Fluorinase variant

Abstract

There is provided a fluorinase variant thereof having at least 70% sequence identity to a sequence MSDLGX6TDDSVAQCKGLMLSICPX24VX26IX28DX30CHX33MTPX37DVVEGARYIVDLPR X52FPEGTVFATTTYPATGTX70X71RSVAX76RX78KX80AALGGARGQX90AGSGX95GX97E RAEGX103YIYIAPNNGLLTX116VIEEHGYX124EAYEVSX131TX133VIPX137X138PEPTFYSR EMVAIPSAHLAAGFPLX163X164VGRX168LX170DX172EIVRFEX179X180KX182X183X184VX186G X188X189LX191GX193X194X195X196X197DHPFGNX204WTNX208HRTDLEKAGIX219YX221TX223X 224KX226VX228DGVLX233FX235LPLX239PTFADAX246X247X248GX250PVX253YX255NSRGYLX2 62X263ARNAAX269LAYPYNLX277AGX280SVX283VTX286A (SEQ ID NO: 1), wherein X is a natural amino acid, and wherein the variant comprises one or more mutations at a site selected from the group consisting of an ion-egress site, a S-Adenosyl-L-Methionine (SAM) binding site, an ion-binding site (IBS), and a conserved site. Also disclosed are polynucleotides encoding the variants, vectors comprising the polynucleotides encoding the variants, and host cells comprising the vectors thereof. Also disclosed are Methods for producing the variants, methods of catalyzing the fluorination of a compound, uses of the fluorinase variants, and methods of treatment using the fluorinase variants thereof.

Description

[0001] FLUORINASE VARIANT

[0002] TECHNICAL FIELD

[0003] The present disclosure relates broadly to fluorinase variants.

[0004] BACKGROUND

[0005] Hydrofluorocarbons (HFCs) or organofluorines are fluorinated organic compounds that consist of one or more fluorine atoms. In recent years. The HFCs have garnered a lot of attentions due to their unique physicochemical properties, such as high thermal and chemical stability and enhanced pharmacokinetic parameters such as metabolic stability, bioavailability and membrane permeability as compared to their non-fluorinated counterparts. Presently, these compounds are widely used in various industries, including agrochemicals, electronics and pharmaceuticals. The global Fluorochemicals market size is estimated to be worth 25.9 billion USD in 2019 and is forecast to a size of 36.7 billion USD in 2030. So far, HFCs have been synthesized utilizing organic chemistry methods. However, the conventional chemical synthesis methods required precious metals, toxic and contaminating chemical reagents, and extreme conditions. This is not conducive to the sustainable development of green chemicals and the global economy.

[0006] Accordingly, there is a need to provide an alternative fluorinase that does not require conventional chemical synthesis requiring previous metals, toxic and contaminating chemical reagents, and extreme conditions. In some examples, the present disclosure aims to provide fluorinases derived from naturally occurring enzymes capable of catalyzing the fluorination of specific compounds, while also providing the versatility needed to customize them for industrial applications. The present disclosure provides fluorinase variants.

[0007] SUMMARY

[0008] In one aspect, there is provided a fluorinase variant thereof having at least 70% sequence identity to a sequence MSDLGX6TDDSVAQCKGLMLSICPX24VX26IX28DX30CHX33MTPX37DVVEGARYIVDLPR X52FPEGTVFATTTYPATGTX70X71 RSVAX76RX78KX80AALGGARGQX90AGSGX95GX97E RAEGX103YIYIAPNNGLLTX116VIEEHGYXi24EAYEVSXi3iTX133VIPXi37Xi38PEPTFYSR EMVAIPSAHLAAGFPLXl63X164VGRXl68LXl70DXl72EIVRFEXl79Xl80KXl82Xl83Xl84VXl86G Xl88Xl89LXl9lGXl93Xl94Xl95X19sXl97DHPFGNX204WTNX208HRTDLEKAGIX219YX22lTX223X 224KX226VX228DGVLX233FX235LPLX239PTFADAX246X247X248GX250PVX253YX255NSRGYLX2 62X283ARNAAX269LAYPYNLX277AGX28OSVX283VTX286A (SEQ ID NO: 1), wherein X is a natural amino acid, and wherein the variant comprises one or more mutations at a site selected from the group consisting of an ion-egress site, a S-Adenosyl-L-Methionine (SAM) binding site, an ion-binding site (IBS), and a conserved site.

[0009] In some examples, the fluorinase variant is a SAM-dependent fluorinase.

[0010] In some examples, the fluorinase variant is selective for fluorination over chlorination.

[0011] In some examples, the variant is capable of facilitating the nucleophilic displacement of L-methionine (L-met) from SAM.

[0012] In some examples, the variant comprises a p3-p4 loop that aids in the dehydrating halogen ions in the IBS.

[0013] In some examples, the variant comprises a p3-p4 loop that has an abundance of threonine (Thr), arginine (Arg), and serine (Ser) residues.

[0014] In some examples, the variant comprises a p3-p4 loop at positions 76 to 86.

[0015] In some examples, Xs is a V or T, X24 is a D or S, X26 is an N or T, X28 is an I or V, X30 is an I or V, X33 is a T or S, X37 is an F or W, X52 is a Y or F, X70 is a T or G, X71 is an A or T, X76 is an I or L, X78 is an L or I , Xso is an R or Q, X90 is an L or W, X95 is a K or A, X97 is an F or L, X103 is an A or S, Xus is an R or T, X124 is an L or I, X131 is an S or N, X133 is a D or K, X137 is an E or A, Xns is an R or E, Xws is an E or N, Xie4is a K or E, Xies is a K or Q, X170 is an E or S, X172 is a H or D, X179 is a Q or R, Xi89is a K or P, X182 is a G or A, Xws is no residue or an S, Xw4 is an A or T, Xi8e is an A or S, Xiss is an E or K, and optionally the N-terminal residue comprising MX2ANGSX7RPX10IAF (SEQ ID NO: 13), where X2 is T or A, X7 is an R or Q, Xw is a T or I. In some examples, the variant further comprises an N-terminal extension comprising MX2ANGSX7RPX10IAF (SEQ ID NO: 13).

[0016] In some examples, the variant comprises the sequence

[0017] MTANGSRRPTIAFMSDLGVTDDSVAQCKGLMLSICPDVNIIDICHTMTPFDVVEGAR

[0018] YIVDLPRYFPEGTVFATTTYPATGTTARSVAIRLKRAALGGARGQLAGSGKGFERAE GAYIYIAPNNGLLTRVIEEHGYLEAYEVSSTDVIPERPEPTFYSREMVAIPSAHLAAG FPLEKVGRKLEDHEIVRFEQKKGAVAGEALVGEVSAIDHPFGNVWTNLHRTDLEKA

[0019] GIKYGTPMKIVVDGVLPFELPLSPTFADAEGPGAPVAYLNSRGYLSVARNAASLAYP YNLNAGMSVRVTTA (SIBER_F1WSEQ ID NO: 2), or

[0020] MAANGSQRPIIAFMSDLGTTDDSVAQCKGLMLSICPSVTIVDVCHSMTPWDVVEG ARYIVDLPRFFPEGTVFATTTYPATGTGTRSVALRIKQAALGGARGQWAGSGAGLE RAEGSYIYIAPNNGLLTTVIEEHGYIEAYEVSNTKVIPAEPEPTFYSREMVAIPSAHLA

[0021] AGFPLNEVGRQLSDDEIVRFERPKASTVSGGVLSGTITNVDHPFGNLWTNIHRTDL EKAGIGYQTQLKLVLDGVLTFDLPLVPTFADAGKIGDPVIYINSRGYLALARNAAPLA YPYNLKAGISVAVTKA (SIBER_F3Nter, SEQ ID NO: 9), or at least 70% sequence identity thereto, or one or two amino acid difference thereto.

[0022] In some examples, the mutation at the ion-egress site is a mutation at a position selected from the group consisting of position 82, position 83, position 84, and / or position 85, optionally the mutation is a substitution.

[0023] In some examples, the mutation at the SAM binding site and / or the ion-binding site is a mutation at position 26 and / or position 156.

[0024] In some examples, the conserved site is at position 210, position 211 , and / or position 212.

[0025] In some examples, the variant comprises one or more mutation at a position selected from the group consisting of residues T82, D210, H211 , R85, F156, and Q26.

[0026] In some examples, the variant comprises one of the sequences selected from the group consisting of:

[0027] Name Protein Sequence

[0028] MTPFDVVEGARYIVDLPRYFPEGTVFATTTYPATGATARSVAIRL KRAALGGARGQLAGSGKGFERAEGAYIYIAPNNGLLTRVIEEHGY LEAYEVSSTDVIPERPEPTFYSREMVAIPSAHLAAGFPLEKVGRKL EDHEIVRFEQKKGAVAGEALVGEVSAIDHPFGNVWTNLHRTDLE KAGIKYGTPMKIVVDGVLPFELPLSPTFADAEGPGAPVAYLNSRG

[0029] YLSVARNAASLAYPYNLNAGMSVRVTTA (SEQ ID NO: 6)

[0030] SIBER F1D210AMTANGSRRPTIAFMSDLGVTDDSVAQCKGLMLSICPDVNIIDICHT

[0031] MTPFDVVEGARYIVDLPRYFPEGTVFATTTYPATGTTARSVAIRLK

[0032] RAALGGARGQLAGSGKGFERAEGAYIYIAPNNGLLTRVIEEHGYL EAYEVSSTDVIPERPEPTFYSREMVAIPSAHLAAGFPLEKVGRKL EDHEIVRFEQKKGAVAGEALVGEVSAIAHPFGNVWTNLHRTDLE

[0033] KAGIKYGTPMKIVVDGVLPFELPLSPTFADAEGPGAPVAYLNSRG YLSVARNAASLAYPYNLNAGMSVRVTTA (SEQ ID NO: 4)

[0034] SIBER F1"211RMTANGSRRPTIAFMSDLGVTDDSVAQCKGLMLSICPDVNIIDICHT MTPFDVVEGARYIVDLPRYFPEGTVFATTTYPATGTTARSVAIRLK RAALGGARGQLAGSGKGFERAEGAYIYIAPNNGLLTRVIEEHGYL

[0035] EAYEVSSTDVIPERPEPTFYSREMVAIPSAHLAAGFPLEKVGRKL

[0036] EDHEIVRFEQKKGAVAGEALVGEVSAIDRPFGNVWTNLHRTDLE

[0037] KAGIKYGTPMKIVVDGVLPFELPLSPTFADAEGPGAPVAYLNSRG

[0038] YLSVARNAASLAYPYNLNAGMSVRVTTA (SEQ ID NO: 5)

[0039] 2A SMTPWDVVEGARYIVDLPRFFPEGTVFATTTYPATGAGTRSVAL

[0040] RIKQAALGGARGQWAGSGAGLERAEGSYIYIAPNNGLLTTVIEEH GYIEAYEVSNTKVIPAEPEPTFYSREMVAIPSAHLAAGFPLNEVGR QLSDDEIVRFERPKASTVSGGVLSGTITNVDHPFGNLWTNIHRTD

[0041] LEKAGIGYQTQLKLVLDGVLTFDLPLVPTFADAGKIGDPVIYINSRG YLALARNAAPLAYPYNLKAGISVAVTKA (SEQ ID NO: 12) SIBER_F3N,er-H2MAANGSQRPIIAFMSDLGTTDDSVAQCKGLMLSICPSVTIVDVCH 12RSMTPWDVVEGARYIVDLPRFFPEGTVFATTTYPATGTGTRSVALR

