NAD (Nicotinamide Adenine Dinucleotide) analogue and preparation method and application thereof

Abstract

The invention discloses an NAD (nicotinamide adenine dinucleotide) analogue as well as a preparation method and application thereof, and belongs to the technical field of preparation of nicotinamide adenine dinucleotide analogues. The NAD analogue provided by the invention is named as nicotinamide pseudouracil dinucleotide, called NPUD for short, and the NPUD is obtained by replacing an adenine part with pseudouracil on the basis of nicotinamide adenine dinucleotide. The NAD analogue NPUD provided by the invention has relatively good biological stability, can be recognized by various oxidoreductases and mutants depending on nicotinamide cofactors, further mutates the oxidoreductases, improves the activity of the oxidoreductases to the analogue, and provides a new thought and a new tool for constructing a biological orthogonal oxidation-reduction system. According to the preparation method of the NAD analogue, the substrate naturally exists in cells, the structure is stable, and the synthesis route is simple.

Description

Technical Field

[0001] The present application belongs to the technical field of preparation of nicotinamide adenine dinucleotide analogs, and in particular to an NAD analog and a preparation method and application thereof. Background Art

[0002] A coenzyme is a non-protein compound that can be covalently or tightly bound to a protein as a cofactor, or only briefly bound and used as a cosubstrate, to perform functions such as transferring electrons, atoms or groups, assisting enzymes in catalytic reactions, and acting as carriers of energy transfer. The most common coenzymes (cofactors) of oxidoreductases are: nicotinamide adenine dinucleotide NAD(P), flavin adenine dinucleotide FAD, and flavin mononucleotide FMN. Among them, the redox cofactors NAD / NADH and their phosphorylated forms NADP / NADPH are most relevant to the product formation of microbial cell factories. They have important applications in in vitro chemical synthesis and in vivo metabolic engineering. For in vitro catalytic processes, NAD has high costs, complex synthesis processes, and poor stability. For in vivo metabolic engineering: About 80% of known oxidoreductases in cells require nicotinamide adenine dinucleotide as a cofactor (Current opinion in biotechnology 2020, 66, 217-226.). That is to say, the electron transfer process mediated by the redox cofactor NAD will cause the electrons to be dispersed throughout the metabolic network, making it difficult to regulate a single NAD-dependent enzyme catalytic process without affecting other metabolic processes.

[0003] Therefore, with the development of synthetic biology, in order to reduce the production cost of biocatalysis industry and reduce the interference with intracellular metabolism, the development of NAD analogs has gradually attracted attention. Since the NAD-mediated electron transfer process is mainly achieved through the nicotinamide ring, the development of NAD analogs is mainly based on retaining the nicotinamide ring and modifying and replacing the rest. Such as benzyl nicotinamide BNA, n-butyl nicotinamide BuNA, carbamoylmethyl nicotinamide AmNA, etc. used in extracellular biocatalysis (Chemcatchem 2020, 12 (5), 1368-1375.), these analogs have stable structures and lower costs. At the same time, researchers have obtained a series of enzymes that can utilize analogs through protein modification (Chembiochem: a European journal of chemical biology 2023.). However, there are still few NAD analogs used in intracellular biocatalytic reactions, mainly limited by transmembrane transport or intracellular synthesis. Currently, the most reported NAD analogs used in cells are: nicotinamide riboside monophosphate NMN, which removes the adenine mononucleotide part from the original NAD (Nature Chemical Biology 2020, 16 (1), 87-94.); nicotinamide cytosine dinucleotide NCD, which replaces the adenine part with cytosine while retaining the NAD skeleton (J Am Chem Soc 2011, 133 (51), 20857-62.). Both can achieve intracellular self-sufficiency and have been successfully used in intracellular catalytic reactions (Journal of Energy Chemistry 2023, 79, 31-36.; Nature Communications 2022, 13 (1).), but there is still room for improvement in terms of bioorthogonality and stability.

[0004] Therefore, how to design NAD analogs with different structures is of great significance for expanding the analog library and enriching the methods of constructing bioorthogonal redox systems. Summary of the invention

[0005] In order to solve the above technical problems, the present application provides a NAD analogue and a preparation method and application thereof.

