Orotate phosphoribosyltransferase from Thermus thermophilus: overexpression in Escherichia coli, purification and characterization.
Orotate phosphoribosyltransferase (OPRTase, EC2.4.2.10) plays a role in de novo synthesis of pyrimidine nucleotide and transfers orotate to 5-phosphoribosyl-1-pyrophosphate (PRPP) to form orotidine-5'-monophosphate (OMP). To obtain heat-stable OPRTase and to elucidate the mechanism of heat stability, this enzyme from Thermus thermophilus was expressed in Escherichia coli and purified. The pyrE gene of T. thermophilus which encodes OPRTase, contains an open reading frame of 549 base pairs with 69% G+C content. Since this gene expressed itself inefficiently 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 major codons for E. coli. Introduction of the modified gene fragments into a plasmid having a tac promoter resulted in production of a polypeptide of molecular weight (M(r)) 20,000 in the presence of isopropyl-beta-D-thiogalactopyranoside (IPTG) in E. coli. This protein represented as much as 16% of the bacterial total protein and showed the OPRTase activity. Three purification steps, consisting of heat treatment at 65 degrees C, 40% ammonium sulfate fractionation, and KCl gradient elution from DEAE-Sephadex A-50, resulted in highly purified single polypeptide. The optimum activity of the purified OPRTase was observed at 150 mM KCl, pH 9.0, 75-80 degrees C, and in the presence of 100 microM PRPP. The activation energy of this enzyme reaction was 20.3 kJ/mol. The Km of this enzyme for orotate as a substrate was 75 microM and the maximum specific activity was 300 units/mg protein under the optimum conditions. The purified OPRTase was stable for 20 min at 85 degrees C.
Bunnak
J
J
Department of Biological and Chemical Engineering, Gunma University.
Hamana
H
H
Ogino
Y
Y
Saheki
T
T
Yamagishi
A
A
Oshima
T
T
Date
T
T
Shinozawa
T
T
D000595:Amino Acid Sequence; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli; D005798:Genes, Bacterial; D006358:Hot Temperature; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009962:Orotate Phosphoribosyltransferase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D009962:Orotate Phosphoribosyltransferase