Bunnak J, Hamana H, Ogino Y, Saheki T, Yamagishi A, Oshima T, Date T, Shinozawa T
Department of Biological and Chemical Engineering, Gunma University.
J Biochem. 1995 Dec;118(6):1261-7. doi: 10.1093/oxfordjournals.jbchem.a125016.
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.
乳清酸磷酸核糖基转移酶（OPRTase，EC2.4.2.10）在嘧啶核苷酸的从头合成中发挥作用，它将乳清酸转移至5-磷酸核糖-1-焦磷酸（PRPP），形成乳清苷-5'-单磷酸（OMP）。为了获得热稳定的OPRTase并阐明其热稳定性机制，对嗜热栖热菌中的这种酶进行了大肠杆菌表达及纯化。嗜热栖热菌中编码OPRTase的pyrE基因含有一个549个碱基对的开放阅读框，G+C含量为69%。由于该基因在大肠杆菌中表达效率较低，因此将编码区的5'端和3'端替换为含有更多A+T且对应于大肠杆菌主要密码子的同义密码子。将修饰后的基因片段导入具有tac启动子的质粒中，在异丙基-β-D-硫代半乳糖苷（IPTG）存在的情况下，大肠杆菌中产生了分子量（M(r)）为20,000的多肽。该蛋白占细菌总蛋白的16%，并表现出OPRTase活性。通过65℃热处理、40%硫酸铵分级分离以及从DEAE-葡聚糖A-50进行KCl梯度洗脱这三个纯化步骤，得到了高度纯化的单一多肽。纯化后的OPRTase在150 mM KCl、pH 9.0、75 - 80℃以及100 microM PRPP存在的条件下表现出最佳活性。该酶反应的活化能为20.3 kJ/mol。在最佳条件下，该酶以乳清酸为底物的Km为75 microM，最大比活性为300单位/毫克蛋白。纯化后的OPRTase在85℃下可稳定20分钟。