[0006] In order to achieve the above-mentioned invention object, this application provides the following technical solutions:

[0007] On the one hand, the present application provides an NAD analogue, named nicotinamide pseudouracil dinucleotide, abbreviated as NPUD, wherein the NPUD is based on nicotinamide adenine dinucleotide, with pseudouracil replacing the adenine part;

[0008] The structural formula of the NPUD is:

[0009]

[0010] In a second aspect, the present application provides a method for preparing the above-mentioned NAD analog, comprising the following steps:

[0011] A mixture containing pseudouridine triphosphate, nicotinamide mononucleotide and a catalyst is reacted to obtain nicotinamide pseudouridine dinucleotide, which is the NAD analogue.

[0012] Preferably, the method comprises the following steps:

[0013] Pseudouridine triphosphate and nicotinamide mononucleotide are mixed, the pH value is adjusted to 5-9 with NaOH, and then a catalyst is added to react to obtain nicotinamide pseudouridine dinucleotide, which is the NAD analog.

[0014] Optionally, the catalyst comprises a mutant of nicotinamide mononucleotide adenylyltransferase.

[0015] Optionally, the mutant of nicotinamide mononucleotide adenylyltransferase includes at least one of 11B4 (Y84V / Y118D), 11B4-1C1 (Y84V / Y118D / P22K / C132L / W176L), 11B4-4G3 (Y84V / Y118D / P22A / C132I / P175W / W176S), 11B4-5G4 (Y84V / Y118D / C132I / P175W / W176S), 11B4-8F10 (Y84V / Y118D / P22G / C132I / P175W / W176S), and 11B4-3G8 (Y84V / Y118D / V23Q / W176E).

[0016] Optionally, the molar equivalent ratio of the pseudouridine triphosphate to nicotinamide mononucleotide is 1.2 to 3:1.

[0017] Optionally, the molar equivalent ratio of pseudouridine triphosphate to nicotinamide mononucleotide is independently selected from any value of 1.2:1, 1.5:1, 1.7:1, 2:1, 2.2:1, 2.5:1, 2.7:1, 3:1, or any range between two of them.

[0018] Optionally, the added amount of the catalyst is 0.1 to 5 mg / mL.

[0019] Optionally, the added amount of the catalyst is independently selected from any value among 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL or any range value therebetween.

[0020] Optionally, the reaction temperature is 20-40°C.

[0021] Optionally, the reaction temperature is independently selected from any value among 20°C, 25°C, 30°C, 35°C, 40°C, or any range therebetween.

[0022] Optionally, inorganic pyrophosphatase is added after the reaction has been carried out for 2 to 10 hours.

[0023] Optionally, the inorganic pyrophosphatase is added in an amount of 0.1 to 1 mg / mL.

[0024] Optionally, the added amount of the inorganic pyrophosphatase is independently selected from any value among 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL or any range value therebetween.

[0025] In the present application, the role of adding inorganic pyrophosphatase is to promote the conversion of substrate.

[0026] In the present application, nicotinamide mononucleotide adenylyltransferase (NadD) is derived from Escherichia coli K12, UniprotPrimary accession: P0A752, and the mutation sites of its mutant 11B4 (Y84V / Y118D) are that the 84th amino acid changes from Y to V, and the 118th amino acid changes from Y to D; the mutation sites of the mutant 11B4-1C1 (Y84V / Y118D / P22K / C132L / W176L) are that the 84th amino acid changes from Y to V, the 118th amino acid changes from Y to D, the 22nd amino acid changes from P to K, and the The mutation sites of mutant 11B4-4G3 (Y84V / Y118D / P22A / C132I / P175W / W176S) are as follows: the 84th amino acid changes from Y to V, the 118th amino acid changes from Y to D, the 22nd amino acid changes from P to A, the 132nd amino acid changes from C to I, the 175th amino acid changes from P to W, and the 176th amino acid changes from W to S; the mutation sites of mutant 11B4-5G The mutation sites of mutant 11B4-8F10 (Y84V / Y118D / P22G / C132I / P175W / W176S) are as follows: the 84th amino acid changes from Y to V, the 118th amino acid changes from Y to D, the 132nd amino acid changes from C to I, the 175th amino acid changes from P to W, and the 176th amino acid changes from W to S; the mutation sites of mutant 11B4-8F10 (Y84V / Y118D / P22G / C132I / P175W / W176S) are as follows: the 84th amino acid changes from Y to V, the 118th amino acid changes from Y to D, the 132nd amino acid changes from C to I, the 175th amino acid changes from P to W, and the 176th amino acid changes from W to S. The mutation sites of the mutant 11B4-3G8 (Y84V / Y118D / V23Q / W176E) are as follows: the amino acid at position 84 changes from Y to V, the amino acid at position 118 changes from Y to D, the amino acid at position 22 changes from P to G, the amino acid at position 132 changes from C to I, the amino acid at position 175 changes from P to W, and the amino acid at position 176 changes from W to S; the mutation sites of the mutant 11B4-3G8 (Y84V / Y118D / V23Q / W176E) are as follows: the amino acid at position 84 changes from Y to V, the amino acid at position 118 changes from Y to D, the amino acid at position 23 changes from V to Q, and the amino acid at position 176 changes from W to S.

