Recombinant purine nucleoside phosphorylases from thermophiles: preparation, properties and activity towards purine and pyrimidine nucleosides

Authors

  • Xinrui Zhou,

    1. Laboratory of Bioprocess Engineering, Department of Biotechnology, Technische Universität Berlin, Germany
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  • Kathleen Szeker,

    1. Laboratory of Bioprocess Engineering, Department of Biotechnology, Technische Universität Berlin, Germany
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  • Bernd Janocha,

    1. Laboratory of Bioprocess Engineering, Department of Biotechnology, Technische Universität Berlin, Germany
    2. Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany
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  • Thomas Böhme,

    1. Laboratory of Bioprocess Engineering, Department of Biotechnology, Technische Universität Berlin, Germany
    2. Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany
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  • Dirk Albrecht,

    1. Institute of Microbiology, Ernst-Moritz-Arndt-Universität Greifswald, Germany
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  • Igor A. Mikhailopulo,

    Corresponding author
    1. Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Minsk, Belarus
    • Laboratory of Bioprocess Engineering, Department of Biotechnology, Technische Universität Berlin, Germany
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  • Peter Neubauer

    Corresponding author
    • Laboratory of Bioprocess Engineering, Department of Biotechnology, Technische Universität Berlin, Germany
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Correspondence

P. Neubauer, Laboratory of Bioprocess Engineering, Department of Biotechnology, Technische Universität Berlin, Ackerstr. 71–76, ACK24, D–13355 Berlin, Germany

Fax: +49 3031 427577

Tel: +49 3031 472269

E-mail: peter.neubauer@tu-berlin.de

Website: http://www.bioprocess.tu-berlin.de

I. A. Mikhailopulo, Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Acad. Kuprevicha 5/2, 220141 Minsk, Belarus

Fax: +375 1726 78161

Tel: +375 1726 78148

E-mail: imikhailopulo@gmail.com

Abstract

Thermostable nucleoside phosphorylases are attractive biocatalysts for the synthesis of modified nucleosides. Hence we report on the recombinant expression of three ‘high molecular mass’ purine nucleoside phosphorylases (PNPs) derived from the thermophilic bacteria Deinococcus geothermalis, Geobacillus thermoglucosidasius and from the hyperthermophilic archaeon Aeropyrum pernix (5′–methythioadenosine phosphorylase; ApMTAP). Thermostability studies, kinetic analysis and substrate specificities are reported. The PNPs were stable at their optimal temperatures (DgPNP, 55 °C; GtPNP, 70 °C; ApMTAP, activity rising to 99 °C). Substrate properties were investigated for natural purine nucleosides [adenosine, inosine and their C2′-deoxy counterparts (activity within 50–500 U·mg−1)], analogues with 2′-amino modified 2′-deoxy-adenosine and -inosine (within 0.1–3 U·mg−1) as well as 2′-deoxy-2′-fluoroadenosine (9) and its C2′-arabino diastereomer (10, within 0.01–0.03 U·mg−1). Our results reveal that the structure of the heterocyclic base (e.g. adenine or hypoxanthine) can play a critical role in the phosphorolysis reaction. The implications of this finding may be helpful for reaction mechanism studies or optimization of reaction conditions. Unexpectedly, the diastereomeric 2′-deoxyfluoro adenine ribo- and arabino-nucleosides displayed similar substrate properties. Moreover, cytidine and 2′-deoxycytidine were found to be moderate substrates of the prepared PNPs, with substrate activities in a range similar to those determined for 2′-deoxyfluoro adenine nucleosides 9 and 10. C2′-modified nucleosides are accepted as substrates by all recombinant enzymes studied, making these enzymes promising biocatalysts for the synthesis of modified nucleosides. Indeed, the prepared PNPs performed well in preliminary transglycosylation reactions resulting in the synthesis of 2′-deoxyfluoro adenine ribo- and arabino- nucleosides in moderate yield (24%).

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