{"paper_id":"3fb14653-e18d-4dcf-824d-9985569027c6","body_text":"Improved nucleoside (2'-deoxy)ribosyltransferases maximize enzyme \npromiscuity while maintaining catalytic efficiency \n \nPeijun (Gary) Tanga, Greice M. Zickuhrb, Alison L. Dicksonb, \nChristopher J. Hardingc, Suneeta Devid, Tomas Leble, David J. \nHarrisonb,f, Rafael G. da Silvaa and Clarissa M. Czekster a,* \naSchool of Biology, Biomedical Sciences Research Complex, University of St Andrews, St Andrews, Fife KY16 9ST, United \nKingdom; bSchool of Medicine, University of St Andrews, North Haugh, St Andrews, KY16 9TF, UK; c Institute of Infec-\ntion, Veterinary and Ecological Sciences, University of Liverpool, L69 3BX, UK; d School of Physical Sciences, University \nof Liverpool, L69 7ZF, UK; eSchool of Chemistry and Biomedical Sciences Research Complex, University of St Andrews \nand EaStCHEM, North Haugh, St Andrews, Fife, KY16 9ST, United Kingdom; fNuCana Plc, Edinburgh, EH12 9DT, United \nKingdom \nNucleoside, enzymology, protein engineering, nucleoside 2'-deoxyribosyltransferase, extremophile \nABSTRACT: Nucleoside analogues have been extensively used to treat viral and bacterial infections \nand cancer for the past 60 years. However, their chemical synthesis is complex and often requires \nmultiple steps and a dedicated synthetic route for every new nucleoside to be produced. Wild type \nnucleoside 2′ -deoxyribosyltransferase enzymes are promising for biocatalysis. Guided by the structure \nof the enzyme from the thermophilic organism Chroococcidiopsis thermalis PCC 7203 (CtNDT) bound \nto the ribonucleoside analogue Immucillin-H, we designed mutants of CtNDT and the psychrotolerant \nBacillus psychrosaccharolyticus (BpNDT) to improve catalytic efficiency with 3 ′ -deoxynucleosides and \nribonucleosides, while maintaining nucleobase promiscuity to generate over 100 distinct nucleoside \nproducts. Enhanced catalytic efficiency towards ribonucleosides and 3 ′ -deoxyribonucleosides occurred \nvia gains in turnover rate, rather than improved substrate binding. We determined crystal structures of \ntwo engineered variants as well as kinetic parameters with different substrates, unveiling molecular \ndetails underlying their expanded substrate scope. Our rational approach generated robust enzymes and \na roadmap for reaction conditions applicable to a wide variety of substrates. \nNucleoside analogues (NAs) are used to treat \ncancer, viral and bacterial infections. 1 They are \nchallenging to produce synthetically, and \nenzymatic routes are an alternative to access \nnovel analogues.2 Merck’s biocatalytic synthesis \nof the islatravir demonstrated the feasibility of an \nin vitro  fully biocatalytic cascade for the \nsynthesis of an anti-HIV NA. 3 Moreover, \nincorporating nucleoside 2 ′ -\ndeoxyribosyltransferase (NDT) enzymes for the \nfully biocatalytic production of the NA anti-Sars-\nCov-2 drug Molnupiravir has been proposed. 4  \nNovel nucleoside and nucleotide analogues \nchemically synthesized for targeting human \ncancers and bypassing resistance to common \ntreatments have been proposed, demonstrating \nscope for future work on nucleoside \ndevelopment.\n5  Importantly, recent work \ndemonstrated the versatility of NDTs and other \nenzymes from nucleoside salvage pathways to \ngenerate new NAs.\n6-7 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 11, 2025. ; https://doi.org/10.1101/2025.03.05.641663doi: bioRxiv preprint \n\n \nFigure 1: Nucleoside and nucleobase substrate scope and possibilities for novel nucleosides using \nCtNDT or BpNDT. Left: 1) A nucleoside donor is chosen between a ribonucleoside, a 2 ′deoxy-\nribonucleoside or 3 ′deoxyribonucleoside. 