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.
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
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
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
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
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
Becker J, Brendel M (1996) Rapid purification and characterization of two distinct N-deoxyribosyltransferases of Lactobacillus leichmannii. Biol Chem Hoppe Seyler 377:357–362
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
Boryski J (2008) Reactions of transglycosylation in the nucleoside chemistry. Curr Org Chem 12:309–325
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
Brown PH, Schuck P (2006) Macromolecular size-and-shape distributions by sedimentation velocity analytical ultracentrifugation. Biophys J 90:4651–4661
Carson DA, Wasson DB (1988) Synthesis of 2′,3′-dideoxynucleosides by enzymatic trans-glycosylation. Biochem Biophys Res Commun 155:829–834
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
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
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
De Clercq E (2005b) Recent highlights in the development of new antiviral drugs. Curr Opin Microbiol 8:552–560
DeLano WL (2002) The PyMOL molecular graphics system. DeLano Scientific, San Carlos
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
el Kouni MH (2003) Potential chemotherapeutic targets in the purine metabolism of parasites. Pharm Ther 99:283–309
Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. Acta Crystallogr Sect D Biol Crystallogr 66:486–501
Evans PR (2011) An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr Sect D Biol Crystallogr 67:282–292
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
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
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
Galmarini CM, Mackey JR, Dumontet C (2002) Nucleoside analogues and nucleobases in cancer treatment. Lancet Oncol 3:415–424
Gill SC, Von Hippel PH (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem 182:319–326
Goodsell DS, Olson AJ (2000) Structural symmetry and protein function. Annu Rev Biophys Biomol Struct 29:105–153
Holm L, Rosenström P (2010) Dali server: conservation mapping in 3D. Nucleic Acids Res 38:W545–W549
Kabsch W (2010) Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr Sect D Biol Crystallogr 66:133–144
Kaminski PA (2002) Functional cloning, heterologous expression, and purification of two different N-deoxyribosyltransferases from Lactobacillus helveticus. J Biol Chem 277:14400–14407
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
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
Krissinel E, Henrick K (2007) Inference of macromolecular assemblies from crystalline state. J Mol Biol 372:774–797
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
Lewkowicz E, Iribarren A (2006) Nucleoside phosphorylases. Curr Org Chem 10:1197–1215
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
McCoy AJ (2007) Solving structures of protein complexes by molecular replacement with Phaser. Acta Crystallogr Sect D Biol Crystallogr 63:32–41
Mikhailopulo IA (2007) Biotechnology of nucleic acid constituents-state of the art and perspectives. Curr Org Chem 11:317–335
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
Okuyama K, Shibuya S, Hamamoto T, Noguchi T (2003) Enzymatic synthesis of 2′-deoxyguanosine with nucleoside deoxyribosyltransferase-II. Biosci Biotechnol Biochem 67:989–995
Parker WB (2009) Enzymology of purine and pyrimidine antimetabolites used in the treatment of cancer. Chem Rev 109:2880–2893
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
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
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
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
Steenkamp DJ, Hälbich TJF (1992) Substrate specificity of the purine-2′-deoxyribonucleosidase of Crithidia luciliae. Biochem J 287:125–129
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
Tuttle JV, Tisdale M, Krenitsky TA (1993) Purine 2′-deoxy-2′-fluororibosides as antiinfluenza virus agents. J Med Chem 36:119–125
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
Versées W, Steyaert J (2003) Catalysis by nucleoside hydrolases. Curr Opin Struct Biol 13:731–738
Wilhelmus KR (2015) Antiviral treatment and other therapeutic interventions for herpes simplex virus epithelial keratitis. Cochrane Database Syst Rev 1:CD00289
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
Yokozeki K, Tsuji T (2000) A novel enzymatic method for the production of purine-2′-deoxyribonucleosides. J Mol Catal B Enzym 10:207–213
Yukiko M, Taheharu M, Shigeru C (2007) Characterization of N-deoxyribosyltransferase from Lactococcus lactis subsp. Lactis. Biochim Biophys Acta 1774:1323–1330
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
Corresponding authors
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.).
Rights and permissions
About this article
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
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-017-8450-y