] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set T37E, T37Q, I47L, V52L, A53S, K54V, Q55S, N56A, N56E, K89D, K89F, K89S, K89W, E92S, I96L, E115H, V125L, D132R, V135E, V135N, K146A, G168C, E179A, E180A, K187R, A220G, Y15A, P40T, A53T, A53V, K54G, K54R, Q55G, Q55N, Q55R, K89A, K89G, K89L, K89P, K89Q, K89V, C91L, K93G, K93L, K93V, F95W, E115N, Q117A, Q117E, Q117V, T122S, D166E, K178A, K178F, K178I, K178M, K178N, K178R, K178T, K178V, K178Y, E180P, K203Q, T37H, T37H / P74H / K93G / V125L, T37H / P74H / I205Q, T37H / K93G / V125L / I205Q, T37H / V125L, T37R / A53M / P74H / K93G / I205Q T37R / P74H / K93G, T37R / D166E, I47L / Q55S / L126I / K178G, I47L / K89G / L126I / K178G, I47L / K89G / K146G / K178L, I47L / Q117T / L126I / K178G, I47L / K178G, A53M / P74H / K93G / D166E, A53M / P74H / V125L, A53M / K93G / V125L / E133W / D166E / I205Q, Q55S / L126I / K178L, P74H, P74H / K93G / E133W, P74H / V125L, P74H / I205Q, K89G / Q117T / K178G, K89G / L126I, K89G / K146G / K178G, K89G / K178L, Q117T, Q117T / L126I, Q117T / L126I / K178G, Q117T / L126I / K178L, Q117T / K178L, L126I / K146G / K178G, L126I / K178G, E133W, K146G, K146G / K178G, K178G, I205Q, T37H / A53M, T37R, T37R / A53M, T37R / A53M / P74H / V125L / I205Q, I47L / K54R / K178G, I47L / K146G / K178G, I47L / K178L, A53M, A53M / P74H / K93G, A53M / K93G / V125L / D166E, A53M / E133W, K54R / Q117T / L126I / K146G, K89G / Q117T / K146G / K178L, K89G / L126I / K178G, K93G / V125L, D166E / I205Q, or K178L, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0225] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution at an amino acid position set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2 or 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0226] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least one substitution set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2 or 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.DocketNo. 5244.286WO3

[0227] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set at amino acid position(s) set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2 or 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ. ID NO: 16.

[0228] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set of an engineered adenylate kinase variant set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2 or 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0229] In some embodiments, the engineered adenylate kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference amino acid sequence comprising a substitution or substitution set of an engineered adenylate kinase variant set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2 or 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0230] In some embodiments, the engineered adenylate kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0231] In some embodiments, the engineered adenylate kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12-231 of an even-numbered SEQ ID NO. of SEQ ID NOs: 18-360 and 372-1018, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 18-360 and 372-1018.

[0232] In some embodiments, the engineered adenylate kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-Docket No. 5244.286WO3 231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0233] In some embodiments, the engineered adenylate kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-231 of an even-numbered SEQ ID NO. of SEQ ID NOs: 18-360 and 372-1018, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 18-360 and 372-1018, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0234] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution at amino acid position 4, 14, 15, 16, 17, 18, 31, 33, 35, 37, 38, 40, 46, 47, 52, 53, 54, 55, 56, 59, 61, 62, 66, 68, 69, 74, 80, 81, 82, 83, 84, 87, 89, 91, 92, 93, 95, 96, 104, 105, 109, 111, 112, 115, 117, 121, 122, 125, 126, 132, 133, 135, 136, 139, 142, 143, 146, 148, 149, 154, 157, 159, 162, 163, 166, 168, 169, 170, 178, 179, 180, 182, 183, 184, 187, 192, 194, 199, 202, 203, 204, 205, 207, 212, 214, 215, 217, 218, 219, 220, 222, 223, 225, 227, 228, and 229, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0235] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution, or amino acid residue 4N, 14P, 15A / H / T, 16C / G / M / T, 171, 18V, 31F / V / W, 33L, 35C / E, 37M, 37E / H / Q / R, 38D, 40T, 46E, 47L, 52L, 53D / M / S / T / V, 54G / R / V, 55Q, 55E / G / N / R / S, 56A / E, 59G, 61G, 62H, 66D, 68V, 69V, 74H, SOS, 81P, 82L, 83R, 84A / T, 87G / R / S, 89A / D / F / G / L / P / Q / S / V / W, 91L, 92S, 93E, 93G / L / V, 95W, 96L, 104L / R, 104Q, 105L / Q / S, 109A, 111N, 112C / T, 115C / H / N, 117A / E / G / R / T / V, 121V, 122S, 125L, 126C / I, 132R, 133W, 135E / N, 136L / R, 139A / H, 142V, 1431, 146A / G / H, 148Q, 149A / L / M / Q, 154E, 157R, 159V, 162D / M / V, 163E / R, 166E, 168C, 169E, 170K / M, 178A / F / G / I / L / M / N / R / T / V / Y, 179A, 180A / P / Q, 182L, 183L, 184P / Q / S, 187D / R, 192L / M / V, 194L, 199F, 202E / G / N, 203a 204D, 205Q, 2051, 205C / L, 2071 / N, 212A, 214K / P, 215V, 217A / E, 218L / V, 219L / T, 220G, 222A, 223D / M / V, 225H / T, 225A / a 227S, 228L / S, and 229G, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0236] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution at amino acid position 37, 93, 125, or 205, or combinations thereof, wherein theDocket No. 5244.286WO3 amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0237] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or amino acid residue 37H / M, 93G, 125L, or 205Q, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0238] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution at amino acid position 4, 47, 95, or 96; or substitution 14P, 15A / T, 16C / G / T, 37E / H / M / Q, 52L, 53T / V, 54V, 55N / R, 56A, 69V, 74H, 89D / F / G / S / W, 93L, 115C / H / N, 117A / E / T / V, 125L, 1261, 132R, 133W, 135E / N, 146A / G, 157R, 166E, 168C, 170M, 178A / F / G / I / L / M / N / R / T / V / Y, 180A / Q, 187D / R, 203Q, 205C / Q, 214K, 217E, or 220G; or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0239] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set, or amino acid residue(s) 4N, 14P, 15A / H / T, 16C / G / M / T, 171, 18V, 31F / V / W, 33L, 35C / E, 37M, 37E / H / Q / R, 38D, 40T, 46E, 47L, 52L, 53D / M / S / T / V, 54G / R / V, 55Q, 55E / G / N / R / S, 56A / E, 59G, 61G, 62H, 66D, 68V, 69V, 74H, 80S, 81P, 82L, 83R, 84A / T, 87G / R / S, 89A / D / F / G / L / P / Q / S / V / W, 91L, 92S, 93E, 93G / L / V, 95W, 96L, 104L / R, 104a 105L / Q / S, 109A, 111N, 112C / T, 115C / H / N, 117A / E / G / R / T / V, 121V, 122S, 125L, 126C / I, 132R, 133W, 135E / N, 136L / R, 139A / H, 142V, 1431, 146A / G / H, 148 149A / L / M / a 154E, 157R, 159V, 162D / M / V, 163E / R, 166E, 168C, 169E, 170K / M, 178A / F / G / I / L / M / N / R / T / V / Y, 179A, 180A / P / a 182L, 183L, 184P / Q / S, 187D / R, 192L / M / V, 194L, 199F, 202E / G / N, 203a 204D, 205a 2051, 205C / L, 2071 / N, 212A, 214K / P, 215V, 217A / E, 218L / V, 219L / T, 220G, 222A, 223D / M / V, 225H / T, 225A / a 227S, 228L / S, and 229G, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0240] In some embodiments, where the amino acid sequence of the engineered adenylate kinase comprises at least a substitution at amino acid position 4, 47, 95, or 96; or substitution 14P, 15A / T, 16C / G / T, 37E / H / M / a 52L, 53T / V, 54V, 55N / R, 56A, 69V, 74H, 89D / F / G / S / W, 93L, 115C / H / N, 117A / E / T / V, 125L, 1261, 132R, 133W, 135E / N, 146A / G, 157R, 166E, 168C, 170M, 178A / F / G / I / L / M / N / R / T / V / Y, 180A / a 187D / R, 203Q, 205C / a 214K, 217E, or 220G; or combinationsDocket No. 5244.286WO3 thereof, the amino acid sequence can further comprise at least a substitution at amino acid position 11, 13, 14, 15, 16, 18, 20, 21, 22, 26, 27, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 97, 98, 99, 100, 101, 102, 104, 105, 108, 109, 110, 111, 112, 113, 115, 116, 117, 118, 119, 120, 122, 123, 124, 125, 126, 127, 128, 129, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 146, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 161, 162, 163, 166, 168, 169, 170, 172, 173, 175, 178, 179, 180, 181, 182, 183, 184, 186, 187, 188, 190, 191, 192, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, or 231, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0241] In some embodiments, the amino acid sequence of the engineered adenylate kinase can further comprise at least a substitution or amino acid residue 11D, 13G / S, 14C / E, 15F, 16M, 18C / E / L, 20A / P / R / T / V, 21L / N / R / S / T / V, 22A / S, 26L, 27C / S, 29Q / R, 30G / L / S / Y, 31R, 32F / I / L / N, 34A / E / S / V, 35A / E / F / L / S, 36A / E / I / L / M / Q / R / S / V / Y, 37F / G / I / L / R / Y, 38C / F / L / R, 39A / M / Q / T, 40C / F / L / S / T, 42T, 43A / C / G / H / N, 44S, 46E / F / L / M / Y, 48F / L, 50A / C / L / N / Q / R / S / V, 51F / G / K / L / M / R / T, 52A / H, 53A / M / S, 54G / H / I / M / Q / R / T, 55G / L / Q / S, 56E / Q / T, 57H / L / P / S, 58C / G / Y, 59E / G / H / M / P / R / T / V / W / Y, 60A / C / P / S / W, 61A / E / N / P / S / V, 62A / E / G / I / L / S, 63S, 64R, 65A / D / F / K / P / T, 66A / C / D / E / F / G / H / N / P / Q / R / S / T / V / W, 67R, 68A / D / E / G / I / L / Q / R / S / T / V / W / Y, 69F / L / Q / R / Y, 70R, 71T, 731 / R, 74C / G / Q / S, 75L, 76G / L / P, 77A / I / M / Q / S, 78C / G / N / T, 79A / K / L / P / W, 80A / D / G / L / N / P / R / S / V / W, 81F / I / L / Q / S, 821 / L / P / Q / T, 83G / H / L / S / T, 84A / E / H / M / S, 87A / E / I / K / L / M / R / V / Y, 88N / R / T, 89A / H / I / L / P / Q / T / V, 90C / E / F / K / S / T / V, 91A / F / G / L / S / V, 92L / S, 93A / E / G / P / S / T / V / Y, 94A / C / F / M / Q / S / T / V / Y, 971, 98A / G / Q, 99A / C, 100F / S / V, 101-, 102A / C / N, 104A / F / H / I / L / Q / R / S / W, 105G / K / L / M / R / S, 108M / R / S / V, 109E / Y, 110C / F, 111E / P / R, 112A / C / E / K / M / N / Q / R / T, 113E / W / Y, 115G / K / R, 116A / E / F / L, 117L / N / S, 118A / G / L / R / S, 119F / K / L / P / R / S, 120G / K / L / R / S / T, 122A / H / S, 123S, 124V, 1251, 126A / E / S / V, 1271 / L / P / S, 128C / E / I / K / N / R / S, 1291 / L / P / S, 131A / G / V, 132G / K / L / T / V, 133A / E / F / L / Q / S / V / W, 134L, 1351 / K / M / P / R / S, 136A / I / L / S / V / Y, 137L, 138C / I / M / V, 139A / H / L / R, 140G, 141G / V, 142F / L / M / W, 143A / C / G / P / R / S / T / V, 146D / H / N / R / V, 148F / H / M / P / Q / R / S / T, 149L, 150C / G / L / P / S / V, 151A / F / R / Y, 152F / H, 153C / K / S / V, 154Q / R, 155A / F / T / W, 156A / C / N / T / V, 157P / V, 161G / L, 162G, 163G / Q / S, 166C / F / L / P / S, 168G / L / N / Q / S, 169A / D / I / Y, 170A / G / H / P / R / S, 172A / H / M / S / T, 173F / K / R / S / T / V, 175F / L / S, 178E / G / N / R, 179A / C / G / I / L / P / V, 180G / H / P, 181C / I / V, 182A / G / I / L / Q / S, 183A / L / Q, 1841 / K / M / N / R / S / T / V, 186L, 187G / Y, 188G / L, 190A / C / E / G / H / N / Q / R, 191D / K, 192A / H / I / W, 194F / L / R / V / Y, 195G / I / M / R, 1961, 197A / L / Q / V, 198G, 199R, 200A, 201A / F / K / L / S, 202E / G / M / S, 203A / E / L / R, 204R / S, 205L / P, 206W, 207A / L / T, 208E, 210V, 211A, 212C / H / I / L / M / N / P / Q / R / S / W, 213L / P / Q / S, 214A / E / L / M / P / R / T / W, 215A / P / V,Docket No. 5244.286WO3 216A / D / E / G / H / L / M / N / P / R / S / T / V, 217E / G / H / P / T, 218A / I / L, 219A / F / G / L / P / S / T / W, 220E / V, 221D, 2221 / T, 223T, 224G / S / T, 225A / L / Q / R / T / V, 226K / L / P / R / S / T, 227P, 228D / F / G / I / L / P / Q / R / S / T / V, 229C / I, 230A / M / P / Q, or 231A / Q / R / T, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0242] In some embodiments, the engineered adenylate kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, or to the reference sequence corresponding to SEQ ID NO: 144, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, or relative to the reference sequence corresponding to SEQ ID NO: 144.

[0243] In some embodiments, the engineered adenylate kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-231 of an even-numbered SEQ ID NO. of SEQ ID NOs: 252-286, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 252-286, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, or relative to the reference sequence corresponding to SEQ ID NO: 144.

[0244] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set at amino acid position(s) 15, 16, 37, 69, 80, 82, 112, 115, 170, 178, 187, 205, or 217, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, or relative to the reference sequence corresponding to SEQ ID NO: 144.

[0245] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set, or amino acid residue(s) 15T, 16C, 16G, 16T, 37M, 69V, 80S, 82L, 112C, 112T, 115C, 170M, 178A, 178L, 178V, 187D, 205C, or 217E, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, or relative to the reference sequence corresponding to SEQ ID NO: 144.

[0246] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set Y15T, L16C, L16G, L16T, H37M, K69V, P80S, V82L, S112C, S112T, E115C, S170M, K178A, K178L, K178V, K187D, Q205C, or P217E, or combinations thereof,Docket No. 5244.286WO3 wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, or relative to the reference sequence corresponding to SEQ ID NO: 144.

[0247] In some embodiments, the engineered adenylate kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-231 of SEQ. ID NO: 252, or to the reference sequence corresponding to SEQ ID NO: 252, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 252, or relative to the reference sequence corresponding to SEQ ID NO: 252.

[0248] In some embodiments, the engineered adenylate kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-231 of an even-numbered SEQ ID NO. of SEQ ID NOs: 288-360, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 288-360, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 252, or relative to the reference sequence corresponding to SEQ ID NO: 252.

[0249] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set at amino acid position(s) 4 / 15, 14 / 15 / 93, 14 / 170 / 178, 14 / 214, 15 / 93 / 157 / 178 / 205 / 214, 15 / 115 / 157, 15 / 115 / 170, 15 / 170 / 180, 15 / 214, 93, 93 / 157 / 170 / 214, 93 / 205, 115, 157 / 170, 157 / 205 / 214, 178 / 180 / 214, 180, 214, 14, 14 / 15 / 157 / 170 / 178 / 180 / 214, 14 / 15 / 157 / 205, 14 / 157 / 170 / 178 / 180 / 205, 14 / 205, 15, 15 / 93 / 157 / 214, 15 / 93 / 180, 15 / 93 / 205, 15 / 157, 15 / 157 / 205, 15 / 178, 15 / 205, 93 / 157 / 205, 93 / 157 / 214, 93 / 170 / 205, 93 / 178 / 205, 93 / 180, or 205, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 252, or relative to the reference sequence corresponding to SEQ ID NO: 252.

[0250] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set, or amino acid residue(s) 4N / 15T, 14P / 15T / 93E, 14P / 170M / 178T, 14P / 214P, 15T / 93E / 157R / 178T / 205L / 214K, 15T / 115H / 157R, 15T / 115H / 170M, 15T / 170M / 180Q, 15T / 214P, 93E, 93E / 157R / 170M / 214K, 93E / 205L, 115H, 157R / 170M, 157R / 205L / 214P, 178T / 180Q / 214P, 180Q, 214P, 14P, 14P / 15T / 157R / 170M / 178T / 180Q / 214K, 14P / 15T / 157R / 205L, 14P / 157R / 170M / 178T / 180Q / 205L, 14P / 205L, 15T, 15T / 93E / 157R / 214P, 15T / 93E / 180Q, 15T / 93E / 205L, 15T / 157R, 15T / 157R / 205L, 15T / 178T, 15T / 205L, 93E / 157R / 205L, 93E / 157R / 214K, 93E / 170M / 205L, 93E / 178T / 205L, 93E / 180Q, or 205L, wherein the amino acid positionsDocket No. 5244.286WO3 are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 252, or relative to the reference sequence corresponding to SEQ ID NO: 252.

[0251] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set H4N / Y15T, A14P / Y15T / G93E, A14P / S170M / K178T, A14P / E214P, Y15T / G93E / P157R / K178T / Q205L / E214K, Y15T / E115H / P157R, Y15T / E115H / S170M, Y15T / S170M / E180Q, Y15T / E214P, G93E, G93E / P157R / S170M / E214K, G93E / Q205L, E115H, P157R / S170M, P157R / Q205L / E214P, K178T / E180Q / E214P, E180Q, E214P, A14P, A14P / Y15T / P157R / S170M / K178T / E180Q / E214K, A14P / Y15T / P157R / Q205L, A14P / P157R / S170M / K178T / E180Q / Q205L, A14P / Q205L, Y15T, Y15T / G93E / P157R / E214P, Y15T / G93E / E180Q, Y15T / G93E / Q205L, Y15T / P157R, Y15T / P157R / Q205L, Y15T / K178T, Y15T / Q205L, G93E / P157R / Q205L, G93E / P157R / E214K, G93E / S170M / Q205L, G93E / K178T / Q205L, G93E / E180Q, or Q205L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 252, or relative to the reference sequence corresponding to SEQ ID NO: 252.

[0252] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution at an amino acid position set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2 or 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0253] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least one substitution set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2 or 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0254] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set at amino acid position(s) set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2. 12.2, 13.2, 14.2, 15.2, 16.2, 17.2 or 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0255] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set of an engineered adenylate kinase variant set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2 or 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, 252, 402, 446,Docket No. 5244.286WO3 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0256] In some embodiments, the engineered adenylate kinase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference amino acid sequence comprising a substitution or substitution set of an engineered adenylate kinase variant set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2 or 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ. ID NO: 144 , 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0257] In some embodiments, the engineered adenylate kinase comprises an amino acid sequence comprising residues 12-231 of an even-numbered SEQ ID NO. of SEQ ID NOs: 18-360 and 372-1018, or comprises an even-numbered SEQ ID NO. of SEQ ID NOs: 18-360 and 372-1018. In some embodiments, the amino acid sequence of the engineered adenylate kinase optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the engineered adenylate kinase includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions. In some embodiments, the amino acid sequence of the engineered adenylate kinase optionally includes 1, 2, 3, 4, or 5 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the engineered adenylate kinase optionally includes 1, 2, 3, 4, or 5 substitutions.

[0258] In some embodiments, the engineered adenylate kinase comprises an amino acid sequence comprising residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or wherein the amino acid sequence comprises SEQ ID NO: 144, 252, 402, 446, 710, or 808. In some embodiments, the amino acid sequence of the engineered adenylate kinase optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the engineered adenylate kinase includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions. In some embodiments, the amino acid sequence of the engineered adenylate kinase optionally includes 1, 2, 3, 4, or 5 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the engineered adenylate kinase optionally includes 1, 2, 3, 4, or 5 substitutions.

[0259] In some embodiments, the engineered adenylate kinase polypeptide has 1, 2, 3, 4, or up to 5 substitutions in the amino acid sequence. In some embodiments, the engineered adenylate kinase polypeptide has 1, 2, 3, or 4 substitutions in the amino acid sequence.

[0260] In some embodiments, the engineered adenylate kinase described herein has adenylate kinase activity and at least one improved property as compared to a reference adenylate kinase.Docket No. 5244.286WO3

[0261] In some embodiments, the engineered adenylate kinase has increased activity on NMP substrate as compared to the reference adenylate kinase. In some embodiments, the NMP substrate is AMP, CMP, GMP, UMP, or TMP. In some embodiments, the engineered adenylate kinase has at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 10, or more fold activity compared to the reference adenylate kinase.

[0262] In some embodiments, the engineered adenylate kinase is capable of converting NMP substrate to NDP product at greater than 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the engineered adenylate kinase is capable of converting substrate AMP, GMP, CMP, UMP, orTMP to product ADP, GDP, CDP, UDP, orTDP, respectively, at greater than 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0263] In some embodiments, the engineered adenylate kinase has increased activity on 2'-fluoro modified nucleoside monophosphate and / or increased activity on 2'-O-methyl modified nucleoside monophosphate. In some embodiments, the engineered adenylate kinase has at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 10, or more fold activity with 2'-fluoro modified nucleoside monophosphate and / or 2'-O-methyl modified nucleoside monophosphate as compared to the reference adenylate kinase. In some embodiments, the 2'-fluoro modified nucleoside monophosphate substrate is 2'-F-AMP, 2'-F-GMP, 2'-F-CMP, 2'-F-UMP, or 2'-F-TMP. In some embodiments, the 2'-O-methyl modified nucleoside monophosphate substrate is 2'-O-methyl-AMP, 2'-O-methyl-GMP, 2'-O-methyl-CMP, 2'-O-methyl-UMP, or 2'-O-methyl-TMP. Exemplary increases in activity with 2'-modified nucleoside monophosphate substrates are provided in the Examples.

[0264] In some embodiments, the engineered adenylate kinase is capable of converting substrate 2'-fluoro-NMP to product 2'-fluoro-NDP at greater than 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the engineered adenylate kinase is capable of converting substrate 2'-fluoro-AMP, 2'-fluoro-GMP, 2'-fluoro-CMP, 2'-fluoro-UMP, or 2'-fluoro-TMP to product 2'-fluoro-ADP, 2'-fluoro-GDP, 2'-fluoro-CDP, 2'-fluoro-UDP, or 2'-fluoro-TDP, respectively, at greater than 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0265] In some embodiments, the engineered adenylate kinase is capable of converting substrate 2'-O-methyl-NMP substrate to product 2'-O-methyl-NDP at greater than 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the engineered adenylate kinase is capable of converting substrate 2'-O-methyl-AMP, 2'-O-methyl-GMP, 2'-O-methyl-CMP, 2'-O-methyl-UMP, or 2'-O-methyl-TMP to product 2'-O-methyl-ADP, 2'-O-methyl-GDP, 2'-O-methyl-CDP, 2'-O-methyl-UDP, or 2'-O-methyl-TDP, respectively, at greater than 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.Docket No. 5244.286WO3

[0266] Exemplary conditions for the conversions are provided in the Examples, including for NMP substrates, 2'-fluoro NDP substrates, and 2'-O-methyl NDP substrates.