2) An enzyme catalyst is chosen based on nucleoside donor \nidentity and reaction conditions since Ct NDT operates at high temperatures, while BpNDT operates at \nlow temperatures.  3) A base acceptor is also chosen. Right: Nucleosides produced with a combination \nof CtNDT or BpNDT variants. HRMS data for each product generated are on Figure S1 and Table S2.  \nEnzymes that display broad substrate scope and \nthe capacity to catalyse more than one type of \nreaction are desirable, albeit less explored. 8 Here \nwe employed two nucleoside 2 ′ -\ndeoxyribosyltransferases, the enzyme from the \nthermophilic organism Chroococcidiopsis \nthermalis PCC 7203 ( CtNDT) and the enzyme \nfrom the psychrotolerant organism Bacillus \npsychrosaccharolyticus ( BpNDT) to produce a \nseries of over 50 NAs, several of which \nunprecedented (Figure 1). Furthermore, we \nexplored the effects of reaction conditions, \nincluding pH, temperature, substrate \nconcentrations and usage of a couple enzyme in \nefforts to drive the reaction equilibrium towards \ndesired products and decrease nucleoside \nhydrolysis uncoupled from nucleoside transfer. \nWe established a workflow to optimize reaction \nconditions and determine factors affecting \nreaction yield, with implications for others \nworking in nucleoside biocatalysis. We also \ndetermined the structure of a double mutant with \nimproved catalytic efficiency towards 3\n′ -\ndeoxynucleosides and ribonucleosides, \nestablishing a path to guide future NDT \nengineering which relies on modulating the \nsterics and electrostatics surrounding a conserved \nand essential catalytic glutamate residue. \nInspired by the structures of Trypanossoma \nbrucei (TbNDT)\n9, BpNDT10 and our recent work \non  CtNDT11, we performed structure-based \nenzyme engineering to produce modified CtNDT \nenzyme variants with expanded substrate scope.  \nAccording to reports with other NDT enzymes 7, \n12, we first explored the natural promiscuity of \nCtNDT and BpNDT. CtNDT has a preference for \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 11, 2025. ; https://doi.org/10.1101/2025.03.05.641663doi: bioRxiv preprint \n\n \npurine-based 2′ -deoxynucleosides, while Bp NDT \nprefers pyrimidine-based 2 ′ -deoxynucleosides \n(Figure S1). Both enzymes can utilize a vast \narray of non-canonical nucleobases as substrates, \nwith CtNDT possessing a broader substrate \nscope. Figure 1 depicts the NAs produced here, \nemploying wild type and enzyme variants \ndiscussed below. Figure S1 and Table S2 show \nmore details on substrates tested and products \nobtained.  Importantly, wild type CtNDT can \nutilize ribonucleoside and 3\n′ -deoxynucleoside \nsubstrates, albeit with lower efficiency, \ndemonstrating less strict selection of 2\n′ -\ndeoxynucleosides, and a possible strategy to \nproduce 3 ′ -deoxynucleosides.7, 13  As proof of \nconcept, we determined cordycepin (3 ′ -\ndeoxyadenosine), a natural product currently \nemployed to treat various types of cancers, can be \nused as a substrate by CtNDT.