Applied Microbiology and Biotechnology

, Volume 101, Issue 19, pp 7187–7200 | Cite as

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

  • N. Crespo
  • P. A. Sánchez-Murcia
  • F. Gago
  • J. Cejudo-Sanches
  • M. A. Galmes
  • Jesús Fernández-Lucas
  • José Miguel Mancheño
Biotechnologically Relevant Enzymes and Proteins


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.


2′-deoxyribosyltransferase Enzymatic synthesis Industrial biocatalyst Protein crystallography Molecular docking Purine nucleoside analogues 



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.

Compliance with ethical standards

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.


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.).

Supplementary material

253_2017_8450_MOESM1_ESM.gif (38.4 mb)
ESM 1 (GIF 39311 kb)
253_2017_8450_MOESM2_ESM.pdf (387 kb)
ESM 2 (PDF 386 kb)


  1. 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 66:213–221CrossRefGoogle Scholar
  2. 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 68:352–367CrossRefGoogle Scholar
  3. 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 43:2384–2393CrossRefPubMedGoogle Scholar
  4. 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–W541CrossRefPubMedPubMedCentralGoogle Scholar
  5. 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 4:97–107CrossRefPubMedGoogle Scholar
  6. Becker J, Brendel M (1996) Rapid purification and characterization of two distinct N-deoxyribosyltransferases of Lactobacillus leichmannii. Biol Chem Hoppe Seyler 377:357–362CrossRefPubMedGoogle Scholar
  7. 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 42:525–530PubMedGoogle Scholar
  8. Boryski J (2008) Reactions of transglycosylation in the nucleoside chemistry. Curr Org Chem 12:309–325CrossRefGoogle Scholar
  9. 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 49:5939–5946CrossRefPubMedGoogle Scholar
  10. Brown PH, Schuck P (2006) Macromolecular size-and-shape distributions by sedimentation velocity analytical ultracentrifugation. Biophys J 90:4651–4661CrossRefPubMedPubMedCentralGoogle Scholar
  11. Carson DA, Wasson DB (1988) Synthesis of 2′,3′-dideoxynucleosides by enzymatic trans-glycosylation. Biochem Biophys Res Commun 155:829–834CrossRefPubMedGoogle Scholar
  12. 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 FranciscoGoogle Scholar
  13. 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 89–100Google Scholar
  14. Datta AK, Datta R, Sen B (2008) In: Majumder HK (ed) Drug targets in Kinetoplastid parasites. Springer New York, New York, NY., pp 116–132Google Scholar
  15. De Clercq E (2005a) Antiviral drug discovery and development: where chemistry meets with biomedicine. Antivir Res 67:56–75CrossRefPubMedGoogle Scholar
  16. De Clercq E (2005b) Recent highlights in the development of new antiviral drugs. Curr Opin Microbiol 8:552–560CrossRefPubMedGoogle Scholar
  17. DeLano WL (2002) The PyMOL molecular graphics system. DeLano Scientific, San CarlosGoogle Scholar
  18. 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–W118CrossRefPubMedPubMedCentralGoogle Scholar
  19. el Kouni MH (2003) Potential chemotherapeutic targets in the purine metabolism of parasites. Pharm Ther 99:283–309CrossRefGoogle Scholar
  20. Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. Acta Crystallogr Sect D Biol Crystallogr 66:486–501CrossRefGoogle Scholar
  21. Evans PR (2011) An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr Sect D Biol Crystallogr 67:282–292CrossRefGoogle Scholar
  22. 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 76:1462–1470CrossRefPubMedPubMedCentralGoogle Scholar
  23. 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 115:63–69CrossRefPubMedGoogle Scholar
  24. 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):3773–3785CrossRefPubMedGoogle Scholar
  25. Galmarini CM, Mackey JR, Dumontet C (2002) Nucleoside analogues and nucleobases in cancer treatment. Lancet Oncol 3:415–424CrossRefPubMedGoogle Scholar
  26. Gill SC, Von Hippel PH (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem 182:319–326CrossRefPubMedGoogle Scholar
  27. Goodsell DS, Olson AJ (2000) Structural symmetry and protein function. Annu Rev Biophys Biomol Struct 29:105–153CrossRefPubMedGoogle Scholar
  28. Holm L, Rosenström P (2010) Dali server: conservation mapping in 3D. Nucleic Acids Res 38:W545–W549CrossRefPubMedPubMedCentralGoogle Scholar
