Production, characterization and synthetic application of a purine nucleoside phosphorylase from Aeromonas hydrophila

Nucleoside phosphorylases (NPs; E.C. 2.4.2) catalyze the reversible cleavage of the glycosidic bond of (deoxy)ribonucleosides in the presence of inorganic orthophosphate (Pi) to generate the nucleobase and α-D-(deoxy)ribose-1-phosphate (R-1-P). If a second nucleobase is added to the reaction medium the formation of a new nucleoside can result (transglycosylation). Thus, NPs can be used for chemoenzymatic synthesis of both natural and unnatural nucleosides, as an alternative to conventional chemical methods which are generally plagued by low stereoselectivity, multi-step procedures and modest yields. Starting from the results of a microbiological screening which highlighted the broad specificity of Aeromonas hydrophila, also towards 6-modified purine nucleosides with antiviral and antitumor activity, we decided to investigate the potential of A. hydrophila purine phosphorylases PNPs (E.C. 2.4.2.1) as biocatalysts for practical uses. We have cloned and over-expressed one of A. hydrophila PNPs, tested its substrate specificity, and used it for synthetic applications, also at a preparative scale. To this aim, PNP encoded by deoD gene was expressed as a fusion protein (His6 tag) and recovered in high purity and concentration. Substrate specificity was firstly assayed towards natural nucleosides (according to Eq. 1) revealing that this PNP catalyzes both the phosphorolysis of 6-oxo and 6-amino purine (deoxy)ribonucleosides. Then, a library of nucleoside analogs was synthesized and then submitted to enzymatic phosphorolysis as well. This assay revealed that 1-, 2-, 6- and 7-modified nucleosides are accepted as substrates, whereas 8-substituted nucleosides are not. To explore the synthetic potential of PNP, a few transglycosylations were carried out using 7-methylguanosine iodide as a D-ribose donor and 6-substituted purines as acceptor. This procedure takes advantage of both the irreversible phosphorolysis of the 7-methyl riboside and the ready preparation of this nucleoside from guanosine. Following this approach, 6-chloro- and 6-methoxy-purine riboside were synthesized in quantitative yield and high purity