[0042] IKQAALGGARGQWAGSGAGLERAEGSYIYIAPNNGLLTTVIEEHG YIEAYEVSNTKVIPAEPEPTFYSREMVAIPSAHLAAGFPLNEVGRQ LSDDEIVRFERPKASTVSGGVLSGTITNVDRPFGNLWTNIHRTDL EKAGIGYQTQLKLVLDGVLTFDLPLVPTFADAGKIGDPVIYINSRG YLALARNAAPLAYPYNLKAGISVAVTKA (SEQ ID NO: 10) SIBER_FIQ2GI+F1MTANGSRRPTIAFMSDLGVTDDSVAICKGLMLSICPDVNIIDICHT 56WMTPFDVVEGARYIVDLPRYFPEGTVFATTTYPATGTTARSVAIRLK

[0043] RAALGGARGQLAGSGKGFERAEGAYIYIAPNNGLLTRVIEEHGYL EAYEVSSTDVIPERPEPTWYSREMVAIPSAHLAAGFPLEKVGRKL EDHEIVRFEQKKGAVAGEALVGEVSAIDHPFGNVWTNLHRTDLE KAGIKYGTPMKIVVDGVLPFELPLSPTFADAEGPGAPVAYLNSRG YLSVARNAASLAYPYNLNAGMSVRVTTA (SEQ ID NO: 3)

[0044] SIBER_F1R®®AMTANGSRRPTIAFMSDLGVTDDSVAQCKGLM MTPFDVVEGARYIVDLPRYFPEGTVFATTTYPATGTTAASVAIRLK RAALGGARGQLAGSGKGFERAEGAYIYIAPNNGLLTRVIEEHGYL EAYEVSSTDVIPERPEPTFYSREMVAIPSAHLAAGFPLEKVGRKL EDHEIVRFEQKKGAVAGEALVGEVSAIDHPFGNVWTNLHRTDLE KAGIKYGTPMKIVVDGVLPFELPLSPTFADAEGPGAPVAYLNSRG YLSVARNAASLAYPYNLNAGMSVRVTTA (SEQ ID NO: 7)

[0045] SIBER F3Nter-D2IVIAANGSQRPIIAFMSDLGTTDDSVAQCKGLMLSICPSVTIVDVCH 11ASMTPWDVVEGARYIVDLPRFFPEGTVFATTTYPATGTGTRSVALR

[0046] IKQAALGGARGQWAGSGAGLERAEGSYIYIAPNNGLLTTVIEEHG YIEAYEVSNTKVIPAEPEPTFYSREMVAIPSAHLAAGFPLNEVGRQ LSDDEIVRFERPKASTVSGGVLSGTITNVAHPFGNLWTNIHRTDL EKAGIGYQTQLKLVLDGVLTFDLPLVPTFADAGKIGDPVIYINSRG YLALARNAAPLAYPYNLKAGISVAVTKA (SEQ ID NO: 11)

[0047] In yet another aspect, there is provided a polynucleotide encoding the variant of the present disclosure.

[0048] In yet another aspect, there is provided a vector comprising a polynucleotide encoding a variant of the present disclosure.

[0049] In yet another aspect, there is provided a host cell comprising a vector of the present disclosure. In yet another aspect, there is provided a method of producing a fluorinase variant, comprising expressing the fluorinase variant of the present disclosure in a suitable expression system.

[0050] In yet another aspect, there is provided a method of catalysing the fluorination of a compound, the method comprising the providing the fluorinase of the present disclosure.

[0051] In yet another aspect, there is provided a fluorinase variant the present disclosure for use in therapy or as medicine.

[0052] In yet another aspect, there is provided a method of treating a disease in a subject in need thereof, the method comprising administering a composition comprising a fluorinase variant of the present disclosure.

[0053] DESCRIPTION OF EMBODIMENTS

[0054] In recent years, organofluorines have garnered a lot of attention due to their unique physicochemical properties, such as high thermal and chemical stability and enhanced pharmacokinetic parameters such as metabolic stability, bioavailability and membrane permeability, when compared to their non-fluorinated counterparts. Presently, organofluorine compounds are widely used in various industries, including agrochemicals, electronics, and pharmaceutical industries. So far, organofluorines have been synthesized using organic chemistry methods, however, the use of hazardous and noxious reagents used in organofluorine synthesis has compelled researchers to hunt for safer, less toxic alternatives. In this context, fluorinase enzymes, belonging to “S- adenosyl methionine (SAM)-Dependent Hydrolases / Halogenases” superfamily, have emerged as an environmentally friendly and sustainable alternative to traditional organic chemistry methods for organofluorine synthesis. SAM-dependent fluorinases are trimeric enzymes (Fig. 1 A), with an ability to introduce fluorine atoms at the C-5' position of their natural substrate SAM, demonstrating their unique and valuable enzymatic function (Fig. 1 B). Notably, fluorinases share key structural characteristics with chlorinases and hydrolases, all members of the "SAM-Dependent Hydrolases / Halogenases" (Fig. 1C).

[0055] By identifying and characterizing additional SAM-dependent fluorinases, the inventors of the present disclosure can unlock a broader range of enzymatic tools for fluorination, enabling the synthesis of new fluorinated molecules with tailored properties. This, in turn, holds immense potential for advancing drug discovery, chemical synthesis, and various technological fields that rely on the unique properties of fluorine-containing compounds. Therefore, exemplary, non-limiting embodiments of fluorinase variants are disclosed hereinafter.

[0056] In one aspect, there is provided a fluorinase variant thereof having at least 70% sequence identity to a sequence MSDLGX6TDDSVAQCKGLMLSICPX24VX26IX28DX30CHX33MTPX37DVVEGARYIVDLPR X52FPEGTVFATTTYPATGTX7OX7I RSVAX76RX78KX8OAALGGARGQX9OAGSGX95GX97E RAEGX103YIYIAPNNGLLTX116VIEEHGYXi24EAYEVSXi3iTX133VIPXi37Xi38PEPTFYSR EMVAIPSAHLAAGFPLXi63X164VGRXi68LXi7oDXi72EIVRFEXi79Xi8oKXi82Xi83Xi84VXi86G Xl88Xl89LXl9lGXl93Xl94Xl95Xl96Xl87DHPFGNX204WrNX208HRTDLEKAGIX219YX22lTX223X 224KX226VX228DGVLX233FX235LPLX239PTFADAX246X247X248GX250PVX253YX255NSRGYLX2 62X263ARNAAX269LAYPYNLX277AGX280SVX283VTX286A (SEQ ID NO: 1), wherein X is a natural amino acid, and wherein the variant comprises one or more mutations at a site selected from the group consisting of an ion-egress site, a S-Adenosyl-L-Methionine (SAM) binding site, an ion-binding site (IBS), and a conserved site.

[0057] As used herein, a fluorinase refers to a naturally occurring enzyme capable of catalysing the fluorination of specific compounds. In some examples, the fluorinase may be a S-Adenosyl-L-Methionine (SAM) hydrolases or a SAM-dependent halogenases. As would be recognized by the skilled person in the art, SAM hydrolases or SAM-dependent halogenases may be categorized into two main groups based on their halide preferences of fluorinases or chlorinases. In some examples, the SAM-hydrolase as disclosed herein is a SAM-dependent fluorinase. Therefore, the fluorinase variant as described herein is capable of catalyzing the fluorination of a target compound.

[0058] Without wishing to be bound by theory, the inventors of the present disclosure believe that, unlike chlorinases and hydrolases, which typically catalyze single reactions, fluorinases exhibit a remarkable degree of promiscuity in their enzymatic activities. Therefore, SAM-dependent fluorinases has the ability to both fluorinate as well as chlorinate.

[0059] In some examples, the fluorinase variant is a SAM-dependent fluorinase.

[0060] In some examples, the fluorinase may be capable of both fluorination and chlorination. However, the fluorinase as described herein has enhanced selectivity for fluorination over chlorination. In some examples, the fluorinase variant is selective for fluorination over chlorination.

[0061] In some examples, the variant comprises substantially the same structure as a wild type fluorinase. In some examples, the variant is capable of facilitating the nucleophilic displacement of L-methionine (L-met) from SAM.

[0062] In some examples, the variant has substantially the same (or similar) secondary, tertiary, and / or quaternary structures as a classical fluorinase (such as FI_AScat, a known fluorinase from Streptomyces cattleya), which allows the variant to have the same (or improved) biological function as the classical fluorinase or a reference fluorinase. In some examples, the variant is capable of facilitating the nucleophilic displacement of L- methionine (L-met) from SAM that results in (1) the formation of halogenated 5'- deoxyadenosine (5 -XDA) when reacted with halides and (2) hydrolysis leading to the production of adenosine when engaged with hydroxyl groups. The general structure of the fluorinases as disclosed herein is as described in Fig. 1 and Fig. 2.

[0063] In some examples, the variant comprises a p3-p4 loop that aids in the dehydrating halogen ions in the IBS.

[0064] The term “ion-egress site” refers to a common path that F' or Cl' ions may follow to exit the ion-binding site (IBS). This ion-egress site is comprised of two loops adjacent to the IBS, specifically the p3-p4 loop and the p6-H4 loop, as shown in Figure 2 and Figure 5A. Without wishing to be bound by theory, it is believed that the separation of these two loops creates the pathway through which Cl' ions are excluded from the chlorine-bound trajectories. The inventors hypothesize that ions enter the IBS utilizing a similar route.

[0065] In some examples, the variant comprises a P3-P4 loop that has an abundance of threonine (Thr), arginine (Arg), and serine (Ser) residues.

[0066] In some examples, the variant comprises a P3-P4 loop at positions 76 to 86.

[0067] As understood by the person skilled in the art, the residue positions for p3-p4 loop are 76-86. As such, the P3-P4 loop nomenclature suggests that this loop exists between beta strand p3 (at residues 71-75) and p4 (at residues 87-91).

[0068] In some examples, the variant further comprises an N-terminal extension comprising MX2ANGSX7RPX10IAF (SEQ ID NO: 13).

[0069] In some examples, the variant may comprise X170 is an E or S, X172 is a H or D, X179 is a Q or R, X o is a K or P, X182 is a G or A, Xias is no residue or an S, Xi84 is an A or T, X e is an A or S, Xiss is an E or G, Xi89 is an A or V, X191 is a V or S, X193 is an E or T, X194 is a V or I, X195 is an S or T, X e is an A or N, X197 is an I or V, X204 is a V or L, X208 is an L or I, X219 is a K or G, X221 is a G or Q, X223 is a P or Q, X224 is an M or L, X226 is an I or L, X228 is a V or L, X233 is a P or T, X235 is an E or D, X239 is an S or V, X246 is an E or G, X247 is a G or K, X248 is a P or I, X250 is an A or D, X253 is an A or I, X255 is an L or I, X262 is an S or A, X263 is a V or L, X269 is an S or P, X277 is an N or K, X280 is an M or I, X283 is an R or A, and X288 is a T or K, and optionally further having N-terminal residue comprising MX2ANGSX7RPX10IAF, where X2 is T or A, X7 is an R or Q, X10 is a T or I (SEQ ID NO: 1).

[0070] The variants as disclosed herein are two naturally occurring fluorinases that the inventors of the present disclosure have discovered from the “SAM Hydrolases / SAM- Dependent Halogenases” superfamily. Upon discovery, the inventors set out to identify novel sequential hotspots and develop mutants of these hotspots with notable enhancement of fluorination activity / specificity. This is a result of a combination of computational modeling and experimental validation. The two naturally occurring fluroinases designated as SIBER_F1WTand SIBER_F3WTare from Streptosporangiales bacterium and Actinoplanes digitatis, respectively.

