Low-molecular-mass purine nucleoside phosphorylase: characterization and application in enzymatic synthesis of nucleoside antiviral drugs

Abstract

Purine nucleoside phosphorylase (PNP) that catalyzes the reversible phosphorolysis of various purine nucleosides is widely distributed in prokaryotes and eukaryotes. Four pnp genes from Bacillus subtilis 168, Escherichia coli K-12 and Pseudoalteromonas sp. XM2107 were cloned by PCR and expressed in E. coli XL1-Blue. Recombinant PNPs (rPNPs) were purified by Ni(2+)-NTA chromatography. Compared with other rPNPs, PNP(816) was a low-molecular-mass homotrimer, which exhibited 11-, 4- and 1.5-fold higher values in k (cat)/K (m) using inosine as the substrate at 37°C. The PNP(816) or engineered strain XBlue (pQE-816) had a higher catalytic activity than other rPNPs or engineered strains during the enzymatic synthesis of ribavirin, which suggested that the low-molecular-mass homotrimer derived from microorganisms has higher catalytic activity for synthesis of nucleoside antiviral drugs.