\n14 This enabled the \ngeneration of other 3 ′ -deoxynucleoside \nderivatives using CtNDT variants. Importantly, \nbulky nucleobases such as 6-(benzyloxy)-9H-\npurine were accepted as substrates, as well as 3-\naminopyridin-2(1H)-one, which is not typically \nconsidered a nucleobase. CtNDT has an average \nsized substrate binding pocket (Figure S3), with a \nsolvent accessible volume of ~ 150 \n/i1 3, smaller \nthan Lactobacillus leichmannii  (LlNDT, ~ 170 \n/i2 3), which was also shown to accept many \ndifferent nucleobases and 2 ′ -deoxynucleoside as \nsubstrates.7, 15 A flexible loop that acts as a “lid” \nand could potentially allow bulkier substrates to \nbe used was observed in an “open” or “closed” \nconformation (Figure 2a and Figure S2). The \nsynthesis of purine nucleosides with bulkier, \nexpanded bases using traditional chemical \nsynthesis has been challenging, despite some \nshowing promise as anticancer and antiviral \ncompounds\n16. \nAn asparagine close to the 3 ′ -OH group of \nribonucleosides has been proposed as a \n“gatekeeping residue” controlling acceptance of \nribonucleoside substrates, and it is replaced by an \naspartate on other enzymes that do not utilize \nribonucleosides as substrates. CtNDT lacks this \n“gatekeeping” asparagine (N53 on TbNDT and \nD62 on Ct NDT). Recent work has exploited a \nFigure 2: Structural features of en-\nzyme variants characterized here.  \na )  O v e rl ay  o f  s tr uc tur e s  o f  w il d ty pe \nCtNDT (pdb 8PQP, purple/pink) and \nCtNDT\nY7F bound to ImmucillinH \n(teal/green). Surface colored to depict \nelectrostatic potential. Loop covering \nactive site and Gln46 occupy different \npositions in the open and closed con-\nformation shown. b) Same overlay as \ndepicted in a, rotated to display the \nnucleobase and sugar conformations. \nc) Overlay of  structures of CtNDT\nY7F \n(teal/green) and CtNDTY7F_A9S (blue) \nbound to ImmH and cordycepin, re-\nspectively). d) Details of interactions \nbetween  Ct NDT\nY7F_A9S and cordy-\ncepin. Key distances are shown, and \nresidues mutated (Tyr7, Ser9) are \nshown in dark blue. Loop covering \nthe active site depicted for reference.  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 11, 2025. ; https://doi.org/10.1101/2025.03.05.641663doi: bioRxiv preprint \n\n \nFigure 3: Kinetic characterization of enzyme variants with Ino and Ade as substrates: a) Le\nraw HPLC data used to obtain initial rates at different substrate concentrations (inset) and genera\nMichaelis-Menten plots (right). b) Summary of kinetic parameters obtained for each enzyme varia\nExperiments were conducted in trip licate and data are shown as average and spread error deriv\nfrom standard error of fits. c) Residues mutated in variants under study. On the top the over\ntetramer is shown, and in the bottom a zoomed- in image of the substrate (cordycepin, dark blue) a\nmutated residues (marine blue), depicted here from CtNDTY7F-A9S.   \ndouble mutant (residues equivalent to Y7F/D62N \nin CtNDT) in the enzyme from L. leichmannii 15  \nto improve acceptance of ribonucleoside \nsubstrates, but an improvement in product \nconversion was not observed for this mutant. A \ncomparison between the structures of CtNDT \nmutants and LhNDT and Ll NDT is shown on \nFigure S3. The mutant Ct NDT\nD62N does not \ndisplay improved kinetic parameters with \nribonucleoside substrates (Figure 3), hence a \ndifferent nucleoside selection strategy is likely \ntaking place. \nPrior work carried out the mutation of a tyrosine \nto phenylalanine in the vicinity of the sugar \nbinding pocket of NDT enzymes to improve \nribonucleoside substrate utilization.\n17 We have \npreviously shown that this tyrosine residue in \nCtNDT (Y7) does not directly interact with the \n2′ -OH group, and instead positions the catalytic \nE88 for reaction. 