[0267] In some embodiments, the engineered adenylate kinase exhibits increased stability as compared to the reference adenylate kinase. In some embodiments, the engineered adenylate kinase exhibis increased thermostability, e.g., at 50 °C to 70 °C. Exemplary stability and thermostability conditions are provided in the Examples.

[0268] In some embodiments, the engineered adenylate kinase exhibits an improved property selected from i) increased activity on unmodified nucleoside monophosphate (NMP), ii) increased stability, iii) increased thermostability, iv) increased activity on 2'-fluoro modified nucleoside monophosphate, and v) increased activity on 2'-O-methyl modified nucleoside monophosphate, or any combinations of i), ii), iii), iv), and v), as compared to a reference adenylate kinase.

[0269] In some embodiments, the reference adenylate kinase for comparison of an improved property has an amino acid sequence corresponding to residues 12-231 of SEQ ID NO: 16, 144, 252, 402, 446, 710, or 808, or an amino acid sequence corresponding to SEQ ID NO: 16, 144, 252, 402, 446, 710, or 808. In some embodiments, the reference adenylate kinase has an amino acid sequence corresponding to residues 12-231 of SEQ ID NO: 16, or an amino acid sequence corresponding to SEQ ID NO: 16.

[0270] In some embodiments, the engineered adenylate kinase is provided in the form of a fusion polypeptide. In some embodiments, the engineered adenylate kinase is fused to variety of polypeptide sequences, such as, by way of example and not limitation, polypeptide tags that can be used for detection and / or purification. In some embodiments, the fusion protein of the engineered adenylate kinase comprises a glycine-histidine or histidine-tag (His-tag). In some embodiments, the fusion protein of the engineered adenylate kinase comprises a one or more lysine residues, e.g., 1-12 residues of lysine, including polylysine, such as for conjugation to a support medium. In some embodiments, the fusion protein of the engineered adenylate kinase comprises an epitope tag, such as c-myc, FLAG, V5, or hemagglutinin (HA). In some embodiments, the fusion protein of the engineered adenylate kinase comprises a GST, SUMO, Strep, MBP, or GFP tag. In some embodiments, the fusion is to the amino (N-) terminus of engineered adenylate kinase polypeptide. In some embodiments, the fusion is to the carboxy (C-) terminus of the adenylate kinase polypeptide.

[0271] In some embodiments, the present disclosure further provides functional fragments or biologically active fragments of engineered adenylate kinase polypeptides described herein. Thus, for each and every embodiment herein of an engineered adenylate kinase, a functional fragment or biologically active fragment of the engineered adenylate kinase is provided herewith. In some embodiments, a functional fragment or biologically active fragments of an engineered adenylate kinase comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the activity of theDocket No. 5244.286WO3 adenylate kinase polypeptide from which it was derived (i.e., the parent adenylate kinase). In some embodiments, functional fragments or biologically active fragments comprise at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the parent sequence of the adenylate kinase. In some embodiments the functional fragment will be truncated by less than 5, less than 10, less than 15, less than 10, less than 25, less than 30, less than 35, less than 40, less than 45, and less than 50 amino acids.

[0272] In some embodiments, a functional fragment of an engineered adenylate kinase herein comprises at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the parent sequence of the engineered adenylate kinase. In some embodiments, the functional fragment will be truncated by less than 5, less than 10, less than 15, less than 10, less than 25, less than 30, less than 35, less than 40, less than 45, less than 50, less than 55, less than 60, less than 65, or less than 70 amino acids.

[0273] In some embodiments, the functional fragments or biologically active fragments of the engineered adenylate kinase polypeptide described herein include at least a mutation or mutation set in the amino acid sequence of an engineered adenylate kinase described herein. Accordingly, in some embodiments, the functional fragments or biologically active fragments of the engineered adenylate kinase displays the enhanced or improved property associated with the mutation or mutation set in the parent adenylate kinase.

[0274] In some embodiments, the engineered adenylate kinase is purified, as described herein. In some embodiments, the purified preparation has the engineered adenylate kinase at least 60%, 70%, 80%, 85%, 90%, or 95% greater of the protein content of the preparation.

[0275] In some embodiments, an engineered adenylate kinase described herein is provided immobilized on a substrate or support medium, such as a solid substrate, a porous substrate, a membrane, or particles. The polypeptide can be entrapped in matrixes or membranes. In some embodiments, matrices include polymeric materials such as calcium-alginate, agar, k-carrageenin, polyacrylamide, and collagen. In some embodiments, the solid matrices, includes, among others, activated carbon, porous ceramic, and diatomaceous earth. In some embodiments, the matrix is a particle, a membrane, or a fiber. Types of membranes include, among others, nylon, cellulose, polysulfone, or polyacrylate.

[0276] In some embodiments, the engineered adenylate kinase is immobilized on the surface of a support material. In some embodiments, the polypeptide is adsorbed on the support material. In some embodiments, the polypeptide is immobilized on the support material by covalent attachment.Support materials include, among others, inorganic materials, such as alumina, silica, porous glass, ceramics, diatomaceous earth, clay, and bentonite, or organic materials, such as cellulose (CMC, DEAE-cellulose), starch, activated carbon, polyacrylamide, polystyrene, polymethacrylate, polyacrylate, andDocket No. 5244.286WO3 ion-exchange resins, such as Amberlite, Sephadex, and Dowex. Various functional groups known in the art may be used to conjugate the engineered adenylate kinase to the substrate or support medium, e.g., epoxide, imide, etc.Polynucleotides Encoding Engineered Polypeptides, Expression Vectors and Host Cells

[0277] In another aspect, the present disclosure provides recombinant polynucleotides encoding the engineered adenylate kinases described herein. In some embodiments, the recombinant polynucleotides are operably linked to one or more heterologous regulatory sequences that control gene expression to create a recombinant polynucleotide construct capable of expressing the engineered adenylate kinase. In some embodiments, an expression construct containing at least one heterologous polynucleotide encoding the engineered adenylate kinase polypeptide(s) is introduced into appropriate host cells to express the corresponding adenylate kinase polypeptide(s).

[0278] As will be apparent to the skilled artisan, availability of a protein sequence and the knowledge of the codons corresponding to the various amino acids provide a description of all the polynucleotides capable of encoding the subject polypeptides. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons, allows an extremely large number of nucleic acids to be made, all of which encode an engineered adenylate kinase of the present disclosure. Thus, the present disclosure provides methods and compositions for the production of each and every possible variation of polynucleotides that could be made that encode the engineered adenylate kinase polypeptides described herein by selecting combinations based on the possible codon choices, and all such polynucleotide variants are to be considered specifically disclosed for any polypeptide described herein, including the amino acid sequences presented in the Examples (e.g., Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2 or 18.2) and in the Sequence Listing.

[0279] In some embodiments, the codons are preferably optimized for utilization by the chosen host cell for protein production. In some embodiments, preferred codons in bacteria are used for expression in bacteria. In some embodiments, preferred codons in fungal cells are used for expression in fungal cells. In some embodiments, preferred codons in insect cells are used for expression in insect cells. In some embodiments, preferred codons in mammalian cells are used for expression in mammalian cells. In some embodiments, codon optimized polynucleotides encoding an engineered adenylate kinase polypeptide described herein contain preferred codons at about 40%, 50%, 60%, 70%, 80%, 90%, or greater than 90% of the codon positions in the full-length coding region.

[0280] Accordingly, in some embodiments, a recombinant polynucleotide of the present disclosure comprises a polynucleotide sequence encoding an engineered adenylate kinase polypeptide described herein. In some embodiments, the polynucleotide sequence of the recombinant polynucleotide has preferred codons for expression (e.g., codon optimized). In some embodiments, the polynucleotideDocket No. 5244.286WO3 sequence of the recombinant polynucleotide is codon optimized for expression in eukaryotic or prokaryotic cells, as further discussed herein.

[0281] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-231 of an even-numbered SEQ ID NO. of SEQ ID NOs: 16-360, or to a reference sequence corresponding to an even-numbered SEQ. ID NO. of SEQ ID NOs: 16-360, wherein the amino acid sequence comprises one or more substitutions relative to a reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, 144, 252, 402, 446, 710, or 808, or relative to a reference sequence corresponding to SEQ ID NO: 16, 144, 252, 402, 446, 710, or 808.

[0282] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, 144, 252, 402, 446, 710, or 808, or to a reference sequence corresponding to SEQ ID NO: 16, 144, 252, 402, 446, 710, or 808, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 16, 144, 252, 402, 446, 710, or 808.

[0283] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence having at least 0%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or to the reference sequence corresponding to SEQ ID NO: 16, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0284] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808, wherein the amino acid sequence comprises one or moreDocketNo. 5244.286WO3 substitutions relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0285] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-231 of an even-numbered SEQ ID NO. of SEQ ID NOs: 18-360 and 372-1018, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 18-360 and 372-1018, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0286] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a engineered adenylate kinase comprising an amino acid sequence comprising at least a substitution at amino acid position 4, 14, 15, 16, 17, 18, 31, 33, 35, 37, 38, 40, 46, 47, 52, 53, 54, 55, 56, 59, 61, 62, 66, 68, 69, 74, 80, 81, 82, 83, 84, 87, 89, 91, 92, 93, 95, 96, 104, 105, 109, 111, 112, 115, 117, 121, 122, 125, 126, 132, 133, 135, 136, 139, 142, 143, 146, 148, 149, 154, 157, 159, 162, 163, 166, 168, 169, 170, 178, 179, 180, 182, 183, 184, 187, 192, 194, 199, 202, 203, 204, 205, 207, 212, 214, 215, 217, 218, 219, 220, 222, 223, 225, 2 1 , 228, and 229, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0287] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence comprising at least a substitution at amino acid position 37, 93, 125, or 205, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0288] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position 4, 47, 95, or 96; or substitution 14P, 15A / T, 16C / G / T, 37E / H / M / Q, 52L, 53T / V, 54V, 55N / R, 56A, 69V, 74H, 89D / F / G / S / W, 93L, 115C / H / N, 117A / E / T / V, 125L, 1261, 132R, 133W, 135E / N, 146A / G, 157R, 166E, 168C, 170M, 178A / F / G / I / L / M / N / R / T / V / Y, 180A / Q, 187D / R, 203Q, 205C / Q, 214K, 217E, or 220G; or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.Docket No. 5244.286WO3

[0289] In some embodiments, the amino acid sequence of the engineered adenylate kinase further comprises at least a substitution at amino acid position 11, 13, 14, 15, 16, 18, 20, 21, 22, 26, 27, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 97, 98, 99, 100, 101, 102, 104, 105, 108, 109, 110, 111, 112, 113, 115, 116, 117, 118, 119, 120, 122, 123, 124, 125, 126, 127, 128, 129, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 146, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 161, 162, 163, 166, 168, 169, 170, 172, 173, 175, 178, 179, 180, 181, 182, 183, 184, 186, 187, 188, 190, 191, 192, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, or 231, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ. ID NO: 16.

[0290] In some embodiments, the amino acid sequence of the engineered adenylate kinase comprises or further comprises at least a substitution or substitution set at amino acid position(s) 37, 47, 52, 53, 54, 55, 56, 89, 92, 96, 115, 125, 132, 135, 146, 168, 179, 180, 187, 220, 15, 40, 91, 93, 95, 117, 122, 166, 178, 203, 37 / 53 / 74 / 93 / 205, 37 / 74 / 93, 37 / 74 / 93 / 125, 37 / 74 / 205, 37 / 93 / 125 / 205, 37 / 125, 37 / 166, 47 / 55 / 126 / 178, 47 / 89 / 126 / 178, 47 / 89 / 146 / 178, 47 / 117 / 126 / 178, 47 / 178, 53 / 74 / 93 / 166, 53 / 74 / 125, 53 / 93 / 125 / 133 / 166 / 205, 55 / 126 / 178, 74, 74 / 93 / 133, 74 / 125, 74 / 205, 89 / 117 / 178, 89 / 126, 89 / 146 / 178, 89 / 178, 117 / 126, 117 / 126 / 178, 117 / 178, 126 / 146 / 178, 126 / 178, 133, 146 / 178, 205, 37 / 53, 37 / 53 / 74 / 125 / 205, 47 / 54 / 178, 47 / 146 / 178, 53 / 74 / 93, 53 / 93 / 125 / 166, 53 / 133, 54 / 117 / 126 / 146, 89 / 117 / 146 / 178, 89 / 126 / 178, 93 / 125, or 166 / 205, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0291] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence comprising at least a substitution at an amino acid position set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2 or 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0292] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence comprising at least one substitution set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2 or 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acidDocket No. 5244.286WO3 residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0293] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2. 13.2. 14.2. 15.2. 16.2. 17.2 or 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ. ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0294] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence comprising at least a substitution or substitution set of an engineered adenylate kinase variant set forth in Tables 7.2, 8.2, 9.2. 10.2. 11.2. 12.2. 13.2. 14.2. 15.2. 16.2. 17.2 or 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0295] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference amino acid sequence comprising a substitution or substitution set of an engineered adenylate kinase variant set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2. 13.2. 14.2. 15.2. 16.2. 17.2 or 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0296] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0297] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12-231 of an even-numbered SEQ ID NO. of SEQ ID NOs: 18-360 and 372-1018, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 18-360 and 372-1018.Docket No. 5244.286WO3

[0298] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or to the reference sequence corresponding to SEQ. ID NO: 144, 252, 402, 446, 710, or 808, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0299] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-231 of an even-numbered SEQ ID NO. of SEQ ID NOs: 18-360 and 372-1018, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 18-360 and 372-1018, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144 , 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0300] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence comprising at least a substitution at amino acid position 4, 14, 15, 16, 17, 18, 31, 33, 35, 37, 38, 40, 46, 47, 52, 53, 54, 55, 56, 59, 61, 62, 66, 68, 69, 74, 80, 81, 82, 83, 84, 87, 89, 91, 92, 93, 95, 96, 104, 105, 109, 111, 112, 115, 117, 121, 122, 125, 126, 132, 133, 135, 136, 139, 142, 143, 146, 148, 149, 154, 157, 159, 162, 163, 166, 168, 169, 170, 178, 179, 180, 182, 183, 184, 187, 192, 194, 199, 202, 203, 204, 205, 207, 212, 214, 215, 217, 218, 219, 220, 222, 223, 225, 227, 228, and 229, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0301] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence comprising at least a substitution at amino acid position 37, 93, 125, or 205, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.Docket No. 5244.286WO3

[0302] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence comprising at least a substitution at amino acid position 4, 47, 95, or 96; or substitution 14P, 15A / T, 16C / G / T, 37E / H / M / Q, 52L, 53T / V, 54V, 55N / R, 56A, 69V, 74H, 89D / F / G / S / W, 93L, 115C / H / N, 117A / E / T / V, 125L, 1261, 132R, 133W, 135E / N, 146A / G, 157R, 166E, 168C, 170M, 178A / F / G / I / L / M / N / R / T / V / Y, 180A / Q, 187D / R, 203Q, 205C / Q, 214K, 217E, or 220G; or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0303] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence further comprising a substitution at amino acid position 11, 13, 14, 15, 16, 18, 20, 21, 22, 26, 27, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 97, 98, 99, 100, 101, 102, 104, 105, 108, 109, 110, 111, 112, 113, 115, 116, 117, 118, 119, 120, 122, 123, 124, 125, 126, 127, 128, 129, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 146, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 161, 162, 163, 166, 168, 169, 170, 172, 173, 175, 178, 179, 180, 181, 182, 183, 184, 186, 187, 188, 190, 191, 192, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, or 231, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0304] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, or to the reference sequence corresponding to SEQ ID NO: 144, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, or relative to the reference sequence corresponding to SEQ ID NO: 144.

[0305] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,Docket No. 5244.286WO3 99%, or more sequence identity to the reference sequence corresponding to residues 12-231 of an even-numbered SEQ ID NO. of SEQ ID NOs: 252-286, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 252-286, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, or relative to the reference sequence corresponding to SEQ ID NO: 144.

[0306] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 15, 16, 37, 69, 80, 82, 112, 115, 170, 178, 187, 205, or 217, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, or relative to the reference sequence corresponding to SEQ ID NO: 144.

[0307] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 252, or to the reference sequence corresponding to SEQ ID NO: 252, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 252, or relative to the reference sequence corresponding to SEQ ID NO: 252.

[0308] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-231 of an even-numbered SEQ ID NO. of SEQ ID NOs: 288-360, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 288-360, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 252, or relative to the reference sequence corresponding to SEQ ID NO: 252.

[0309] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 4 / 15, 14 / 15 / 93, 14 / 170 / 178, 14 / 214, 15 / 93 / 157 / 178 / 205 / 214, 15 / 115 / 157, 15 / 115 / 170, 15 / 170 / 180, 15 / 214, 93, 93 / 157 / 170 / 214, 93 / 205, 115, 157 / 170, 157 / 205 / 214, 178 / 180 / 214, 180, 214, 14, 14 / 15 / 157 / 170 / 178 / 180 / 214, 14 / 15 / 157 / 205, 14 / 157 / 170 / 178 / 180 / 205, 14 / 205, 15, 15 / 93 / 157 / 214, 15 / 93 / 180, 15 / 93 / 205, 15 / 157, 15 / 157 / 205, 15 / 178, 15 / 205, 93 / 157 / 205, 93 / 157 / 214, 93 / 170 / 205, 93 / 178 / 205, 93 / 180, or 205, wherein the aminoDocket No. 5244.286WO3 acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 252, or relative to the reference sequence corresponding to SEQ ID NO: 252.

[0310] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence comprising at least a substitution at an amino acid position set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2. 17.2 or 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0311] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence comprising at least one substitution set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2 or 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144 , 252, 402, 446, 710, or 808.

[0312] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2. 13.2. 14.2. 15.2. 16.2. 17.2 or 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0313] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence comprising at least a substitution or substitution set of an engineered adenylate kinase variant set forth in Tables 7.2, 8.2, 9.2. 10.2. 11.2. 12.2. 13.2. 14.2. 15.2. 16.2. 17.2 or 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0314] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference amino acid sequence comprising a substitution or substitution set of an engineered adenylate kinase variant set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2. 13.2. 14.2. 15.2. 16.2. 17.2 or 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.Docket No. 5244.286WO3

[0315] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence comprising residues 12-231 of an even-numbered SEQ ID NO. of SEQ ID NOs: 18-360 and 372-1018, or comprises an even-numbered SEQ ID NO. of SEQ ID NOs: 18-360 and 372-1018.

[0316] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered adenylate kinase comprising an amino acid sequence comprising residues 12-231 of SEQ. ID NO: 144, 252, 402, 446, 710, or 808, or wherein the amino acid sequence comprises SEQ ID NO: 144, 252, 402, 446, 710, or 808.

[0317] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34-693 of SEQ ID NO: 143, 251, 401, 445, 709, or 807, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 143, 251, 401, 445, 709, or 807, wherein the recombinant polynucleotide encodes an adenylate kinase.

[0318] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34-693 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 17-359 and 371-1017, or to a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO. of SEQ ID NOs: 17-359 and 371-1017, wherein the recombinant polynucleotide encodes an engineered adenylate kinase.

[0319] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 34-693 of SEQ ID NO: 143, 251, 401, 445, 709, or 807, or comprising SEQ ID NO: 143, 251, 401, 445, 709, or 807.|0320| In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 34-693 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 17-359 and 371-1017, or comprises SEQ ID NO: 17-359 and 371-1017, or comprising an odd-numbered SEQ ID NO. of SEQ ID NOs: 17-359 and 371-1017, or comprises SEQ ID NO: 17-359 and 371-1017.

[0321] In some embodiments, the present disclosure provides a recombinant polynucleotide capable of hybridizing under highly stringent conditions to a reference polynucleotide encoding an engineered adenylate kinase polypeptide described herein, e.g., a recombinant polynucleotide provided in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, and 13.2, or a reverse complement thereof. In some embodiments, the recombinant polynucleotide hybridizes under highly stringent conditions to a reference polynucleotideDocket No. 5244.286WO3 corresponding to nucleotide residues 34-693 of SEQ ID NO: 143, 251, 401, 445, 709, or 807, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 143, 251, 401, 445, 709, or 807, or a reverse complement thereof. In some embodiments, the recombinant polynucleotide hybridizes under highly stringent conditions to a reference polynucleotide corresponding to nucleotide residues 34-693 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 17-359 and 371-1017, or corresponding to an odd-numbered SEQ ID NO. of SEQ ID NOs: 17-359 and 371-1017, or a reverse complement thereof.

[0322] In some embodiments, the present disclosure provides a recombinant polynucleotide capable of hybridizing under highly stringent conditions to a reverse complement of a reference polynucleotide encoding an engineered adenylate kinase polypeptide described herein, wherein the recombinant polynucleotide hybridizing under stringent conditions encodes an adenylate kinase polypeptide comprising an amino acid sequence having one or more amino acid differences as compared to SEQ ID NO: 16, 144, 252, 402, 446, 710, or 808, at residue positions selected from any positions as set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, and 13.2. In some embodiments, the recombinant polynucleotide that hybridizes under highly stringent conditions to a reverse complement of a reference polynucleotide encoding an engineered adenylate kinase polypeptide described herein encodes an adenylate kinase polypeptide having one or more amino acid differences present in an engineered adenylate kinase having an amino acid sequence corresponding to residues 12-231 of an even-numbered SEQ ID NO. of SEQ ID NOs: 18-360 and 372-1018, or an amino acid sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 18-360 and 372-1018, wherein the amino acid differences are relative to SEQ ID NO: 16, 144, 252, 402, 446, 710, or 808.

[0323] In some embodiments, the recombinant polynucleotide that hybridizes under highly stringent conditions comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34-693 of SEQ ID NO: 143, 251, 401, 445, 709, or 807, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 143, 251, 401, 445, 709, or 807, or a reverse complement thereof. In some embodiments, the recombinant polynucleotide that hybridizes under highly stringent conditions comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34-693 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 17-359 and 371-1017, or an odd-numbered SEQ ID NO. of SEQ ID NOs: 17-359 and 371-1017, or a reverse complement thereof.

[0324] In some additional embodiments, the polynucleotide hybridizing under highly stringent conditions comprises a polynucleotide sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%,DocketNo. 5244.286WO3 86% 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reverse complement of a polynucleotide reference sequence corresponding to nucleotide residues 34-693 of SEQ ID NO: 143, 251, 401, 445, 709, or 807, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 143, 251, 401, 445, 709, or 807 encodes an engineered adenylate kinase polypeptide. In some additional embodiments, the polynucleotide hybridizing under highly stringent conditions comprises a polynucleotide sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reverse complement of a polynucleotide reference sequence corresponding to nucleotide residues 34-693 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 17-359 and 371-1017, or an odd-numbered SEQ ID NO. of SEQ ID NOs: 17-359 and 371-1017 encodes an engineered adenylate kinase polypeptide.

