Abstract
Orotidine-5′-monophosphate decarboxylase (OMPdecase) catalyzes the final step in pyrimidine biosynthesis, the conversion of orotidine-5′-monophosphate (OMP) to uridine-5′-monophosphate. ThepyrF gene, encoding OMPdecase, was isolated from a chromosomal library ofPseudomonas aeruginosa PAO1 by screening for complementation of anEscherichia coli and aP. aeruginosa pyrF mutant. The nucleotide sequence of a 2510-bp chromosomal DNA fragment, complementing both strains, was determined (EMBL accession number X65613). On this a 696-bp open reading frame capable of encoding the 24 kDa OMPdecase was identified. Despite a generally good correspondence to other OMPdecase sequences, theP. aeruginosa gene was unique in that it did not constitute part of an operon. ThepyrF gene was amplified by polymerase chain reaction, overexpressed in the pT7-7/E. coli BL21(DE3) system and purified to near electrophoretic homogeneity by anion exchange chromatography. Characterization of the purified enzyme revealed the following data, aK m value for OMP of 9.91 μM and an isoelectric point of 6.65. No major decrease in enzyme activity was observed in a pH range between 7.8 and 10.2. Gel electrophoresis under nondenaturing conditions suggested that the native form of OMPdecase is the dimer.
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Strych, U., Wohlfarth, S. & Winkler, U.K. Orotidine-5′-monophosphate decarboxylase fromPseudomonas aeruginosa PAO1: Cloning, overexpression, and enzyme characterization. Current Microbiology 29, 353–359 (1994). https://doi.org/10.1007/BF01570229
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DOI: https://doi.org/10.1007/BF01570229