[0071] In some examples, the variant comprises the sequence

[0072] MTANGSRRPTIAFMSDLGVTDDSVAQCKGLMLSICPDVNIIDICHTMTPFDVVEGAR YIVDLPRYFPEGTVFATTTYPATGTTARSVAIRLKRAALGGARGQLAGSGKGFERAE GAYIYIAPNNGLLTRVIEEHGYLEAYEVSSTDVIPERPEPTFYSREMVAIPSAHLAAG FPLEKVGRKLEDHEIVRFEQKKGAVAGEALVGEVSAIDHPFGNVWTNLHRTDLEKA GIKYGTPMKIVVDGVLPFELPLSPTFADAEGPGAPVAYLNSRGYLSVARNAASLAYP YNLNAGMSVRVTTA (SIBER_F1w), or MAANGSQRPIIAFMSDLGTTDDSVAQCKGLMLSICPSVTIVDVCHSMTPWDVVEG ARYIVDLPRFFPEGTVFATTTYPATGTGTRSVALRIKQAALGGARGQWAGSGAGLE RAEGSYI Yl APN NGLLTTVI EEHGYI EAYEVSNTKVI PAEPEPTFYSREM VAI PSAH LA AGFPLNEVGRQLSDDEIVRFERPKASTVSGGVLSGTITNVDHPFGNLWTNIHRTDL EKAGIGYQTQLKLVLDGVLTFDLPLVPTFADAGKIGDPVIYINSRGYLALARNAAPLA YPYNLKAGISVAVTKA (SIBER_F3Nter), or a sequence having at least 70% sequence identity thereto, or one or two amino acid difference thereto.

[0073] In some examples, the variant may comprise a sequence having at least 71 %, or 72% or 73% or 74%, or 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88% ,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequences as disclosed herein.

[0074] In some examples, the variant comprises a sequence having at least 85%, 86%, 87%, 88% ,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0075] As used herein, the term “mutation” refers to alterations in amino acids (or gene sequences encoding for the amino acid sequences). Mutations may include one or more alterations known in the art. In some examples, the mutation may be a substitution, a deletion, or an insertion. In some examples, the mutation is a substitution and / or a deletion.

[0076] In some examples, the mutation is a substitution, a deletion, and / or an insertion.

[0077] In some examples, the mutation is a substitution.

[0078] Unless otherwise specified, SEQ ID NO: 1 serves as a reference point for any position number of a particular amino acid residue. For example, mutation at position 26 is a mutation at an amino acid position 26thfrom the first residue at the N-terminus of SEQ ID NO: 1.

[0079] In some examples, the mutation at the ion-egress site is a mutation at a position selected from the group consisting of position 82, position 83, position 84, and / or position 85.

[0080] In some examples, the mutation at the SAM binding site and / or the ion-binding site is a mutation at position 26 and / or position 156.

[0081] In some examples, the conserved site is at position 210, position 211 , and / or position 212.

[0082] In some examples, the variant comprises one or more mutations at a position selected from the group consisting of residues at position 82, position 210, position 211 , position 212, position 26, position 83, position 84, position 85, and position 156.

[0083] In some examples, the variant comprises one or more mutations at a position selected from the group consisting of residues T82, D210, H211 , R85, F156, and Q26.

[0084] In some examples, the variant may comprise one mutation, two mutations, three mutations, four mutations, five mutations, six mutations, seven mutations, eight mutations, nine mutations, ten mutations, or more.

[0085] In some examples, the variant may comprise a mutation such as, but is not limited to, T82A, D210A, H211R, Q26I, R85A, F156W, and the like.

[0086] In some examples, the variant comprises any one of the following sequences: Name Protein Sequence

[0087] MTPFDVVEGARYIVDLPRYFPEGTVFATTTYPATGTTARSVAIRLK RAALGGARGQLAGSGKGFERAEGAYIYIAPNNGLLTRVIEEHGYL EAYEVSSTDVIPERPEPTFYSREMVAIPSAHLAAGFPLEKVGRKL EDHEIVRFEQKKGAVAGEALVGEVSAIAHPFGNVWTNLHRTDLE KAGIKYGTPMKIVVDGVLPFELPLSPTFADAEGPGAPVAYLNSRG YLSVARNAASLAYPYNLNAGMSVRVTTA (SEQ ID NO: 4)

[0088] SIBER_F1H211RMTANGSRRPTIAFMSDLGVTDDSVAQCKGLMLSICPDVNIIDICHT MTPFDVVEGARYIVDLPRYFPEGTVFATTTYPATGTTARSVAIRLK RAALGGARGQLAGSGKGFERAEGAYIYIAPNNGLLTRVIEEHGYL EAYEVSSTDVIPERPEPTFYSREMVAIPSAHLAAGFPLEKVGRKL EDHEIVRFEQKKGAVAGEALVGEVSAIDRPFGNVWTNLHRTDLE KAGIKYGTPMKIVVDGVLPFELPLSPTFADAEGPGAPVAYLNSRG YLSVARNAASLAYPYNLNAGMSVRVTTA (SEQ ID NO: 5)

[0089] SIBER_F3N,erMAANGSQRPIIAFMSDLGTTDDSVAQCKGLMLSICPSVTIVDVCH SMTPWDVVEGARYIVDLPRFFPEGTVFATTTYPATGTGTRSVALR IKQAALGGARGQWAGSGAGLERAEGSYIYIAPNNGLLTTVIEEHG YIEAYEVSNTKVIPAEPEPTFYSREMVAIPSAHLAAGFPLNEVGRQ LSDDEIVRFERPKASTVSGGVLSGTITNVDHPFGNLWTNIHRTDL EKAGIGYQTQLKLVLDGVLTFDLPLVPTFADAGKIGDPVIYINSRG YLALARNAAPLAYPYNLKAGISVAVTKA (SEQ ID NO: 9)

[0090] SIBER_F3N,er-T8IVIAANGSQRPIIAFMSDLGTTDDSVAQCKGLMLSICPSVTIVDVCH

[0091] “ SMTPWDVVEGARYIVDLPRFFPEGTVFATTTYPATGAGTRSVAL

[0092] RIKQAALGGARGQWAGSGAGLERAEGSYIYIAPNNGLLTTVIEEH GYIEAYEVSNTKVIPAEPEPTFYSREMVAIPSAHLAAGFPLNEVGR QLSDDEIVRFERPKASTVSGGVLSGTITNVDHPFGNLWTNIHRTD LEKAGIGYQTQLKLVLDGVLTFDLPLVPTFADAGKIGDPVIYINSRG

[0093] YLALARNAAPLAYPYNLKAGISVAVTKA (SEQ ID N0:12)

[0094] SIBER F3Nter ii2MAANGSQRp||AFMSDLGTTDDSVAQCKGLMLSICPSVTIVDVCH 12RSMTPWDVVEGARYIVDLPRFFPEGTVFATTTYPATGTGTRSVALR

[0095] IKQAALGGARGQWAGSGAGLERAEGSYIYIAPNNGLLTTVIEEHG YIEAYEVSNTKVIPAEPEPTFYSREMVAIPSAHLAAGFPLNEVGRQ LSDDEIVRFERPKASTVSGGVLSGTITNVDRPFGNLWTNIHRTDL EKAGIGYQTQLKLVLDGVLTFDLPLVPTFADAGKIGDPVIYINSRG YLALARNAAPLAYPYNLKAGISVAVTKA (SEQ ID NO: 10) SIBER_FIQ2GI+F1MTANGSRRPTIAFMSDLGVTDDSVAICKGLMLSICPDVNIIDICHT 56WMTPFDVVEGARYIVDLPRYFPEGTVFATTTYPATGTTARSVAIRLK

[0096] RAALGGARGQLAGSGKGFERAEGAYIYIAPNNGLLTRVIEEHGYL EAYEVSSTDVIPERPEPTWYSREMVAIPSAHLAAGFPLEKVGRKL EDHEIVRFEQKKGAVAGEALVGEVSAIDHPFGNVWTNLHRTDLE KAGIKYGTPMKIVVDGVLPFELPLSPTFADAEGPGAPVAYLNSRG YLSVARNAASLAYPYNLNAGMSVRVTTA (SEQ ID NO: 3)

[0097] MTPFDVVEGARYIVDLPRYFPEGTVFATTTYPATGTTAASVAIRLK RAALGGARGQLAGSGKGFERAEGAYIYIAPNNGLLTRVIEEHGYL EAYEVSSTDVIPERPEPTFYSREMVAIPSAHLAAGFPLEKVGRKL EDHEIVRFEQKKGAVAGEALVGEVSAIDHPFGNVWTNLHRTDLE KAGIKYGTPMKIVVDGVLPFELPLSPTFADAEGPGAPVAYLNSRG YLSVARNAASLAYPYNLNAGMSVRVTTA (SEQ ID NO: 7)

[0098] SIBER_F3N,er-D2IVIAANGSQRPIIAFMSDLGTTDDSVAQCKGLMLSICPSVTIVDVCH

[0099] 11ASMTPWDVVEGARYIVDLPRFFPEGTVFATTTYPATGTGTRSVALR

[0100] IKQAALGGARGQWAGSGAGLERAEGSYIYIAPNNGLLTTVIEEHG

[0101] YIEAYEVSNTKVIPAEPEPTFYSREMVAIPSAHLAAGFPLNEVGRQ

[0102] LSDDEIVRFERPKASTVSGGVLSGTITNVAHPFGNLWTNIHRTDL

[0103] EKAGIGYQTQLKLVLDGVLTFDLPLVPTFADAGKIGDPVIYINSRG

[0104] YLALARNAAPLAYPYNLKAGISVAVTKA (SEQ ID NO: 11) At the same time, the significance of Ser158, a residue in the binding site of FLAScathas been extensively explored in the art using mutagenesis and protein structure determination. Interestingly, this residue is conserved in all known fluorinases and is substituted to glycine in known chlorinase and hydrolases. S158G and S158A substitution in FLAScatresulted in 8% and 38% activity compared to the native enzyme implying the role of side chain hydroxyl of S158 in fluoride ion desolvation. Therefore, in some examples, the variants as disclosed herein may further comprise a mutation at position 158 (for example S158G or S158A). In some examples, the variants may comprise S158G or S158A mutation. In some examples, the variants as disclosed herein may not comprise the mutation at position 158. In some examples, the variants may not comprise S158G or S158A mutation.

[0105] As further disclosed in the Experimental section, the variants of SIBER_F1WTas disclosed herein advantageously results in infinite-fold enhancement in selectivity for fluorination over chlorination (no chlorinase activity detected) compared to SIBER_F1WT(e.g. fluorinase comprising mutations at different residue positions (R85A, D210A, H211 R, Q26I+F156W)).

[0106] Further, four beneficial mutations D210A, H211 R, F156W+Q26I, R85A on SIBER_F1WTare found to increase the specificity to fluorination by 18.75 (0.75 / 0.04), 2.81 (0.87 / 0.31), infinite (0.22 / 0), infinite (0.01 / 0) fold, respectively.

[0107] One mutant of SIBER_F1WT(T82A) has 1.12-fold enhancement in fluorinase activity and one mutant of SIBER_F3WT(SIBER_F3Nter) has infinite-fold enhancement in fluorinase activity.

[0108] Three SI BER_F1mmutants (T82A, D210A, H211 R) were also tested in FLAMA37and SIBER_F3Nter, the outcomes of these mutations yielded comparable results, underscoring a consistent pattern across all fluorinases.

[0109] Thus, the fluorinases as disclosed herein clearly provide for enhanced selectivity for fluorination over chlorination and enhancement in fluorinase activity.

[0110] In another aspect, there is provided a polynucleotide encoding the variant as disclosed herein.

[0111] The term “polynucleotide” refers to a linear polymer whose molecule is composed of many nucleotide units, constituting a section of a nucleic acid molecule. Polynucleotides are made up of long chains of nucleotides like deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0112] In yet another aspect, there is provided a vector comprising a polynucleotide encoding a variant as described herein. As described herein, a "vector" is any molecule or composition that has the ability to carry a nucleic acid sequence into a suitable host cell where e.g., synthesis of the encoded polypeptide can take place. Typically, and preferably, a vector is a nucleic acid that has been engineered, using recombinant DNA techniques that are known in the art, to incorporate a desired nucleic acid sequence (e g., a nucleic acid of the present disclosure). Expression vectors typically contain one or more of the following components (if they are not already provided by the nucleic acid molecules): a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a leader sequence for secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element.