[0027] In a third aspect, the present application provides the use of the above-mentioned NAD analog as a coenzyme of NAD(P)-dependent oxidoreductase.

[0028] Optionally, the NAD(P)-dependent oxidoreductase includes at least one of malic enzyme, phosphite dehydrogenase, lactate dehydrogenase, formate dehydrogenase, formaldehyde dehydrogenase, and methanol dehydrogenase.

[0029] In a fourth aspect, the present application provides the use of the above-mentioned NAD analog in catalyzing the conversion of substrates into products.

[0030] Preferably, the substrate comprises at least one of malic acid, malate, phosphorous acid, phosphite, lactic acid, lactate, formic acid, formate, formaldehyde and methanol.

[0031] Compared with the prior art, this application has the following beneficial effects:

[0032] (1) The NAD analog NPUD provided in this application has good biological stability and can be recognized by a variety of nicotinamide cofactor-dependent oxidoreductases and mutants. Further mutation of the oxidoreductase can improve the activity of the enzyme towards the analog, providing new ideas and new tools for constructing a bioorthogonal redox system.

[0033] (2) The preparation method of NAD analogs provided in this application has a substrate that exists naturally in cells, has a stable structure, and a simple synthetic route. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 The TLC spot plate results of the control group without enzyme addition and the experimental group in Example 2 of the present application (Note: 0 represents the control group without adding NADH pyrophosphatase (i.e., pure NudC enzyme); 1 represents the experimental group with adding pure NudC enzyme; the spot plate results were performed in triplicate). DETAILED DESCRIPTION

[0036] The present application is further described below in conjunction with specific embodiments. The following are only a few embodiments of the present application, and are not intended to limit the present application in any form. Although the present application discloses the following preferred embodiments, they are not intended to limit the present application. Any technician familiar with the profession, without departing from the scope of the technical solution of the present application, using the above disclosed technical content to make some changes or modifications are equivalent to equivalent implementation cases and are within the scope of the technical solution.

[0037] Unless otherwise specified, the raw materials in the examples of the present application were purchased from commercial sources and used directly without any special treatment.

[0038] Unless otherwise specified, the analysis methods in the examples all adopt conventional settings and conventional analysis methods of instruments or equipment.

[0039] The inorganic pyrophosphatase PPase used in this application is derived from Escherichia coli K12, Uniprot Primary accession: P0A7A9.

[0040] The NADH pyrophosphatase NudC used in this application is derived from Escherichia coli K12, Uniprot Primary accession: P32664.

[0041] The malic enzyme ME used in the present application is derived from Escherichia coli K12, UniProt Primary accession: P26616, and the mutation site of its mutant ME-L310R is that the amino acid at position 310 changes from L to R; the mutation site of the mutant ME-L310R / Q401V is that the amino acid at position 310 changes from L to R, and the amino acid at position 401 changes from Q to V; the mutation site of the mutant ME-L310R / Q401C is that the amino acid at position 310 changes from L to R, and the amino acid at position 401 changes from Q to C; the mutation site of the mutant ME-L310R / Q401S is that the amino acid at position 310 changes from L to R, and the amino acid at position 401 changes from Q to S; the mutation site of the mutant ME-L310R / Q401G is that the amino acid at position 310 changes from L to R, and the amino acid at position 401 changes from Q to G.