11 This tyrosine residue is \nconserved in NDT enzymes, and further explored \nbelow.  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 11, 2025. ; https://doi.org/10.1101/2025.03.05.641663doi: bioRxiv preprint \n\n \nFigure 4: Steps to optimize reaction conditions \nand determine kinetic parameters, while also im-\nproving reaction yield. After choosing a CtNDT \nvariant to test (depending on the nucleoside sub-\nstrate as wild type, CtNDT\nY7F and CtNDTY7F-A9S \nwere shown to possess different preferences), \nstep 1 is to determine the best pH and tempera-\nture to monitor the reaction, step 2 is to investi-\ngate whether xanthine oxidase can improve reac-\ntion yield, step 3 is to determine optimal substrate \nratio, and step 4 is to determine the optimal reac-\ntion time, if the objective is to improve yield. \nWhen time is evaluated, one must also consider \nenzyme stability in the pH/temperature range. In \nthe bottom right, optimal conditions were estab-\nlished.  \n Crucial to our engineering efforts, as well as \nfuture applications of NDTs as biocatalysts, we \nobtained crystal structures of engineered mutants \nbound to ribonucleoside analogues, providing \natomic-level detail into how changes in the \nribosyl-binding pocket accommodate these \nmodified substrates (Figure 2 and Tables S4 and \nS5). To understand the interaction between \nCtNDT and ribonucleosides, we co-crystallized \nthe variant CtNDT\nY7F, which possesses higher \ncatalytic efficiency with ribonucleoside \nsubstrates than CtNDT\nWT with the nucleoside \nanalogue Immucillin-H (ImmH). This analogue is \na purine nucleoside phosphorylase inhibitor, \nrationally designed to mimic the transition state \nof the reaction catalysed by that enzyme. \nImportantly, it is a non-hydrolysable analogue \ncontaining hydroxyl groups on positions 2 ′  and \n3′ . Figure 2a compares the structures of  \nCtNDTWT and CtNDTY7F, depicting key residues \nparticipating in the reaction and substrate \nselection. No major differences are present in the \nprotein backbone, but the wild-type enzyme \nbinds ImmH in a distorted conformation in \nrelation to the ribose ring, bringing the 5\n′ -OH \ncloser to D82, likely to avoid clashes with the 2 ′ -\nOH of ImmH (Figure 2a). A Dali search indicates \nthe closest NDT homologues in the PDB are the \nproteins from Trypanosoma cruzi (pdb 2f67\n9, \nrmsd 0.85 Å), Leishmania mexicana (6qai 18, \nrmsd 0.87 Å), and Lactobacillus helveticus  \n(1s2g19, rmsd 0.89 Å – henceforth referred to as \nLhNDT). LhNDT complex with 2 ′ -\ndeoxyadenosine allows a comparison between \nresidues interacting with base and nucleoside \nmoieties (Figure S3a). Following our interaction \nmap with ImmH, a ribonucleoside analogue, we \ndesigned mutants targeting residues surrounding \nthe 2\n′ -OH  group, aimed at further tailoring \nsubstrate selection towards modified sugars. Our \nrationale was centred in ( i) opening up the \nsubstrate binding pocket by mutating P40 and Y7 \nand ( ii) and exploring additional hydrogen \nbonding interactions to better position a 2\n′ -OH \ngroup by mutating A9 and V85. Single mutants \nCtNDT\nA9S, CtNDTV85S, CtNDTY7A, CtNDTP40A, \ndouble mutant CtNDTY7F-A9S and triple mutants \nCtNDTY7F-A9S-V85 and CtNDTY7F-A9S-P40A were \nevaluated with inosine as sugar donor and \nadenine as sugar acceptor. These mutants had \nmodest changes on K M-inosine, and the most \npronounced effects were driven by kcat (Figure 3 \nand Figure S4) Figure 2b shows the complex \nstructure of CtNDT\nY7F-A9S and Cordycepin. A \npotential additional interaction between S9 and \nthe catalytic E88 (distance 2.5 Å) is formed. This \ncould be important to compensate for the loss in \npositioning previously conferred by Y7, mutated \nto a phenylalanine in this variant. In agreement \nwith this, CtNDT\nY7F-A9S showed an improvement \non kcat/KM-inosine of eight-fold in comparison to \nwild type, and a two-fold improvement towards \nCtNDT\nY7F. Furthermore, as depicted in Figure 2, \nCtNDTY7F and CtNDTY7F-A9S ligands harbouring \na 2 ′ -OH adopt a substrate-like conformation, \nunlike what is observed in CtNDTWT bound to \nImmH, which binds the ribosyl moiety in a \ndistorted conformation.  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 11, 2025. ; https://doi.org/10.1101/2025.03.05.641663doi: bioRxiv preprint \n\n \nSince pyrimidines were not efficient substrates \nfor CtNDT, we turned to BpNDT as it was \npreviously shown to use 2'-deoxynucleoside \npyrimidine substrates.\n10, 20  In our enzymatic \nassays, analogues of 2-deoxyuridine were \nproduced. Given 5-fluoro-2 ′ -deoxyuridine \n(Floxuridin) and capecitabine are FDA approved \nto treat different cancers, new analogues are \ndesirable. We generated the mutant BpNDTY5F, \nwhich is equivalent to CtNDTY7F since \nBpNDTWT cannot utilize ribonucleosides as \nsubstrates. BpNDTY5F uses guanosine and uracil \nas substrates to produce 5-fluorouridine as a \nproduct (Figure S1, Table S2). Cordycepin and \nclofarabine were not substrates for Bp NDTWT or \nBpNDTY5F, demonstrating this enzyme has a \nnarrower substrate scope than CtNDT. Both \nBpNDT21 and CtNDT22 have been previously \nimmobilized, further illustrating the potential of \nthese enzymes in nucleoside production.  \nTo demonstrate the applicability of employing \nCtNDT, CtNDTY7F-A9S and BpNDT to produce \nnovel compounds, we developed a substrate \nscope and reaction condition testing matrix as \nsummarized on Figure 1 and detailed on Figure 4. \nDepending on the enzyme variant, an optimal \nnucleoside is chosen to act as “sugar donor”, \nwhile nucleobase is varied. When sufficient \nquantities of nucleobase are available, increasing \nthe ratio nucleobase/nucleoside can increase \nreaction yields to up to 99% when considering \nthe limiting substrate. Other factors influence \nreaction yields, including pH, temperature and \nreaction times, and we hypothesize this is due to \nreaction kinetics x reaction equilibrium when \ndifferent substrates are employed. In cases where \n2′-deoxyinosine acts as nucleoside sugar donor, \nadding xanthine oxidase increases product \nformation specially in earlier time points, but \nsince uric acid is a substrate for Ct NDT with a  \nk\ncat/KM-uric_acid = 0.22 mM-1s-1 in the same range as \nobserved for guanine for example (0.33 mM-1s-1), \nover time it is consumed as a nucleobase \nsubstrate, shifting the equilibrium towards initial \nconditions.  \nApplying our condition matrix, we determined \nyield and developed a purification protocol for 2-\nfluoro-3\n′ -deoxyadenosine (or 2F-3 ′ d-Ado or 2F-\ncordycepin, compound 3'deoxy-3), and 2-\nfluoroadenosine (compound ribo-3) using both \nsubstrates at a 1:1 ratio, and producing N-(9H-\npurin-6-yl)benzamide (compound ribo-6) and 2\n′ -\ndeoxy-2-amino-adenosine (compound 2'deoxy-\n54) using a 10:1 nucleobase to nucleoside \nsubstrate ratio. Yields were 13%, 80%, 78%, and \n73% respectively. 