[0113] Vectors are typically selected to be functional in the host cell in which the vector will be used (the vector is compatible with the host cell machinery such that amplification of the gene and / or expression of the gene can occur. The vector as described herein may be an expression vector and / or a cloning vector.

[0114] In yet another aspect, there is provided a host cell comprising a vector as described herein.

[0115] The term “host cell,” as used herein, is intended to refer to a cell into which an expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.

[0116] In yet another aspect, there is provided a method of producing a fluorinase variant, comprising expressing the fluorinase variant as described herein in a suitable expression system.

[0117] In yet another aspect, there is provided a method of catalyzing the fluorination of a compound, the method comprising the providing the fluorinase as described herein.

[0118] In yet another aspect, there is provided a fluorinase variant as described herein for use in therapy or as medicine.

[0119] In yet another aspect, there is provided a method of treating or preventing a disease in a subject in need thereof comprising administering the fluorinase variant as described herein to the subject. The terms “treating", "treat" and “therapy”, and synonyms thereof refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) a medical condition, which includes but is not limited to diseases, symptoms and disorders. A medical condition also includes a body’s response to a disease or disorder, e g. inflammation. Those in need of such treatment include those already with a medical condition as well as those prone to getting the medical condition or those in whom a medical condition is to be prevented.

[0120] The term “subject” as used herein includes patients and non-patients. The term “patient” refers to individuals suffering or are likely to suffer from a medical condition, while “non-patients” refer to individuals not suffering and are likely to not suffer from the medical condition. “Non-patients” include healthy individuals, non-diseased individuals and / or an individual free from the medical condition. The term “subject” includes humans and animals. Animals may include, but is not limited to, mammals (for example nonhuman primates, canine, murine and the like), and the like. “Murine” refers to any mammal from the family Muridae and / or Leporidae, such as mouse, rat, rabbit, and the like.

[0121] The term “preventing” and / or “reducing the severity of symptoms” as used herein refers to process of delaying the onset, reducing the severity of symptoms, reducing and / or preventing weight loss, preventing death, inhibiting deterioration, inhibiting further deterioration, and / or ameliorating at least one sign or symptom of a disease.

[0122] Also disclosed are enzymes as described herein. Also disclosed are fluorinases as described herein.

[0123] Also disclosed are SAM Hydrolases / SAM-Dependent Halogenases as described herein.

[0124] Also disclosed are method as described herein. Also disclosed are products or kits comprising the fluorinases as described herein.

[0125] The term "and / or", e.g., "X and / or Y" is understood to mean either "X and Y" or "X or Y" and should be taken to provide explicit support for both meanings or for either meaning.

[0126] Further, in the description herein, the word “substantially” whenever used is understood to include, but not restricted to, "entirely" or “completely” and the like. In addition, terms such as "comprising", "comprise", and the like whenever used, are intended to be non-restricting descriptive language in that they broadly include elements / components recited after such terms, in addition to other components not explicitly recited. For example, when “comprising” is used, reference to a “one” feature is also intended to be a reference to “at least one” of that feature. Terms such as “consisting”, “consist”, and the like, may in the appropriate context, be considered as a subset of terms such as "comprising", "comprise", and the like. Therefore, in embodiments disclosed herein using the terms such as "comprising", "comprise", and the like, it will be appreciated that these embodiments provide teaching for corresponding embodiments using terms such as “consisting”, “consist”, and the like. Further, terms such as "about", "approximately" and the like whenever used, typically means a reasonable variation, for example a variation of + / - 5% of the disclosed value, or a variance of 4% of the disclosed value, or a variance of 3% of the disclosed value, a variance of 2% of the disclosed value or a variance of 1% of the disclosed value.

[0127] Furthermore, in the description herein, certain values may be disclosed in a range. The values showing the end points of a range are intended to illustrate a preferred range. Whenever a range has been described, it is intended that the range covers and teaches all possible sub-ranges as well as individual numerical values within that range. That is, the end points of a range should not be interpreted as inflexible limitations. For example, a description of a range of 1% to 5% is intended to have specifically disclosed sub-ranges 1% to 2%, 1% to 3%, 1 % to 4%, 2% to 3% etc., as well as individually, values within that range such as 1%, 2%, 3%, 4% and 5%. It is to be appreciated that the individual numerical values within the range also include integers, fractions and decimals. Furthermore, whenever a range has been described, it is also intended that the range covers and teaches values of up to 2 additional decimal places or significant figures (where appropriate) from the shown numerical end points. For example, a description of a range of 1% to 5% is intended to have specifically disclosed the ranges 1 .00% to 5.00% and also 1 .0% to 5.0% and all their intermediate values (such as 1 .01 %, 1.02% ... 4.98%, 4.99%, 5.00% and 1.1%, 1.2% ... 4.8%, 4.9%, 5.0% etc.,) spanning the ranges. The intention of the above specific disclosure is applicable to any depth / breadth of a range.

[0128] Additionally, when describing some embodiments, the disclosure may have disclosed a method and / or process as a particular sequence of steps. However, unless otherwise required, it will be appreciated that the method or process should not be limited to the particular sequence of steps disclosed. Other sequences of steps may be possible. The particular order of the steps disclosed herein should not be construed as undue limitations. Unless otherwise required, a method and / or process disclosed herein should not be limited to the steps being carried out in the order written. The sequence of steps may be varied and still remain within the scope of the disclosure. Furthermore, it will be appreciated that while the present disclosure provides embodiments having one or more of the features / characteristics discussed herein, one or more of these features / characteristics may also be disclaimed in other alternative embodiments and the present disclosure provides support for such disclaimers and these associated alternative embodiments.

[0129] DETAILED DESCRIPTION OF FIGURES

[0130] Example embodiments of the disclosure will be better understood and readily apparent to one of ordinary skill in the art from the following discussions and if applicable, in conjunction with the figures. It should be appreciated that other modifications may be made without deviating from the scope of the invention. Example embodiments are not necessarily mutually exclusive as some may be combined with one or more embodiments to form new exemplary embodiments. The example embodiments should not be construed as limiting the scope of the disclosure.

[0131] FIG. 1A shows a ribbon diagram of the biological unit of SAM Hydrolases / SAM- Dependent Halogenases" superfamily members (PDB 1 RQP).

[0132] FIG. 1B shows a diagram of the diversity of the reaction mechanisms in "SAM Hydrolases / SAM-Dependent Halogenases" superfamily.

[0133] FIG. 1C shows a ribbon diagram of identical structural fold "SAM Hydrolases / SAM- Dependent Halogenases" superfamily. The crystal-structures of fluorinase (PDB 1 RQP), chlorinase (PDB 6RYZ), hydrolase (PDB 2WR8) are superimposed. The bound SAM is shown in sticks. The IBS bound chloride ion in chlorinase is (PDB 6RYZ) is shown in sphere.

[0134] FIG. 2A, FIG. 2C, FIG. 2E and FIG. 2G shows bar graphs with maximum sequence identities between subnetwork members and known fluorinases.

[0135] FIG. 2B, FIG. 2D, FIG. 2F and FIG. 2H shows bar graphs with distribution of ionbinding site (IBS) residues among subnetwork members.

[0136] FIG. 3 shows comparative sequence alignment of newly discovered fluorinases alongside previously characterized counterparts. -strand and helical segments are shown. The residues constituting ion-binding site and ion-egress site are shown in light grey and dark grey boxes, respectively. Residue positions where mutations were are marked with a star symbol. FIG. 4 shows a table with a heatmap of HTP screening. Fold change is percentage (%) conversion measured against FLAMA37for both fluorination and chlorination.

[0137] FIG. 5A and 5B shows a bar graph with enzymatic activity. FIG. 5A shows a bar graph with the fluorinase activity of SIBER_F1m(ID 121) & SIBER_F3Nter(ID 260) against FLAMA37at 1.5 h and 24 h reaction. FIG. 5B shows a bar graph with Chlorinase activity of SIBER-FI^ (ID 121) and SIBER_F3Nter(ID 260) against FLAMA37at 1.5 h reaction.

[0138] FIG. 6 shows a bar graph with the effect of temperature on fluorinase activity for FLAMA37, SIBER_F1mand SIBER_F3Nterat 1.5 h reaction.

[0139] FIG. 7 shows genome-neighbourhood diagrams.

[0140] FIG. 8A shows a ribbon diagram of the relative location of D210 and H211 in SIBER_F1WT. The salt-bridge interaction between D210 and SAM is shown in dashed line.

[0141] FIG. 8B Ligplot+ generated 2D ligand interaction plot illustrating the type of molecular interactions between SAM and SAM-binding residues in the SIBER_F1WT. SIBER_F1WThomology model, was constructed using FLAScat(PDB 1 RQP) as the template.

[0142] FIG. 8C shows a ribbon diagram of the relative location of ion-egress site (IES) with respect to ion-binding site (IBS) in SIBER_F1WT. Loops constituting ion-egress site are shown in dark grey. The residues in ion-egress site that can help in displacement of the hydration sphere of halogen ions and the IBS S158 are shown in sticks.

[0143] Experimental Section

[0144] Material and Methods

[0145] Chemicals

[0146] All chemicals were purchased from Sigma-Aldrich and used as received unless otherwise stated. S-adenosyl-L-methionine (SAM) was purchased from Santa Cruz Biotechnology. 5’-fluoro-5’-deoxyadenosine (5’-FDA) was synthesized as previously mentioned.

[0147] Identification of novel fluorinases using sequence-based approaches

[0148] The protein sequence of fluorinase from Streptomyces cattleya, FLAScat(UniProt Q70GK9), was downloaded from the UniProt sequence database. Using this sequence, a BLAST search was performed using an E-value threshold of 10-5 against the UniProt sequence database and a set of 7074 sequences from Bacteria, Archaea, and metagenomic samples was identified. The inventors of the present disclosure employed the sequence similarity network (SSN) to identify conserved features and decipher the potential functional relationship among the computed protein sequence dataset. In SSN, protein sequences are represented as nodes in a network graph that are connected to other nodes by edges when their pairwise sequence similarity is greater than a predetermined threshold. Enzyme Function Initiative-Enzyme Similarity Tool (EFI-EST), with a permissive edge detection threshold equal to 40% sequence identity, was used to compute the SSN. SSN was visualized in Cytoscape 3.9.1 and a cut-off of 60% sequence identity was employed to remove edges and divide SSN into smaller subnetworks. For each subnetwork, a genome neighborhood diagram (GND) was generated using EFI- GNT tool. GNDs were then used to identify possible functional linkage, e.g., metabolic pathways, between the subnetwork members.

[0149] Cloning of fluorinase genes into different vectors

[0150] DNA sequences optimized for E. coli codons were synthesized by Twist Biosciences, Singapore. Subsequently, these sequences were assembled into the pET28a (+) vector using the golden gate assembly. The assembled constructs, which carry a C-terminal 6xHis tag for protein purification, were verified by sanger sequencing.

[0151] New variants construction

[0152] Single amino acid mutant expression plasmids were generated through the QuikChange site-directed mutagenesis protocol (Agilent, USA), utilizing the respective expression plasmids for the wild-type genes as the template. To replace N-terminal regions, NEBuilder HiFi DNA assembly was used to assemble the N-terminal portion of the FLAMA37with SIBER_F3Nter. Sequences of resulting constructs were confirmed through Sanger sequencing.