  29. Kabsch W (2010) Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr Sect D Biol Crystallogr 66:133–144CrossRefGoogle Scholar
  30. Kaminski PA (2002) Functional cloning, heterologous expression, and purification of two different N-deoxyribosyltransferases from Lactobacillus helveticus. J Biol Chem 277:14400–14407CrossRefPubMedGoogle Scholar
  31. 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 283:20053–20059CrossRefPubMedGoogle Scholar
  32. 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 8:3395–3408CrossRefPubMedGoogle Scholar
  33. Krissinel E, Henrick K (2007) Inference of macromolecular assemblies from crystalline state. J Mol Biol 372:774–797CrossRefPubMedGoogle Scholar
  34. 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 90–125Google Scholar
  35. 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 72:1517–1529CrossRefPubMedPubMedCentralGoogle Scholar
  36. Lewkowicz E, Iribarren A (2006) Nucleoside phosphorylases. Curr Org Chem 10:1197–1215CrossRefGoogle Scholar
  37. 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 40:1451–1463CrossRefGoogle Scholar
  38. McCoy AJ (2007) Solving structures of protein complexes by molecular replacement with Phaser. Acta Crystallogr Sect D Biol Crystallogr 63:32–41CrossRefGoogle Scholar
  39. Mikhailopulo IA (2007) Biotechnology of nucleic acid constituents-state of the art and perspectives. Curr Org Chem 11:317–335CrossRefGoogle Scholar
  40. 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 9:1140–1144CrossRefPubMedGoogle Scholar
  41. Okuyama K, Shibuya S, Hamamoto T, Noguchi T (2003) Enzymatic synthesis of 2′-deoxyguanosine with nucleoside deoxyribosyltransferase-II. Biosci Biotechnol Biochem 67:989–995CrossRefPubMedGoogle Scholar
  42. Parker WB (2009) Enzymology of purine and pyrimidine antimetabolites used in the treatment of cancer. Chem Rev 109:2880–2893CrossRefPubMedPubMedCentralGoogle Scholar
  43. 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 13:3165–3189CrossRefPubMedGoogle Scholar
  44. 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 55:5854–5864CrossRefPubMedGoogle Scholar
  45. 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 274:21114–21120CrossRefPubMedGoogle Scholar
  46. 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 271:4978–4987CrossRefPubMedGoogle Scholar
  47. Steenkamp DJ, Hälbich TJF (1992) Substrate specificity of the purine-2′-deoxyribonucleosidase of Crithidia luciliae. Biochem J 287:125–129CrossRefPubMedPubMedCentralGoogle Scholar
  48. 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–D368CrossRefPubMedGoogle Scholar
  49. Tuttle JV, Tisdale M, Krenitsky TA (1993) Purine 2′-deoxy-2′-fluororibosides as antiinfluenza virus agents. J Med Chem 36:119–125CrossRefPubMedGoogle Scholar
  50. 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–W517CrossRefPubMedPubMedCentralGoogle Scholar
  51. Versées W, Steyaert J (2003) Catalysis by nucleoside hydrolases. Curr Opin Struct Biol 13:731–738CrossRefPubMedGoogle Scholar
  52. Wilhelmus KR (2015) Antiviral treatment and other therapeutic interventions for herpes simplex virus epithelial keratitis. Cochrane Database Syst Rev 1:CD00289Google Scholar
  53. World Health Organization (2011) WHO model list of essential medicines: 17th list, MarchGoogle Scholar
  54. Ye Y, Godzik A (2003) Flexible structure alignment by chaining aligned fragment pairs allowing twists. Bioinformatics 19:ii246–ii255PubMedGoogle Scholar
  55. Yokozeki K, Tsuji T (2000) A novel enzymatic method for the production of purine-2′-deoxyribonucleosides. J Mol Catal B Enzym 10:207–213CrossRefGoogle Scholar
  56. Yukiko M, Taheharu M, Shigeru C (2007) Characterization of N-deoxyribosyltransferase from Lactococcus lactis subsp. Lactis. Biochim Biophys Acta 1774:1323–1330Google Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  1. 1.Department of Crystallography and Structural BiologyInstitute Rocasolano (CSIC)MadridSpain
  2. 2.Applied Biotechnology GroupEuropean University of MadridVillaviciosa de OdónSpain
  3. 3.Institute of Theoretical Chemistry, Faculty of ChemistryUniversity of ViennaViennaAustria
  4. 4.Department of Biomedical Sciences and “Unidad Asociada IQM-CSIC”, School of Medicine and Health SciencesUniversity of AlcaláAlcalá de HenaresSpain
  5. 5.Grupo de Investigación en Desarrollo Agroindustrial Sostenible, Department of Agroindustrial Engineering, School of Environmental SciencesUniversidad de la CostaBarranquillaColombia

Personalised recommendations