[0042] The D-lactate dehydrogenase DLDH used in the present application is derived from Lactobacillus helveticus, UniProtPrimary accession: P30901, and the mutation sites of its mutant DLDH-V152R / I177K / N213I are that the 152nd amino acid changes from V to R, the 177th amino acid changes from I to K, and the 213th amino acid changes from N to I.

[0043] The phosphite dehydrogenase PDH used in the present application is derived from Ralstonia sp. strain 4506, UniProt Primary accession: G4XDR8, and the mutation site of its mutant PDH-I151R / P176E is that the 151st amino acid changes from I to R, and the 176th amino acid changes from P to E; the mutation site of the mutant PDH-I151R / P176E / M207A is that the 151st amino acid changes from I to R, the 176th amino acid changes from P to E, and the 207th amino acid changes from M to A; the mutation site of the mutant PDH-I151R / P176R / M207A is that the 151st amino acid changes from I to R, the 176th amino acid changes from P to R, and the 207th amino acid changes from M to A.

[0044] The formate dehydrogenase FDH used in the present application is derived from Pseudomonas sp.101, Uniprot Primary accession: P33160, and the mutation sites of its mutant FDH-V198I / C256I / P260S / E261P / S381N / S383F are: the 198th amino acid changes from V to I, the 256th amino acid changes from C to I, the 260th amino acid changes from P to S, the 261st amino acid changes from E to P, the 381st amino acid changes from S to N, and the 383rd amino acid changes from S to F.

[0045] The formaldehyde dehydrogenase FADH used in the present application is derived from Pseudomonas putida, Uniprot Primary accession: P46154, and the mutation sites of its mutant FADH-A192R / L223V / L236V are: the 192nd amino acid changes from A to R, the 223rd amino acid changes from L to V, and the 236th amino acid changes from L to V.

[0046] The methanol dehydrogenase MDH used in the present application is derived from Bacillus stearothermophilus, Uniprot Primary accession: P42327, and the mutation sites of its mutant MDH-Y171R / I196V / V237T / N240E / K241A are: the 171st amino acid changes from Y to R, the 196th amino acid changes from I to V, the 237th amino acid changes from V to T, the 240th amino acid changes from N to E, and the 241st amino acid changes from E to A.

[0047] The mutant dehydrogenase used in this application is obtained by The amino acid mutations were introduced using a single-site mutation kit.

[0048] The pure enzymes used in this application were expressed and purified according to the method in the literature (Protein Expression and Purification, 2007, 53, 97-103). The reagents and biological materials used in the specific examples can be obtained from commercial channels unless otherwise specified.

[0049] In the experiment of verifying the biological activity of the analogue NPUD, the method of obtaining each crude enzyme solution is as follows: the glycerol bacteria are activated and streaked on the plate overnight for protein expression, and the activated bacteria are selected and induced in a 24-well plate for 48 hours (2.5mL culture medium per well, LB culture medium plus 50μg / mL kanamycin and 0.5mM IPTG) and centrifuged at 4000g for 5min to pour off the supernatant, add 250μL of cell lysis solution, freeze at -80℃ for more than 1h to assist lysis, and then lyse at 37℃ and 200rpm for 2h. Centrifuge at 4000g for 5min to obtain the supernatant of whole cell lysate. Cell lysate composition: Tris-HCl buffer (pH 8.0) 10mM, MgCl2 1mM, lysozyme 1mg / mL, DNase I 0.1mg / mL.