[0153] High throughput (HTP) growth and lysis

[0154] The plasmids harbouring the fluorinase genes were transformed into E. coli BL21(DE3) APNP strain. 84 colonies of variants, 6 colonies of FIA1 variant, and 6 colonies of negative control (pET28a) were inoculated into a 96-well plate. Individual colony was inoculated in 180 pL LB medium containing kanamycin (50 pg / mL) and cultured overnight at 37°C. 20 pL of cultures were transferred to 380 pL 2xYT medium containing 50 pg / mL kanamycin and grown until an absorbance of 0.5 at 600 nm was reached. Overexpression was induced by adding 0.2 mM isopropylthiogalactoside (IPTG) and incubation was continued at 22°C for 20 h. Cells were harvested by centrifugation (2,182 x g for 10 min at 4°C). The cell pellets were lysed with 250 pL lysis buffer (50 mM sodium phosphate buffer, pH 7.8, 10% glycerol, 0.1 % Triton-X) at room temperature for 3h at speed 6.5 on titer plate shaker (Barnstead). The lysates were obtained by centrifugation (2,182 x g for 20 min at 4°C).

[0155] Large scale fluorinase expression in E. coli and purification

[0156] The E. coli containing the plasmid was grown in 500 mL 2xYT medium with 50 pg / mL kanamycin at 37°C until an absorbance of 0.5 at 600 nm was reached. Overexpression was induced by adding 0.2 mM IPTG and incubation was continued at 22°C for 20 h. Cells were harvested by centrifugation (3,830 x g for 20 min). The cell pellet was resuspended in 20 mL binding buffer (50 mM sodium phosphate buffer, pH 7.8, 10% glycerol, 300 mM NaCI, 5 mM imidazole) and the cells were lysed using a cell disruptor (Constant Systems) at 40 kPSI. The lysate was then centrifuged, and the clarified supernatant was added to TALON metal affinity resin (Clontech). The supernatant-resin mixture was incubated at 4°C with rotation for 3 h. The His-tagged protein bound resin was washed with the binding buffer and eluted with the elution buffer (50 mM sodium phosphate buffer, pH 7.8, 10% glycerol, 300 mM NaCI, 250 mM imidazole). The elution was desalted using PD-10 desalting column (GE Healthcare) in storage buffer (50 mM sodium phosphate buffer, pH 7.8, 10% glycerol). The eluted protein was then concentrated using a 30 kDa concentrator (Merck Millipore) with centrifugation at 5000 x g. The protein concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific). The extinction coefficient was determined using the ExPAsy ProtParam tool.

[0157] Enzymatic reaction

[0158] HTP enzymatic reaction conditions: SAM (1 mM), NaF (200 mM), and fluorinase lysates (182 pL) in a final volume of 260 pL. HTP enzymatic reaction was carried out in a 96-well deepwell plate at 37°C for 4 h and reaction was stopped by quenching with 1 :1 volume of methanol. The quenched reaction mixtures were centrifuged (2,182 x g for 20 min) and 10 pL was used for HPLC-UV analysis.

[0159] All reactions using purified proteins were carried out in triplicates in 1.5 mL tubes in shaking incubator at 250 rpm. Reactions were stopped by heating the samples at 95°C for 1 min (using a PCR machine). The precipitated protein was then removed by centrifugation (20,238 x g for 2 min). 10 pL of the reaction mixture was used for HPLC- UV analysis.

[0160] Tube enzymatic reaction conditions (activity): SAM (1 mM), NaF or NaCI (200 mM), and fluorinase purified proteins (20 pM) in a final volume of 100 pL at 37°C for 1.5 h and 24h.

[0161] Tube enzymatic reaction conditions (thermal stability): SAM (1 mM), NaF or NaCI (200 mM), and fluorinase purified proteins (20 pM) in a final volume of 100 pL at 37°C for 1.5 h at either 47°C or 60°C.

[0162] Kinetic assay reaction conditions: Various concentrations of SAM, NaF (200 mM), and fluorinase purified proteins (20 pM) in a final volume of 100 pL at 37°C. Reactions were carried out at various time points. All reactions were carried out in triplicates, in 200 pL PCR tubes using the PCR machine. Reactions were stopped by heating the samples at 95°C for 1 min (using a PCR machine). The precipitated protein was then removed by centrifugation (20,238 x g for 2 min). 10 pL of the reaction mixture was used for HPLC- UV analysis. Kinetic parameters were obtained by the best-fit model of initial velocity against substrate concentrations based on Michaelis-Menten equation using GraphPad Prism 9 (GraphPad Software).

[0163] HPLC analysis

[0164] HPLC analysis was carried out using Agilent HPLC 1200 Infinity series paired with CTC Analytics HTS PAL LC Autosampler.

[0165] Mobile phase A: 0.1% formic acid in water; B: 0.1 % formic acid in methanol, Phenomenex, Kinetex® 2.6 pm Biphenyl 100 A, LC Column 150 x 4.6 mm, 0.6 mUmin flow rate, isocratic elution, 23% B for 15 min. The retention time of 5’-FDA is approximately 3.95 min. The retention time of 5’-CIDA is approximately 5.93 min.

[0166] Protein thermal shift assay

[0167] Protein thermal shift assay conditions: 0.25 pg / pL Purified proteins, 1x GloMelt dye, 20 pL total volume, SYBR Green channel, 95°C for 1 min, melting curve of 10°C - 95°C with increment of 0.1 °C / s, 95°C for 1.5 min. Protein thermal shift assay were carried out using Biorad CFX96™ Real-Time System and melting temperatures were obtained from analysis using its respective software. Features of the present disclosure

[0168] 1. Dataset Acquisition: Sequence similarity-based searches to acquire a large dataset of -7000 protein sequences, which were used to construct a sequence similarity network (SSN). This dataset served as the foundation for subsequent analyses.

[0169] 2. Network Construction: Utilizing the acquired protein sequences, a Sequence Similarity Network (SSN) was constructed. This network was further processed to identify smaller subnetworks, potentially containing functionally related sequences. This filtration process employed the classification number (EC: 2.5.1 .63) associated with the fluorinase enzyme, resulting in the identification of four distinct subnetworks.

[0170] 3. Residue Analysis: Within these four subnetworks, a sequence alignment was performed, leading to the identification of key residues constituting ion-binding sites (IBS). The study also involved an examination of the gene neighbourhoods of these sequences, providing insights into their genomic context.

[0171] 4. Putative Enzyme Selection: From the four subnetworks, a set of 23 putative fluorinases was extracted. These selections were based on the presence of IBS regions and their respective gene neighbourhoods, making them prime candidates for experimental testing.

[0172] 5. Experimental testing of putative fluorinases: The putative fluorinases were tested against well-known FLAMA37enzyme at two distinct time points 1.5 h and 24 h reaction. Additionally, their chlorination activity was also determined and compared. SIBER_F1WTshowed lower activity than FLAMA37(-0.7-fold) while SIBER_F3WTdid not show substantial fluorination activity.

[0173] 6. Experimental testing of the i) SIBER_F1 mutants: a) Selective mutations that increase the specificity of newly discovered SIBER_F1mtowards fluorination: D210A, H211R, F156W+Q26I, R85A. b) Selective mutations that increase the overall catalysis in SIBER_F1WT: T82A ii) SIBER_F3 mutant: a) SIBER_F3Nterengineered by incorporating the N-terminal sequence from FLAMA37within SIBER_F3WT, exhibited activity comparable to that of F L A37

[0174] 7. Experimental testing of residues (T82, D210, and H211) in FLAMA37and SIBER_F3Nter: The residues T82, D210, and H211 associated with SIBER_FTArmutants were similarly altered in the previously known FLAMA37enzyme, as well as in the engineered SIBER_F3Nterenzyme. The outcomes of these mutations yielded comparable results, underscoring a consistent pattern across all fluorinases. i) FLAMA37mutants: T82, D210, and H211 increase the specificity towards fluorination ii) SIBER_F3Ntermutants: T82 and D210 increase the specificity towards fluorination

[0175] Identification of novel fluorinases

[0176] In current study, the inventors of the present disclosure aimed to (1) identify new naturally occurring fluorinases (FLAs) from the SAM Hydrolases / SAM-Dependent Halogenases superfamily to facilitate the biological production of organofluorines (2) to uncover key structural features that impart fluorinases with their remarkable versatility, and (3) to engineer fluorinase mutants with enhanced fluorination activity.

[0177] Dataset Acquisition: With the goal of expanding the known FLA dataset to aid biological production of organofluorines, the inventors of the present disclosure conducted sequence similarity based search using FLAScat(UniProt Q70GK9), a known fluorinase from Streptomyces cattleya, protein sequence to scan the sequence space in entire UNIPROT database using BLAST. The obtained large dataset of 7074 sequences were used as the foundation for subsequent analyses.

[0178] Network Construction: The inventors of the present disclosure then employed this dataset to construct a sequence similarity network (SSN), which are graphical representations that help visualize the relationships and clusters of similar sequences. Further refinement of these SSNs were then subjected to further processing to extract smaller subnetworks that might contain functionally related sequences. To focus on sequences with potential fluorinase activity, the inventors of the present disclosure used fluorinase enzyme classification number (EC: 2.5.1.63) as a filter. Using this filter, the inventors of the present disclosure identified 4 distinct subnetworks (subnetwork 1 to 4), each containing at least one sequence annotated as a fluorinase. The FIG. 2 likely represents the visual representation of the four identified subnetworks and their relationships within the broader SSN.

[0179] Sequence analysis of putative fluorinases in identified four subnetworks. Creating experimental dataset:

[0180] The inventors of the present disclosure generated a multiple sequence alignment of sequences belonging to subnetwork 1 to 4 and extracted the ion-binding site (IBS) residues based on the insights gained from previously known fluorinases. Subsequently, the inventors of the present disclosure also examined the gene neighbourhoods associated with the members of subnetwork 1 to 4, providing insights into their genomic context. Based on the comprehensive analysis, which considered sequence similarity to known fluorinases, the IBS residues, and the context of neighbouring genes, the inventors of the present disclosure selected 23 sequences for the experimental testing.

[0181] Subnetwork-1 consists of 18 unique and non-redundant sequences, 14 of which have been previously identified as fluorinase sequences (Table 1). Two of these known fluorinases, namely FLAScatand FI_AMA37, already have their protein crystal structures determined. However, there are four additional members in Subnetwork-1 , namely SIBER_FT“'r, SIBER_F2“'rSIBER_F3“'r. and SIBER_F4“'r(see Table 1), which have not been characterized as fluorinases before.

[0182] The sequence analysis revealed that SIBER_FT“'rSIBER_F2v' , SIBER_F3“' , and SIBER_F4WTshare 79.8%, 68.9%, 93.7%, and 97.9% sequence identity, respectively, with previously identified fluorinases. This indicates that these newly discovered members may belong to the fluorinase family and potentially possess similar catalytic properties. Moreover, the analysis of the ion binding sites (IBS) of these newly discovered members shows that they share similarities with the IBS of the known fluorinases (as shown in Table 1). In particular, the IBS of SIBER_F1WT, SIBER_F3WT, and SIBER_F4WTare identical to that of FLAScatand FLAMA37. On the other hand, the ion binding site of SIBER_F2 is identical to the IBS found in FIACbac, FIAPbac, and FIATnor.

[0183] These findings suggest that the newly identified members in subnetwork-1 may also function as fluorinases due to their sequence similarities and the presence of similar ion binding sites. To confirm the fluorinase activity of newly identified subnetwork-1 sequences, the inventors of the present disclosure conducted experimental characterizations of SIBER_F1m, SIBER_F2WT, and SIBER_F3v. The sequence SIBER_F4WTwithin subnetwork-1 displayed exceptionally high sequence identity of 97.9% sequence identity with a previously identified fluorinase, FLAMA37. Consequently, SIBER_F4 was not subjected to experimental characterization. While among the remaining 20 sequences, 15, 4, and 1 sequence were associated with subnetwork-2, subnetwork-3, and subnetwork-4, respectively (data not shown). Notably, the sequence analysis of the present disclosure revealed the absence of the N-terminus in SIBER_F2WTand SIBER_F3“'r. Therefore, the inventors of the present disclosure engineered mutants of these two proteins, termed SIBER_F2Nter, SIBER_F3Nter(FIG. 3), by incorporating the initial 13 N-terminal residues from the well- established FLAMA37enzyme, as found in Streptomyces sp. MA37. Subsequently, these engineered mutants were also experimentally tested for fluorinase activity.

