Sequence and phylogenetic analysis of the Rhizobium leguminosarum biovar trifolii pyrE gene, overproduction, purification and characterization of orotate phosphoribosyltransferase.

The pyrE gene of Rhizobium leguminosarum biovar trifolii (Rl) was subcloned and its sequence is presented. The nucleotide sequence analysis suggests that this gene is not regulated by transcriptional attenuation as seen for the pyrE and pyrB genes of Escherichia coli (Ec) and Salmonella typhimurium. The Rl pyrE gene was subcloned into Ec AT2538 pyrE60 where the Rl pyrE gene product, orotate phosphoribosyltransferase (OPRTase), was overproduced. Using Ec AT2538 pyrE60 overproducing Rl OPRTase, the enzyme was purified to homogeneity utilizing ammonium sulfate fractionation and affinity chromatography with an orotate monophosphate agarose matrix. The electrophoretically pure OPRTase was characterized and found to be a 24.7 +/- 0.3-kDa protein with a K(m) of 27.6 micromol l(-1). The deduced amino acid sequence for OPRTase was compared with OPRTases from other organisms and found to be most similar to that of Bacillus subtilis (Bs). The Rl OPRTase exhibits 37% identity and 46% similarity to the Bs OPRTase.