[0050] Example 1

[0051] Synthesis and purification of NPUD:

[0052] The synthesis steps of NPUD are as follows: 30mM nicotinamide mononucleotide (NMN), 36mM pseudouridine triphosphate (pUTP), 10mM MgCl2, 2mg / mL11B4-4G3 (P22A / C132I / P175W / W176S) pure enzyme were added to the system, and the pH was adjusted to 8.0 with NaOH before adding the enzyme. The reaction was carried out at 37°C. During the reaction, TLC (developing agent: 95% ethanol: 1M ammonium acetate = 7:3) was used to monitor the progress of the reaction. Enzymes or substrates were added according to the results to make NMN react completely. 0.1mg / mL inorganic pyrophosphatase PPase was added in the middle and late stages of the reaction to promote the forward movement of the reaction. When the reaction was terminated, 4M HCl was added to the pH value of 2.5, and some proteins were removed by centrifugation. The supernatant was transferred to a 10KDa ultrafiltration tube for ultrafiltration to further remove proteins. Then, the system was concentrated using a vacuum concentrator at 30°C for 2h, 90% pre-cooled anhydrous ethanol was added for alcohol precipitation, the supernatant was removed by centrifugation, and the product was dried using a vacuum concentrator. The dried product was separated and purified by 50WX8 cation exchange resin and DEAE-cellulose anion exchange resin, respectively. Finally, white powder NPUD was obtained.

[0053] The nuclear magnetic resonance spectrum of the NPUD is: 1 H-NMR (D2O, 700MHz, H +form): δ9.36(s,1H),9.21(d,J=6.23Hz,1H),8.89(d,J=8.12Hz,1H),8.22(q,J=4.78Hz,1H),7.65(s,1H),6.11(d,J=5.53Hz,1H),4.68(m,1 H), 4.54 (t, J = 2.28Hz, 1H), 4.48 (t, J = 5.25Hz, 1H), 4.39 (q, J = 2.54Hz, 1H), 4.31 (m, 1H), 4.18 (m, 1H), 4.08-4.16 (m, 3H), 3.98-4.07 (m, 2H). 13 CNMR(D2O,176MHz): δ165.60,165.18,152.79,146.00,142.53,140.66,139.81,133.85, 128.56,111.35,99.84,87.11,80.71,78.48,77.53,74.12,70.75,69.60,64.84,64.59. 31 P NMR (D2O, 400MHz, H + form), δ-11.08,-11.20,-11.57,-11.70. HRMS calculated forC 20 H 26 N4O 16 P2(MH) - 639.07, found 639.07.

[0054] Example 2

[0055] Biological stability of NPUD:

[0056] The biological stability of the analogs was verified by the main NAD degrading enzymes. The main NAD degrading enzyme in the cytoplasm is NADH pyrophosphatase NudC, which acts on the pyrophosphate bond of NAD. Use NudC to verify the biological stability of the analogs. Add 50mM Tris-HCl (pH 8.0), 5mM substrate NAD or NPUD, 20mM MgCl2, and 2mg / mL of NudC pure enzyme, and incubate at 37°C for 1.0h. The degradation of the analogs was determined by TLC spot plate and grayscale analysis of the control group without enzyme and the experimental group. TLC spot plate results are shown in Figure 1 , the grayscale analysis results are shown in Table 1. The results show that under the action of pyrophosphatase NudC, the analog NPUD is more stable than NAD.

[0057] Table 1 Remaining percentage of NAD and NPUD after NudC pure enzyme treatment

[0058]

[0059] Example 3

[0060] Biological Activity of NPUD - Methanol Dehydrogenase:

[0061] Activity determination of methanol dehydrogenase MDH and mutants: prepare 100 μL of reaction system, add 50 mM HEPES (pH 7.5), 800 mM methanol, 0.4 mM MTT, 1 mM PES, 100 μM NAD or NPUD, add 10 μL of methanol dehydrogenase and its mutant pure enzyme, mix well and place in a microplate reader, measure the change of absorbance at 570 nm to reflect the enzyme activity. The results are shown in Table 2.

[0062] Table 2 Enzyme activities of methanol dehydrogenase and its mutants

[0063]

[0064] As can be seen from Table 2, when the analog NPUD was used as a cofactor, methanol dehydrogenase MDH and its mutant MDH-Y171R / I196V / V237T / N240E / K241A both showed partial activity, and the activity of the mutant with NPUD as a cofactor was higher than that with NAD as a cofactor, indicating that methanol dehydrogenase MDH and its mutant MDH-Y171R / I196V / V237T / N240E / K241A can use NPUD as a cofactor.