[0184] Table 1 : List of subnetwork- 1 members.

[0185] The largest subnetwork, denoted as subnetwork-2, encompasses a total of 370 distinct sequences. However, three sequences, namely Uniprot ID A0A355ACP7, A0A3N4NQI7, and A0A2G6LQ02, were excluded from analysis due to their deletion within the ion-binding site region. Among the remaining 367 sequences, a sizable portion corresponds to proteins without well-established functions. Additionally, 78 sequences were identified as SAM-dependent halogenases, and 5 sequences were classified as SAM-dependent hydrolases, as detailed in Table 2. Notably, the sequences within subnetwork-2 show pronounced variability in their ion-binding residues, as illustrated in FIG. 2D.

[0186] The third largest subnetwork has 10 sequences, out of which 6 lack definitive functional annotations, while 2 are designated as SAM-dependent hydrolases, and the remaining 2 are categorized as SAM-dependent halogenases, as detailed in Table 2. Lastly, the fourth subnetwork comprises a duo of sequences, with one sequence being uncharacterized, and the other being annotated as a SAM-dependent fluorinase (Table 2). Table 1 : Distribution of Uniport assigned annotations.

[0187] Experimental high-throughput screening

[0188] Utilizing the information derived from ion-binding (IBS) site residues, the inventors of the present disclosure meticulously identified 23 potential fluorinases for subsequent experimental validation, as delineated in Table 3. This selection encompassed representatives from each of the four previously expounded subnetworks. To discern promising fluorinase candidates, a comprehensive high-throughput screening process was executed, allowing for the pinpointing of specific fluorinases that warranted further in-depth investigation. Briefly the genes for these representative fluroinases were individually cloned into the pET28a vector, each tagged with distinct solubility tags, as illustrated in FIG. 4. Table 2: List of putative fluorinases selected for experimental testing.

[0189] The inventors of the present disclosure found that all the tested sequences from subnetwork-2, subnetwork-3, and subnetwork-4 exhibited no detectable fluorinase activity. Consequently, these latter sequences were excluded from further testing and analysis. Remarkably, it was observed that only the sequences from subnetwork-1 exhibited fluorinase activity. All SIBER_F1WTconstructs (IDs 121 , 122, 123, 228, 229, 230, 232, 233, and 236) from the subnetwork-1 demonstrated discernible fluorinase activity (Fig. 4). Interestingly, SIBER_F2WTdisplayed no protein expression during high- throughput screening, whereas SIBER_F3WTexhibited no fluorinase activity (IDs 97, 98, 99, 241, 243, 248, 249).

[0190] Notably, the homology-based 3D structure models of subnetwork-1 members revealed absence of a crucial beta-strand at the N-terminus in SIBER_F2WT, and SIBER_F3WT(FIG. 3). In response, the inventors of the present disclosure engineered a mutant variant of these proteins, termed SIBER_F2Nter, SIBER_F3Nter(FIG. 3). This involved the incorporation of the initial 13 N-terminal residues from the well-established FLAMA37enzyme, found in Streptomyces sp. MA37. The genes SIBER_F1WTand SIBER_F3UVTwere cloned into the pET28a vector, each with distinct solubility tags. Notably, owing to the absence of an N-terminus segment in SIBER_F3WT, the inventors of the present disclosure integrated the N-terminus DNA sequence from FLAMA37to engineer the SIBER_F3Ntermutant (IDs 260, 261 , 262).

[0191] The experimental details for SIBER_F1'm, SIBER_F3“"rand SIBER_F3Nterare given below. Subsequently, all the constructs underwent high-throughput screening to initially assess their fluorinase and chlorinase activity, as depicted in FIG. 4 and Table 10. All SIBER_F1WTconstructs exhibited fluorinase activity.

[0192] An intriguing observation was made with the SIBER_F3WT, where the wildtype DNA sequence, in the absence of the N-terminus segment, displayed no fluorinase activity (IDs 99, 241). Subsequent experimental testing of these engineered mutants where the N-terminus DNA sequence from FLAMA37was incorporated for fluorinase activity revealed confirmed fluorination activity in SIBER_F3Nter(IDs 260, 261 , 262) during high throughput screening. Similar to SIBER_F2WT, SIBER_F2Nterexhibited no protein expression during high-throughput screening, leading to their exclusion from further analysis. Based on these HTP screening results, the top performing variant for SIBER_F1WTand SIBER_F3Ntergenes were then selected for large scale protein purification, ID 121 for SIBER_F1Wand ID 260 for SIBER_F3Nter.

[0193] Fluorinase activity in purified proteins

[0194] The inventors of the present disclosure conducted a comparative analysis of the fluorinase activity of the purified SIBER_F1WT(ID 121) and SIBER_F3Nter(ID 260) against the well-established fluorinase FLAMA37at two time points, namely 1.5 hours and 24 hours, as depicted in FIG. 5A. SIBER_F3Ntershowed almost comparable activity to FLAMA37while SIBER_F1WTshowed lower activity than FLAMA37(~0.7-fold). To further understand these variants, kinetic assays against SAM substrate were performed as illustrated in Table 4. SIBER_F1WThas similar affinity (KM value) for SAM substrate and lower turnover (kcat value) as compared to FLAMA37. These resulted in slightly lower catalytic efficiency (lower kcat / KM value). SIBER_F3Nterhas lower affinity (higher KM value) for SAM substrate and higher turnover (kcat value) as compared to FLAMA37. These resulted in almost similar catalytic efficiency as FLMA37(kcat / KM value).

[0195] Table 3: Kinetic (kcat and KM) values for FLAMA37, SIBER_F1 (ID 121) and SIBER_F3Nter(ID 260) using SAM substrate.

[0196] Chlorinase activity in purified proteins

[0197] In a study of chlorinase activity, SIBER_F1m(ID 121) and SIBER_F3Nter(ID 260) were assessed alongside FLAMA37at a 1.5-hour reaction period (FIG. 5B). Unexpectedly, SIBER_F3Nterexhibited a significant ~1.5-fold improvement in chlorinase activity compared to FLAMA37, indicating potential enhancements in its molecular structure. Conversely, SIBER_F1WTdisplayed diminished chlorinase activity, approximately ~0.7- fold lower than FU\MA37. The reduced activity of SIBER_FT“,rhints at potential variations in its molecular composition or structural elements, resulting in a less efficient chlorinase function during the 1.5-hour reaction period. These findings underscore the nuanced interplay of molecular features in regulating chlorinase activity, and prompt further exploration into the specific factors influencing these surprising outcomes.

[0198] 4.7 Effect of temperature on fluorinase activity:

[0199] The inventors of the present disclosure additionally investigated the impact of temperature on fluorinase activity. The study encompassed three distinct temperature settings: 37°C, 47°C, and 60°C. By examining fluorinase performance across this temperature spectrum, the inventors of the present disclosure aimed to discern any temperature-dependent variations in enzymatic activity. The results obtained at these different temperatures are presented in FIG. 6, providing insights into how the fluorinase responds to variations in temperature and shedding light on its thermal sensitivity.

[0200] The investigation of the present disclosure unveiled distinct temperature preferences in the fluorinase activities of SIBER_F3Nterand SIBER_F1“,r. SIBER_F3Nterexhibited the best activity at 60°C, followed by 47°C, then 37°C, mirroring the temperature trend observed for FU\MA37. Intriguingly, SIBER_F1WTexhibited optimal activity at both 47°C and 60°C, followed by 37°C, with a notably lower efficiency compared to SIBER_F3Nterand FLAMA37at these temperatures. This suggests that SIBER_F1WTis not as thermal stable as FLAMA37and SIBER_F3Nter. Notably, the increment in temperature from 47°C to 60°C for SIBER_F1WTdid not yield substantial improvement in activity, underscoring the intricate interplay between temperature, protein stability, and enzymatic functionality. A protein thermal shift assay using GloMelt dye (Biotium) was also carried out to measure the melting temperature (Tm) of each variant (Table 5). The protein thermal shift assay revealed that SIBER_F1WTpossesses the lowest melting temperature (Tm) among the variants, at 41.5°C (Table 5). The protein's melting temperature (Tm) serves as a crucial indicator of thermostability, representing the temperature at which 50% of the protein undergoes unfolding. Hence the increment of temperature from 47°C to 60°C for SIBER_F1WTdid not show much improvement in activity. This suggests that the SIBER_F1“iris less thermostable than the other two proteins.

[0201] Table 4. Melting temperature of FU\MA37, SIBER_FTmand SIBER_F3Nterusing GloMelt

[0202] Gene neighborhood analysis of the newly identified fluorinases

[0203] The inventors of the present disclosure conducted a thorough analysis of the genomic surroundings of the recently discovered and well-defined enzymes, namely SIBER_F1WTand SIBER_F3WT, in comparison with fluorinases that were previously identified. The detailed findings and comparisons are presented in FIG. 8A and 8B.

[0204] In a prior study by Pardo et al., several distinctive characteristics of FLA gene clusters were delineated. One pivotal attribute is the presence of genes integral to the biosynthesis of fluorinated metabolites. Notably, these include genes encoding enzymes like 5-FDA phosphorylase, 5-fluoro-5-deoxy-d-ribose 1-phosphate isomerase, and 4- fluoro-l-threonine transaldolase. These enzymes collectively contribute to the formation of fluorinated compounds. Another noteworthy trait observed within these gene clusters is the inclusion of genes implicated in transport mechanisms. These genes encompass diverse functionalities such as cation:H+ antiporters, MFS transporter / permease proteins, and members of the EamA family transporter, potentially facilitating the transport of fluorometabolites across cellular membranes. Furthermore, a recurring feature of these gene clusters is the presence of genes encoding regulatory elements. These encompass DNA-binding proteins and transcriptional regulators, suggesting intricate control mechanisms governing the expression of the genes involved in fluorometabolite production. Beyond these shared attributes, there is a consistent occurrence of genes coding for proteins participating in S-adenosylmethionine (SAM) biosynthesis. These include S-adenosylmethionine synthase, which is vital in SAM production, a crucial co-substrate in numerous biochemical reactions. Additionally, genes involved in biodegradation processes, such as S-adenosyl-L-homocysteinase, are also commonly present in the genetic vicinity of previously identified fluorinase gene clusters. The inventors of the present disclosure’s investigation revealed similar genomic context for SIBER_FT“'rand SIBER_F3' / / Tas seen for previously characterized fluorinases. The examination highlighted the consistent presence of genes linked to fluorometabolite biosynthesis, transport mechanisms, regulatory functions, SAM biosynthesis, and biodegradation in the gene neighborhoods newly discovered FLAs. These findings collectively contribute to a more comprehensive understanding of the genetic underpinnings and potential roles of these enzymes within their biological contexts.

