Skip to main content

Advertisement

Log in

2′-Deoxyribosyltransferase from Leishmania mexicana, an efficient biocatalyst for one-pot, one-step synthesis of nucleosides from poorly soluble purine bases

  • Biotechnologically Relevant Enzymes and Proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Processes catalyzed by enzymes offer numerous advantages over chemical methods although in many occasions the stability of the biocatalysts becomes a serious concern. Traditionally, synthesis of nucleosides using poorly water-soluble purine bases, such as guanine, xanthine, or hypoxanthine, requires alkaline pH and/or high temperatures in order to solubilize the substrate. In this work, we demonstrate that the 2′-deoxyribosyltransferase from Leishmania mexicana (LmPDT) exhibits an unusually high activity and stability under alkaline conditions (pH 8–10) across a broad range of temperatures (30–70 °C) and ionic strengths (0–500 mM NaCl). Conversely, analysis of the crystal structure of LmPDT together with comparisons with hexameric, bacterial homologues revealed the importance of the relationships between the oligomeric state and the active site architecture within this family of enzymes. Moreover, molecular dynamics and docking approaches provided structural insights into the substrate-binding mode. Biochemical characterization of LmPDT identifies the enzyme as a type I NDT (PDT), exhibiting excellent activity, with specific activity values 100- and 4000-fold higher than the ones reported for other PDTs. Interestingly, LmPDT remained stable during 36 h at different pH values at 40 °C. In order to explore the potential of LmPDT as an industrial biocatalyst, enzymatic production of several natural and non-natural therapeutic nucleosides, such as vidarabine (ara A), didanosine (ddI), ddG, or 2′-fluoro-2′-deoxyguanosine, was carried out using poorly water-soluble purines. Noteworthy, this is the first time that the enzymatic synthesis of 2′-fluoro-2′-deoxyguanosine, ara G, and ara H by a 2′-deoxyribosyltransferase is reported.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Adams PD, Afonine PV, Bunkóczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW, McCoy AJ, Moriarty NW, Oeffner R, Read RJ, Richardson DC, Richardson JS, Terwilliger TC, Zwart PH (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr Sect D Biol Crystallogr 66:213–221

    Article  CAS  Google Scholar 

  • Afonine PV, Grosse-Kunstleve RW, Echols N, Headd JJ, Moriarty NW, Mustyakimov M, Terwilliger TC, Urzhumtsev A, Zwart PH, Adams PD (2012) Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr Sect D Biol Crystallogr 68:352–367

    Article  CAS  Google Scholar 

  • Anand R, Kaminski PA, Ealick SE (2004) Structures of purine 2′-deoxyribosyltransferase, substrate complexes, and the ribosylated enzyme intermediate at 2.0 Å resolution. Biochemistry 43:2384–2393

    Article  CAS  PubMed  Google Scholar 

  • Anandakrishnan R, Aguilar B, Onufriev AV (2012) H++ 3.0: automating pK prediction and the preparation of biomolecular structures for atomistic molecular modeling and simulations. Nucleic Acids Res 40:W537–W541

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Armstrong SR, Cook WJ, Short SA, Ealick SE (1996) Crystal structures of nucleoside 2′-deoxyribosyltransferase in native and ligand-bound forms reveal architecture of the active site. Structure 4:97–107

    Article  CAS  PubMed  Google Scholar 

  • Becker J, Brendel M (1996) Rapid purification and characterization of two distinct N-deoxyribosyltransferases of Lactobacillus leichmannii. Biol Chem Hoppe Seyler 377:357–362

    Article  CAS  PubMed  Google Scholar 

  • Bondoc LL, Ahluwalia G, Cooney DA, Hartman NR, Johns DG, Fridland A (1992) Metabolic pathways for the activation of the antiviral agent 2′,3′-dideoxyguanosine in human lymphoid cells. Mol Pharmacol 42:525–530

    CAS  PubMed  Google Scholar 

  • Boryski J (2008) Reactions of transglycosylation in the nucleoside chemistry. Curr Org Chem 12:309–325