[0325] In some embodiments, a recombinant polynucleotide encoding any of the adenylate kinase provided herein is manipulated in a variety of ways to provide for expression of the polypeptide. In some embodiments, the recombinant polynucleotide encoding the polypeptides are provided as expression vectors where one or more control sequences is operably linked to the recombinant polynucleotide to regulate the expression of the polynucleotide and / or encoded polypeptide. The techniques for modifying polynucleotides and nucleic acid sequences utilizing recombinant DNA methods are known in the art.

[0326] In some embodiments, the control sequences include, among others, promoter sequences, Kozak sequence, leader sequences, polyadenylation sequences, pro-peptide sequences, signal peptide sequences, regulatory elements, and transcription terminators. As known in the art, suitable promoters can be selected based on the host cells used. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure, include, but are not limited to promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (See e.g., Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA, 1978, 75:3727-3731), as well as the tac promoter (See e.g., DeBoer et al., Proc. Natl Acad. Sci. USA, 1983, 80: 21-25). Exemplary promoters for filamentous fungal host cells, include, but are not limited to promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha- amylase, Aspergillus niger or Aspergillus awamori glucoamylase (gla A), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (See e.g., WO 96 / 00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and AspergillusDocket No. 5244.286WO3 oryzae triose phosphate isomerase), and mutant, truncated, and hybrid promoters thereof. Exemplary yeast cell promoters can be from the genes can be from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GALI), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. In some embodiments, promoters effective in Pichia cells are used. Other useful promoters for yeast host cells are known in the art (See e.g., Romanos et al., Yeast, 1992, 8:423-488). In some embodiments, for insect host cells, suitable promoters include, among others, baculovirus promoters (e.g., PIO and polyhedron promoters), OplE2 promoter, and Nephotettix cincticeps actin promoters. In some embodiments, promoters effective in Pichia cells are used. In some embodiments, for insect host cells, suitable promoters include, among others, baculovirus promoters (e.g., PIO and polyhedron promoters), OplE2 promoter, and Nephotettix cincticeps actin promoters. In some embodiments, for mammalian host cells, suitable promoters include, among others, promoters of cytomegalovirus (CMV), chicken -actin promoter fused with the CMV enhancer, simian virus 40 (SV40), human phosphoglycerate kinase, beta actin, elongation factor-la or glyceraldehyde-3-phosphate dehydrogenase, or Gallus 0-actin.

[0327] In some embodiments, the control sequence is also a suitable transcription terminator sequence (i.e., a sequence recognized by a host cell to terminate transcription). In some embodiments, the terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the leucine decarboxylase polypeptide. Any suitable terminator which is functional in the host cell of choice finds use in the present invention. For bacterial expression, the transcription terminators can be a Rho-dependent terminators that rely on a Rho transcription factor, or a Rho-independent, or intrinsic terminators, which do not require a transcription factor. Exemplary bacterial transcription terminators are described in Peters et al., J Mol BioL, 2011, 412(5) :793-813. Exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alphaglucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (See e.g., Romanos et aL, supra). Exemplary terminators for mammalian cells include, but are not limited to those from cytomegalovirus (CMV), Simian virus 40 (SV40), from Homo sapiens growth hormone hGH, from bovine growth hormone BGH, and from human or rabbit beta globulin.

[0328] In some embodiments, the control sequence is also a suitable leader sequence (i.e., a nontranslated region of an mRNA that is important for translation by the host cell). In some embodiments, the leader sequence is operably linked to the 51terminus of the nucleic acid sequence encoding theDocket No. 5244.286WO3 adenylate kinase polypeptide. Any suitable leader sequence that is functional in the host cell of choice find use in the present invention. Exemplary leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, and Aspergillus nidulans triose phosphate isomerase. Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP). Suitable leaders for mammalian host cells include but are not limited to the 5'-UTR element present in orthopoxvirus mRNA.

[0329] In some embodiments, the control sequence comprises a 3' untranslated nucleic acid region and polyadenylation tail nucleic acid sequence, sequences operably linked to the 31terminus of the protein coding nucleic acid sequence which mediate binding to proteins involved in mRNA trafficking and translation and mRNA half-life. Any polyadenylation sequence and 3' UTR which is functional in the host cell of choice may be used in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to those from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are also known in the art (See e.g., Guo and Sherman, Mol. Cell. Biol., 1995, 15:5983-5990). Useful polyadenylation and 3' UTR sequences for mammalian host cells include, but are not limited to, the 3'-UTRs of a- and (3-globin mRNAs that harbor several sequence elements that increase the stability and translation of mRNA.

[0330] In some embodiments, the control sequence is a signal peptide (i.e., a coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway). In some embodiments, the 5' end of the coding sequence of the nucleic acid sequence inherently contains a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, in some embodiments, the 5' end of the coding sequence contains a signal peptide coding region that is foreign to the coding sequence. Any suitable signal peptide coding region which directs the expressed polypeptide into the secretory pathway of a host cell of choice finds use for expression of the engineered polypeptide(s). Effective signal peptide coding regions for bacterial host cells are the signal peptide coding regions include, but are not limited to those obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus stearothermophilus alpha- amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are known in the art (See e.g., Simonen and Palva, Microbiol. Rev., 1993, 57:109-137). In some embodiments, effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to the signalDocket No. 5244.286WO3 peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. Useful signal peptides for yeast host cells include, but are not limited to those from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Useful signal peptides for mammalian host cells include but are not limited to, those from the genes for immunoglobulin gamma ( IgG ) and the signal peptide in a human secreted protein, such as human beta-galactosidase polypeptide.

[0331] In some embodiments, the control sequence is a regulatory sequence that facilitates the regulation of the expression of the recombinant polynucleotide and / or encoded polypeptide. Examples of regulatory systems are those that cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. In prokaryotic host cells, suitable regulatory sequences include, but are not limited to the lac, tac, and trp operator systems. In yeast host cells, suitable regulatory systems include, but are not limited to the ADH2 system or GALI system. In filamentous fungi, suitable regulatory sequences include, but are not limited to the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter. In mammalian cells, suitable regulatory systems include, among others, zinc-inducible sheep metallothionine (MT) promoter, dexamethasone (Dex)-inducible promoter, mouse mammary tumor virus (MMTV) promoter; ecdysone insect promoter, tetracycline-inducible promoter system, RU486-inducible promoter system, and the rapamycin-inducible promoter system.

[0332] In another aspect, the present disclosure provides an expression vector comprising a recombinant polynucleotide encoding an adenylate kinase polypeptide, where the recombinant polynucleotide is operably or operatively linked to a control sequence, such as a promoter and a terminator, a replication origin, etc., depending on the type of hosts into which they are to be introduced. The recombinant expression vector may be any suitable vector (e.g., a plasmid or virus), that can be conveniently subjected to recombinant DNA procedures and bring about the expression of the adenylate kinase polynucleotide sequence. The choice of the vector typically depends on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids.

[0333] In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, such as a plasmid, an extra-chromosomal element, a minichromosome, or an artificial chromosome). The vector may contain any means for assuring self-replication. In some alternative embodiments, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has beenDocket No. 5244.286WO3 integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.

[0334] In some embodiment, recombinant polynucleotides may be provided on a non-replicating expression vector or plasmid. In some embodiments, the non-replicating expression vector or plasmid can be based on viral vectors defective in replication (see, e.g., Travieso et al., npj Vaccines, 2022, Vol.7, Article 75).

[0335] In some embodiments, the expression vector contains one or more selectable markers, which permit easy selection of transformed cells. A "selectable marker" is a gene, the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers include, but are not limited to the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers, which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase; e.g., from A. nidulans or A. orzyae), argB (ornithine carbamoyltransferases), bar (phosphinothricin acetyltransferase; e.g., from S. hygroscopicus), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase; e.g., from A. nidulans or A. orzyae), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Selectable marker for mammalian cells include, among others, chloramphenicol acetyl transferase (CAT), nourseothricin N-acetyl transferase, blasticidin-S deaminase, blastcidin S acetyltransferase, Sh ble (Zeocin® resistance), aminoglycoside 3'-phosphotransferase (neomycin resistance), hph (hygromycin resistance), thymidine kinase, and puromycin N-acetyl-transferase.

[0336] In another aspect, the present disclosure provides a host cell comprising at least one recombinant polynucleotide encoding an adenylate kinase polypeptide of the present disclosure, the recombinant polynucleotide(s) being operatively linked to one or more control sequences for expression of the adenylate kinase polypeptide. In some embodiments, the host cells suitable for use in expressing the polypeptides encoded by the expression vectors is a prokaryotic cell or eukaryotic cell known in the art. In some embodiments, the host cell is a bacterial cell, including, among others, E. coli, B. subtilis, Vibrio fluvialis, Streptomyces and Salmonella typhimurium cell. Exemplary bacterial host cells also include various Escherichia coli strains (e.g., W3110 (AfhuA) and BL21). In some embodiments, the host cell is a fungal cell, such as filamentous fungal cell or yeast cell, n some embodiments, suitable fungal host cells include, among others, Pichia, Saccharomyces, Yarrowia, Kluyveromyces, Aspergillus, Trichoderma, Neurospora, Mucor, Penicillium T. Trichoderma, or Myceliophthora fungal cell. Exemplary fungal host cell includes, among others, Pichia pastoris, YarrowiaDocket No. 5244.286WO3 lipolytica, Kluyveromyces marxianus, Kluyveromyces lactis, Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus Trichoderma reesei. Neurospora crassa, Mucor circinelloides, Penicillium chrysogenum T. reesei, Trichoderma harzianum, Saccharomyces cerevisiae, or Myceliophthora thermophile. In some embodiments, the host cell is an insect cell. In some embodiments, a suitable insect host cell is a lepidopteran or dipteran insect cell. Exemplary insect host cell includes, among others, Sf9 cell, Sf21 cell, Schneider 2 cell, and BTI-TN-5B1-4 (High Five) cell. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a human cell or rodent cell.Exemplary mammalian cells include, among others, Expi293, HeLa, U2OS, A549, HT1080, CAD, P19, NIH 3T3, L929, Hek 293, 293F, 293E, 293T, COS, Vero, NSO, Sp2 / 0 cell, DUKX-X11, MCF-7, Y79, SO-Rb50, Hep G2, J558L, and CHO cell.

[0337] In another aspect, the present disclosure provides a method of producing the adenylate kinase polypeptides, where the method comprises culturing a host cell comprising an expression vector capable of expressing or producing the adenylate kinase polypeptide under suitable culture conditions such that the adenylate kinase polypeptide is expressed or produced. In some embodiments, the method comprises a step of isolating the adenylate kinase from the culture medium and / or host cell, as described herein. In some further embodiments, the method further comprises a step of purifying the expressed adenylate kinase polypeptide.

[0338] In some embodiments, the adenylate kinase polypeptide expressed in a host cell is recovered from the cells and / or the culture medium using any one or more of the well-known techniques for protein purification, including, among others, lysozyme or detergent treatment, sonication, filtration, salting-out, ultra-centrifugation, and chromatography, such as described herein.

[0339] Chromatographic techniques for isolation / purification of the adenylate kinase polypeptides include, among others, reverse phase chromatography, high-performance liquid chromatography, ionexchange chromatography, hydrophobic-interaction chromatography, size-exclusion chromatography, gel electrophoresis, and affinity chromatography. Conditions for purifying the adenylate kinase depends, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and will be apparent to those having skill in the art. In some embodiments, affinity techniques may be used to isolate the adenylate kinase. For affinity chromatography purification, an antibody that specifically binds adenylate kinase polypeptide may be used. In some embodiments, an affinity tag, e.g., His-tag, can be introduced into the adenylate kinase polypeptide for purposes of isolation / purification.

[0340] Appropriate culture media and growth conditions for the above-described host cells are well known in the art. Polynucleotides for expression of the adenylate kinases may be introduced into cells by various methods known in the art. Techniques include, among others, electroporation, biolisticDocket No. 5244.286WO3 particle bombardment, liposome mediated transfection, calcium chloride transfection, and protoplast fusion.

[0341] In some embodiments, the polynucleotides encoding the adenylate kinase polypeptide can be prepared by standard solid-phase methods, according to known synthetic methods. In some embodiments, polynucleotide fragments can be individually synthesized, then joined (e.g., by enzymatic or chemical litigation methods, or polymerase mediated methods) to form any desired continuous sequence. For example, polynucleotides and oligonucleotides disclosed herein can be prepared by chemical synthesis using the classical phosphoramidite method (See e.g., Beaucage et aL, Tetra. Lett., 1981, 22:1859-69; and Matthes et al., EMBO J., 1984, 3:801-05), as it is typically practiced in automated synthetic methods.

[0342] In some embodiments, a method for preparing the adenylate kinase can comprise: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the amino acid sequence of an adenylate kinase, such as described in the Tables of the Examples, and (b) expressing the engineered adenylate kinase encoded by the polynucleotide. In some embodiments of the method, the amino acid sequence encoded by the polynucleotide can optionally have one or several (e.g., up to 3, 4, 5, or up to 10) amino acid residue deletions, insertions and / or substitutions. In some embodiments, the amino acid sequence has optionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the substitutions can be conservative or non-conservative substitutions. The expressed polypeptide can be assessed for the desired property, e.g., adenylate kinase activity on one or more NMPs.Compositions

[0343] In a further aspect, the present disclosure provides compositions of the engineered adenylate kinases disclosed herein. In some embodiments, the engineered adenylate kinase polypeptide in the composition is isolated or purified. In some embodiments, the adenylate kinase is combined with other components and compounds to provide compositions and formulations comprising the engineered adenylate kinase polypeptide as appropriate for different applications and uses.

[0344] In some embodiments, the composition comprises at least one adenylate kinase described herein. For example, a composition comprises at least one engineered adenylate kinase exemplified in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, and 13.2, and the Sequence Listing. In some embodiments, theDocket No. 5244.286WO3 composition comprising an engineered adenylate kinase is an aqueous solution. In some embodiments, the composition comprising an engineered adenylate kinase is a lyophilizate. In some embodiments, the composition comprising an engineered adenylate kinase is in a powder form.

[0345] In some embodiments, the composition further comprises at least a buffer, including the suitable buffers described herein, e.g., MOPS, triethanolamine, etc. In some embodiments, the buffer is present at a concentration of about 1 mM-500 mM, 5 mM-450 mM, 10 mM-400 mM, 20 mM-350 mM, 30 mM-300 mM, 40 mM-250 mM, or 50 mM-200 mM. In some embodiments, the buffer is present at a concentration of about 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, or 500 mM.

[0346] In some embodiments, the composition further comprises an additional enzyme, including, among others, an adenosine kinase, acetate kinase, pyruvate oxidase, or 3'-O-kinase, or combinations thereof.

[0347] In some embodiments, the composition further comprises an NMP substrate. In some embodiments, the NMP substrate is a modified NMP. In some embodiments, the modified NMP substrate is modified on the 2'- and / or 3'-position of the sugar moiety (e.g., 2'-halo, 2'-O-CH3), the nucleobase, and / or the phosphate group (e.g., NMP-S, NMPaS), as further described herein.

[0348] In some embodiments, the NMP substrate is an unmodified NMP. In some embodiments, the NMP substrate is AMP, GMP, CMP, UMP, orTMP. In some embodiments, the concentration of NMP in the composition is greater than that found in cells, e.g., bacterial cells or mammalian cells. In some embodiments, the concentration of NMP in the composition is at least 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 7 mM, or 10 mM, or greater. In some embodiments, the NMP substrate concentration is about 0.1 mM to 200 mM, 0.5 mM to 180 mM, 1 mM to 150 mM, 2 mM to 120 mM, 5 mM to 100 mM, 10 mM to 80 mM, or 20 mM to 60 mM, as further described herein. In some embodiments, the NMP substrate concentration is about 100 mM-200 mM. In some embodiments, the NMP substrate concentration is about 0.1 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, 10 mM, 20 mM, 60 mM, 80 mM, 100 mM, 120 mM, 150 mM, 180 mM, or 200 mM.

[0349] In some embodiments, the concentration of AMP in the composition is at least 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 7 mM, or 10 mM, or greater. In some embodiments, the concentration of AMP in the composition is about 0.1 mM to 200 mM, 0.5 mM to 180 mM, 1 mM to 150 mM, 2 mM to 120 mM, 5 mM to 100 mM, 10 mM to 80 mM, or 20 mM to 60 mM. In some embodiments, the AMP substrate concentration is about 100 mM-200 mM. In some embodiments, the concentration of AMP in the composition is at least 0.1 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 7 mM, 10 mM, 20 mM, 60 mM, 80 mM, 100 mM, 120 mM, 150 mM, 180 mM, or 200 mM, or greater.Docket No. 5244.286WO3

[0350] In some embodiments, the concentration of GMP in the composition is greater than 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 7 mM, or 10 mM, or greater. In some embodiments, the concentration of GMP in the composition is about 0.1 mM to 200 mM, 0.5 mM to 180 mM, 1 mM to 150 mM, 2 mM to 120 mM, 5 mM to 100 mM, 10 mM to 80 mM, or 20 mM to 60 mM. In some embodiments, the GMP substrate concentration is about 100 mM-200 mM. In some embodiments, the concentration of GMP in the composition is at least 0.1 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 7 mM, 10 mM, 20 mM, 60 mM, 80 mM, 100 mM, 120 mM, 150 mM, 180 mM, or 200 mM, or greater.

[0351] In some embodiments, the concentration of CMP in the composition is at least 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 7 mM, or 10 mM, or greater. In some embodiments, the concentration of CMP in the composition is about 0.1 mM to 200 mM, 0.5 mM to 180 mM, 1 mM to 150 mM, 2 mM to 120 mM, 5 mM to 100 mM, 10 mM to 80 mM, or 20 mM to 60 mM. In some embodiments, the CMP substrate concentration is about 100 mM-200 mM. In some embodiments, the concentration of CMP in the composition is at least 0.1 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 7 mM, 10 mM, 20 mM, 60 mM, 80 mM, 100 mM, 120 mM, 150 mM, 180 mM, or 200 mM, or greater.

[0352] In some embodiments, the concentration of UMP in the composition is at least 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 7 mM, or 10 mM or greater. In some embodiments, the concentration of UMP in the composition is about 0.1 mM to 200 mM, 0.5 mM to 180 mM, 1 mM to 150 mM, 2 mM to 120 mM, 5 mM to 100 mM, 10 mM to 80 mM, or 20 mM to 60 mM. In some embodiments, the UMP substrate concentration is about 100 mM-200 mM. In some embodiments, the concentration of UMP in the composition is at least 0.1 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 7 mM, 10 mM, 20 mM, 60 mM, 80 mM, 100 mM, 120 mM, 150 mM, 180 mM, or 200 mM, or greater.

[0353] In some embodiments, the concentration of TMP in the composition is at least 0.1 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 7 mM, or 10 mM, or greater. In some embodiments, the concentration of TMP in the composition is about 0.1 mM to 200 mM, 0.5 mM to 180 mM, 1 mM to 150 mM, 2 mM to 120 mM, 5 mM to 100 mM, 10 mM to 80 mM, or 20 mM to 60 mM. In some embodiments, the TMP substrate concentration is about 100 mM-200 mM. In some embodiments, the concentration of TMP in the composition is at least 0.1 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 7 mM, 10 mM, 20 mM, 60 mM, 80 mM, 100 mM, 120 mM, 150 mM, 180 mM, or 200 mM, or greater.

[0354] In some embodiments, the NMP substrate is a modified NMP. In some embodiments, the modified NMP substrate is modified on the 2'- and / or 3'-position of the sugar moiety (e.g., 2'-halo, 2'-O-methyl, 3'-0-phosphate, etc.), the nucleobase, the phosphate group (e.g., NMPaS), or any combinations thereof. In some embodiments, the modified NMP substrate concentration is about 0.1 mM to 200 mM, 0.5 mM to 180 mM, 1 mM to 150 mM, 2 mM to 120 mM, 5 mM to 100 mM, 10 mM to 80 mM, or 20 mM to 60 mM, as further described herein. In some embodiments, the modified NMPDocketNo. 5244.286WO3 substrate concentration is about 100 mM-200mM. In some embodiments, the modified NMP substrate concentration is about 0.1 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, 10 mM, 20 mM, 60 mM, 80 mM, 100 mM, 120 mM, 150 mM, 180 mM, or 200 mM.

[0355] In some embodiments, the modified NMP substrate comprises a 2'-fluoro modified NMP. In some embodiments, the 2'-F modified nucleoside is 2'-F-AMP, 2'-F-GMP, 2'-F-CMP, 2'-F-2'-UMP, and / or 2-F-TMP. In some embodiments, the modified nucleoside comprises a 2'-O-CH3modified NMP. In some embodiments, the 2'-O-CH3modified nucleoside is 2'-O-CH3AMP, 2'-O-CH3GMP, 2'-O-CH3CMP, 2'-O-CH3UMP, and / or 2'-O-CH3TMP. Other 2'- modifications are described below.

[0356] In some embodiments, the modified NMP is modified at the 3'-position of the sugar moiety. In some embodiments, the 3'-position of the sugar moiety is modified with a blocking group, preferably a reversible blocking group. In some embodiments, the blocking group is a formate, benzoylformate, acetate, propionate, isobutyrate, aminoxy (-ONH2), O-methyl, O-methoxymethyl, 0-methylthiomethyl, O-benzyloxymethyl, O-allyl, O-(2-nitrobenzyl), O-azidomethyl (O-CH2N3), O-tert-butyldithiomethyl, phosphate, diphosphate, or triphosphate. Reversible 3'-blocked nucleoside / nucleotides are described in Chen et aL, Genomics, Proteomics & Bioinformatics, 2013, ll(l):34-40, Metzker et al., Nucleic Acids Res., 1994, 22 (20): 4259-4267; Sabat et aL, Front Chem. 2023; 11: 1161462; and patent publications US5763594, US9650406, US20200216891; W02004 / 018497; and WO 2014 / 139596; all of which are incorporated by reference) In some embodiments, the modified sugar moiety is a 3'-O-phosphate.

[0357] In some embodiments, the modified NMP substrate comprises modified 2'- and 3'-positions of the sugar moiety, such as described herein. Exemplary modified NNP substrates with modified 2'- and 3'-positions include, among others, 2'-fluoro or 2'-O-methyl, and 3'-O-allyl, 3'-0-(2-nitrobenzyl), 3'-O-azidomethyl (O-CH2N3), 3'-0-tert-butyldithiomethyl, or 3'-phosphate. Other such modifications are described herein.

[0358] In some embodiments, the modified sugar moiety of the NMP substrate in the compositions is a "locked" nucleotide (e.g., locked NMP). In some embodiments, the locked NMP is a locked AMP, locked GMP, locked CMP, locked TMP, or locked UMP.

