OROTATE PHOSPHORIBOSYLTRANSFERASE FROM THERMUS-THERMOPHILUS - OVEREXPRESSION IN ESCHERICHIA-COLI, PURIFICATION AND CHARACTERIZATION

Orotate phosphoribosyltransferase (OPRTase, EC 2.4.2.10) plays a role in de novo synthesis of pyrimidine nucleotide and transfers orotate to 5-phosphoribosyl-1-pyrophosphate (PRPP) to form orotidine-5'-monophos phate (OR9P), To obtain heat-stable OPRTase and to elucidate the mecha nism of heat stability, this enzyme from Thermus thermophilus was expr essed in Escherichia coli and purified, The pyrE gene of T. thermophil us which encodes OPRTase, contains an open reading frame of 549 base p airs with 69% G+C content, Since this gene expressed itself inefficien tly in E. coli, the 5' and 3' ends of the coding regions were replaced with synonymous codons which contain more A+T and corresponds to majo r codons for E. coli. Introduction of the modified gene fragments into a plasmid having a toe promoter resulted in production of a polypepti de of molecular weight (M(r)) 20,000 in the presence of isopropyl-beta -D-thiogalactopyranoside (IPTG) in E. coli. This protein represented a s much as 16% of the bacterial total protein and showed the OPRTase ac tivity, Three purification steps, consisting of heat treatment at 65 d egrees C, 40% ammonium sulfate fractionation, and KCl gradient elution from DEAE-Sephadex A-50, resulted in highly purified single polypepti de, The optimum activity of the purified OPRTase was observed at 150mM KCl, pH 9.0, 75-80 degrees C, and in the presence of 100 mu M PRPP. T he activation energy of this enzyme reaction was 20.3 kJ/mol. The K-m of this enzyme for orotate as a substrate was 75 mu M and the maximum specific activity was 300 units/mg protein under the optimum condition s. The purified OPRTase was stable for 20 min at 85 degrees C.