Recombinant purine nucleoside phosphorylases from thermophiles: preparation, properties and activity towards purine and pyrimidine nucleosides
DNA, Bacterial
0301 basic medicine
0303 health sciences
Glycosylation
Deoxyadenosines
Molecular Sequence Data
Geobacillus
Aeropyrum
Purine Nucleosides
Pyrimidine Nucleosides
Recombinant Proteins
3. Good health
Enzyme Activation
Kinetics
03 medical and health sciences
Purine-Nucleoside Phosphorylase
Enzyme Stability
Escherichia coli
Amino Acid Sequence
Deinococcus
Cloning, Molecular
Phosphorylation
Sequence Alignment
Enzyme Assays
DOI:
10.1111/febs.12143
Publication Date:
2013-01-19T13:32:40Z
AUTHORS (7)
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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