Purifications and Properties of Orotidine-phosphorolyzing Enzyme and Purine Nucleoside Phosphorylase from Erwinia carotovora AJ 2992

Abstract

An orotidine-phosphorolyzing enzyme and a purine nucleoside phosphorylase (PNPase) of Erwinia carotovora AJ 2992, which is a potent producer of ribavirin (1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), an antiviral agent, from orotidine and 1,2,4-triazole-3-carboxamide (TCA), were purified 23-fold and 103-fold, respectively. At each purification step, the orotidine-phosphorolyzing enzyme was always co-purified with an uridine phosphorylase (UPase) and its activity could not be separated from that of the UPase after it showed as a single band on SDS-polyacrylamide gel electrophoresis. These results suggest that this enzyme may be identical with UPase. The purified enzyme had a molecular weight of 68,000 ± 2,000, and seemed to be a dimer. The optimal temperatures and pH values were 60°C and 6.0 for orotidine phosphorolysis, and 70°C and 7.0 for uridine phosphorolysis. The Michaelis constants for uridine and orotidine were 0.75 mM and 10.87 mM, respectively, at 40°C. The PNPase of E. carotovora AJ 2992 had a molecular weight of 58,000 ± 2,000 and seemed to be a dimer. The Michaelis constants for inosine and guanosine were 1.92 mM and 1.85 mM, respectively, at 40°C. The PNPase was completely inactivated by p-chloromercuribenzoate. © 1991, Japan Society for Bioscience, Biotechnology, and Agrochemistry. All rights reserved.