[0359] In some embodiments of the composition, the modified NMP substrate comprises a modified phosphate. In some embodiments, the modified nucleoside diphosphate comprises an alphathiodiphosphate (NMPaS). In some embodiments, the modified NMP substrate with a modified phosphate also has a modified sugar moiety and / or modified nucleobase.

[0360] In some embodiments, the modified NMP substrate comprises a modified nucleobase. In some embodiments, the modified nucleobases is 5-bromo-uracil, 5-iodo-uracil, 6-mCEPh-purine, 6-Docket No. 5244.286WO3 phenylpyrrolocytidine, N2-alkyl 8-oxoguanosine, difluorotoluene, difluorobenzene, dichlorobenzene, imidazole, or benzimidazole. Other nucleobases are described herein.

[0361] In some embodiments, the composition further comprises a phosphate donor for the adenylate kinase, for example, NTP. In some embodiments, the phosphate donor NTP has the same nucleoside structure as the NMP substrate. By way of example and not limitation, if the NMP substrate is modified at the 2'-position of the sugar moiety, the donor NTP has the same modified 2'-position of the sugar moiety. Additionally, the phosphate donor NTP has the same nucleobase as the NMP substrate.

[0362] In some embodiments, the composition comprises an engineered adenylate kinase and one or more components of a NTP regenerating system. In some embodiments, the components of the NTP regeneration system includes, among others, pyruvate kinase, creatine kinase, adenylate kinase, and / or polyphosphate kinase. In some embodiments, the components of the NTP regeneration system includes a substrate (i.e., phosphate donor) for the enzyme in the NTP regenerating system, for example, phosphoenolpyruvate, creatine phosphate, and polyphosphate, respectively.

[0363] In some embodiments, where the NTP regenerating system uses an acetate kinase, the NTP regenerating system further comprises a pyruvate oxidase, pyruvate, and phosphate, for converting the acetate to acetyl phosphate.

[0364] In some embodiments, the composition comprises an immobilized engineered adenylate kinase, as described herein. In some embodiments, the immobilized adenylate kinase is immobilized with other enzymes, e.g., an adenosine kinase, acetate kinase, and / or 3'-O-kinase.Uses and Methods

[0365] In another aspect, the present disclosure provides uses of the engineered adenylate kinase enzymes, either alone or in combination with other enzymes. In some embodiments, the engineered adenylate kinase is used in the production of nucleoside diphosphate (NDP) from nucleoside monophosphate (NMP). In some embodiments, a method of converting nucleoside monophosphate to nucleoside diphosphate comprises contacting a nucleoside monophosphate with an engineered adenylate kinase described herein in the presence of phosphate donor under suitable reaction conditions to convert the nucleoside monophosphate to the corresponding product nucleoside diphosphate.

[0366] In some embodiments, the nucleoside monophosphate substrate is a naturally occurring or unmodified nucleoside monophosphate. In some embodiments, unmodified nucleoside diphosphate refers to nucleosides present in naturally occurring DNA or mRNA, where the nucleosides have a 5'-phosphate.Docket No. 5244.286WO3

[0367] In some embodiments, the nucleoside monophosphate has at the 2'-position of the sugar moiety a H or OH.

[0368] In some embodiments, the nucleoside monophosphate comprises a nucleobase selected from adenine, cytosine, guanine, thymine, uracil, xanthine, hypoxanthine, 2,6-diaminopurine, purine, 6,8-diaminopurine, 5-methylcytosine (m5C), 2-thiouridine, pseudouridine, dihydrouridine, inosine, and 7-methylguanosine (m7G).

[0369] In some embodiments, the unmodified nucleoside monophosphate is AMP, GMP, UMP, CMP, or TMP, and wherein the nucleoside has at the 2'-position of the sugar moiety an OH, thereby resulting in corresponding product rADP, rGDP, rllDP, rCDP, or rTDP, respectively.

[0370] In some embodiments, the unmodified nucleoside monophosphate is AMP, GMP, UMP, CMP, or TMP, and wherein the nucleoside has at the 2'-position of the sugar moiety an H, thereby by resulting in corresponding product dADP, dGDP, dUDP, dCDP, or dTDP, respectively.

[0371] In some embodiments of the method, the nucleoside monophosphate substrate is a modified nucleoside monophosphate, thereby resulting in production of the corresponding modified nucleotide diphosphate. In some embodiments, the modified nucleotide is modified in the sugar moiety, the nucleobase, or phosphate, or any combination of modified sugar moiety, nucleobase, and phosphate.

[0372] In some embodiments of the method, the modified nucleoside monophosphate comprises a modified sugar moiety. In some embodiments, the modified sugar moiety is modified at the 2'-position and / or 3'-position of the sugar moiety.

[0373] In some embodiments, the modified sugar moiety is a modified at the 2'-position of the sugar moiety. In some embodiments, the 2'-position of the sugar moiety is halo, 2'-O-R', or 2'-O-COR', where R' is an alkyl, alkyloxyalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkyl, heterocyclylalkyl, arylalkyl, or heteroarylalkyl. In some embodiments, R' is a Ci-C4alkyl. In some embodiments, the modified 2'-position is a 2'-O-R', wherein in R' is alkyloxyalkyl, alkylamine, cyanoalkyl, or -C(O)-a Ikyl. In some embodiments, the 2'-position of the sugar moiety of the nucleoside substrate is -O-R', wherein R' is -CH3or -CH2CH3 or -CH2CH2OCH3. In some embodiments, the modified 2'-position is 2'-O-(2-methoxyethyl), 2'-O-allyl, 2'-O-propargyl, 2'-O-ethylamine, 2'-O-cyanoethyl, or 2'-O-acetalester.

[0374] In some embodiments, the 2'-position of the sugar moiety is halo. In some embodiments, the halo is F (2'-F) or Br (2'-Br).

[0375] In some embodiments, the modified nucleoside monophosphate substrate is a locked nucleoside. In some embodiments, the locked nucleoside is a locked AMP, locked GMP, locked CMP, locked TMP, or locked UMP. In some embodiments, the ribose moiety of the locked nucleoside is in the C3'-endo (beta-D) or C2'-endo (alpha-L) conformation. In some embodiments, the locked nucleosideDocket No. 5244.286WO3 has a methylene bridge. In some embodiments, the locked nucleoside has an ethylene bridge. Various locked nucleotides are described in, for example, WO 99 / 014226, WO 00 / 66604, WO 98 / 039352, WO 2004 / 046160, WO 00 / 047599, WO 2007 / 134181, WO 2010 / 077578, WO 2010 / 036698, WO 2007 / 090071, WO 2009 / 006478, WO 2011 / 156202, WO 2008 / 154401, WO 2009 / 067647, WO 2008 / 150729, Morita et al., Bioorganic & Med. Chem. Lett. 2002, 12, 73-76, Seth et al. J. Org. Chem. 2010, Vol 75(5) pp. 1569-81, and Mitsuoka et al., Nucleic Acids Research 2009, 37(4), 1225-1238, and Wan and Seth, J. Medical Chemistry 2016, 59, 9645-9667.

[0376] In some embodiments of the method, the modified sugar moiety is modified at the 3'-position of the sugar moiety. In some embodiments, the 3'-position of the sugar residue is modified with a blocking group, preferably a reversible blocking group. In some embodiments, the blocking group is a formate, benzoylformate, acetate, propionate, isobutyrate, aminoxy (-ONHZ), O-methyl, O-methoxymethyl, O-methylthiomethyl, O-benzyloxymethyl, O-allyl, 0-(2-nitrobenzyl), O-azidomethyl (O-CH2N3), O-tert-butyldithiomethyl, sulfate, phosphate, diphosphate, or triphosphate. Reversible 3'-blocked nucleoside / nucleotides are described in Chen et aL, Genomics, Proteomics & Bioinformatics, 2013, ll(l):34-40, Metzker et al., Nucleic Acids Res., 1994, 22 (20): 4259-4267; and patent publications US5763594, US9650406, US20200216891; W02004 / 018497; and WO 2014 / 139596; all of which are incorporated by reference) In some embodiments, the modified sugar moiety is a 3'-O-phosphate. For example, a modified NMP substrate with a 3'-0-phosphate has the structure pNp, where the 3'-OH of the sugar moiety is modified with a phosphate.

[0377] In some embodiments, the modified NMP substrate comprises a modified nucleobase with a modification at the 2'- and the 3'-position of the sugar moiety. In some embodiments, any modification at the 2'-position of the sugar moiety is combined with any compatible modification at the 3'-position of the sugar moiety. In some embodiments, the modified NMP substrate a 2'-modification, including the 2'-modifications described herein, and a 3'-modification comprising a 3'-blocking group, particularly a 3'-reversible blocking group. In some embodiments, the 2'-modification is -2'-O-(2-methoxyethyl), 2'-O-allyl, 2'-0-propargyl, 2'-O-ethylamine, 2'-O-cyanoethyl, 2'-O-acetalester, 2'-O-methyl, 2'-O-ethyl, or 2'-fluoro, and the 3'-modification is 3'-O-methyl, 3'-0-methoxymethyl, 3'-0-methylthiomethyl, 3'-O-benzyloxymethyl, 3'-O-allyl, 3'-0-(2-nitrobenzyl), 3'-0-azidomethyl (O-CH2N3), 3'-0-tert-butyldithiomethyl, phosphate, or triphosphate. Exemplary modified NMP substrates with a 2'- and 3'-modified sugar moiety, include among others, 2'-fluoro-AMP-3'-phosphate, 2'-fluoro-GMP-3'-phosphate, 2'-fluoro-UMP-3'-phosphate, 2'-fluoro-CMP-3'-phosphate, 2'-fluoro-TMP-3'-phosphate, 2'-0-methyl-AMP-3'-phosphate, 2'-0-methyl-GMP-3'-phosphate, 2'-0-methyl-UMP-3'-phosphate, 2'-O-methyl-CMP-3'-phosphate, and 2'-O-methyl-TMP-3'-phosphate.DocketNo. 5244.286WO3

[0378] In some embodiments of the method, the modified nucleoside monophosphate comprises a modified nucleobase. In some embodiments, the modified nucleobase is 5-bromo-uracil, 5-iodo-uracil, 6-mCEPh-purine, 6-phenylpyrrolocytidine, N2-a Ikyl 8-oxoguanosine, difluorotoluene, difluorobenzene, dichlorobenzene, imidazole, or benzimidazole.

[0379] In some embodiments, the nucleobase of the nucleoside monophosphate substrate is, among others, 5-methylcytosine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5- alkylcytidines, 5-alkyluridines, 5-halouridines, 6-azapyrimidines, 6-alkylpyrimidines, 5-(l-propynyl)-uridine, 5-(l-propynyl)-cytidine, quesosine, 2-thiouridine, 4-thiouridine, 4-acetyltidine, 5- (carboxyhydroxymethyl)uridine, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluridine, -D-galactosylqueosine, 1-methyladenosine, 1- methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2- methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2- thiouridine, 5-methylaminomethy luridine, 5-methylcarbon ylmethyluridine, 5- methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine, -D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine, Nl-methyl-adenine, N6-methyl- adenine, 8'-azido-adenine, N,N-dimethyl-adenosine, aminoallyl-adenosine, 5'-methyl-uridine, pseudouridine, Nl- methyl-pseudouridine, 5'-hydroxy-methyl-uridine, 2'-th io-uridine, 4'-thio- uridine, hypoxanthine, xanthine, 5'-methyl-cytidine, 5'-hydroxy-methyl-cytidine, 6'-th io- guanine, or N7-methyl-guanine.

[0380] In some embodiments of the method, the modified nucleoside monophosphate comprises a modified phosphate. In some embodiments, the modified nucleoside monophosphate comprises a 5'-thiophosphate (NMP-S), thereby resulting in product nucleoside alpha-thiodiphosphate (NDPaS). In some embodiments, the NDPaS product is (Rp)-NDPaS, (Sp)-NDPaS, or a mixture of (Rp)-NDPaS and (Sp)-NDPaS diastereomers. In some embodiments, the method further comprises the step of separating the (Rp)-NDPaS and (Sp)-NDPaS diastereomeric products.

[0381] It is to be understood that the NMPaS can have a modified nucleoside, e.g., modification of the sugar residue at the 2'- and / or 3'-positions, a modified nucleobase, or any combinations thereof as described herein. Exemplary modified NMPaS substrates with a 2'-modified sugar moiety, include among others, 2'-fluoro-AMPaS, 2'-fluoro-GMPaS, 2'-fluoro-UMPaS, 2'-fluoro-CMPaS, 2'-fluoro-TMPaS, 2'-O-methyl-AMPaS, 2'-O-methyl-GMPaS, 2'-O-methyl-UMPaS, 2'-O-methyl-CMPaS, and 2'-O-methyl-TMPaS. Exemplary modified NMPaS substrates with a 3'-modified sugar moiety, include among others, AMPaS-3'-phosphate, GMPaS-3'-phosphate, UMPaS-3'-phosphate, CMPaS-3'-phosphate, and TMPaS-3'-phosphate. Exemplary modified NMPaS substrates with a 2'- and 3'-modified sugar moiety, include among others, 2'-fluoro-AMPaS-3'-phosphate, 2'-fluoro-GMPaS-3'-phosphate, 2'-fluoro-UMPaS-3'-phosphate, 2'-fluoro-CMPaS-3'-phosphate, 2'-fluoro-TMPaS-3'-phosphate, 2'-O-methyl-AMPaS-3'-Docket No. 5244.286WO3 phosphate, 2'-O-methyl-GMPaS-3'-phosphate, 2'-O-methyl-UMPaS-3'-phosphate, 2'-0-methyl-CMPaS-3'-phosphate, and 2'-O-methyl-TMPaS-3'-phosphate.

[0382] In some embodiments of the method, the phosphate donor is a nucleotide triphosphate ( NTP) . In some embodiments, in some embodiments, the phosphate donor NTP is rATP or dATP. In some embodiments, the phosphate donor NTP has the same nucleotide or nucleoside structure as the NMP substrate. For example, if the NMP substrate is modified at the 2'-position of the sugar moiety, the phosphate donor NTP has the same modification in the sugar moiety. Additionally, the nucleobase of the NTP donor has the same nucleobase as the NMP substrate.

[0383] In some embodiments, the phosphate donor NTP is present at a concentration of about 0.001 mM to 1 mM, 0.005 mM to 0.5 mM, 0.01 mM to 0.4 mM, 0.05 mM to 0.3 mM, or 0.1 mM to 0.2 mM. In some embodiments, the phosphate donor NTP is present at a concentration of about 0.001 mM, 0.005 mM, 0.01 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.5 mM, or 1 mM.

[0384] In some embodiments, the method further comprises regenerating the NTP donor using a NTP regenerating or recycling system to regenerate the NTP from the NDP produced by the adenylate kinase reaction. In some embodiments of the method, the NTP regenerating system comprises a creatine kinase, a pyruvate kinase, a polyphosphate kinase, and / or an acetate-kinase. In the description of enzymes herein, the database accession numbers (e.g., UniProt, GenBank, NCBI, etc.) disclosing the amino acid sequence are provided in parentheses.

[0385] In some embodiments, the NTP regenerating system is pyruvate kinase and substrate phosphoenolpyruvate. Various suitable pyruvate kinases are known in the art. In some embodiments, the pyruvate kinase for use in the NTP regenerating system is the pyruvate kinase of, among others, Escherichia coll str. K-12 substr. MG1655 (A0A3L1NNV5), Geobacillus stearothermophilus ATCC 7953 (Q02499), Schizosaccharomyces pombe (Q10208), Saccharomyces cerevisiae S288C (D6VPH8), Gallus gallus liver (F1NW43), Solanum tuberosum (P22200), Oryctolagus cuniculus Ml / 2 (018919), Mus musculus muscle (P52480), Rattus norvegicus Ml / 2 (A0A8L2Q.7B9), or Homo sapiens muscle (A0A804F729).

[0386] In some embodiments, the NTP regenerating system is creatine kinase and substrate creatine phosphate. Various suitable creatine kinases are known in the art. In some embodiments, the creatine kinase for use in the NTP regenerating system is the creatine kinase of, among others, Danio rerio (A8E5L0), Gallus gallus mitochondrial (P 11009), Mus musculus (P07310), Bos taurus (Q5E9Y4), Oryctolagus cuniculus (P00563), or Homo sapiens M-type (P06732).

[0387] In some embodiments, the NTP regenerating system is polyphosphate kinase and substrate polyphosphate. Various suitable polyphosphate kinases are known in the art. In a preferredDocket No. 5244.286WO3 embodiment, the polyphosphate kinase is PPK1. In some embodiments, the PPK1 for use in the NTP regenerating system is the PPK1 of, among others, Pseudomonas putida DOT-TIE (AFO50238.1), Escherichia coli K-12 P0A7B1 (AAC75554.1), Clostridium acetobutylicum ATCC 824 (NP_347259.1), Thermosynechococcus elongatus (WP_011056068), Acidithiobacillus ferrooxidans (WP_064219446), Acidithiobacillus thiooxidans (WP_031572361), Bacillus acidicola (WP_066264350), Acetobacter aceti (GAN58028), Acetobacter aceti (WP_077811826.1), Thioalkalivibrio denitrificans (WP_077277945.1), or Psychromonas ingrahamii (WP_041766473.1).

[0388] In some embodiments, the NTP regenerating system is an acetate kinase and its substrate acetyl phosphate and / or propionyl phosphate. Various suitable acetates kinases are known in the art and also describe herein. In some embodiments, the acetate kinase for use in the NTP regeneration system is an acetate kinase of Escherichia coli str. K-12 substr. MG1655 (NP_416799.1), Corynebacterium jeikeium K411 (WP_011272972.1), Lactococcus cremoris subsp. cremoris KW2 (WP_011835968.1), Lactococcus lactis (WP_004254593.1), Marinitoga sp. 38H-ov (WP_165147355.1), Thermotoga sp. K0L6 (WP_101510533.1), Thermosipho melaniensis (WP_012057479.1), Thermotoga sp. RQ7 (WP_041844042.1), or Thermosipho africanus (WP_004102380.1).

[0389] In some embodiments, where the NTP regenerating system is an acetate kinase, the method further comprises a pyruvate oxidase and substrate pyruvate and inorganic phosphate. The pyruvate oxidase converts acetate to acetyl phosphate, which is then used as a phosphate donor by the acetate kinase in the NTP regenerating system. Various pyruvate oxidases are known in the art. In some embodiments, the pyruvate oxidases are homologs of pyruvate oxidases. In some embodiments, the pyruvate oxidase is a pyruvate oxidase of Bifidobacterium mongoliense (A0A087C4V4), Alkalibacterium subtropicum (A0A1I1KLE2), Pisciglobus halotolerans (A0A1I3CCM7), Jeotgalibaca sp PTS2502 (A0A1U7E9W7), Vagococcus fluvialis (A0A8I2AXT4), Candidatus Gracilibacteria bacterium (A0A2M7FGE0), Bavariicoccus seileri (A0A3D4S346), Bifidobacterium aquikefiri (A0A261G4D1), Aerococcus urinae (F2I8Y6), or Aerococcus suis (A0A1W1YA59).

[0390] In some embodiments, the method further comprises a catalase, which converts H2O2 produced by the pyruvate oxidase to H2O. Various suitable catalases are known in the art. In some embodiments, the catalase can be the catalase of Archaeoglobus fulgidus, Bacillus stearothermophilus, E. coli, Mycobacterium intracellulare, Synechococcus sp PCC7942, Arabidopsis thaliana, Pisum sativum, or Saccharomyces cerevisiae (see, e.g., Zamocky, M., Progress in Biophysics and Molecular Biology, 1999, 72(l):19-66); see also WO1992017571). In some embodiments, the catalase is a catalase of Bos taurus (P00432), Aspergillus niger (A0A254TZH3), Helicobacter pylori (J0N6C6), Drosophila melanogaster (P17336) and Rattus norvegicus ( P04762).Docket No. 5244.286WO3

[0391] In the embodiments as provided herein and illustrated in the Examples, various ranges of suitable reaction conditions that can be used in the processes, including but not limited to, substrate loading, co-substrate loading, pH, temperature, buffer, solvent system, cofactor, polypeptide loading, and reaction time. Further suitable reaction conditions for carrying out the process for biocatalytic conversion of substrate compounds to product compounds using the enzymes described herein can be supplemented in view of the guidance provided herein to the skilled artisan, including, but not limited to, contacting the enzymes and one or more substrate compounds under experimental reaction conditions of concentration, pH, temperature, and solvent conditions, and detecting the product compound. The present disclosure contemplates any suitable reaction conditions that may find use in the methods described herein.

[0392] In some embodiments, the substrate compound in the reaction mixtures can be varied, taking into consideration, for example, the desired amount of product compound, the effect of each substrate concentration on enzyme activity, stability of enzyme under reaction conditions, and the percent conversion of each substrate to product. In some embodiments, the suitable reaction conditions comprise a substrate compound (e.g., NMP) loading of at least about 0.1 uM to 1 uM, 1 uM to 2 uM, 2 uM to 3 uM, 3 uM to 5 uM, 5 uM to 10 uM, or 10 uM to 100 uM or greater. In some embodiments, the suitable reaction conditions comprise a substrate compound (e.g., NMP) loading of about 0.1 mM to 200 mM, 0.5 mM to 180 mM, 1 mM to 150 mM, 2 mM to 120 mM, 5 mM to 100 mM, 10 mM to 80 mM, or 20 mM to 60 mM. In some embodiments, the suitable reactions conditions comprise a nucleoside substrate compound loading of about 0.1 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, 10 mM, 20 mM, 60 mM, 80 mM, 100 mM, 120 mM, 150 mM, 180 mM, or 200 mM.

[0393] In some embodiments, the suitable reaction conditions comprise a substrate compound loading of at least about 0.5 to about 25 g / L, 1 to about 25 g / L, 5 to about 25 g / L, about 10 to about 25 g / L, about 20 to about 25 g / L, or about 30 to about 60 g / L. In some embodiments, the suitable reaction conditions comprise a substrate compound loading of at least about 0.5 g / L, at least about 1 g / L, at least about 5 g / L, at least about 10 g / L, at least about 15 g / L, at least about 20 g / L, at least about 30 g / L, at least about 40 g / L, at least about 50 g / L, or at least about 60 g / L, or even greater.

[0394] In carrying out the processes described herein, the enzymes may be added to the reaction mixture in the form of a purified enzyme, partially purified enzyme, whole cells transformed with gene(s) encoding the enzyme, as cell extracts and / or lysates of such cells, and / or as an enzyme immobilized on a solid support. Whole cells transformed with gene(s) encoding the enzyme(s) or cell extracts, lysates thereof, and isolated enzymes may be employed in a variety of different forms, including solid (e.g., lyophilized, spray-dried, and the like) or semisolid (e.g., a crude paste). The cell extracts or cell lysates may be partially purified by precipitation (ammonium sulfate, polyethyleneimine,Docket No. 5244.286WO3 heat treatment or the like, followed by a desalting procedure prior to lyophilization (e.g., ultrafiltration, dialysis, etc.). Any of the enzyme preparations (including whole cell preparations) may be stabilized by crosslinking using known crosslinking agents, such as, for example, glutaraldehyde or immobilization to a solid phase (e.g., Eupergit C, and the like).

