Kim S S, Choi I G, Kim S H, Yu Y G
Structural Biology Center, Korea Institute of Science and Technology, Seoul.
Extremophiles. 1999 Aug;3(3):175-83. doi: 10.1007/s007920050114.
A gene encoding glutamate racemase has been cloned from Aquifex pyrophilus, a hyperthermophilic bacterium, and expressed in Escherichia coli. The A. pyrophilus glutamate racemase is composed of 254 amino acids and shows high homology with glutamate racemase from Escherichia coli, Bacillus subtilis, or Lactobacillus brevis. This racemase converts L- or D-glutamate to D- or L-glutamate, respectively, but not other amino acids such as alanine, aspartate, and glutamine. The cloned gene was expressed and the protein was purified to homogeneity. The A. pyrophilus racemase is present as a dimer but it oligomerizes as the concentration of salt is increased. The Km and k(cat) values of the overexpressed A. pyrophilus glutamate racemase for the racemization of L-glutamate to the D-form and the conversion of D-glutamate to the L-form were measured as 1.8 +/- 0.4mM and 0.79 +/- 0.06s(-1) or 0.50 +/- 0.07mM and 0.25 +/- 0.01s(-1), respectively. Complete inactivation of the racemase activity by treatment with cysteine-modifying reagents suggests that cysteine residues may be important for activity. The protein shows strong thermostability in the presence of phosphate ion, and it retains more than 50% of its activity after incubation at 85 degrees C for 90 min.
已从嗜热栖热菌（一种嗜热细菌）中克隆出编码谷氨酸消旋酶的基因，并在大肠杆菌中表达。嗜热栖热菌谷氨酸消旋酶由254个氨基酸组成，与大肠杆菌、枯草芽孢杆菌或短乳杆菌的谷氨酸消旋酶具有高度同源性。这种消旋酶分别将L-或D-谷氨酸转化为D-或L-谷氨酸，但不能转化其他氨基酸，如丙氨酸、天冬氨酸和谷氨酰胺。克隆的基因得以表达，蛋白质被纯化至同质。嗜热栖热菌消旋酶以二聚体形式存在，但随着盐浓度的增加会发生寡聚化。过量表达的嗜热栖热菌谷氨酸消旋酶将L-谷氨酸外消旋化为D-型以及将D-谷氨酸转化为L-型的米氏常数（Km）和催化常数（k(cat)）分别测定为1.8±0.4mM和0.79±0.06s(-1)或0.50±0.07mM和0.25±0.01s(-1)。用半胱氨酸修饰试剂处理使消旋酶活性完全失活，这表明半胱氨酸残基可能对活性很重要。该蛋白质在磷酸根离子存在下表现出很强的热稳定性，并在85℃孵育90分钟后仍保留其活性的50%以上。