[0065] Example 4

[0066] Biological Activity of NPUD - Formaldehyde Dehydrogenase:

[0067] Activity of formaldehyde dehydrogenase FADH and its mutants to analogs. Prepare 100 μL of reaction system, add 50 mM HEPES (pH 7.5), 10 mM formaldehyde, 0.4 mM MTT, 1 mM PES, 100 μM NAD or NPUD, add 10 μL of methanol dehydrogenase and its mutant pure enzyme, mix well and place in a microplate reader, measure the change of absorbance at 570 nm to reflect the enzyme activity. The measured results are shown in Table 3.

[0068] Table 3 Enzyme activity of formaldehyde dehydrogenase and its mutants

[0069]

[0070] As shown in Table 3, when NPUD was used as a cofactor, partial activity of formaldehyde dehydrogenase FADH and its mutant FADH-A192R / L223V / L236V could be detected, indicating that formaldehyde dehydrogenase FADH and its mutant FADH-A192R / L223V / L236V could use NPUD as a cofactor.

[0071] Example 5

[0072] Biological Activity of NPUD - Formate Dehydrogenase:

[0073] Activity of formate dehydrogenase FDH and its mutants on analogs. Prepare 100 μL of reaction system, add 50 mM HEPES (pH 7.5), 10 mM sodium formate, 0.4 mM MTT, 1 mM PES, 100 μM NAD or NPUD, add 10 μL of formate dehydrogenase and its mutant crude enzyme solution, mix well and place in a microplate reader, measure the change of absorbance at 570 nm to reflect the crude enzyme activity. The measured results are shown in Table 4.

[0074] Table 4 Crude enzyme activity of formate dehydrogenase and its mutants

[0075]

[0076] It can be seen from Table 4 that when NPUD is used as a cofactor, partial activity can be detected for formate dehydrogenase FDH and its mutant FDH-V198I / C256I / P260S / E261P / S381N / S383F, indicating that formate dehydrogenase FDH and its mutant FDH-V198I / C256I / P260S / E261P / S381N / S383F can use NPUD as a cofactor.

[0077] Example 6

[0078] Biological Activity of NPUD - Malic Enzyme:

[0079] Activity of malate dehydrogenase ME and its mutants against analogs. Prepare a 100 μL reaction system, add 50 mM HEPES (pH 7.5), 5 mM malic acid, 10 mM MgCl2, 0.4 mM MTT, 1 mM PES, 100 μM NAD or NPUD, add 10 μL malate dehydrogenase and its mutant crude enzyme solution, mix well and place in a microplate reader, measure the change of absorbance at 570 nm to reflect the crude enzyme activity. The measured results are shown in Table 5.

[0080] Table 5 Crude enzyme activity of malic enzyme and its mutants

[0081]

[0082]

[0083] It can be seen from Table 5 that when NPUD is used as a cofactor, partial activity can be detected in malic enzyme ME and its mutants ME-L310R, ME-L310R / Q401V, ME-L310R / Q401C, ME-L310R / Q401S, and ME-L310R / Q401G, indicating that malic enzyme ME and its mutants ME-L310R, ME-L310R / Q401V, ME-L310R / Q401C, ME-L310R / Q401S, and ME-L310R / Q401G can utilize NPUD as a cofactor.

[0084] Example 7

[0085] Biological Activity of NPUD - Lactate Dehydrogenase:

[0086] Activity of lactate dehydrogenase DLDH and mutants against analogs. Prepare 100 μL of reaction system, add 50 mM HEPES (pH 7.5), 5 mM sodium D-lactate, 0.4 mM MTT, 1 mM PES, 100 μM NAD or NPUD, add 10 μL of lactate dehydrogenase and its mutant crude enzyme solution, mix well and place in a microplate reader, measure the change of absorbance at 570 nm to reflect the crude enzyme activity. The measured results are shown in Table 6.

[0087] Table 6 Crude enzyme activity of lactate dehydrogenase and its mutants

[0088]

[0089] As shown in Table 6, when NPUD was used as a cofactor, partial activity of lactate dehydrogenase DLDH and its mutant DLDH-V152R / I177K / N213I could be detected, indicating that lactate dehydrogenase DLDH and its mutant DLDH-V152R / I177K / N213I could use NPUD as a cofactor.