[0205] Alteration of ion specificity in SIBER_F1WTmutants / Hotspot residues predicted in SIBER_F1WT

[0206] Previously, Sun et al., established that mutations in SAM binding residues within FLAMA37can impact the fluorinase activity. Prior research, encompassing both crystal structure analysis and subsequent theoretical investigations of FLAScat, has compellingly demonstrated that a threonine residue at position 80 (denoted by * in FIG. 2), referred to as Thr80, plays a pivotal role not only in shaping the IBS but also in stabilizing fluorine ions (F-). The sequence and structure analysis of the present disclosure unveiled a common path that F- or Cl- ions may follow to exit the IBS, which the inventors of the present disclosure term the "ion-egress site". This ion-egress site is comprised of two loops adjacent to the IBS, specifically the p3-p4 loop and the p6-H4 loop, highlighted in red in both FIG. 2 and FIG. 8A. The separation of these two loops creates the pathway through which Cl- ions are excluded from the chloride-bound trajectories. The inventors of the present disclosure speculate that ions enter the IBS utilizing a similar route. Of particular interest, the p3-p4 loop contains an abundance of threonine (Thr), arginine (Arg), and serine (Ser) residues, which may aid in dehydrating the halogen ions as they enter the IBS. Building on the insights gained from the inventors of the present disclosure’s MD simulation findings, the inventors of the present disclosure identified several key residue positions that either participate in (1) SAM or Ion binding or (2) are involved in the ion-egress / are situated within the ion-egress site. The inventors of the present disclosure hypothesized that introducing mutations at these specific positions would also bring about significant changes in enzyme catalysis, potentially resulting in an increase or decrease in both fluorinase and chlorinase activity. These mutations were subsequently incorporated into the SIBER_F1WT(variant ID 121) enzyme, as outlined in Table 6. The inventors of the present disclosure’s findings reveal notable changes in both fluorinase and chlorinase activities for specific mutants. In the inventors of the present disclosure’s previous findings, FLAptaU1emerged as a distinctive fluorinase that exclusively functions in fluorination. This enzyme displayed an unconventional ion-binding site, featuring elements characteristic of both fluorinase and chlorinase enzymes. Specifically, the second ion-binding residue in FLAptaU1is a Trp resembling chlorinases, while the fourth residue is Ser as in other known fluorinases. The inventors of the present disclosure’s comparison of Cl" bound FLAptaU1bound trajectories revealed a unique trend compared to other systems (FIG. 3).

[0207] Halogenation activity in SIBER_F1WTmutants

[0208] The inventors of the present disclosure reveal notable changes in both fluorinase and chlorinase activities for specific mutants. To elucidate the impact of the F->W substitution on fluorination / chlorination specificity, the inventors of the present disclosure engineered a SIBER_F1Q26I+F156Wmutant (ID 278), where Q26 lies in the Helix-1 (FIG. 3) and interacts with F156. Drawing from the sequence comparison between FI_AptaU1and other fluorinases, an additional mutant, ID 279 (SIBER_F1H211 R), was also created. Notably, Mutant ID 278, denoted as SIBER_F1Q26I+F156W, displayed a marked reduction in both fluorinase and chlorinase activities. Intriguingly, this mutant demonstrated a mere 0.22- fold fluorination activity in comparison to the SIBER_F1WT, with no substantial chlorination activity observed (Table 6). It is imperative to highlight that F156 plays a dual role in both SAM binding and forms a crucial part of the ion-binding site (IBS). Another compelling mutant, identified as ID 279 (SIBER_F1H211R), also showcased decreased activities in both enzymatic functions, as outlined in Table 4. It is to be noted that while the previous reports establish FLptaU1as a better fluorinase than FLAMA37, in the hands of the inventors of the present disclosure it showed a limited fluorinase activity. Strikingly, the H211 R mutation resulted in a substantial decrease in chlorination activity, registering at 0.31-fold compared to the SIBER_F1WT(Table 6). Intriguingly, this mutant maintained a significant fluorination activity at 0.87-fold compared to the SIBER_F1WT. The inventors of the present disclosure note that H211 does not interact with the SAM or ion in the IBS (Fig. 8A) but lies next to D210. The inventors of the present disclosure hypothesize that H211 R mutation could affect the interactions between D210 and SAM and leading to similar effects.

[0209] To create SAM binding site mutants, the analysis of MD trajectories by the inventors of the present disclosure focused on computing specific interactions between SAM and SAM binding-site residues. We identified a set of seven residue positions within the SAM-binding site that form specific interactions with SAM, each occurring with > 60% frequency (SIBER-FI^ sequence used as the reference for residue numbering) (Fig. 8B). Among these seven positions, F50 (W50 in FI_AMA37and SIBER_F3Nter), F213, and F254 form pi-pi interactions, while N215 forms a hydrogen bond with the adenosine moiety of SAM. The methionine moiety of SAM is stabilized by R270, D21 , and D210 through salt-bridges.

[0210] Given Sun et a / .'s previous success in generating mutants that stabilize the adenosine moiety and hence resulting in altered functionality, the attention of the inventors of the present disclosure shifted to the D210 position, crucial for stabilizing the methionine group through salt-bridge interactions (FIG.8B-C). To discern the significance of D210, the inventors of the present disclosure generated a D210A mutant (ID 293), aimed at abolishing the robust salt-bridge interaction that stabilizes the methionine moiety of SAM within the binding site. Intriguingly, the inventors of the present disclosure observed decreased activity in both functions in SIBER_F1D210Amutant (ID 293). However, a more pronounced impact was observed in chlorination, with activity reduced to 0.04-fold of the SIBER_F1WT(Table 6). This suggests that the D210A mutation has a more substantial effect on chlorinase activity while maintaining a significant fluorinase activity at 0.75-fold of the SIBER_F1WT. As D210 is highly conserved in all fluorinases, this suggests that mutations at D210 can drastically change halogenation activity and some mutants can be more selective for F- ions than Cl- ions. This could result from the fact that D210 forms salt-bridge interaction with the amine group of methionine moiety in SAM (Figure 8B-C) and does not directly interact with the ion in the IBS.

[0211] To establish the location of ion-egress site and to prove the role of polar and charged residues from p3-p4 loop in this process, the inventors of the present disclosure created of ion-egress site mutants two additional mutants namely, SIBER_F1T82A(ID 294) and SIBER_F1R85A(ID 294) were created. Noteworthy, these mutations are far away from both SAM-binding site and IBS. The inventors of the present disclosure hypothesized that these mutations will significantly reduce the halogenation activity by affecting the ion dehydration process. Interestingly, the T82A mutation (ID 294), positioned within the ion-egress site, demonstrated an unexpected reverse trend, where both the fluorination and chlorination activity are significantly enhanced (Table 6). It was found that this mutant showed a notable, ~1.1-fold, activity for both these reactions. The inventors of the present disclosure speculate that T82 is not that important for the dehydration and possibly the Threonine or Serine residue in its vicinity compensate for its absence. Additionally, the extra flexibility of the p3-|34 loop due to the incorporation of alanine residue could be the reason behind the improved enzymatic activity. Another mutant (SIBER_F1R85A), also positioned within the ion-egress site, showed drastic reduction in both fluorinase and chlorinase activity (no substantial chlorination activity observed), establishing p3-p4 loop and the p6-H4 loop as the ion egress site.

[0212] In summary, all the reported mutations (Q26I+F156W, H211 R, D210A, T82A, R85A) showed up to infinite-fold enhancement in selectivity for fluorination over chlorination for SIBER_F1'jXr(Table 6).

[0213] Table 6. Predicted mutations and the screening results of fold comparison against SIBER_F1W(ID 121) [).= 0.75 - 0.99-fold, J,|= 0.25 - 0.75-fold, J J = 0 - 0.25-fold, INF = infinite], and F / CI selectivity at 1.5 h reaction. F / CI selectivity = % conversion of fluorination reaction / % conversion of chlorination reaction.

[0214] Halogenation activity of mutations (T82A, D210A, H211R) in FLAMA37and SIBER_F3Nter

[0215] To assess the generalizability of the functional effects observed in mutations within SIBER_F1WT, the inventors of the present disclosure extended the investigation to other established fluorinases. Analogous mutants were introduced not only in the well- known FLAMA37enzyme and the engineered SIBER_F3Nterenzyme. The residues associated with SIBER_F1WTmutants were similarly altered in the previously known FLAMA37enzyme, as well as in the engineered SI BER_F3Nterenzyme.

[0216] The outcomes of these mutations yielded comparable results, underscoring a consistent pattern across all fluorinases, with the exception of T82A which showed a decreased in activity in chlorination for FLAMA37and SIBER_F3Nteras opposed to slight improvement in activity for SIBER_F1UVT(Table 7). It is also interesting to note that H211 R showed a slight improvement in fluorination for 24 h reaction only for FI_AMA37(Table 8). Apart from H211R in SIBER_F3Nter, all other mutants (T82A, D210A, H211R) showed up to infinite-fold enhancement in selectivity for fluorination over chlorination for SIBER_F1“,rand SIBER_F3Nter(Table 7).

[0217] Overall, by examining multiple enzymes, gained a comprehensive understanding of the broader implications and potential universality of the observed functional changes resulting from these mutations.

[0218] Table 7: Predicted mutations and the screening results of fold comparison against the respective wild-type (WT) of FLMA37and SIBER_F3Nterand F7CT selectivity at 1.5 h reaction for both fluorination and chlorination activity. F / CI selectivity = % conversion of fluorination reaction / % conversion of chlorination reaction [INF = infinite]

[0219] Table 8: Predicted mutations and the screening results of fold comparison against respective wild-type (WT) at 24 h reaction for fluorination activity. The protein sequences of SIBER_F1m, SIBER_F3Wand their respective mutants are listed in Table 9.

[0220] Table 9: Protein sequences of SIBER_F1WTand SIBER_F3WTand their respective mutants. The mutations are marked in bold and underlined.

[0221] Table 10: High throughput combinatorial screening of N and C- terminus tag construct design for fluorinases. These constructs were screened at 400 L culture scale for fluorination and chlorination activity.

[0222] APPLICATIONS Embodiments of the fluorinase variants as disclosed herein provide variants that convey superior enhancement in fluorination activity and / or specificity. For example, the variants of SIBER_F1WTcomprising mutations at different residue positions (R85A, D210A, H211 R, Q26I+F156W) are found to have up to infinite-fold enhancement in selectivity for fluorination over chlorination (no chlorinase activity detected) compared to SIBER_F1WT. Four mutations D210A, H211 R, F156W+Q26I, R85A on SIBER_F1WTare found to increase the specificity to fluorination by 18.75 (0.75 / 0.04), 2.81 (0.87 / 0.31), infinite (0.22 / 0), infinite (0.01 / 0) fold, respectively. One mutant of SIBER_F1WT(T82A) has 1.12-fold enhancement in fluorinase activity and one mutant of SIBER_F3WT(SIBER_F3Nter) has infinite-fold enhancement in fluorinase activity. Three SIBER_F1WTmutants (T82A, D210A, H211R) are also tested in FLAMA37and SIBER_F3Nter, the outcomes of these mutations yielded comparable results, underscoring a consistent pattern across all fluorinases. Thus, the disclosure herein provides pathways and insights into the ways of improving the activity and selectivity of any natural fluorinases.

[0223] It will be appreciated by a person skilled in the art that other variations and / or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the disclosure as broadly described. For example, in the description herein, features of different exemplary embodiments may be mixed, combined, interchanged, incorporated, adopted, modified, included etc. or the like across different exemplary embodiments. The present embodiments are, therefore, to be considered in all respects to be illustrative and not restrictive.

Claims

CLAIMS1 . A fluorinase variant thereof having at least 70% sequence identity to a sequence MSDLGXSTDDSVAQCKGLMLSICPX24VX26IX28DX30CHX33MTPX37DVVEGARYIVDLPR X52FPEGTVFATTTYPATGTX7OX7I RSVAX76RX78KX8OAALGGARGQX9OAGSGX95GX97E RAEGX103YIYIAPNNGLLTX116VIEEHGYXi24EAYEVSXi3iTX133VIPXi37Xi38PEPTFYSR EMVAIPSAHLAAGFPLXl63X164VGRXl68LXl70DXl72EIVRFEXl79Xl80KXl82Xl83Xl84VXl86G Xl88Xl89LXl9lGXl93Xl84Xl95X19eXl87DHPFGNX204WrNX208HRTDLEKAGIX219YX22lTX223X224KX226VX228DGVLX233FX235LPLX239PTFADAX246X247X248GX250PVX253YX255NSRGYLX262X263ARNAAX269LAYPYNLX277AGX280SVX283VTX286A(SEQ ID NO: 1), wherein X is a natural amino acid, and wherein the variant comprises one or more mutations at a site selected from the group consisting of an ion-egress site, a S-Adenosyl-L-Methionine (SAM) binding site, an ion-binding site (IBS), and a conserved site.