[0395] In some embodiments, the gene(s) encoding the polypeptides can be transformed into host cell separately or together into the same host cell. For example, in some embodiments one set of host cells can be transformed with gene(s) encoding one polypeptide and another set can be transformed with gene(s) encoding another polypeptide. Both sets of transformed cells can be utilized together in the reaction mixture in the form of whole cells, or in the form of lysates or extracts derived therefrom. In other embodiments, a host cell can be transformed with gene(s) encoding multiple polypeptides. In some embodiments the polypeptides can be expressed in the form of secreted polypeptides and the culture medium containing the secreted polypeptides can be used for the synthesis reaction.

[0396] In some embodiments, the improved activity of the engineered adenylate kinase polypeptides disclosed herein provides for processes wherein higher percentage conversion can be achieved with lower concentrations of the engineered polypeptide. In some embodiments of the process, the suitable reaction conditions comprise an engineered polypeptide amount of about 0.1% w / w, 0.2% (w / w), 0.5% (w / w), 1% (w / w), 2% (w / w), 5% (w / w), 10% (w / w), 20% (w / w), 30% (w / w), 40% (w / w), 50% (w / w), 75% (w / w), 100% (w / w) or more of substrate compound loading.

[0397] In some embodiments, the engineered polypeptide is present at a molar ratio of engineered polypeptide to substrate of about 50 to 1, 25 to 1, 10 to 1, 5 to 1, 1 to 1, 1 to 5, 1 to 10, 1 to 25 or 1 to 50. In some embodiments, the engineered polypeptide is present at a molar ratio of engineered polypeptide to substrate from a range of about 50 to 1 to a range of about 1 to 50 or 1 to 100.

[0398] In some embodiments, the engineered polypeptide is present at about 0.01 g / L to about 50 g / L; about 0.01 to about 0.1 g / L; about 0.05 g / L to about 50 g / L; about 0.1 g / L to about 40 g / L; about 1 g / L to about 40 g / L; about 2 g / L to about 40 g / L; about 5 g / L to about 40 g / L; about 5 g / L to about 30 g / L; about 0.1 g / L to about 10 g / L; about 0.5 g / L to about 10 g / L; about 1 g / L to about 10 g / L; about 0.1 g / L to about 5 g / L; about 0.5 g / L to about 5 g / L; or about 0.1 g / L to about 2 g / L. In some embodiments, the adenylate kinase polypeptide is present at about 0.01 g / L, 0.05 g / L, 0.1 g / L, 0.2 g / L, 0.5 g / L, 1, 2 g / L, 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, or 50 g / L.

[0399] In the embodiments of the process, the reaction conditions comprise a suitable pH. The desired pH or desired pH range can be maintained by use of an acid or base, an appropriate buffer, or a combination of buffering and acid or base addition. The pH of the reaction mixture can be controlled before and / or during the course of the reaction. In some embodiments, the suitable reaction conditions comprise a solution pH from about 4 to about 10, pH from about 5 to about 10, pH from about 5 toDocket No. 5244.286WO3 about 9, pH from about 6 to about 9, pH from about 6 to about 8. In some embodiments, the reaction conditions comprise a solution pH of about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10.

[0400] In some embodiments, in the course of the reaction, the pH of the reaction mixture may change. The pH of the reaction mixture may be maintained at a desired pH or within a desired pH range, for example, among others, by the addition of an acid or a base, before and / or during the course of the reaction. Alternatively, the pH may be controlled by using a buffer. Accordingly, in some embodiments, the reaction condition comprises a buffer. Suitable buffers to maintain desired pH ranges are known in the art and include, by way of example and not limitation, borate, potassium phosphate, 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), acetate, triethanolamine, and 2-amino-2-hydroxymethyl-propane-l,3-diol (Tris), and the like. In some embodiments, the buffer is present in a concentration of 1 mM-500 mM, 5 mM to 450 mM, 10 mM to 400 mM, 20 mM to 350 mM, 30 mM to 300 mM, 40 mM to 200 mM, or 50 mM to 100 mM. In some embodiments, the buffer is present in the composition at about 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM or 500 mM. In some embodiments, the reaction conditions comprise water as a suitable solvent with no buffer present.

[0401] In the embodiments of the processes herein, a suitable temperature is used for the reaction conditions, for example, taking into consideration the increase in reaction rate at higher temperatures, and the activity of the enzyme during the reaction time period. In some embodiments, the suitable reaction conditions comprise a temperature of about 10 °C to about 95 °C, about 10 °C to about 75 °C, about 15 °C to about 95 °C, about 20 °C to about 95 °C, about 20 °C to about 65 °C, about 25 °C to about 70 °C, or about 50 °C to about 70 °C. In some embodiments, the suitable reaction conditions comprise a temperature of about 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C or 95 °C. In some embodiments, the temperature during the enzymatic reaction can be maintained at a specific temperature throughout the course of the reaction. In some embodiments, the temperature during the enzymatic reaction can be adjusted over a temperature profile during the course of the reaction.

[0402] In some embodiments, the processes are carried out in a solvent. Suitable solvents include water, aqueous buffer solutions, organic solvents, polymeric solvents, and / or co-solvent systems, which generally comprise aqueous solvents, organic solvents and / or polymeric solvents. The aqueous solvent (water or aqueous co-solvent system) may be pH-buffered or unbuffered. In some embodiments, the processes using the engineered adenylate kinase polypeptides can be carried out in an aqueous cosolvent system comprising an organic solvent (e.g., ethanol, isopropanol (IPA), dimethyl sulfoxide (DMSO), dimethylformamide (DMF) ethyl acetate, butyl acetate, 1-octanol, heptane, octane, methyl tDocket No. 5244.286WO3 butyl ether (MTBE), toluene, and the like), ionic or polar solvents (e.g., 1-ethyl 4 methylimidazolium tetrafluoroborate, l-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl 3 methylimidazolium hexafluorophosphate, glycerol, polyethylene glycol, and the like). In some embodiments, the co-solvent can be a polar solvent, such as a polyol, dimethylsulfoxide (DMSO), or lower alcohol. The non-aqueous co- solvent component of an aqueous co-solvent system may be miscible with the aqueous component, providing a single liquid phase, or may be partly miscible or immiscible with the aqueous component, providing two liquid phases. Exemplary aqueous co-solvent systems can comprise water and one or more co-solvents selected from an organic solvent, polar solvent, and polyol solvent. In general, the cosolvent component of an aqueous co-solvent system is chosen such that it does not adversely inactivate the enzymes under the reaction conditions. Appropriate co-solvent systems can be readily identified by measuring the enzymatic activity of the specified enzymes with a defined substrate of interest in the candidate solvent system, utilizing an enzyme activity assay, such as those described herein.

[0403] In some embodiments of the process, the suitable reaction conditions comprise an aqueous cosolvent, where the co-solvent comprises DMSO at about 1% to about 50% (v / v), about 1 to about 40% (v / v), about 2% to about 40% (v / v), about 5% to about 30% (v / v), about 10% to about 30% (v / v), or about 10% to about 20% (v / v). In some embodiments of the process, the suitable reaction conditions can comprise an aqueous co-solvent comprising ethanol at about 1% (v / v), about 5% (v / v), about 10% (v / v), about 15% (v / v), about 20% (v / v), about 25% (v / v), about 30% (v / v), about 35% (v / v), about 40% (v / v), about 45% (v / v), or about 50% (v / v).

[0404] In some embodiments, the reaction conditions comprise a surfactant for stabilizing or enhancing the reaction. Surfactants can comprise non-ionic, cationic, anionic and / or amphiphilic surfactants. Exemplary surfactants, include by way of example and not limitation, nonyl phenoxypolyethoxylethanol (NP40), TRITON™ X-100 polyethylene glycol tert-octylphenyl ether, polyoxyethylene-stearylamine, cetyltrimethylammonium bromide, sodium oleylamidosulfate, polyoxyethylene-sorbitanmonostearate, hexadecyldimethylamine, etc. Any surfactant that may stabilize or enhance the reaction may be employed. The concentration of the surfactant to be employed in the reaction may be generally from 0.1 to 50 mg / mL, particularly from 1 to 20 mg / mL.

[0405] In some embodiments, the reaction conditions include an antifoam agent, which aids in reducing or preventing formation of foam in the reaction solution, such as when the reaction solutions are mixed or sparged. Anti-foam agents include non-polar oils (e.g., minerals, silicones, etc.), polar oils (e.g., fatty acids, alkyl amines, alkyl amides, alkyl sulfates, etc.), and hydrophobic (e.g., treated silica, polypropylene, etc.), some of which also function as surfactants. Exemplary anti-foam agents include Y-30* (Dow Corning), poly-glycol copolymers, oxy / ethoxylated alcohols, and polydimethylsiloxanes. In some embodiments, the anti-foam can be present at about 0.001% (v / v) to about 5% (v / v), about 0.01%Docket No. 5244.286WO3 (v / v) to about 5% v / v , about 0.1% (v / v) to about 5% (v / v), or about 0.1% (v / v) to about 2% (v / v). In some embodiments, the anti-foam agent can be present at about 0.001% (v / v), about 0.01% (v / v), about 0.1% (v / v), about 0.5% (v / v), about 1% (v / v), about 2% (v / v), about 3% (v / v), about 4% (v / v), or about 5% (v / v) or more as desirable to promote the reaction.

[0406] In some embodiments, the quantities of reactants used in the synthesis reaction will generally vary depending on the quantities of product desired, and concomitantly the amount of substrates employed. Those having ordinary skill in the art will readily understand how to vary these quantities to tailor them to the desired level of productivity and scale of production.

[0407] In some embodiments, the order of addition of reactants is not critical. The reactants may be added together at the same time to a solvent (e.g., monophasic solvent, biphasic aqueous co-solvent system, and the like), or alternatively, some of the reactants may be added separately, and some together at different time points. For example, where applicable, the cofactor, co-substrate and substrate may be added first to the solvent.

[0408] In some embodiments, the reaction components (e.g., enzyme, salts, etc.) may be provided to the reaction in a variety of different forms, including powder (e.g., lyophilized, spray dried, and the like), solution, emulsion, suspension, and the like. The reactants can be readily lyophilized or spray dried using methods and equipment that are known to those having ordinary skill in the art. For example, the protein solution can be frozen at -80 °C in small aliquots, then added to a pre-chilled lyophilization chamber, followed by the application of a vacuum.

[0409] For improved mixing efficiency when an aqueous co-solvent system is used, the polypeptide(s), and co-substrate may be added and mixed into the aqueous phase first. The substrate may be added and mixed in, followed by the organic phase or the substrate may be dissolved in the organic phase and mixed in. Alternatively, the substrate may be premixed in the organic phase, prior to addition to the aqueous phase.|0410| The processes of the present invention are generally allowed to proceed until further conversion of substrate to product does not change significantly with reaction time (e.g., less than 10% of substrate being converted, or less than 5% of substrate being converted). In some embodiments, the reaction is allowed to proceed until there is complete or near complete conversion of substrate to product. Transformation of substrate to product can be monitored using known methods by detecting substrate and / or product, with or without derivatization. Suitable analytical methods include gas chromatography, HPLC, MS, and the like. In some embodiments, after suitable conversion to product, the reactants are separated from the product and additional reactants are added.Docket No. 5244.286WO3

[0411] Any of the processes disclosed herein using the polypeptides for the preparation of products can be carried out under a range of suitable reaction conditions, including but not limited to ranges of substrates, temperature, pH, solvent system, substrate loading, polypeptide loading, cofactor loading, and reaction time. In one example, the suitable reaction conditions for the conversion of a NMP to a NDP comprise: (a) substrate loading of about 1-200 mM NMP; (b) about 0.01 g / L to 5 g / L engineered adenylate kinase polypeptide; (c) 1-100 mM MgCL; (e) 5 to 100 mM of buffer, e.g., Tris-HCI; (f) 0.01-100 mM NTP (e.g., ATP); (g) pH at 5-9; and (h) temperature of about 15 °C to 70 °C. In some embodiments, additional reaction components or additional techniques carried out to supplement the reaction conditions. These can include taking measures to stabilize or prevent inactivation of the enzyme, reduce product inhibition, shift reaction equilibrium to formation of the desired product.

[0412] As described herein, the methods of using the polypeptides described herein can be carried out using the polypeptides bound or immobilized on a solid support. Methods of polypeptide immobilization are known in the art. In some embodiments, the engineered polypeptides can be bound non-covalently or covalently. Various methods for conjugation and immobilization of enzymes to solid supports (e.g., resins, membranes, beads, glass, etc.) are described in, for example, Yi et al., Proc. Biochem., 2007, 42(5):895-898; Martin et al., Appl. Microbiol. Biotechnol., 2007, 76(4): 843-851;Koszelewski et aL, J. Mol. Cat. B: Enzymatic, 2010, 63: 39-44; Truppo et aL, Org. Proc. Res. Dev., published online: dx.doi.org / 10.1021 / op200157c; Hermanson, "Bioconjugate Techniques," 2nd Ed., Academic Press, Cambridge, MA (2008); Mateo et al., Biotechnol. Prog., 2002, 18(3) :629-34; and "Bioconjugation Protocols: Strategies and Methods," In "Methods in Molecular Biology," Niemeyer (ed.), Humana Press, New York, NY (2004); the disclosures of each which are incorporated by reference herein). Solid supports useful for immobilizing the engineered polypeptides of the present disclosure include, but are not limited to, beads or resins comprising polymethacrylate with epoxide functional groups, polymethacrylate with amino epoxide functional groups, styrene / DVB copolymer or polymethacrylate with octadecyl functional groups. In some embodiments, exemplary solid supports useful for immobilizing the engineered polypeptides of the present invention include, but are not limited to, EnginZyme (including, EziG-1, EziG-1, and EziG-3), chitosan beads, Eupergit C, and SEPABEADs (Mitsubishi) (including EC-EP, EC-HFA / S, EXA252, EXE119 and EXE120).

[0413] In further embodiments, any of the above described processes for the conversion of one or more substrate compounds to product compound can further comprise one or more steps selected from: extraction; isolation; purification; and crystall...

Claims

Docket No. 5244.286WO3CLAIMSWhat is claimed is:

1. An engineered adenylate kinase comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-231 of an even-numbered SEQ ID NO. of SEQ ID NOs: 16-360 and 372-1018, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 16-360 and 372-1018, wherein the amino acid sequence comprises one or more substitutions relative to a reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, 144, 252, 402, 446, 710, or 808 or relative to a reference sequence corresponding to SEQ ID NO: 16, 144, 252, 402, 446, 710, or 808.

2. The engineered adenylate kinase of claim 1, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or to the reference sequence corresponding to SEQ ID NO: 16, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

3. The engineered adenylate kinase of claim 1, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

4. The engineered adenylate kinase of claim 1, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-231 of an even-numbered SEQ ID NO. of SEQ ID NOs: 18-360 and 372-1018, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 18-360 and 372-1018, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.Docket No. 5244.286WO3 5. The engineered adenylate kinase of any one of claims 1-4, wherein the amino acid sequence of the engineered adenylate kinase comprises at least a substitution at amino acid position 4, 14, 15, 16, 17, 18, 31, 33, 35, 37, 38, 40, 46, 47, 52, 53, 54, 55, 56, 59, 61, 62, 66, 68, 69, 74, 80, 81, 82, 83, 84, 87, 89, 91, 92, 93, 95, 96, 104, 105, 109, 111, 112, 115, 117, 121, 122, 125, 126, 132, 133, 135, 136, 139, 142, 143, 146, 148, 149, 154, 157, 159, 162, 163, 166, 168, 169, 170, 178, 179, 180, 182, 183, 184, 187, 192, 194, 199, 202, 203, 204, 205, 207, 212, 214, 215, 217, 218, 219, 220, 222, 223, 225, 227, 228, and 229 or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

6. The engineered adenylate kinase of any one of claims 1-5, wherein the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or amino acid residue 4N, 14P, 15A / H / T, 16C / G / M / T, 171, 18V, 31F / V / W, 33L, 35C / E, 37M, 37E / H / Q / R, 38D, 40T, 46E, 47L, 52L, 53D / M / S / T / V, 54G / R / V, 55Q, 55E / G / N / R / S, 56A / E, 59G, 61G, 62H, 66D, 68V, 69V, 74H, 80S, 81P, 82L, 83R, 84A / T, 87G / R / S, 89A / D / F / G / L / P / Q / S / V / W, 91L, 92S, 93E, 93G / L / V, 95W, 96L, 104L / R, 104Q, 105L / Q / S, 109A, 111N, 112C / T, 115C / H / N, 117 A / E / G / R / T / V , 121V, 122S, 125 L, 126C / 1, 132R, 133W, 135E / N, 136L / R, 139A / H, 142V, 1431, 146A / G / H, 148Q, 149A / L / M / Q, 154E, 157R, 159V, 162D / M / V, 163E / R, 166E, 168C, 169E, 170K / M, 178A / F / G / I / L / M / N / R / T / V / Y, 179A, 180A / P / Q, 182L, 183L, 184P / Q / S, 187D / R, 192L / M / V, 194L, 199F, 202E / G / N, 203Q, 204D, 205Q, 2051, 205C / L, 2071 / N, 212A, 214K / P, 215V, 217A / E, 218L / V, 219L / T, 220G, 222A, 223D / M / V, 225H / T, 225A / Q, 227S, 228L / S, and 229G, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

7. The engineered adenylate kinase of any one of claims 1-5, wherein the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or amino acid residue H4N, A14P, Y15A / H / T, L16C / G / M / T, V17I, L18V, R31F / V / W, I33L, A35C / E, H37M, T37E / H / Q / R, G38D, P40T, D46E, I47L, V52L, A53D / M / S / T / V, K54G / R / V, E55Q, Q55E / G / N / R / S, N56A / E, V59G, S61G, S62H, W66D, L68V, K69V, P74H, P80S, V81P, V82L, L83R, R84A / T, K87G / R / S, K89A / D / F / G / L / P / Q / S / V / W, C91L, E92S, G93E, K93G / L / V, F95W, I96L, Q104L / R, R104Q, R105L / Q / S, Y109A, D111N, S112C / T, E115C / H / N, Q117A / E / G / R / T / V, L121V, T122S, V125L, L126C / I, D132R, E133W, V135E / N, V136L / R, L139A / H, R142V, V143I, K146A / G / H, G148Q, R149A / L / M / Q, I154E, P157R, K159V, E162D / M / V, L163E / R, D166E, G168C, V169E, S170K / M, K178A / F / G / I / L / M / N / R / T / V / Y, E179A, E180A / P / Q, V182L, R183L, M184P / Q / S, K187D / R, K192L / M / V, Q194L, Y199F, K202E / G / N, K203Q, G204D, I205Q, L205I, Q205C / L, A207I / N, S212A, E214K / P, 1215V, P217A / E, I218L / V, V219L / T, A220G, V222A, L223D / M / V, A225H / T, I225A / Q, G227S, V228L / S, and I229G, or combinations thereof, wherein the amino acid positions are relative toDocket No. 5244.286WO3 the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

8. The engineered adenylate kinase of any one of claims 1-7, wherein the amino acid sequence of the engineered adenylate kinase comprises at least a substitution at amino acid position 37, 93, 125, or 205, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

9. The engineered adenylate kinase of any one of claims 1-8, wherein the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or amino acid residue 37H / M, 93G, 125L, or 205Q, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

10. The engineered adenylate kinase of any one of claims 1-9, wherein the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or amino acid residue T37H / M, K93G, V125L, or I205Q, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

11. The engineered adenylate kinase of any one of claims 1-10, wherein the amino acid sequence of the engineered adenylate kinase comprises at least a substitution at amino acid position 4, 47, 95, or 96; or substitution 14P, 15A / T, 16C / G / T, 37E / H / M / Q, 52L, 53T / V, 54V, 55N / R, 56A, 69V, 74H, 89D / F / G / S / W, 93L, 115C / H / N, 117A / E / T / V, 125L, 1261, 132R, 133W, 135E / N, 146A / G, 157R, 166E, 168C, 170M, 178A / F / G / I / L / M / N / R / T / V / Y, 180A / Q, 187D / R, 203Q, 205C / Q, 214K, 217E, or 220G; or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

12. The engineered adenylate kinase of claim 11, wherein the amino acid sequence of the engineered adenylate kinase comprises at least a substitution 4N, 14P, 15A / H / T, 16C / G / M / T, 171, 18V, 31F / V / W, 33L, 35C / E, 37M, 37E / H / Q / R, 38D, 40T, 46E, 47L, 52L, 53D / M / S / T / V, 54G / R / V, 55Q, 55E / G / N / R / S, 56A / E, 59G, 61G, 62H, 66D, 68V, 69V, 74H, 80S, 81P, 82L, 83R, 84A / T, 87G / R / S, 89A / D / F / G / L / P / Q / S / V / W, 91L, 92S, 93E, 93G / L / V, 95W, 96L, 104L / R, 104Q, 105L / Q / S, 109A, 111N, 112C / T, 115C / H / N, 117A / E / G / R / T / V, 121V, 122S, 125L, 126C / 1, 132R, 133W, 135E / N, 136L / R, 139A / H, 142V, 1431, 146A / G / H, 148Q, 149A / L / M / Q, 154E, 157R, 159V, 162D / M / V, 163E / R, 166E, 168C, 169E,Docket No. 5244.286WO3 170K / M, 178A / F / G / I / L / M / N / R / T / V / Y, 179A, 180A / P / Q, 182L, 183L, 184P / Q / S, 187D / R, 192L / M / V, 194L, 199F, 202E / G / N, 203Q, 204D, 205Q, 2051, 205C / L, 2071 / N, 212A, 214K / P, 215V, 217A / E, 218L / V, 219L / T, 220G, 222A, 223D / M / V, 225H / T, 225A / Q, 227S, 228L / S, and 229G, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

13. The engineered adenylate kinase of claim 11, wherein the amino acid sequence of the engineered adenylate kinase comprises at least a substitution H4N, A14P, Y15A / H / T, L16C / G / M / T, V17I, L18V, R31F / V / W, I33L, A35C / E, H37M, T37E / H / Q / R, G38D, P40T, D46E, I47L, V52L, A53D / M / S / T / V, K54G / R / V, E55Q, Q55E / G / N / R / S, N56A / E, V59G, S61G, S62H, W66D, L68V, K69V, P74H, P80S, V81P, V82L, L83R, R84A / T, K87G / R / S, K89A / D / F / G / L / P / Q / S / V / W, C91L, E92S, G93E, K93G / L / V, F95W, I96L, Q104L / R, R104Q, R105L / Q / S, Y109A, D111N, S112C / T, E115C / H / N, Q117A / E / G / R / T / V, L121V, T122S, V125L, L126C / I, D132R, E133W, V135E / N, V136L / R, L139A / H, R142V, V143I, K146A / G / H, G148Q, R149A / L / M / Q, I154E, P157R, K159V, E162D / M / V, L163E / R, D166E, G168C, V169E, S170K / M, K178A / F / G / I / L / M / N / R / T / V / Y, E179A, E180A / P / Q, V182L, R183L, M184P / Q / S, K187D / R, K192L / M / V, Q194L, Y199F, K202E / G / N, K203Q, G204D, I205Q, L205I, Q205C / L, A207I / N, S212A, E214K / P, 1215V, P217A / E, I218L / V, V219L / T, A220G, V222A, L223D / M / V, A225H / T, I225A / Q, G227S, V228L / S, and I229G, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