[0090] Example 8

[0091] Biological Activity of NPUD - Phosphite Dehydrogenase:

[0092] Activity of phosphite dehydrogenase PDH and mutants against analogs. Prepare 100 μL of reaction system, add 50 mM HEPES (pH 7.5), 5 mM phosphorous acid, 0.4 mM MTT, 1 mM PES, 100 μM NAD or NPUD, add 10 μL of crude enzyme solution of phosphite dehydrogenase and its mutants, mix well and place in a microplate reader, measure the change of absorbance at 570 nm to reflect the crude enzyme activity. The measured results are shown in Table 7.

[0093] Table 7 Enzyme activity of formaldehyde dehydrogenase and its mutants

[0094]

[0095] It can be seen from Table 7 that when NPUD is used as a cofactor, partial activity can be detected for the phosphite dehydrogenase mutants PDH-I151R / P176E, PDH-I151R / P176E / M207A, and PDH-I151R / P176R / M207A, indicating that the phosphite dehydrogenase mutants PDH-I151R / P176E, PDH-I151R / P176E / M207A, and PDH-I151R / P176R / M207A can utilize NPUD as a cofactor.

[0096] In summary, the NAD analogs synthesized by the present invention have good biological stability, a simple synthesis route, and the substrates used exist naturally in cells. They can be recognized by a variety of nicotinamide cofactor-dependent oxidoreductases and mutants. Further mutation of the oxidoreductase can improve the activity of the enzyme towards the analogs, providing new ideas and new tools for constructing a bioorthogonal redox system.

[0097] The above are only a few embodiments of the present application and do not constitute any form of limitation to the present application. Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the present application. Any technician familiar with the profession, without departing from the scope of the technical solution of the present application, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A NAD analogue, characterized in that Named as nicotinamide pseudouracil dinucleotide, abbreviated as NPUD, the NPUD is based on nicotinamide adenine dinucleotide, with pseudouracil replacing the adenine part; The structural formula of the NPUD is:

2. The method for preparing a NAD analogue according to claim 1, characterized in that: The steps include: A mixture containing pseudouridine triphosphate, nicotinamide mononucleotide and a catalyst is reacted to obtain nicotinamide pseudouridine dinucleotide, which is the NAD analogue.

3. The method for preparing a NAD analogue according to claim 2, characterized in that: The catalyst includes a mutant of nicotinamide mononucleotide adenylyltransferase.

4. The method for preparing a NAD analogue according to claim 3, characterized in that: The mutants of nicotinamide mononucleotide adenylyltransferase include at least one of 11B4 (Y84V / Y118D), 11B4-1C1 (Y84V / Y118D / P22K / C132L / W176L), 11B4-4G3 (Y84V / Y118D / P22A / C132I / P175W / W176S), 11B4-5G4 (Y84V / Y118D / C132I / P175W / W176S), 11B4-8F10 (Y84V / Y118D / P22G / C132I / P175W / W176S), and 11B4-3G8 (Y84V / Y118D / V23Q / W176E).

5. The method for preparing a NAD analogue according to claim 2, characterized in that: The molar equivalent ratio of the pseudouridine triphosphate to nicotinamide mononucleotide is 1.2 to 3:1; Preferably, the added amount of the catalyst is 0.1-5 mg / mL.

6. The method for preparing a NAD analogue according to claim 2, characterized in that: The reaction temperature is 20-40°C.

7. The method for preparing a NAD analogue according to claim 2, characterized in that: After the reaction has been going on for 2 to 10 hours, inorganic pyrophosphatase is added.

8. The method for preparing a NAD analogue according to claim 7, characterized in that: The added amount of the inorganic pyrophosphatase is 0.1-1 mg / mL.

9. Use of a NAD analogue according to claim 1 as a coenzyme of NAD(P)-dependent oxidoreductase; Preferably, the NAD(P)-dependent oxidoreductase comprises at least one of malic enzyme, phosphite dehydrogenase, lactate dehydrogenase, formate dehydrogenase, formaldehyde dehydrogenase and methanol dehydrogenase.

10. Use of a NAD analogue according to claim 1 in catalyzing the conversion of a substrate into a product; Preferably, the substrate comprises at least one of malic acid, malate, phosphorous acid, phosphite, lactic acid, lactate, formic acid, formate, formaldehyde and methanol.

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