SEQUENCE AND PHYLOGENETIC ANALYSIS OF THE RHIZOBIUM-LEGUMINOSARUM BIOVAR TRIFOLII PYRE GENE, OVERPRODUCTION, PURIFICATION AND CHARACTERIZATION OF OROTATE PHOSPHORIBOSYLTRANSFERASE

The pyr E gene of Rhizobium leguminosarum biovar trifolii (RI) was sub cloned and its sequence is presented. The nucleotide sequence analysis suggests that this gene is not regulated by transcriptional attenuati on as seen for the pyr E and pyr B genes of Escherichia coli (Ec) and Salmonella typhimurium. The Rl pyr E gene was subcloned into Ec AT2538 pyr E60 where the RI pyr E gene product, orotate phosphoribosyltransf erase (OPRTase), was overproduced. Using Ec AT2538 pyr E60 overproduci ng RE OPRTase, the enzyme was purified to homogeneity utilizing ammoni um sulfate fractionation and affinity chromatography with an orotate m onophosphate 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 mu mol l(-1). The deduced amino acid sequence for OPRTase was co mpared with OPRTases from other organisms and found to be most similar to that of Bacillus subtilis (Bs). The RI OPRTase exhibits 37% identi ty and 46% similarity to the Bs OPRTase. (C) 1997 Elsevier Science B.V .