2F-cordycepin was previously \nsynthesized with 2% overall final yield starting \nfrom adenosine and shown to be a potent anti-\ntrypanosomal compound 23. Furthermore, NDTs \ncan also act as nucleoside hydrolases in the \nabsence or under limiting concentrations of the \nnucleobase substrate in the second half reaction. \nWe evaluated nucleoside substrate hydrolysis to \nhave a variable effect on reaction yield (from no \nhydrolysis to up to 23% hydrolysis of 3'deoxy-\nadenosine when CtNDT\nY7F-A9S was employed \n(Figure S5).  \nIn summary, we engineered NDT enzymes to \nbroaden substrate scope and generate over 50 \nnucleoside analogues, several of which currently \nhave no synthetic route proposed. We established \na workflow to determine optimal reaction \nconditions and generated a mutant ( CtNDT\nY7F-\nA9S) with altered nucleoside substrate specificity \ntowards 3'-deoxynucleosides and ribonucleoside \nsubstrates.  \nASSOCIATED CONTENT  \nSupporting Information \nThe Supporting Information is available free of charge on the \nACS Publications website. Materials and methods, raw data for \nLC-MS and HPLC traces, intact mass for proteins employed here, \nsaturation curves and additional figures and tables are available.  \nAUTHOR INFORMATION \nCorresponding Author \n*Clarissa Melo Czekster – School of Biology, Biomedical \nSciences Research Complex, University of St Andrews, St \nAndrews, Fife KY16 9ST, United Kingdom; orcid \nhttps://orcid.org/0000-0002-7163-4057; Email: cmc27@st-\nandrews.ac.uk \nAuthor Contributions \nC.M. Czekster, R.G. da Silva, and D.J. Harrison conceptualized \nthe project and discussed data, discussed and proofread the article; \nT. Lebl provided support with compound characterisation; P. \nTang, A. Dickson, C.J. Harding, G.M. Zickuhr, and S. Devi \nperformed experiments, wrote and proofread the manuscript. \nFunding Sources \nPT was funded by IBioIC (IBioIC 2020-2-1) and by a University \nof St Andrews Impact grant, CMC was funded by the Wellcome \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 11, 2025. ; https://doi.org/10.1101/2025.03.05.641663doi: bioRxiv preprint \n\n \ntrust (217078/Z/19/Z). CMC and D.H. were funded by research \ngrants from NuCana plc. \n \nNotes \nD.J.H. is part-time employed by NuCana plc. A.D. is employed \nby Compass Pathways. \nACKNOWLEDGMENT  \nWe thank the mass spectrometry facility in St Andrews for \nsupport in mass spectrometry.  \nABBREVIATIONS \n2′-deoxyribosyltransferase from Chroococcidiopsis thermalis  \nPCC 7203 ( CtNDT, Uniprot K9TVX3) and Bacillus \npsychrosaccharolyticus ( BpNDT, Uniprot A0A3G5BRZ6), \nImmucillin-H (ImmH).  \nPDB Accession codes \n9EMX - CtNDTY7F-A9S bound to Cordycepin, and 9EMW - \nCtNDTY7F bound to ImmH. \nREFERENCES \n1. Egli, M.; Flavell, A.; Pyle, A. M.; Wilson, W. D.; Haq, \nS. I.; Luisi, B.; Fisher, J.; Laughton, C.; Allen, S.; Engels, J.; \nGrasby, J. A.; Neidle, S., Introduction and Overview. In Nucleic \nAcids in Chemistry and Biology , Blackburn, G. M.; Gait, M. J.; \nLoakes, D.; Williams, D. M., Eds. The Royal Society of \nChemistry: 2006; p 0. \n2. Lapponi, M. J.; Rivero, C. W.; Zinni, M. A.; Britos, C. \nN.; Trelles, J. A., New developments in nucleoside analogues \nbiosynthesis: A review. 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It is made \nThe copyright holder for this preprintthis version posted March 11, 2025. ; https://doi.org/10.1101/2025.03.05.641663doi: bioRxiv preprint \n\n \n \n9\n \nInsert Table of Contents artwork here \n \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 11, 2025. ; https://doi.org/10.1101/2025.03.05.641663doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}