    Article  CAS  Google Scholar 

  • Bosch J, Robien MA, Mehlin C, Boni E, Riechers A, Buckner FS, Van Voorhis WC, Myler PJ, Worthey EA, DeTitta G, Luft JR, Lauricella A, Gulde S, Anderson LA, Kalyuzhniy O, Neely HM, Ross J, Earnest TN, Soltis M, Schoenfeld L, Zucker F, Merritt EA, Fan E, Verlinde CLMJ, Hol WGJ (2006) Using fragment cocktail crystallography to assist inhibitor design of Trypanosoma brucei nucleoside 2-deoxyribosyltransferase. J Med Chem 49:5939–5946

    Article  CAS  PubMed  Google Scholar 

  • Brown PH, Schuck P (2006) Macromolecular size-and-shape distributions by sedimentation velocity analytical ultracentrifugation. Biophys J 90:4651–4661

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Carson DA, Wasson DB (1988) Synthesis of 2′,3′-dideoxynucleosides by enzymatic trans-glycosylation. Biochem Biophys Res Commun 155:829–834

    Article  CAS  PubMed  Google Scholar 

  • Case DA, Babin V, Berryman JT, Betz RM, Cai Q, Cerutti DS, Cheatham TE, Darden TA, Duke RE, Gohlke H, Goetz AW, Gusarov S, Homeyer N, Janowski P, Kaus J, Kolossváry I, Kovalenko A, Lee TS, LeGrand S, Luchko T, Luo R, Madej B, Merz KM, Paesani F, Roe DR, Roitberg A, Sagui C, Salomon-Ferrer R, Seabra G, Simmerling CL, Smith W, Swails J, Walker RC, Wang J, Wolf RM, Wu X, Kollman PA (2014) AMBER 14. University of California, San Francisco

    Google Scholar 

  • Cortés-Cabrera Á, Gago F, Morreale A (2015) A computational fragment-based de novo design protocol guided by ligand efficiency indices. In: Klon AE (ed) Fragment-based methods in drug discovery. Springer, New York, pp 89–100

    Google Scholar 

  • Datta AK, Datta R, Sen B (2008) In: Majumder HK (ed) Drug targets in Kinetoplastid parasites. Springer New York, New York, NY., pp 116–132

  • De Clercq E (2005a) Antiviral drug discovery and development: where chemistry meets with biomedicine. Antivir Res 67:56–75

    Article  PubMed  Google Scholar 

  • De Clercq E (2005b) Recent highlights in the development of new antiviral drugs. Curr Opin Microbiol 8:552–560

    Article  PubMed  Google Scholar 

  • DeLano WL (2002) The PyMOL molecular graphics system. DeLano Scientific, San Carlos

    Google Scholar 

  • Dundas J, Ouyang Z, Tseng J, Binkowski A, Turpaz Y, Liang J (2006) CASTp: computed atlas of surface topography of proteins with structural and topographical mapping of functionally annotated residues. Nucleic Acids Res 34:W116–W118

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • el Kouni MH (2003) Potential chemotherapeutic targets in the purine metabolism of parasites. Pharm Ther 99:283–309

    Article  Google Scholar 

  • Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. Acta Crystallogr Sect D Biol Crystallogr 66:486–501

    Article  CAS  Google Scholar 

  • Evans PR (2011) An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr Sect D Biol Crystallogr 67:282–292

    Article  CAS  Google Scholar 

  • Fernández-Lucas J, Acebal C, Sinisterra JV, Arroyo M, de la Mata I (2010) Lactobacillus reuteri 2′-deoxyribosyltransferase, a novel biocatalyst for tailoring of nucleosides. Appl Environ Microbiol 76:1462–1470

    Article  PubMed  PubMed Central  Google Scholar 

  • Fernández-Lucas J, Fresco-Taboada A, de la Mata I, Arroyo M (2012) One-step enzymatic synthesis of nucleosides from low water-soluble purine bases in non-conventional media. Bioresour Technol 115:63–69

    Article  PubMed  Google Scholar 

  • Fresco-Taboada A, de la Mata I, Arroyo M, Fernández-Lucas J (2013) New insights on nucleoside 2′-deoxyribosyltransferases: a versatile biocatalyst for one-pot one-step synthesis of nucleoside analogs. Appl Microbiol Biotechnol 97(9):3773–3785