14. The engineered adenylate kinase of any one of claims 1-13, wherein the amino acid sequence of the engineered adenylate kinase further comprises at least a substitution at amino acid position 11, 13, 14, 15, 16, 18, 20, 21, 22, 26, 27, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 97, 98, 99, 100, 101, 102, 104, 105, 108, 109, 110, 111, 112, 113, 115, 116, 117, 118, 119, 120, 122, 123, 124, 125, 126, 127, 128, 129, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 146, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 161, 162, 163, 166, 168, 169, 170, 172, 173, 175, 178, 179, 180, 181, 182, 183, 184, 186, 187, 188, 190, 191, 192, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, or 231, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

15. The engineered adenylate kinase of claim 14, wherein the amino acid sequence of theDocket No. 5244.286WO3 engineered adenylate kinase further comprises at least a substitution or amino acid residue 11D, 13G / S, 14C / E, 15F, 16M, 18C / E / L, 20A / P / R / T / V, 21L / N / R / S / T / V, 22A / S, 26L, 27C / S, 29Q / R, 30G / L / S / Y, 31R, 32F / I / L / N, 34A / E / S / V, 35A / E / F / L / S, 36A / E / I / L / M / Q / R / S / V / Y, 37F / G / I / L / R / Y, 38C / F / L / R, 39A / M / Q / T, 40C / F / L / S / T, 42T, 43A / C / G / H / N, 44S, 46E / F / L / M / Y, 48F / L, 50A / C / L / N / Q / R / S / V, 51F / G / K / L / M / R / T, 52A / H, 53A / M / S, 54G / H / I / M / Q / R / T, 55G / L / Q / S, 56E / Q / T, 57H / L / P / S, 58C / G / Y, 59E / G / H / M / P / R / T / V / W / Y, 60A / C / P / S / W, 61A / E / N / P / S / V, 62A / E / G / I / L / S, 63S, 64R, 65A / D / F / K / P / T, 66A / C / D / E / F / G / H / N / P / Q / R / S / T / V / W, 67R, 68A / D / E / G / I / L / Q / R / S / T / V / W / Y, 69F / L / Q / R / Y, 70R, 71T, 731 / R, 74C / G / Q / S, 75 L, 76G / L / P, 77A / I / M / Q / S, 78C / G / N / T, 79A / K / L / P / W, 80A / D / G / L / N / P / R / S / V / W, 81F / I / L / Q / S, 821 / 1 / P / Q / T, 83G / H / L / S / T, 84A / E / H / M / S, 87A / E / I / K / L / M / R / V / Y, 88N / R / T, 89A / H / I / L / P / Q / T / V, 90C / E / F / K / S / T / V, 91A / F / G / L / S / V, 92L / S, 93A / E / G / P / S / T / V / Y, 94A / C / F / M / Q / S / T / V / Y, 971, 98A / G / Q, 99A / C, 100F / S / V, 101-, 102A / C / N, 104A / F / H / I / L / Q / R / S / W, 105G / K / L / M / R / S, 108M / R / S / V, 109E / Y, 11OC / F, 111E / P / R, 112A / C / E / K / M / N / Q / R / T, 113E / W / Y, 115G / K / R, 116A / E / F / L, 117L / N / S, 118A / G / L / R / S, 119F / K / L / P / R / S, 120G / K / L / R / S / T, 122A / H / S, 123S, 124V, 1251, 126A / E / S / V, 1271 / L / P / S, 128C / E / I / K / N / R / S, 1291 / L / P / S, 131A / G / V, 132G / K / L / T / V, 133A / E / F / L / Q / S / V / W, 134L, 1351 / K / M / P / R / S, 136A / I / L / S / V / Y, 137L, 138C / I / M / V, 139A / H / L / R, 140G, 141G / V, 142F / L / M / W, 143A / C / G / P / R / S / T / V, 146D / H / N / R / V, 148F / H / M / P / Q / R / S / T, 149L, 150C / G / L / P / S / V, 151A / F / R / Y, 152F / H, 153C / K / S / V, 154Q / R, 155A / F / T / W, 156A / C / N / T / V, 157P / V, 161G / L, 162G, 163G / Q / S, 166C / F / L / P / S, 168G / L / N / Q / S, 169A / D / I / Y, 170A / G / H / P / R / S, 172A / H / M / S / T, 173F / K / R / S / T / V, 175F / L / S, 178E / G / N / R, 179A / C / G / I / L / P / V, 180G / H / P, 181C / I / V, 182A / G / I / L / Q / S, 183A / L / Q, 1841 / K / M / N / R / S / T / V, 186L, 187G / Y, 188G / L, 190A / C / E / G / H / N / Q / R, 191D / K, 192A / H / I / W, 194F / L / R / V / Y, 195G / I / M / R, 1961, 197A / L / Q / V, 198G, 199R, 200A, 201 A / F / K / L / S, 202E / G / M / S, 203A / E / L / R, 204R / S, 205 L / P, 206W, 207A / L / T, 208 E, 210V, 211A, 212C / H / I / L / M / N / P / Q / R / S / W, 213L / P / Q / S, 214A / E / L / M / P / R / T / W, 215A / P / V, 216A / D / E / G / H / L / M / N / P / R / S / T / V, 217E / G / H / P / T, 218A / I / L, 219A / F / G / L / P / S / T / W, 220E / V, 221D, 2221 / T, 223T, 224G / S / T, 225A / L / Q / R / T / V, 226K / L / P / R / S / T, 227P, 228D / F / G / I / L / P / Q / R / S / T / V, 229C / I, 230A / M / P / Q, or 231A / Q / R / T, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

16. The engineered adenylate kinase of claim 14, wherein the amino acid sequence of the engineered adenylate kinase further comprises at least a substitution or amino acid residue G11D, M13G / S, A14C / E, Y15F, L16M, F18C / E / L, G20A / P / R / T / V, P21L / N / R / S / T / V, P22A / S, K26L, G27C / S, Y29Q / R, A30G / L / S / Y, K31R, I32F / I / L / N, Q34A / E / S / V, E35A / F / L / S, K36A / E / I / L / M / Q / R / S / V / Y, T37F / G / I / L / R / Y, G38C / F / L / R, I39A / M / Q / T, P40C / F / L / S / T, I42T, S43A / C / G / H / N, T44S, D46E / F / L / M / Y, F48L, D50A / C / L / N / Q / R / S / V, I51F / G / K / L / M / R / T, V52A / H, K53A / M / S, K54G / H / I / M / Q / R / T, E55G / L / Q / S, N56E / Q / T, D57H / L / P / S, E58C / G / Y, L59E / G / H / M / P / R / T / V / W / Y, G60A / C / P / S / W, K61A / E / N / P / S / V,Docket No. 5244.286WO3 K62A / E / G / I / L / S, I63S, K64R, E65A / D / F / K / P / T, I66A / C / D / E / F / G / H / N / P / Q / R / S / T / V / W, M67R, E68A / D / G / I / L / Q / R / S / T / V / W / Y, K69F / L / Q / R / Y, G70R, E71T, V73I / R, P74C / G / Q / S, D75L, E76G / L / P, L77A / I / M / Q / S, V78C / G / N / T, N79A / K / L / P / W, E80A / D / G / L / N / P / R / S / V / W, V81F / I / L / Q / S, V82 l / L / P / Q / T, K83G / H / L / S / T, R84A / E / H / M / S, S87A / E / I / K / L / M / R / V / Y, E88N / R / T, K89A / H / I / L / P / Q / T / V, D90C / E / F / K / S / T / V, C91A / F / G / L / S / V, E92L / S, K93A / E / G / P / S / T / V / Y, G94A / C / F / M / Q / S / T / V / Y, L97I, D98A / G / Q, G99A / C, Y100F / S / V, P1O1-, R102A / C / N, V104A / F / H / I / L / Q / R / S / W, A105G / K / L / M / R / S, E108M / R / S / V, F109E / Y, L11OC / F, D111E / P / R, S112A / C / E / K / M / N / Q / R / T, F113E / W / Y, E115G / K / R, S116A / E / F / L, Q117L / N / S, N118A / G / L / R / S, K119F / K / L / P / R / S, Q120G / K / L / R / S / T, T122A / H / S, A123S, A124V, V125I, L126A / E / S / V, F127I / L / P / S, D128C / E / I / K / N / R / S, V129I / L / P / S, E131A / G / V, D132G / K / L / T / V, V133A / E / F / L / Q / S / W, V134L, V135I / K / M / P / R / S, Q136A / I / L / S / V / Y, R137L, L138C / I / M / V, T139A / H / L / R, SMOG, R141G / V, R142F / L / M / W, 1143A / C / G / P / R / S / T / V, K146D / H / N / R / V, G148F / H / M / P / Q / R / S / T, R149L, I150C / G / L / P / S / V, Y151A / F / R, N152F / H, M153C / K / S / V, I154Q / R, S155A / F / T / W, L156A / C / N / T / V, P157V, D161G / L, E162G, L163G / Q / S, D166C / F / L / P / S, K168G / L / N / Q / S, V169A / D / I / Y, K170A / G / H / P / R / S, V172A / H / M / S / T, Q173F / K / R / S / T / V, D175F / L / S, K178E / G / N / R, E179A / C / G / I / L / P / V, E180G / H / P, T181C / I / V, V182A / G / I / L / Q / S, R183A / L / Q, H184I / K / M / N / R / S / T / V, Y186L, K187G / Y, V188G / L, L190A / C / E / G / H / N / Q / R, E191D / K, K192A / H / I / W, Q194F / L / R / V / Y, P195G / I / M / R, V196I, I197A / L / Q / V, D198G, Y199R, Y200A, G201A / F / K / L / S, K202E / G / M / S, K203A / E / L / R, G204R / S, I205L / P, L206W, K207A / L / T, R208E, D210V, G211A, T212C / H / I / L / M / N / P / Q / R / S / W, I213L / P / Q / S, G214A / E / L / M / P / R / T / W, I215A / P / V, D216A / D / E / G / H / L / M / N / P / R / S / T / V, N217E / G / H / P / T, V218A / I / L, V219A / F / G / L / P / S / T / W, A220E / V, E221D, V222I / T, L223T, K224G / S / T, I225A / L / Q / R / T / V, I226K / L / P / R / S / T, G227P, W228D / F / G / I / L / P / Q / R / S / T / V, S229C / I, D230A / M / P / Q, or K231A / Q / R / T, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

17. The engineered adenylate kinase of any one of claims 1-16, wherein the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set at amino acid position(s) 37, 47, 52, 53, 54, 55, 56, 89, 92, 96, 115, 125, 132, 135, 146, 168, 179, 180, 187, 220, 15, 40, 91, 93, 95, 117, 122, 166, 178, 203, 37 / 53 / 74 / 93 / 205, 37 / 74 / 93, 37 / 74 / 93 / 125, 37 / 74 / 205, 37 / 93 / 125 / 205, 37 / 125, 37 / 166, 47 / 55 / 126 / 178, 47 / 89 / 126 / 178, 47 / 89 / 146 / 178, 47 / 117 / 126 / 178, 47 / 178, 53 / 74 / 93 / 166, 53 / 74 / 125, 53 / 93 / 125 / 133 / 166 / 205, 55 / 126 / 178, 74, 74 / 93 / 133, 74 / 125, 74 / 205, 89 / 117 / 178, 89 / 126, 89 / 146 / 178, 89 / 178, 117 / 126, 117 / 126 / 178, 117 / 178, 126 / 146 / 178, 126 / 178, 133, 146 / 178, 205, 37 / 53, 37 / 53 / 74 / 125 / 205, 47 / 54 / 178, 47 / 146 / 178, 53 / 74 / 93, 53 / 93 / 125 / 166, 53 / 133, 54 / 117 / 126 / 146, 89 / 117 / 146 / 178, 89 / 126 / 178, 93 / 125, or 166 / 205, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ. ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.DocketNo. 5244.286WO3 18. The engineered adenylate kinase of any one of claims 1-17, wherein the amino acid sequence of the engineered adenylate kinase further comprises at least a substitution or substitution set 37E, 37Q, 47L, 52L, 53S, 54V, 55S, 56A, 56E, 89D, 89F, 89S, 89W, 92S, 96L, 115H, 125L, 132R, 135E, 135N, 146A, 168C, 179A, 180A, 187R, 220G, 15A, 40T, 53T, 53V, 54G, 54R, 55G, 55N, 55R, 89A, 89G, 89L, 89P, 89Q, 89V, 91L, 93G, 93L, 93V, 95W, 115N, 117A, 117E, 117V, 1225, 166E, 178A, 178F, 1781, 178M, 178N, 178R, 178T, 178V, 178Y, 180P, 203Q, 37H, 37H / 74H / 93G / 125L, 37H / 74H / 205Q, 37H / 93G / 125L / 205Q, 37H / 125L, 37R / 53M / 74H / 93G / 205Q, 37R / 74H / 93G, 37R / 166E, 47L / 55S / 126I / 178G, 47L / 89G / 126I / 178G, 47L / 89G / 146G / 178L, 47L / 117T / 126I / 178G, 47L / 178G, 53M / 74H / 93G / 166E, 53M / 74H / 125L, 53M / 93G / 125L / 133W / 166E / 205Q, 55S / 126I / 178L, 74H, 74H / 93G / 133W, 74H / 125L, 74H / 205Q, 89G / 117T / 178G, 89G / 126I, 89G / 146G / 178G, 89G / 178L, 117T, 117T / 126I, 117T / 126I / 178G, 117T / 126I / 178L, 117T / 178L, 126I / 146G / 178G, 126I / 178G, 133W, 146G, 146G / 178G, 178G, 205Q, 37H / 53M, 37R, 37R / 53M, 37R / 53M / 74H / 125L / 205Q, 47L / 54R / 178G, 47L / 146G / 178G, 47L / 178L, 53M, 53M / 74H / 93G, 53M / 93G / 125L / 166E, 53M / 133W, 54R / 117T / 126I / 146G, 74H, 89G / 117T / 146G / 178L, 89G / 126I / 178G, 93G / 125L, 166E / 205Q, or 178L, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

19. The engineered adenylate kinase of any one of claims 1- 17, wherein the amino acid sequence of the engineered adenylate kinase further comprises at least a substitution or substitution set T37E, T37Q, I47L, V52L, A53S, K54V, Q55S, N56A, N56E, K89D, K89F, K89S, K89W, E92S, I96L, E115H, V125L, D132R, V135E, V135N, K146A, G168C, E179A, E180A, K187R, A220G, Y15A, P40T, A53T, A53V, K54G, K54R, Q55G, Q55N, Q55R, K89A, K89G, K89L, K89P, K89Q, K89V, C91L, K93G, K93L, K93V, F95W, E115N, Q.117A, Q.117E, Q117V, T122S, D166E, K178A, K178F, K178I, K178M, K178N, K178R, K178T, K178V, K178Y, E180P, K203Q, T37H, T37H / P74H / K93G / V125L, T37H / P74H / I205Q, T37H / K93G / V125L / I205Q, T37H / V125L, T37R / A53M / P74H / K93G / I205Q, T37R / P74H / K93G, T37R / D166E, I47L / Q55S / L126I / K178G, I47L / K89G / L126I / K178G, I47L / K89G / K146G / K178L, I47L / Q117T / L126I / K178G, I47L / K178G, A53M / P74H / K93G / D166E, A53M / P74H / V125L, A53M / K93G / V125L / E133W / D166E / I205Q, Q55S / L126I / K178L, P74H, P74H / K93G / E133W, P74H / V125L, P74H / I205Q, K89G / Q117T / K178G, K89G / L126I, K89G / K146G / K178G, K89G / K178L, Q117T, Q117T / L126I, Q117T / L126I / K178G, Q117T / L126I / K178L, Q117T / K178L, L126I / K146G / K178G, L126I / K178G, E133W, K146G, K146G / K178G, K178G, I205Q, T37H / A53M, T37R, T37R / A53M, T37R / A53M / P74H / V125L / I205Q, I47L / K54R / K178G, I47L / K146G / K178G, I47L / K178L, A53M, A53M / P74H / K93G, A53M / K93G / V125L / D166E, A53M / E133W, K54R / Q117T / L126I / K146G, K89G / Q117T / K146G / K178L, K89G / L126I / K178G, K93G / V125L, D166E / I205Q, or K178L, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-Docket No. 5244.286WO3 231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

20. The engineered adenylate kinase of any one of claims 1-4, wherein the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set of an engineered adenylate kinase variant set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2 or 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

21. The engineered adenylate kinase of any one of claims 1-4, wherein the adenylate kinase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference amino acid sequence comprising a substitution or substitution set of an engineered adenylate kinase variant set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2 or 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

22. The engineered adenylate kinase of claim 1, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12-231 of an even-numbered SEQ ID NO. of SEQ ID NOs: 18-360 and 372-1018, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 18-360 and 372-1018.

23. The engineered adenylate kinase of any one of claims 1-22, wherein the amino acid sequence of the engineered adenylate kinase comprises at least a substitution at amino acid position 4, 14, 15, 16, 17, 18, 31, 33, 35, 37, 38, 40, 46, 47, 52, 53, 54, 55, 56, 59, 61, 62, 66, 68, 69, 74, 80, 81, 82, 83, 84, 87, 89, 91, 92, 93, 95, 96, 104, 105, 109, 111, 112, 115, 117, 121, 122, 125, 126, 132, 133, 135, 136, 139, 142, 143, 146, 148, 149, 154, 157, 159, 162, 163, 166, 168, 169, 170, 178, 179, 180, 182, 183, 184, 187, 192, 194, 199, 202, 203, 204, 205, 207, 212, 214, 215, 217, 218, 219, 220, 222, 223, 225, 227, 228, and 229, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

24. The engineered adenylate kinase of any one of claims any one of claims 1-23, wherein the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or amino acid residue 4N, 14P, 15A / H / T, 16C / G / M / T, 171, 18V, 31F / V / W, 33L, 35C / E, 37M, 37E / H / Q / R, 38D, 40T, 46E, 47L, 52L, 53D / M / S / T / V, 54G / R / V, 55Q, 55E / G / N / R / S, 56A / E, 59G, 61G, 62H, 66D, 68V, 69V, 74H,Docket No. 5244.286WO3 80S, 81P, 82L, 83R, 84A / T, 87G / R / S, 89A / D / F / G / L / P / Q / S / V / W, 91 L, 92S, 93E, 93G / L / V, 95W, 96L, 104L / R, 104Q, 105L / Q / S, 109A, 111N, 112C / T, 115C / H / N, 117A / E / G / R / T / V, 121V, 122S, 125 L, 126C / I, 132R, 133W, 135E / N, 136L / R, 139A / H, 142V, 1431, 146A / G / H, 148Q, 149A / L / M / Q, 154E, 157R, 159V, 162D / M / V, 163E / R, 166E, 168C, 169E, 170K / M, 178A / F / G / I / L / M / N / R / T / V / Y, 179A, 180A / P / Q, 182L, 183L, 184P / Q / S, 187D / R, 192L / M / V, 194L, 199F, 202E / G / N, 203Q, 204D, 205Q, 2051, 205C / L, 2071 / N, 212A, 214K / P, 215V, 217A / E, 218L / V, 219L / T, 220G, 222A, 223D / M / V, 225H / T, 225A / Q, 227S, 228L / S, and 229G, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

25. The engineered adenylate kinase of any one of claims any one of claims 1-24, wherein the amino acid sequence of the engineered adenylate kinase comprises at least a substitution at amino acid position 37, 93, 125, or 205, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

26. The engineered adenylate kinase of any one of claims 1-25, wherein the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or amino acid residue 37H / M, 93G, 125L, or 205Q, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

27. The engineered adenylate kinase of any one of claims 1-26, wherein the amino acid sequence of the engineered adenylate kinase comprises at least a substitution at amino acid position 4, 47, 95, or 96; or substitution 4N, 14P, 15A / H / T, 16C / G / M / T, 171, 18V, 31F / V / W, 33L, 35C / E, 37M, 37E / H / Q / R, 38D, 40T, 46E, 47L, 52L, 53D / M / S / T / V, 54G / R / V, 55Q, 55E / G / N / R / S, 56A / E, 59G, 61G, 62H, 66D, 68V, 69V, 74H, 80S, 81P, 82L, 83R, 84A / T, 87G / R / S, 89A / D / F / G / L / P / Q / S / V / W, 91L, 92S, 93E, 93G / L / V, 95W, 96L, 104L / R, 104Q, 105L / Q / S, 109A, 111N, 112C / T, 115C / H / N, 117A / E / G / R / T / V, 121V, 122S, 125L, 126C / 1, 132R, 133W, 135E / N, 136L / R, 139A / H, 142V, 1431, 146A / G / H, 148Q, 149A / L / M / Q, 154E, 157R, 159V, 162D / M / V, 163E / R, 166E, 168C, 169E, 170K / M, 178A / F / G / I / L / M / N / R / T / V / Y, 179A, 180A / P / Q, 182L, 183L, 184P / Q / S, 187D / R, 192L / M / V, 194L, 199F, 202E / G / N, 203Q, 204D, 205Q, 2051, 205C / L, 2071 / N, 212A, 214K / P, 215V, 217A / E, 218L / V, 219L / T, 220G, 222A, 223D / M / V, 225H / T, 225A / Q, 227S, 228L / S, or 229G; or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 144, 252, 402, 446,Docket No. 5244.286WO3 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

28. The engineered adenylate kinase of claim 27 wherein the amino acid sequence of the engineered adenylate kinase comprises at least a substitution 4N, 14P, 15A / H / T, 16C / G / M / T, 171, 18V, 31F / V / W, 33L, 35C / E, 37M, 37E / H / Q / R, 38D, 40T, 46E, 47L, 52L, 53D / M / S / T / V, 54G / R / V, 55Q, 55E / G / N / R / S, 56A / E, 59G, 61G, 62H, 66D, 68V, 69V, 74H, 80S, 81P, 82L, 83R, 84A / T, 87G / R / S, 89A / D / F / G / L / P / Q / S / V / W, 91L, 92S, 93E, 93G / L / V, 95W, 96L, 104L / R, 104a 105L / Q / S, 109A, 111N, 112C / T, 115C / H / N, 117A / E / G / R / T / V, 121V, 122S, 125L, 126C / 1, 132R, 133W, 135E / N, 136L / R, 139A / H, 142V, 1431, 146A / G / H, 148 149A / L / M / a 154E, 157R, 159V, 162D / M / V, 163E / R, 166E, 168C, 169E, 170K / M, 178A / F / G / I / L / M / N / R / T / V / Y, 179A, 180A / P / a 182L, 183L, 184P / Q / S, 187D / R, 192L / M / V, 194L, 199F, 202E / G / N, 203a 204D, 205a 2051, 205C / L, 2071 / N, 212A, 214K / P, 215V, 217A / E, 218L / V, 219L / T, 220G, 222A, 223D / M / V, 225H / T, 225A / a 227S, 228L / S, or 229G, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ. ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

