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

• DOI: 10.1111/febs.12143
• Publication Date: 2013-01-19T13:32:40Z
• Authors: Xinrui Zhou; Kathleen Szeker; Bernd Janocha; Thomas Böhme; Dirk Albrecht; Igor A. Mikhailopulo; Peter Neubauer
• Keywords: Phosphorolysis; Inosine; Hypoxanthine; Thermostability

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