    Article  CAS  PubMed  Google Scholar 

  • Galmarini CM, Mackey JR, Dumontet C (2002) Nucleoside analogues and nucleobases in cancer treatment. Lancet Oncol 3:415–424

    Article  CAS  PubMed  Google Scholar 

  • Gill SC, Von Hippel PH (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem 182:319–326

    Article  CAS  PubMed  Google Scholar 

  • Goodsell DS, Olson AJ (2000) Structural symmetry and protein function. Annu Rev Biophys Biomol Struct 29:105–153

    Article  CAS  PubMed  Google Scholar 

  • Holm L, Rosenström P (2010) Dali server: conservation mapping in 3D. Nucleic Acids Res 38:W545–W549

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kabsch W (2010) Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr Sect D Biol Crystallogr 66:133–144

    Article  CAS  Google Scholar 

  • Kaminski PA (2002) Functional cloning, heterologous expression, and purification of two different N-deoxyribosyltransferases from Lactobacillus helveticus. J Biol Chem 277:14400–14407

    Article  CAS  PubMed  Google Scholar 

  • Kaminski PA, Dacher P, Dugue L, Pochet S (2008) In vivo reshaping the catalytic site of nucleoside 2 '-deoxyribosyltransferase for dideoxy- and didehydronucleosides via a single amino acid substitution. J Biol Chem 283:20053–20059

    Article  CAS  PubMed  Google Scholar 

  • Klett J, Núñez-Salgado A, Dos Santos HG, Cortés-Cabrera A, Perona A, Gil-Redondo R, Abia D, Gago F, Morreale A (2012) MM-ISMSA: an ultrafast and accurate scoring function for protein–protein docking. J Chem Theory Comput 8:3395–3408

    Article  CAS  PubMed  Google Scholar 

  • Krissinel E, Henrick K (2007) Inference of macromolecular assemblies from crystalline state. J Mol Biol 372:774–797

    Article  CAS  PubMed  Google Scholar 

  • Laue TM, Shah BD, Ridgeway TM, Pelletier SL (1992) Computer-aided interpretation of analytical sedimentation data for proteins. In: Harding SE, Rowe AJ, Horton JC (eds) Analytical ultracentrifugation in biochemistry and polymer science. The Royal Society of Chemistry, Cambridge, pp 90–125

  • Lawrence KA, Jewett MW, Rosa PA, Gherardini FC (2009) Borrelia burgdorferi bb0426 encodes a 2′-deoxyribosyltransferase that plays a central role in purine salvage. Mol Microbiol 72:1517–1529

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lewkowicz E, Iribarren A (2006) Nucleoside phosphorylases. Curr Org Chem 10:1197–1215

    Article  CAS  Google Scholar 

  • Mateo C, Palomo JM, Fernandez-Lorente G, Guisan JM, Fernandez-Lafuente R (2007) Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzym Microb Technol 40:1451–1463

    Article  CAS  Google Scholar 

  • McCoy AJ (2007) Solving structures of protein complexes by molecular replacement with Phaser. Acta Crystallogr Sect D Biol Crystallogr 63:32–41

    Article  CAS  Google Scholar 

  • Mikhailopulo IA (2007) Biotechnology of nucleic acid constituents-state of the art and perspectives. Curr Org Chem 11:317–335

    Article  CAS  Google Scholar 

  • Müller M, Hutchinson LK, Guengerich FP (1996) Addition of deoxyribose to guanine and modified DNA bases by Lactobacillus helveticus trans-N-deoxyribosylase. Chem Res Toxicol 9:1140–1144

    Article  PubMed  Google Scholar 

  • Okuyama K, Shibuya S, Hamamoto T, Noguchi T (2003) Enzymatic synthesis of 2′-deoxyguanosine with nucleoside deoxyribosyltransferase-II. Biosci Biotechnol Biochem 67:989–995

    Article  CAS  PubMed  Google Scholar 

  • Parker WB (2009) Enzymology of purine and pyrimidine antimetabolites used in the treatment of cancer. Chem Rev 109:2880–2893

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Robak T, Lech-Maranda E, Korycka A, Robak E (2006) Purine nucleoside analogs as immunosuppressive and antineoplastic agents: mechanism of action and clinical activity. Curr Med Chem 13:3165–3189