29. The engineered adenylate kinase of any one of claims 1-29, wherein the amino acid sequence can further comprise at least a substitution at amino acid position 11, 13, 14, 15, 16, 18, 20, 21, 22, 26, 27, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 97, 98, 99, 100, 101, 102, 104, 105, 108, 109, 110, 111, 112, 113, 115, 116, 117, 118, 119, 120, 122, 123, 124, 125, 126, 127, 128, 129, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 146, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 161, 162, 163, 166, 168, 169, 170, 172, 173, 175, 178, 179, 180, 181, 182, 183, 184, 186, 187, 188, 190, 191, 192, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, or 231, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

30. The engineered adenylate kinase of claim 29, wherein the amino acid sequence can further comprise at least a substitution or amino acid residue 11D, 13G / S, 14C / E, 15F, 16M, 18C / E / L, 20A / P / R / T / V, 21L / N / R / S / T / V, 22A / S, 26L, 27C / S, 29Q / R, 30G / L / S / Y, 31R, 32F / I / L / N, 34A / E / S / V, 35A / E / F / L / S, 36A / E / I / L / M / Q / R / S / V / Y, 37F / G / I / L / R / Y, 38C / F / L / R, 39A / M / Q / T, 40C / F / L / S / T, 42T, 43A / C / G / H / N, 44S, 46E / F / L / M / Y, 48F / L, 50A / C / L / N / Q / R / S / V, 51F / G / K / L / M / R / T, 52A / H, 53A / M / S,Docket No. 5244.286WO3 54G / H / I / M / Q / R / T, 55G / L / Q / S, 56E / Q / T, 57H / L / P / S, 58C / G / Y, 59E / G / H / M / P / R / T / V / W / Y, 60A / C / P / S / W, 61A / E / N / P / S / V, 62A / E / G / I / L / S, 63S, 64R, 65A / D / F / K / P / T, 66A / C / D / E / F / G / H / N / P / Q / R / S / T / V / W, 67R, 68A / D / E / G / I / L / Q / R / S / T / V / W / Y, 69F / L / Q / R / Y, 70R, 71T, 731 / R, 74C / G / Q / S, 75L, 76G / L / P, 77A / I / M / Q / S, 78C / G / N / T, 79A / K / L / P / W, 80A / D / G / L / N / P / R / S / V / W, 81F / I / L / Q / S, 82 l / L / P / Q / T, 83G / H / L / S / T, 84A / E / H / M / S, 87A / E / I / K / L / M / R / V / Y, 88N / R / T, 89A / H / I / L / P / Q / T / V, 90C / E / F / K / S / T / V, 91A / F / G / L / S / V, 92L / S, 93A / E / G / P / S / T / V / Y, 94A / C / F / M / Q / S / T / V / Y, 971, 98A / G / Q, 99A / C, 100F / S / V, 1O1-, 102A / C / N, 104A / F / H / I / L / Q / R / S / W, 105G / K / L / M / R / S, 108M / R / S / V, 109E / Y, 11OC / F, 111E / P / R, 112A / C / E / K / M / N / Q / R / T, 113E / W / Y, 115G / K / R, 116A / E / F / L, 117L / N / S, 118A / G / L / R / S, 119F / K / L / P / R / S, 120G / K / L / R / S / T, 122A / H / S, 123S, 124V, 1251, 126A / E / S / V, 1271 / L / P / S, 128C / E / I / K / N / R / S, 1291 / L / P / S, 131A / G / V, 132G / K / L / T / V, 133A / E / F / L / Q / S / V / W, 134L, 1351 / K / M / P / R / S, 136A / I / L / S / V / Y, 137L, 138C / I / M / V, 139A / H / L / R, 140G, 141G / V, 142F / L / M / W, 143A / C / G / P / R / S / T / V, 146D / H / N / R / V, 148F / H / M / P / Q / R / S / T, 149L, 150C / G / L / P / S / V, 151A / F / R / Y, 152F / H, 153C / K / S / V, 154Q / R, 155A / F / T / W, 156A / C / N / T / V, 157P / V, 161G / L, 162G, 163G / Q / S, 166C / F / L / P / S, 168G / L / N / Q / S, 169A / D / I / Y, 170A / G / H / P / R / S, 172A / H / M / S / T, 173F / K / R / S / T / V, 175F / L / S, 178E / G / N / R, 179A / C / G / I / L / P / V, 180G / H / P, 181C / I / V, 182A / G / I / L / Q / S, 183A / L / Q, 1841 / K / M / N / R / S / T / V, 186L, 187G / Y, 188G / L, 190A / C / E / G / H / N / Q / R, 191D / K, 192A / H / I / W, 194F / L / R / V / Y, 195G / I / M / R, 1961, 197A / L / Q / V, 198G, 199R, 200A, 2O1A / F / K / L / S, 2O2E / G / M / S, 203A / E / L / R, 204R / S, 205L / P, 206W, 207A / L / T, 208E, 210V, 211A, 212C / H / I / L / M / N / P / Q / R / S / W, 213L / P / Q / S, 214A / E / L / M / P / R / T / W, 215A / P / V, 216A / D / E / G / H / L / M / N / P / R / S / T / V, 217E / G / H / P / T, 218A / I / L, 219A / F / G / L / P / S / T / W, 220E / V, 221D, 2221 / T, 223T, 224G / S / T, 225A / L / Q / R / T / V, 226K / L / P / R / S / T, 227P, 228D / F / G / I / L / P / Q / R / S / T / V, 229C / I, 230A / M / P / Q, or 231A / Q / R / T, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

31. The engineered adenylate kinase of claim 29 or 30, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, or to the reference sequence corresponding to SEQ ID NO: 144, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, or relative to the reference sequence corresponding to SEQ ID NO: 144.

32. The engineered adenylate kinase of claim 29 or 30, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residuesDocket No. 5244.286WO3 12-231 of an even-numbered SEQ ID NO. of SEQ ID NOs: 252-286, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 252-286, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, or relative to the reference sequence corresponding to SEQ ID NO: 144.

33. The engineered adenylate kinase of claim 29 or 30, wherein the amino acid sequence of the engineered adenylate kinase comprises at least a substitution at amino acid position 15, 16, 37, 69, 80, 82, 112, 115, 170, 178, 187, 205, or 217, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, or relative to the reference sequence corresponding to SEQ ID NO: 144.

34. The engineered adenylate kinase of any one of claims 1-33, wherein the amino acid sequence of the engineered adenylate kinase comprises at least a substitution set, or amino acid residue(s) 1ST, 16C, 16G, 16T, 37M, 69V, SOS, 82L, 112C, 112T, 115C, 170M, 178A, 178L, 178V, 187D, 205C, or 217E, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, or relative to the reference sequence corresponding to SEQ ID NO: 144.

35. The engineered adenylate kinase of any one of claims 1-34, wherein the amino acid sequence of the engineered adenylate kinase comprises at least a substitution set, or amino acid residue(s) Y15T, L16C, L16G, L16T, H37M, K69V, P80S, V82L, S112C, S112T, E115C, S170M, K178A, K178L, K178V, K187D, Q205C, or P217E, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, or relative to the reference sequence corresponding to SEQ ID NO: 144.

36. The engineered adenylate kinase of claim 33 or 34, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 252, or to the reference sequence corresponding to SEQ ID NO: 252, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 252, or relative to the reference sequence corresponding to SEQ ID NO: 252.

37. The engineered adenylate kinase of any one of claims 1-36, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding toDocket No. 5244.286WO3 residues 12-231 of an even-numbered SEQ ID NO. of SEQ ID NOs: 288-360, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 288-360, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 252, or relative to the reference sequence corresponding to SEQ ID NO: 252.

38. The engineered adenylate kinase of claim any one of claims 1-37, wherein the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set at amino acid position(s) 4 / 15, 14 / 15 / 93, 14 / 170 / 178, 14 / 214, 15 / 93 / 157 / 178 / 205 / 214, 15 / 115 / 157, 15 / 115 / 170, 15 / 170 / 180, 15 / 214, 93, 93 / 157 / 170 / 214, 93 / 205, 115, 157 / 170, 157 / 205 / 214, 178 / 180 / 214, 180, 214, 14, 14 / 15 / 157 / 170 / 178 / 180 / 214, 14 / 15 / 157 / 205, 14 / 157 / 170 / 178 / 180 / 205, 14 / 205, 15, 15 / 93 / 157 / 214, 15 / 93 / 180, 15 / 93 / 205, 15 / 157, 15 / 157 / 205, 15 / 178, 15 / 205, 93 / 157 / 205, 93 / 157 / 214, 93 / 170 / 205, 93 / 178 / 205, 93 / 180, or 205, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 252, or relative to the reference sequence corresponding to SEQ ID NO: 252.

39. The engineered adenylate kinase of any one of claims 37-38, wherein the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set, or amino acid residue(s) 4N / 15T, 14P / 15T / 93E, 14P / 170M / 178T, 14P / 214P, 15T / 93E / 157R / 178T / 205L / 214K, 15T / 115H / 157R, 15T / 115H / 170M, 15T / 170M / 180Q, 15T / 214P, 93E, 93E / 157R / 170M / 214K, 93E / 205L, 115H, 157R / 170M, 157R / 205L / 214P, 178T / 180Q / 214P, 180Q, 214P, 14P, 14P / 15T / 157R / 170M / 178T / 180Q / 214K, 14P / 15T / 157R / 205L, 14P / 157R / 170M / 178T / 180Q / 205L, 14P / 205L, 15T, 15T / 93E / 157R / 214P, 15T / 93E / 180Q, 15T / 93E / 205L, 15T / 157R, 15T / 157R / 205L, 15T / 178T, 15T / 205L, 93E / 157R / 205L, 93E / 157R / 214K, 93E / 170M / 205L, 93E / 178T / 205L, 93E / 180Q, or 205L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 252, or relative to the reference sequence corresponding to SEQ ID NO: 252.

40. The engineered adenylate kinase of any one of claims 37-39, wherein the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set, or amino acid residue(s) H4N / Y15T, A14P / Y15T / G93E, A14P / S170M / K178T, A14P / E214P, Y15T / G93E / P157R / K178T / Q205L / E214K, Y15T / E115H / P157R, Y15T / E115H / S170M, Y15T / S170M / E180Q, Y15T / E214P, G93E, G93E / P157R / S170M / E214K, G93E / Q205L, E115H, P157R / S170M, P157R / Q205L / E214P, K178T / E180Q / E214P, E180Q, E214P, A14P, A14P / Y15T / P157R / S170M / K178T / E180Q / E214K, A14P / Y15T / P157R / Q205L, A14P / P157R / S170M / K178T / E180Q / Q205L, A14P / Q205L, Y15T, Y15T / G93E / P157R / E214P,Docket No. 5244.286WO3 Y15T / G93E / E180Q, Y15T / G93E / Q205L, Y15T / P157R, Y15T / P157R / Q205L, Y15T / K178T, Y15T / Q205L, G93E / P157R / Q205L, G93E / P157R / E214K, G93E / S170M / Q205L, G93E / K178T / Q205L, G93E / E180Q, or Q205L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 252, or relative to the reference sequence corresponding to SEQ ID NO: 252.

41. The engineered adenylate kinase of any one of claims 1-40, wherein the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set at amino acid position(s) 180 / 205, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 252, or relative to the reference sequence corresponding to SEQ ID NO: 252.

42. The engineered adenylate kinase of any one of claims 1-41, wherein the amino acid sequence of the engineered adenylate kinase further comprises at least a substitution or substitution set 180Q / 205L, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 252, or relative to the reference sequence corresponding to SEQ ID NO: 252.

43. The engineered adenylate kinase of any one of claims 1-42, wherein the amino acid sequence of the engineered adenylate kinase further comprises at least a substitution or substitution set E180Q / Q205L wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 252, or relative to the reference sequence corresponding to SEQ ID NO: 252.

44. The engineered adenylate kinase of any one of claims 1-43, wherein the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set at amino acid position(s) 18, 31, 35, 38, 59, 62, 87, 104, 105, 109, 139, 143, 146, 149, 184, 202, 207, 219, 222, 223, 227, 228, and 229, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 402, or relative to the reference sequence corresponding to SEQ ID NO: 402.

45. The engineered adenylate kinase of any one of claims 1-44, wherein the amino acid sequence of the engineered adenylate kinase further comprises at least a substitution or substitution set of 18V, 31F, 31V, 31W, 35C, 35E, 38D, 59G, 62H, 87G, 104Q, 105Q, 109A, 139H, 1431, 146H, 149A, 149L, 149Q, 184P, 202E, 202G, 207N, 219L, 219T, 222A, 223V, 227S, 228L, 228S, and 229G, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 402, or relative to the reference sequence corresponding to SEQ ID NO: 402.Docket No. 5244.286WO3 46. The engineered adenylate kinase of any one of claims 1-45, wherein the amino acid sequence of the engineered adenylate kinase further comprises at least a substitution or substitution set L18V, R31F, R31V, R31W, A35C, A35E, G38D, V59G, S62H, K87G, R104Q, R105Q, Y109A, L139H, V143I, K146H, R149A, R149L, R149Q, M184P, K202E, K202G, A207N, V219L, V219T, V222A, L223V, G227S, V228L, V228S, and I229G wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 402, or relative to the reference sequence corresponding to SEQ ID NO: 402.

47. The engineered adenylate kinase of any one of claims 1-46, wherein the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set at amino acid position(s) 33, 33 / 54, 33 / 54 / 83 / 104 / 109, 33 / 54 / 104, 33 / 54 / 104 / 109, 33 / 54 / 104 / 109 / 207, 33 / 54 / 104 / 109 / 223, 33 / 54 / 104 / 154 / 223, 33 / 54 / 104 / 199 / 223, 33 / 54 / 104 / 223, 33 / 54 / 154, 33 / 54 / 154 / 199, 33 / 54 / 154 / 223, 33 / 54 / 207 / 223, 33 / 54 / 223, 33 / 104, 33 / 104 / 109, 33 / 104 / 109 / 199 / 223, 33 / 104 / 207, 33 / 104 / 223, 33 / 109, 33 / 109 / 199 / 207, 33 / 109 / 199 / 223, 33 / 109 / 202 / 223, 33 / 154, 33 / 154 / 223, 33 / 207, 33 / 223, 54, 54 / 83, 54 / 83 / 223, 54 / 104, 54 / 104 / 109, 54 / 104 / 223, 54 / 154 / 199, 55, 55 / 87, 55 / 87 / 105, 55 / 87 / 105 / 139, 55 / 87 / 105 / 139 / 184, 55 / 87 / 105 / 139 / 225, 55 / 87 / 105 / 184, 55 / 87 / 105 / 184 / 202 / 225, 55 / 87 / 105 / 225, 55 / 87 / 139 / 184, 55 / 87 / 184, 55 / 87 / 184 / 202 / 225, 55 / 87 / 184 / 225, 55 / 87 / 225, 55 / 105, 55 / 105 / 139 / 184, 55 / 105 / 139 / 184 / 225, 55 / 105 / 139 / 225, 55 / 105 / 184, 55 / 105 / 184 / 225, 55 / 105 / 225, 55 / 139, 55 / 139 / 184 / 225, 55 / 139 / 225, 55 / 184, 55 / 184 / 202, 55 / 184 / 202 / 225, 55 / 184 / 225, 55 / 225, 83, 87, 87 / 105, 87 / 105 / 139, 87 / 105 / 182 / 184 / 225, 87 / 105 / 184 / 202, 87 / 105 / 184 / 225, 87 / 139 / 184 / 225, 87 / 139 / 225, 87 / 184, 87 / 184 / 202, 87 / 202 / 204, 87 / 225, 104, 104 / 109, 104 / 109 / 154, 104 / 109 / 154 / 199, 104 / 109 / 199, 104 / 109 / 199 / 207, 104 / 109 / 199 / 207 / 223, 104 / 109 / 207, 104 / 207, 104 / 223, 105, 105 / 111 / 184 / 225, 105 / 139 / 184, 105 / 139 / 184 / 202, 105 / 139 / 184 / 225, 105 / 184, 105 / 184 / 202, 105 / 225, 109 / 199, 139 / 184, 139 / 184 / 225, 139 / 202 / 225, 139 / 225, 154, 154 / 199, 184, 184 / 202, 184 / 225, 199, 199 / 223, 202, 202 / 225, 207, 223, and 225, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 446, or relative to the reference sequence corresponding to SEQ ID NO: 446.

48. The engineered adenylate kinase of any one of claims 1-47, wherein the amino acid sequence of the engineered adenylate kinase further comprises at least a substitution or substitution set 33L, 33L / 54G, 33L / 54G / 83R / 104L / 109A, 33L / 54G / 104L, 33L / 54G / 104L / 109A, 33L / 54G / 104L / 109A / 207I, 33L / 54G / 104L / 154E / 223M, 33L / 54G / 104L / 199F / 223M, 33L / 54G / 104R / 109A / 223M, 33L / 54G / 104R / 223M, 33L / 54G / 154E, 33L / 54G / 154E / 199F, 33L / 54G / 154E / 223M, 33L / 54G / 207I / 223D, 33L / 54G / 223D, 33L / 104L / 223M, 33L / 104R, 33L / 104R / 109A, 33L / 104R / 109A / 199F / 223M, 33L / 104R / 207I, 33L / 109A, 33L / 109A / 199F / 207I, 33L / 109A / 199F / 223D,Docket No. 5244.286WO3 33L / 109A / 199F / 223M, 33L / 109A / 202N / 223D, 33L / 154E, 33L / 154E / 223M, 33L / 207I, 33L / 223M, 54G, 54G / 83R, 54G / 83R / 223D, 54G / 104L / 223M, 54G / 104R, 54G / 104R / 109A, 54G / 154E / 199F, 55E, 55E / 87S, 55E / 87S / 105L / 225A, 55E / 87S / 105Q / 139A, 55E / 87S / 105Q / 139A / 184S, 55E / 87S / 105Q / 184S, 55E / 87S / 105S, 55E / 87S / 105S / 139A / 184Q, 55E / 87S / 105S / 139A / 225Q, 55E / 87S / 105S / 184Q, 55E / 87S / 105S / 184Q / 202E / 225Q, 55E / 87S / 139A / 184Q, 55E / 87S / 184Q, 55E / 87S / 184Q / 225Q, 55E / 87S / 184S, 55E / 87S / 184S / 202E / 225A, 55E / 87S / 184S / 225Q, 55E / 87S / 225Q, 55E / 105L, 55E / 105Q, 55E / 105Q / 139A / 184Q / 225A, 55E / 105Q / 139A / 225Q, 55E / 105Q / 184S, 55E / 105Q / 225Q, 55E / 105S, 55E / 105S / 139A / 184Q, 55E / 105S / 139A / 225A, 55E / 105S / 184Q / 225A, 55E / 105S / 184Q / 225Q, 55E / 105S / 225A, 55E / 139A, 55E / 139A / 184Q / 225A, 55E / 139A / 225A, 55E / 184Q, 55E / 184Q / 202E, 55E / 184Q / 202E / 225A, 55E / 184Q / 202E / 225Q, 55E / 184Q / 225A, 55E / 184Q / 225Q, 55E / 184S, 55E / 184S / 202E, 55E / 184S / 202E / 225Q, 55E / 184S / 225Q, 55E / 225A, 55E / 225Q, 83R, 87S, 87S / 105L / 182L / 184S / 225Q, 87S / 105L / 184Q / 202E, 87S / 105L / 184S / 225Q, 87S / 105Q, 87S / 105Q / 139A, 87S / 105S, 87S / 139A / 184Q / 225Q, 87S / 139A / 184S / 225Q, 87S / 139A / 225Q, 87S / 184Q, 87S / 184S, 87S / 184S / 202E, 87S / 202E / 204D, 87S / 225A, 104L, 104L / 109A, 104L / 109A / 154E, 104L / 109A / 199F, 104L / 109A / 199F / 207I / 223M, 104L / 109A / 2071, 104L / 207I, 104R / 109A, 104R / 109A / 154E / 199F, 104R / 109A / 199F / 207I, 104R / 109A / 2071, 104R / 223M, 105L, 105L / 139A / 184Q / 225Q, 105L / 139A / 184S / 202E, 105Q, 105Q / 111N / 184Q / 225Q, 105Q / 139A / 184S / 225Q, 105Q / 225Q, 105S, 105S / 139A / 184Q, 105S / 139A / 184Q / 225A, 105S / 139A / 184Q / 225Q, 105S / 139A / 184S / 225A, 105S / 184Q, 105S / 184Q / 202E, 105S / 225Q, 109A / 199F, 139A / 184Q, 139A / 184S / 225Q, 139A / 202E / 225A, 139A / 225A, 154E, 154E / 199F, 184Q, 184Q / 202E, 184Q / 225A, 184S, 199F, 199F / 223M, 202E, 202E / 225A, 2071, 223D, 223M, 225A, and 225Q, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 446, or relative to the reference sequence corresponding to SEQ. ID NO: 446.

