Division of Biotechnology, The Catholic University of Korea, Bucheon 420-743, Republic of Korea.
J Microbiol Biotechnol. 2013 Oct 28;23(10):1395-402. doi: 10.4014/jmb.1305.05030.
Reductases convert some achiral ketone compounds into chiral alcohols, which are important materials for the synthesis of chiral drugs. The Saccharomyces cerevisiae reductase YOR120W converts ethyl-4-chloro-3-oxobutanoate (ECOB) enantioselectively into (R)-ethyl-4-chloro-3- hydroxybutanoate ((R)-ECHB), an intermediate of a pharmaceutical. As YOR120W requires NADPH as a cofactor for the reduction reaction, a cofactor recycling system using Bacillus subtilis glucose dehydrogenase was employed. Using this coupling reaction system, 100 mM ECOB was converted to (R)-ECHB. A homology modeling and site-directed mutagenesis experiment were performed to determine the NADPH-binding site of YOR120W. Four residues (Q29, K264, N267, and R270) were suggested by homology and docking modeling to interact directly with 2'-phosphate of NADPH. Among them, two positively charged residues (K264 and R270) were experimentally demonstrated to be necessary for NADPH 2'-phosphate binding. A mutant enzyme (Q29E) showed an enhanced enantiomeric excess value compared with that of the wild-type enzyme.
还原酶将一些手性酮化合物转化为手性醇，这些醇是手性药物合成的重要材料。酿酒酵母还原酶 YOR120W 对手性药物合成的中间体乙基-4-氯-3-氧代丁酸酯（ECOB）进行对映选择性还原，生成（R）-乙基-4-氯-3-羟基丁酸酯（（R）-ECHB）。由于 YOR120W 的还原反应需要 NADPH 作为辅助因子，因此采用了枯草芽孢杆菌葡萄糖脱氢酶的辅助因子再生系统。使用该偶联反应系统，将 100mM 的 ECOB 转化为（R）-ECHB。通过同源建模和定点突变实验确定了 YOR120W 与 NADPH 结合的位点。通过同源性和对接建模，有四个残基（Q29、K264、N267 和 R270）被认为与 NADPH 的 2'-磷酸直接相互作用。其中，两个带正电荷的残基（K264 和 R270）被实验证明是 NADPH 2'-磷酸结合所必需的。与野生型酶相比，突变酶（Q29E）的对映体过量值得到了提高。