    Article  CAS  PubMed  Google Scholar 

  • Sánchez-Murcia PA, Bueren-Calabuig JA, Camacho-Artacho M, Cortés-Cabrera Á, Gago F (2016) Stepwise simulation of 3, 5-dihydro-5-methylidene-4 H-imidazol-4-one (MIO) biogenesis in histidine ammonia-lyase. Biochemistry 55:5854–5864

    Article  PubMed  Google Scholar 

  • Shi W, Schramm VL, Almo SC (1999) Nucleoside hydrolase from Leishmania major: cloning, expression, catalytic properties, transition state inhibitors, and the 2.5 Å structure. J Biol Chem 274:21114–21120

    Article  CAS  PubMed  Google Scholar 

  • Short SA, Armstrong SR, Ealick SE, Porter DJT (1996) Active site amino acids that participate in the catalytic mechanism of nucleoside 2′-deoxyribosyltransferase. J Biol Chem 271:4978–4987

    Article  CAS  PubMed  Google Scholar 

  • Steenkamp DJ, Hälbich TJF (1992) Substrate specificity of the purine-2′-deoxyribonucleosidase of Crithidia luciliae. Biochem J 287:125–129

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Touw WG, Baakman C, Black J, te Beek TA, Krieger E, Joosten RP, Vriend G (2015) A series of PDB-related databanks for everyday needs. Nucleic Acids Res 43:D364–D368

    Article  CAS  PubMed  Google Scholar 

  • Tuttle JV, Tisdale M, Krenitsky TA (1993) Purine 2′-deoxy-2′-fluororibosides as antiinfluenza virus agents. J Med Chem 36:119–125

    Article  CAS  PubMed  Google Scholar 

  • Vanquelef E, Simon S, Marquant G, Garcia E, Klimerak G, Delepine JC, Cieplak FY, Dupradeau FY (2011) RED server: a web service for deriving RESP and ESP charges and building force field libraries for new molecules and molecular fragments. Nucleic Acids Res 39:W511–W517

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Versées W, Steyaert J (2003) Catalysis by nucleoside hydrolases. Curr Opin Struct Biol 13:731–738

    Article  PubMed  Google Scholar 

  • Wilhelmus KR (2015) Antiviral treatment and other therapeutic interventions for herpes simplex virus epithelial keratitis. Cochrane Database Syst Rev 1:CD00289

    Google Scholar 

  • World Health Organization (2011) WHO model list of essential medicines: 17th list, March

  • Ye Y, Godzik A (2003) Flexible structure alignment by chaining aligned fragment pairs allowing twists. Bioinformatics 19:ii246–ii255

    PubMed  Google Scholar 

  • Yokozeki K, Tsuji T (2000) A novel enzymatic method for the production of purine-2′-deoxyribonucleosides. J Mol Catal B Enzym 10:207–213

    Article  CAS  Google Scholar 

  • Yukiko M, Taheharu M, Shigeru C (2007) Characterization of N-deoxyribosyltransferase from Lactococcus lactis subsp. Lactis. Biochim Biophys Acta 1774:1323–1330

Download references

Acknowledgements

This work was supported by grants from the Spanish Ministerio de Economía y Competitividad (BFU2010-17929/BMC to J.M.M. and SAF2015-64629-C2-2-R to F.G.), and SAN151610 from the Santander Foundation (to J.F.L.). J.M.M. thanks the synchrotron ALBA for the access to the radiation source.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Jesús Fernández-Lucas or José Miguel Mancheño.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals by any of the authors.

Funding

This work was supported by grants from the Spanish Ministerio de Economía y Competitividad (BFU2010-17929/BMC to J.M.M. and SAF2015-64629-C2-2-R to F.G.), and SAN151610 from the Santander Foundation (to J.F.L.).

Electronic supplementary material

ESM 1

(GIF 39311 kb)

ESM 2

(PDF 386 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Crespo, N., Sánchez-Murcia, P.A., Gago, F. et al. 2′-Deoxyribosyltransferase from Leishmania mexicana, an efficient biocatalyst for one-pot, one-step synthesis of nucleosides from poorly soluble purine bases. Appl Microbiol Biotechnol 101, 7187–7200 (2017). https://doi.org/10.1007/s00253-017-8450-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-017-8450-y

Keywords

Navigation