title: Recombinant purine nucleoside phosphorylases from thermophiles: preparation, properties and activity towards purine and pyrimidine nucleosides.
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%).
authors: Laboratory of Bioprocess Engineering, Department of Biotechnology, Technische Universität Berlin, Berlin, Germany.
authors: Xinrui
authors: X
authors: Zhou
authors: Kathleen
authors: K
authors: Szeker
authors: Bernd
authors: B
authors: Janocha
authors: Thomas
authors: T
authors: Böhme
authors: Dirk
authors: D
authors: Albrecht
authors: Igor A
authors: IA
authors: Mikhailopulo
authors: Peter
authors: P
authors: Neubauer
mesh_terms: D041522:Aeropyrum / Q000201:enzymology / Q000235:genetics; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D034301:Deinococcus / Q000201:enzymology* / Q000235:genetics; D003839:Deoxyadenosines / Q000378:metabolism; D004789:Enzyme Activation; D057075:Enzyme Assays; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D056506:Geobacillus / Q000201:enzymology* / Q000235:genetics; D006031:Glycosylation; D007700:Kinetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011684:Purine Nucleosides / Q000378:metabolism; D011683:Purine-Nucleoside Phosphorylase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011741:Pyrimidine Nucleosides / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012995:Solubility; D013379:Substrate Specificity; D013696:Temperature
chemical_list: C412233:2'-deoxy-2-fluoroadenosine; D004269:DNA, Bacterial; D003839:Deoxyadenosines; D011684:Purine Nucleosides; D011741:Pyrimidine Nucleosides; D011994:Recombinant Proteins; D011683:Purine-Nucleoside Phosphorylase