49. The engineered adenylate kinase of any one of claims 1-48, wherein the amino acid sequence of the engineered adenylate kinase further comprises at least a substitution or substitution set I33L, I33L / K54G, I33L / K54G / L83R / Q104L / Y109A, I33L / K54G / Q104L, I33L / K54G / Q104L / Y109A, I33L / K54G / Q104L / Y109A / A207I, I33L / K54G / Q104L / I154E / L223M, I33L / K54G / Q104L / Y199F / L223M, I33L / K54G / Q104R / Y109A / L223M, I33L / K54G / Q104R / L223M, I33L / K54G / I154E, I33L / K54G / I154E / Y199F, I33L / K54G / I154E / L223M, I33L / K54G / A207I / L223D, I33L / K54G / L223D, I33L / Q104L / L223M, I33L / Q104R, I33L / Q104R / Y109A, I33L / Q104R / Y109A / Y199F / L223M, I33L / Q104R / A207I, I33L / Y109A, I33L / Y109A / Y199F / A207I, I33L / Y109A / Y199F / L223D, I33L / Y109A / Y199F / L223M, I33L / Y109A / K202N / L223D, I33L / I154E, I33L / I154E / L223M, I33L / A207I, I33L / L223M, K54G, K54G / L83R, K54G / L83R / L223D, K54G / Q104L / L223M, K54G / Q104R, K54G / Q104R / Y109A, K54G / I154E / Y199F, Q55E, Q55E / K87S, Q55E / K87S / R105L / I225A, Q55E / K87S / R105Q / L139A, Q55E / K87S / R105Q / L139A / M184S, Q55E / K87S / R105Q / M184S,DocketNo. 5244.286WO3 Q55E / K87S / R105S, Q55E / K87S / R105S / L139A / M184Q, Q55E / K87S / R105S / L139A / I225Q, Q55E / K87S / R105S / M184Q, Q55E / K87S / R105S / M184Q / K202E / I225Q, Q55E / K87S / L139A / M184Q, Q55E / K87S / M184Q, Q55E / K87S / M184Q / I225Q, Q55E / K87S / M184S, Q55E / K87S / M184S / K202E / I225A, Q55E / K87S / M184S / I225Q, Q55E / K87S / I225Q, Q55E / R105L, Q55E / R105Q, Q55E / R105Q / L139A / M184Q / I225A, Q55E / R105Q / L139A / I225Q, Q55E / R105Q / M184S, Q55E / R105Q / I225Q, Q55E / R105S, Q55E / R105S / L139A / M184Q, Q55E / R105S / L139A / I225A, Q55E / R105S / M184Q / I225A, Q55E / R105S / M184Q / I225Q, Q55E / R105S / I225A, Q55E / L139A, Q55E / L139A / M184Q / I225A, Q55E / L139A / I225A, Q55E / M184Q, Q55E / M184Q / K202E, Q55E / M184Q / K202E / I225A, Q55E / M184Q / K202E / I225Q, Q55E / M184Q / I225A, Q55E / M 1840 / 12250, Q55E / M184S, Q55E / M184S / K202E, Q55E / M184S / K202E / I225Q, Q55E / M184S / I225Q, Q55E / I225A, Q55E / I225Q, L83R, K87S, K87S / R105L / V182L / M184S / I225Q, K87S / R105L / M184Q / K202E, K87S / R105L / M184S / I225Q, K87S / R105Q, K87S / R105Q / L139A, K87S / R105S, K87S / L139A / M184Q / I225Q, K87S / L139A / M184S / I225Q, K87S / L139A / I225Q, K87S / M184Q, K87S / M184S, K87S / M184S / K202E, K87S / K202E / G204D, K87S / I225A, Q104L, Q104L / Y109A, Q104L / Y109A / I154E, Q104L / Y109A / Y199F, Q104L / Y109A / Y199F / A207I / L223M, Q104L / Y109A / A207I, Q104L / A207I, Q104R / Y109A, Q104R / Y109A / I154E / Y199F, Q104R / Y109A / Y199F / A207I, Q104R / Y109A / A207I, Q104R / L223M, R105L, R105L / L139A / M184Q / I225Q, R105L / L139A / M184S / K202E, R105Q, R105Q / D111N / M184Q / I225Q, R105Q / L139A / M184S / I225Q, R105Q / I225Q, R105S, R105S / L139A / M184Q, R105S / L139A / M184Q / I225A, R105S / L139A / M184Q / I225Q, R105S / L139A / M184S / I225A, R105S / M184Q, R105S / M184Q / K202E, R105S / I225Q, Y109A / Y199F, L139A / M184Q, L139A / M184S / I225Q, L139A / K202E / I225A, L139A / I225A, I154E, I154E / Y199F, M184Q, M184Q / K202E, M184Q / I225A, M184S, Y199F, Y199F / L223M, K202E, K202 E / I225A, A207I, L223D, L223M, I225A, and l225Qwherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 446, or relative to the reference sequence corresponding to SEQ ID NO: 446.

50. The engineered adenylate kinase of any one of claims 1-49, wherein the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set at amino acid position(s) 15 / 61 / 136 / 212, 16, 16 / 53, 16 / 53 / 68, 16 / 53 / 68 / 84 / 170 / 223, 16 / 53 / 68 / 170 / 192, 16 / 53 / 170, 16 / 53 / 170 / 218 / 223, 16 / 53 / 223, 16 / 68, 16 / 68 / 84 / 170 / 218, 16 / 68 / 170, 16 / 68 / 170 / 192 / 218 / 223, 16 / 68 / 170 / 218, 16 / 68 / 192 / 218 / 223, 16 / 68 / 218, 16 / 68 / 218 / 223, 16 / 68 / 223, 16 / 84, 16 / 84 / 170, 16 / 84 / 170 / 192, 16 / 84 / 170 / 192 / 218, 16 / 170, 16 / 170 / 192, 16 / 170 / 192 / 218, 16 / 170 / 218, 16 / 170 / 218 / 223, 16 / 170 / 223, 16 / 192, 16 / 192 / 223, 16 / 218 / 223, 16 / 223, 16 / 223 / 225, 17, 17 / 61 / 136, 17 / 61 / 136 / 205 / 212, 17 / 61 / 205, 17 / 66 / 136 / 212, 17 / 66 / 205, 17 / 66 / 205 / 217, 17 / 117 / 212, 17 / 136, 17 / 136 / 205, 17 / 205, 17 / 217, 53, 53 / 68, 53 / 68 / 84 / 170 / 218, 53 / 68 / 218, 53 / 84 / 170 / 218, 53 / 84 / 192 / 223, 53 / 170, 53 / 170 / 218, 53 / 170 / 223, 53 / 192, 53 / 223, 61, 61 / 66, 61 / 205, 66, 68, 68 / 84, 68 / 84 / 218, 68 / 170, 68 / 170 / 192 / 218, 68 / 170 / 218, 68 / 170 / 223, 68 / 192, 68 / 218, 84, 84 / 170 / 192 / 223,Docket No. 5244.286WO3 84 / 170 / 218, 84 / 192, 84 / 192 / 218 / 223, 84 / 192 / 223, 84 / 218, 136, 136 / 205, 136 / 205 / 212, 136 / 212, 170, 170 / 192, 170 / 192 / 218, 170 / 192 / 218 / 223, 170 / 192 / 223, 170 / 218, 170 / 218 / 223, 183, 192, 192 / 218 / 223, 192 / 223, 205, 212 / 217, 217, 218, 218 / 223, and 223, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 710, or relative to the reference sequence corresponding to SEQ. ID NO: 710.

51. The engineered adenylate kinase of any one of claims 1-40, wherein the amino acid sequence of the engineered adenylate kinase further comprises at least a substitution or substitution set 15H / 61G / 136L / 212A, 16M, 16M / 53D, 16M / 53D / 68V, 16M / 53D / 68V / 84A / 170K / 223M, 16M / 53D / 68V / 170K / 192L, 16M / 53D / 170K, 16M / 53D / 170K / 218L / 223M, 16M / 53D / 223M, 16M / 68V, 16M / 68V / 84A / 170K / 218V, 16M / 68V / 170K, 16M / 68V / 170K / 192L / 218V / 223M, 16M / 68V / 170K / 218V, 16M / 68V / 192L / 218L / 223M, 16M / 68V / 218V, 16M / 68V / 218V / 223M, 16M / 68V / 223M, 16M / 84A / 170K, 16M / 84A / 170K / 192L, 16M / 84A / 170K / 192L / 218V, 16M / 84T, 16M / 170K, 16M / 170K / 192L, 16M / 170K / 192L / 218L, 16M / 170K / 218L, 16M / 170K / 218L / 223M, 16M / 170K / 218V, 16M / 170K / 223M, 16M / 192L, 16M / 192L / 223M, 16M / 192M, 16M / 218V / 223M, 16M / 223M, 16M / 223M / 225T, 171, 17I / 61G / 136L, 17I / 61G / 136R / 205I / 212A, 17I / 61G / 205I, 17I / 66D / 136L / 212A, 17I / 66D / 205I, 17I / 66D / 205I / 217A, 17I / 117R / 212A, 17I / 136L / 205I, 17I / 136R, 171 / 2051, 17I / 217A, 53D, 53D / 68V, 53D / 68V / 84A / 170K / 218V, 53D / 68V / 218L, 53D / 84A / 170K / 218L, 53D / 84A / 192L / 223M, 53D / 170K, 53D / 170K / 218V, 53D / 170K / 223M, 53D / 192L, 53D / 223M, 61G, 61G / 66D, 61G / 205I, 66D, 68V, 68V / 84A, 68V / 84T / 218L, 68V / 170K, 68V / 170K / 192L / 218V, 68V / 170K / 218L, 68V / 170K / 223M, 68V / 192L, 68V / 218V, 84A, 84A / 170K / 218V, 84A / 192L, 84A / 192L / 218L / 223M, 84A / 192L / 223M, 84A / 218V, 84T, 84T / 170K / 192L / 223M, 84T / 218L, 136L, 136R, 136R / 205I, 136R / 205I / 212A, 136R / 212A, 170K, 170K / 192L, 170K / 192L / 218L / 223M, 170K / 192L / 218V, 170K / 192L / 218V / 223M, 170K / 192L / 223M, 170K / 218V, 170K / 218V / 223M, 183L, 192L, 192L / 218L / 223M, 192L / 218V / 223M, 192L / 223M, 2051, 212A / 217 A, 217A, 218L, 218L / 223M, 218V, 218V / 223M, and 223M, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 710, or relative to the reference sequence corresponding to SEQ ID NO: 710.

52. The engineered adenylate kinase of any one of claims 1-41, wherein the amino acid sequence of the engineered adenylate kinase further comprises at least a substitution or substitution set Y15H / S61G / V136L / S212A, L16M, L16M / A53D, L16M / A53D / L68V, L16M / A53D / L68V / R84A / S170K / L223M, L16M / A53D / L68V / S170K / K192L, L16M / A53D / S170K, L16M / A53D / S170K / I218L / L223M, L16M / A53D / L223M, L16M / L68V, L16M / L68V / R84A / S170K / I218V, L16M / L68V / S170K, L16M / L68V / S170K / K192L / I218V / L223M, L16M / L68V / S170K / I218V, L16M / L68V / K192L / I218L / L223M, L16M / L68V / I218V, L16M / L68V / I218V / L223M, L16M / L68V / L223M, L16M / R84A / S170K, L16M / R84A / S170K / K192L, L16M / R84A / S170K / K192L / I218V, L16M / R84T,Docket No. 5244.286WO3 L16M / S170K, L16M / S170K / K192L, L16M / S170K / K192L / I218L, L16M / S170K / I218L, L16M / S170K / I218L / L223M, L16M / S170K / I218V, L16M / S170K / L223M, L16M / K192L, L16M / K192L / L223M, L16M / K192M, L16M / I218V / L223M, L16M / L223M, L16M / L223M / A225T, V17I, V17I / S61G / V136L, V17 I / S61G / V136R / L205I / S212A, V17I / S61G / L205I, V17I / W66D / V136L / S212A, V17I / W66D / L205I, V17I / W66D / L205I / P217A, V17I / Q117R / S212A, V17I / V136L / L2051, V17I / V136R, V17I / L2051, V17I / P217A, A53D, A53D / L68V, A53D / L68V / R84A / S170K / I218V, A53D / L68V / I218L, A53D / R84A / S170K / I218L, A53D / R84A / K192L / L223M, A53D / S170K, A53D / S170K / I218V, A53D / S170K / L223M, A53D / K192L, A53D / L223M, S61G, S61G / W66D, S61G / L205I, W66D, L68V, L68V / R84A, L68V / R84T / I218L, L68V / S170K, L68V / S170K / K192L / I218V, L68V / S170K / I218L, L68V / S170K / L223M, L68V / K192L, L68V / I218V, R84A, R84A / S170K / I218V, R84A / K192L, R84A / K192L / I218L / L223M, R84A / K192L / L223M, R84A / I218V, R84T, R84T / S170K / K192L / L223M, R84T / I218L, V136L, V136R, V136R / L205I, V136R / L205I / S212A, V136R / S212A, S170K, S170K / K192L, S170K / K192L / I218L / L223M, S170K / K192L / I218V, S170K / K192L / I218V / L223M, S170K / K192L / L223M, S170K / I218V, S170K / I218V / L223M, R183L, K192L, K192L / I218L / L223M, K192L / I218V / L223M, K192L / L223M, L205I, S212A / P217A, P217A, I218L, I218L / L223M, 1218V, I218V / L223M, and L223M wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 710, or relative to the reference sequence corresponding to SEQ ID NO: 710.

53. The engineered adenylate kinase of any one of claims 1-42, wherein the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set at amino acid position(s) 46, 55, 81, 87, 117, 121, 126, 142, 148 / 202, 149, 159, 162, 163, 169, 192, 194, 215, and 225, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 808, or relative to the reference sequence corresponding to SEQ ID NO: 808.

54. The engineered adenylate kinase of any one of claims 1-43, wherein the amino acid sequence of the engineered adenylate kinase further comprises at least a substitution or substitution set 46E, 55Q, 81P, 87R, 117G, 121V, 126C, 142V, 148Q / 202N, 149M, 159V, 162D, 162M, 162V, 163E, 163R, 169E, 192V, 194L, 215V, and 225H, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 808, or relative to the reference sequence corresponding to SEQ ID NO: 808.

55. The engineered adenylate kinase of any one of claims 1-44, wherein the amino acid sequence of the engineered adenylate kinase further comprises at least a substitution or substitution set D46E, E55Q, V81P, K87R, Q117G, L121V, L126C, R142V, G148Q / K202N, R149M, K159V, E162D,Docket No. 5244.286WO3 E162M, E162V, L163E, L163R, V169E, K192V, Q194L, 1215V, and A225H wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 808, or relative to the reference sequence corresponding to SEQ ID NO: 808.

56. The engineered adenylate kinase of any one of claims 1-4, wherein the amino acid sequence of the engineered adenylate kinase comprises at least a substitution or substitution set of an engineered adenylate kinase variant set forth in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2 or 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or relative to the reference sequence corresponding to SEQ ID NO: 144, 252, 402, 446, 710, or 808.

57. The engineered adenylate kinase of claim 1, wherein the amino acid sequence of the engineered adenylate kinase comprises residues 12-231 of an even-numbered SEQ ID NO. of SEQ ID NOs: 18-360 and 372-1018, or comprises an even-numbered SEQ ID NO. of SEQ ID NOs: 18-360 and 372-1018.

58. The engineered adenylate kinase of claim 1, wherein the amino acid sequence of the engineered adenylate kinase comprises residues 12-231 of SEQ ID NO: 144, 252, 402, 446, 710, or 808, or wherein the amino acid sequence comprises SEQ ID NO: 144, 252, 402, 446, 710, or 808.

59. The engineered adenylate kinase of any one of claims 1-58, having adenylate kinase activity and at least one improved property as compared to a reference adenylate kinase.

60. The engineered adenylate kinase of claim 59, wherein the improved property is selected from i) increased activity on unmodified nucleoside monophosphate (NMP), ii) increased stability, iii) increased thermostability, iv) increased activity on 2'-fluoro modified nucleoside monophosphate, and v) increased activity on 2'-O-methyl modified nucleoside monophosphate, or any combinations of i), ii), iii), iv), and v) as compared to a reference adenylate kinase.

61. The engineered adenylate kinase of any one of claims 1-60, wherein the engineered adenylate kinase is purified.

62. A recombinant polynucleotide comprising a polynucleotide sequence encoding an engineered adenylate kinase of any one of claims 1-60.

63. The recombinant polynucleotide of claim 62, comprising a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequenceDocket No. 5244.286WO3 corresponding to nucleotide residues 34-693 of SEQ ID NO: 143, 251, 401, 445, 709, or 807, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 143, 251, 401, 445, 709, or 807, wherein the recombinant polynucleotide encodes an adenylate kinase.

64. The recombinant polynucleotide of claim 62, comprising a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34-693 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 17-359 and 371-1017, or to a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO. of SEQ ID NOs: 17-359 and 371-1017, wherein the recombinant polynucleotide encodes an engineered adenylate kinase.

65. The recombinant polynucleotide of any one of claims 62-64, wherein the polynucleotide sequence has preferred codons or is codon optimized.

66. The recombinant polynucleotide of claim 62, wherein the polynucleotide sequence comprises nucleotide residues 34-693 of SEQ ID NO: 17-359 and 371-1017, or comprises SEQ ID NO: 17-359 and 371-1017.

67. The recombinant polynucleotide of claim 66, wherein the polynucleotide sequence comprises nucleotide residues 34-693 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 143, 251, 401, 445, 709, or 807, or comprises an odd-numbered SEQ ID NO. of SEQ ID NOs: 143, 251, 401, 445, 709, or 807.

68. An expression vector comprising a recombinant polynucleotide of any one of claims 62-67.

69. The expression vector of claim 68, wherein the recombinant polynucleotide is operably linked to a control sequence.

70. The expression vector of claim 69, wherein the control sequence comprises at least a promoter.

71. A host cell comprising an expression vector of any one of claims 68-70.

72. The host cell of claim 72, wherein the host cell is prokaryotic cell or eukaryotic cell.

73. The host cell of claim 72, comprising a bacterial cell, fungal cell, insect cell, or mammalian cell.Docket No. 5244.286WO3 74. A method of producing an engineered adenylate kinase polypeptide, comprising culturing a host cell of any one of claims 71-73 under suitable culture conditions such that the engineered adenylate kinase polypeptide is produced.

75. The method of claim 74, further comprising recovering the adenylate kinase polypeptide from the culture and / or host cells.

76. The method of claim 74 or 75, further comprising purifying the adenylate kinase polypeptide.

77. A composition comprising an engineered adenylate kinase of any one of claims 1-61.

78. The composition of claim 77, further comprising a nucleoside monophosphate substrate.

79. The composition of claim 78, wherein the nucleoside is a modified nucleoside monophosphate substrate.

80. The composition of claim 79, wherein the modified nucleoside monophosphate has a modified sugar moiety, modified nucleobase, and / or modified phosphate.

81. A method of converting a nucleoside or a modified nucleoside monophosphate to the corresponding nucleoside diphosphate (NMP), comprising contacting a nucleoside monophosphate with an engineered adenylate kinase of any one of claims 1-61 in the presence of phosphate donor NTP under suitable reaction conditions to convert the nucleoside monophosphate to the corresponding nucleoside diphosphate (NMP).

82. The method of claim 81, wherein the nucleoside monophosphate comprises a nucleobase selected from adenine, cytosine, guanine, thymine, uracil, xanthine, hypoxanthine, 2,6-diaminopurine, purine, 6,8-diaminopurine, 5-methylcytosine (m5C), 2-thiouridine, pseudouridine, dihydrouridine, inosine, and 7-methylguanosine (m7G).

83. The method of claim 81, wherein the nucleoside monophosphate has at the 2'-position of the sugar moiety a H or OH.

84. The method of any one of claims 81-83, wherein the nucleoside monophosphate is AMP, GMP, UMP, CMP, or TMP, and wherein the nucleoside monophosphate has at the 2'-position of the sugar moiety an OH, thereby by resulting in corresponding product rADP, rGDP, rlJDP, rCDP, or rTDP, respectively.Docket No. 5244.286WO3 85. The method of any one of claims 81-83, wherein the nucleoside monophosphate is AMP, GMP, UMP, CMP, or TMP, and wherein the nucleoside monophosphate has at the 2'-position of the sugar moiety an H, thereby by resulting in corresponding product dADP, dGDP, dUDP, dCDP, or dTDP, respectively.

86. The method of claim 81, wherein the nucleoside monophosphate is a modified nucleoside, wherein the modified nucleoside monophosphate has a modified sugar moiety, modified nucleobase, and / or modified phosphate.

87. The method of claim 86, wherein the modified nucleoside monophosphate comprises a modified sugar moiety.

88. The method of claim 87, wherein the modified sugar moiety is a modified 2'-position of the sugar moiety.

89. The method of claim 88, wherein the modified 2'-position 2 is a 2'-O-R', wherein in R' is alkyl, aminoalkyl, cyanoalkyl, -C(O)-alkyl, or 2'-halo.

90. The method of claim 88, wherein the modified 2'-position is 2'-O-a Ikyl is 2'-O-CH3, 2'-O-CH2CH3, 2'-0-(2-methoxyethyl), 2'-O-allyl, 2'-0-propargyl, 2'-0-aminoethyl, 2'-0-cyanoethyl, or2'-0-acetalester.

91. The method of claim 89, wherein the 2'-position is 2'-halo.

92. The method of claim 91, wherein the 2'-position is 2'-F, 2'-CL, or 2'-Br.

93. The method of any one of claims 81-87, wherein the modified sugar moiety is modified at the 3'-position.

94. The method of claim 93, wherein the 3'-position of the sugar moiety comprises a blocking group, wherein the blocking group is formate, benzoylformate, acetate, propionate, isobutyrate, aminoxy (-ONH2), O-methyl, O-methoxymethyl, O-methylthiomethyl, O-benzyloxymethyl, O-allyl, 3'-0-(2-nitrobenzyl), O-azidomethyl (O-CH2N3), O-tert-butyldithiomethyl, phosphate, diphosphate, or triphosphate.

95. The method of any one of claims 81-94, wherein the modified nucleoside monophosphate comprises a modified nucleobase.

96. The method of claim 95, wherein the modified nucleobase is selected from 5-bromo-uracil, 5-iodo-uracil, 6-mCEPh-purine, 6-phenylpyrrolocytidine, N2-alkyl 8-oxoguanosine,Docket No. 5244.286WO3 difluorotoluene, difluorobenzene, dichlorobenzene, imidazole, and benzimidazole.

97. The method of any one of claims 81-96, wherein the modified nucleoside monophosphate comprises a modified phosphate.

98. The method of claim 97, wherein the modified nucleoside monophosphate comprises a nucleoside 5'-thiophosphate, thereby resulting in product nucleoside 5'-l-thiodiphosphate (NDPaS).

99. The method of claim 98, wherein product NDPaS is the (Rp)-NDPaS, (Sp)-NDPaS, or a mixture of (Rp)-NDPaS and (Sp)-NDPaS diastereomers.

100. The method of claim 99, further comprising the step of separating the (Rp)-NDPaS and (Sp)-NDPaS diastereomers.

101. The method of any one of claims 81-100, wherein the phosphate donor NTP is rATP or dATP.

102. The method of any one of claims 81-101, wherein the phosphate donor NTP has the same nucleotide structure of the nucleoside monophosphate.

103. The method of any one of claims 81-101, further comprising regenerating NTP using an NTP regenerating system.

104. The method of claim 103, wherein the NTP regenerating system comprises creatine kinase, pyruvate kinase, polyphosphate kinase, and / or acetate-kinase.

105. The method of claim 103, wherein the NTP regenerating system is acetate kinase and substrate acetyl phosphate.

106. The method of claim 103, further comprising pyruvate oxidase and substrates pyruvate and inorganic phosphate.

107. The method of claim 81, further comprising a catalase.

108. A modified nucleoside-5'-0-thiotriphosphate comprising a modified nucleobase produced by an engineered adenylate kinase of any one of claims 1-61.

109. A diastereomeric mixture of nucleotides produced by an engineered adenylate kinase of any one of claims 1-61.

110. An NTP with a phosphate group at the 3' position of the sugar (NQP) produced by anDocket No. 5244.286WO3 engineered adenylate kinase of any one of claims 1-61.

111. The NQP of claim 110, further comprising a modification of the nucleoside, NTP and / or NQP at the 2' position of the sugar.

112. The NQP of any one of claims 110-111 further comprising a modification of the nucleoside, NTP and / or NQP at the nucleobase comprising a noncanonical nucleobase known to those skilled in the art, a removable tag, a cleavable linker, or a radio, a photo, and / or a chemical sensor; a modification of the nucleoside, NTP and / or NQP at the phosphate chain, optionally comprising an a-phosphothioate linkage or other non-phosphodiester linkage; or a combination of a modification at the nucleobase and a modification at the phosphate chain.