OROTIDINE-5'-MONOPHOSPHATE DECARBOXYLASE FROM PSEUDOMONAS-AERUGINOSA PAO1 - CLONING, OVEREXPRESSION, AND ENZYME CHARACTERIZATION

Orotidine-5'-monophosphate decarboxylase (OMPdecase) catalyzes the fin al step in pyrimidine biosynthesis, the conversion of orotidine-5'-mon ophosphate (OMP) to uridine-5'-monophosphate. The pyrF gene, encoding OMPdecase, was isolated from a chromosomal library of Pseudomonas aeru ginosa PAO1 by screening for complementation of an Escherichia coli an d a P. aeruginosa pyrF mutant. The nucleotide sequence of a 2510-bp ch romosomal DNA fragment, complementing both strains, was determined (EM BL accession number X65613). On this a 696-bp open reading frame capab le of encoding the 24 kDa OMPdecase was identified. Despite a generall y good correspondence to other OMPdecase sequences, the P. aeruginosa gene was unique in that it did not constitute part of an operon. The p yrF gene was amplified by polymerase chain reaction, overexpressed in the pT7-7/E. coli BL21(DE3) system and purified to near electrophoreti c homogeneity by anion exchange chromatography. Characterization of th e purified enzyme revealed the following data, a K-m value for OMP of 9.91 mu M and an isoelectric point of 6.65. No major decrease in enzym e activity was observed in a pH range between 7.8 and 10.2. Gel electr ophoresis under nondenaturing conditions suggested that the native for m of OMPdecase is the dimer.