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

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.

Keywords

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

Supplementary material

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ESM 1(GIF 39311 kb)
253_2017_8450_MOESM2_ESM.pdf (387 kb)
ESM 2(PDF 386 kb)

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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

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