2. The fluorinase variant of the preceding claim, wherein the fluorinase variant is a SAM-dependent fluorinase.

3. The fluorinase variant of any one of the preceding claims, wherein the fluorinase variant is selective for fluorination over chlorination.

4. The fluorinase variant of any one of the preceding claims, wherein the variant is capable of facilitating the nucleophilic displacement of L-methionine (L-met) from SAM.

5. The fluorinase variant of any one of the preceding claims, wherein the variant comprises a 03-04 loop that aids in the dehydrating halogen ions in the IBS.

6. The fluorinase variant of any one of the preceding claims, wherein the variant comprises a 03-04 loop that has an abundance of threonine (Thr), arginine (Arg), and serine (Ser) residues.

7. The fluorinase variant of any one of the preceding claims, wherein the variant comprises a 03-04 loop at positions 76 to 86.

8. The fluorinase variant of any one of the preceding claims, whereinX170 is an E or S, X172 is a H or D, Xi7g is a Q or R, Xwo is a K or P, X182 is a G or A, Xies is no residue or an S, Xi84 is an A or T, Xwe is an A or S, Xiss is an E or G, Xis9is an A or V, X191 is a V or S, Xi93 is an E or T, X194 is a V or I, Xi9s is an S or T, Xwe is an A orN, X197 is an I or V, X204 is a V or L, X208 is an L or I, X219 is a K or G, X221 is a G or Q, X223 is a P or Q, X224 is an M or L, X226 is an I or L, X228 is a V or L, X233 is a P or T, X235 is an E or D, X239 is an S or V, X246 is an E or G, X247 is a G or K, X248 is a P or I, X250 is an A or D, X253 is an A or I, X255 is an L or I, X262 is an S or A, X263 is a V or L, X269 is an S or P, X277 is an N or K, X280 is an M or I, X283 is an R or A, and X286 is a T or K, and optionally the N-terminal residue comprises MX2ANGSX7RPX10IAF, where X2 is T or A, X7 is an R or Q, X10 is a T or I (SEQ ID NO: 1).

9. The fluorinase variant of any one of the preceding claims, wherein the variant further comprises an N-terminal extension comprising MX2ANGSX7RPX10IAF (SEQ ID NO: 13).

10. The fluorinase variant of any one of the preceding claims, wherein the variant comprises the sequenceMTANGSRRPTIAFMSDLGVTDDSVAQCKGLMLSICPDVNIIDICHTMTPFDVVEGAR YIVDLPRYFPEGTVFATTTYPATGTTARSVAIRLKRAALGGARGQLAGSGKGFERAE GAYIYIAPNNGLLTRVIEEHGYLEAYEVSSTDVIPERPEPTFYSREMVAIPSAHLAAG FPLEKVGRKLEDHEIVRFEQKKGAVAGEALVGEVSAIDHPFGNVWTNLHRTDLEKA GIKYGTPMKIVVDGVLPFELPLSPTFADAEGPGAPVAYLNSRGYLSVARNAASLAYP YNLNAGMSVRVTTA (SIBER_F1w, SEQ ID NO: 2), or MAANGSQRPIIAFMSDLGTTDDSVAQCKGLMLSICPSVTIVDVCHSMTPWDVVEG ARYIVDLPRFFPEGTVFATTTYPATGTGTRSVALRIKQAALGGARGQWAGSGAGLE RAEGSYI Yl APN NGLLTTVI EEHGYI EAYEVSNTKVI PAEPEPTFYSREM VAI PSAH LA AGFPLNEVGRQLSDDEIVRFERPKASTVSGGVLSGTITNVDHPFGNLWTNIHRTDL EKAGIGYQTQLKLVLDGVLTFDLPLVPTFADAGKIGDPVIYINSRGYLALARNAAPLA YPYNLKAGISVAVTKA (SIBER_F3Nter, SEQ ID NO: 9), or at least 70% sequence identity thereto, or one or two amino acid difference thereto.11 . The fluorinase variant of any one of the preceding claims, wherein the mutation at the ion-egress site is a mutation at a position selected from the group consisting of position 82, position 83, position 84, and / or position 85, optionally the mutation is a substitution.

12. The fluorinase variant of any one of the preceding claims, wherein the mutation at the SAM binding site and / or the ion-binding site is a mutation at position 26 and / or position 156.

13. The fluorinase variant of any one of the preceding claims, wherein the conserved site is at position 210, position 21 1 , and / or position 212.

14. The fluorinase variant of any one of the preceding claims, wherein the variant comprises one or more mutation at a position selected from the group consisting of residues T82, D210, H211 , R85, F156, and Q26.

15. The fluorinase variant of any one of the preceding claims, wherein the variant comprises one of the sequence selected from the group consisting of:Name Protein SequenceSIBER_F1T82AMTANGSRRPTIAFMSDLGVTDDSVAQCKGLMLSICPDVNIIDICHTMTPFDVVEGARYIVDLPRYFPEGTVFATTTYPATGATARSVAIRLKRAALGGARGQLAGSGKGFERAEGAYIYIAPNNGLLTRVIEEHGYLEAYEVSSTDVIPERPEPTFYSREMVAIPSAHLAAGFPLEKVGRKLEDHEIVRFEQKKGAVAGEALVGEVSAIDHPFGNVWTNLHRTDLEKAGIKYGTPMKIVVDGVLPFELPLSPTFADAEGPGAPVAYLNSRGYLSVARNAASLAYPYNLNAGMSVRVTTA (SEQ ID NO: 6)SIBER_F1D210AMTANGSRRPTIAFMSDLGVTDDSVAQCKGLMLSICPDVNIIDICHTMTPFDVVEGARYIVDLPRYFPEGTVFATTTYPATGTTARSVAIRLKRAALGGARGQLAGSGKGFERAEGAYIYIAPNNGLLTRVIEEHGYLEAYEVSSTDVIPERPEPTFYSREMVAIPSAHLAAGFPLEKVGRKLEDHEIVRFEQKKGAVAGEALVGEVSAIAHPFGNVWTNLHRTDLEKAGIKYGTPMKIVVDGVLPFELPLSPTFADAEGPGAPVAYLNSRGYLSVARNAASLAYPYNLNAGMSVRVTTA (SEQ ID NO: 4)SIBER_F1H211RMTANGSRRPTIAFMSDLGVTDDSVAQCKGLMLSICPDVNIIDICHTMTPFDVVEGARYIVDLPRYFPEGTVFATTTYPATGTTARSVAIRLKRAALGGARGQLAGSGKGFERAEGAYIYIAPNNGLLTRVIEEHGYLEAYEVSSTDVIPERPEPTFYSREMVAIPSAHLAAGFPLEKVGRKLEDHEIVRFEQKKGAVAGEALVGEVSAIDRPFGNVWTNLHRTDLEKAGIKYGTPMKIVVDGVLPFELPLSPTFADAEGPGAPVAYLNSRGYLSVARNAASLAYPYNLNAGMSVRVTTA (SEQ ID NO: 5)SIBER_F3N,erIVIAANGSQRPIIAFMSDLGTTDDSVAQCKGLMLSICPSVTIVDVCHSMTPWDVVEGARYIVDLPRFFPEGTVFATTTYPATGTGTRSVALRIKQAALGGARGQWAGSGAGLERAEGSYIYIAPNNGLLTTVIEEHGYIEAYEVSNTKVIPAEPEPTFYSREMVAIPSAHLAAGFPLNEVGRQLSDDEIVRFERPKASTVSGGVLSGTITNVDHPFGNLWTNIHRTDLEKAGIGYQTQLKLVLDGVLTFDLPLVPTFADAGKIGDPVIYINSRGYLALARNAAPLAYPYNLKAGISVAVTKA (SEQ ID NO: 9)SIBER_F3N,er-T8MAANGSQRPIIAFMSDLGTTDDSVAQCKGLMLSICPSVTIVDVCH “ SMTPWDVVEGARYIVDLPRFFPEGTVFATTTYPATGAGTRSVALRIKQAALGGARGQWAGSGAGLERAEGSYIYIAPNNGLLTTVIEEH GYIEAYEVSNTKVIPAEPEPTFYSREMVAIPSAHLAAGFPLNEVGR QLSDDEIVRFERPKASTVSGGVLSGTITNVDHPFGNLWTNIHRTD LEKAGIGYQTQLKLVLDGVLTFDLPLVPTFADAGKIGDPVIYINSRG YLALARNAAPLAYPYNLKAGISVAVTKA (SEQ ID NO: 12)SIBER F3Nter ll2 MAANGSQRp||AFMSDLGTTDDSVAQCKGLMLSICPSVTIVDVCH12RSMTPWDVVEGARYIVDLPRFFPEGTVFATTTYPATGTGTRSVALRIKQAALGGARGQWAGSGAGLERAEGSYIYIAPNNGLLTTVIEEHG YIEAYEVSNTKVIPAEPEPTFYSREMVAIPSAHLAAGFPLNEVGRQ LSDDEIVRFERPKASTVSGGVLSGTITNVDRPFGNLWTNIHRTDL EKAGIGYQTQLKLVLDGVLTFDLPLVPTFADAGKIGDPVIYINSRG YLALARNAAPLAYPYNLKAGISVAVTKA (SEQ ID NO: 10)MTPFDVVEGARYIVDLPRYFPEGTVFATTTYPATGTTAASVAIRLK RAALGGARGQLAGSGKGFERAEGAYIYIAPNNGLLTRVIEEHGYL EAYEVSSTDVIPERPEPTFYSREMVAIPSAHLAAGFPLEKVGRKL EDHEIVRFEQKKGAVAGEALVGEVSAIDHPFGNVWTNLHRTDLE KAGIKYGTPMKIVVDGVLPFELPLSPTFADAEGPGAPVAYLNSRG YLSVARNAASLAYPYNLNAGMSVRVTTA (SEQ ID NO: 7)SIBER F3Nter-D2MAANGSQRPIIAFMSDLGTTDDSVAQCKGLMLSICPSVTIVDVCH11ASMTPWDVVEGARYIVDLPRFFPEGTVFATTTYPATGTGTRSVALRIKQAALGGARGQWAGSGAGLERAEGSYIYIAPNNGLLTTVIEEHG YIEAYEVSNTKVIPAEPEPTFYSREMVAIPSAHLAAGFPLNEVGRQ LSDDEIVRFERPKASTVSGGVLSGTITNVAHPFGNLWTNIHRTDL EKAGIGYQTQLKLVLDGVLTFDLPLVPTFADAGKIGDPVIYINSRG YLALARNAAPLAYPYNLKAGISVAVTKA (SEQ ID NO: 11)16. A polynucleotide encoding the variant of any one of the preceding claims.

17. A vector comprising a polynucleotide encoding a variant of any one of claims 1 to 15.

18. A host cell comprising a vector of the claim 17.

19. A method of producing a fluorinase variant, comprising expressing the fluorinase variant of any one of claims 1 to 15 in a suitable expression system.

20. A method of catalysing the fluorination of a compound, the method comprising the providing the fluorinase of any one of claims 1 to 15.

21. A fluorinase variant of any one of claims 1 to 15 for use in therapy or as medicine.

22. A method of treating a disease in a subject in need thereof, the method comprising administering a composition comprising a fluorinase variant of any one claims 1 to 15.

Images