title: 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.
authors.affiliation: Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457, China.
authors.forename: Xixian
authors.initials: X
authors.lastname: Xie
authors.forename: Jungang
authors.initials: J
authors.lastname: Xia
authors.forename: Kuifu
authors.initials: K
authors.lastname: He
authors.forename: Lining
authors.initials: L
authors.lastname: Lu
authors.forename: Qingyang
authors.initials: Q
authors.lastname: Xu
authors.forename: Ning
authors.initials: N
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001709:Biotechnology; D004795:Enzyme Stability; D048168:Escherichia coli K12 / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015202:Protein Engineering; D020836:Protein Structure, Quaternary; D031161:Pseudoalteromonas / Q000201:enzymology / Q000235:genetics; D011684:Purine Nucleosides / Q000096:biosynthesis; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012254:Ribavirin / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D000998:Antiviral Agents; D001426:Bacterial Proteins; D011684:Purine Nucleosides; D011994:Recombinant Proteins; D012254:Ribavirin; D011683:Purine-Nucleoside Phosphorylase