Nuñez M F, Pellicer M T, Badia J, Aguilar J, Baldoma L
Department of Biochemistry, Faculty of Pharmacy, University of Barcelona, 08028 Barcelona, Spain.
Biochem J. 2001 Mar 15;354(Pt 3):707-15. doi: 10.1042/0264-6021:3540707.
Glyoxylate is an important intermediate of the central microbial metabolism formed from acetate, allantoin or glycolate. Depending on the physiological conditions, glyoxylate is incorporated into the central metabolism by the combined actions of the activity of malate synthase and the D-glycerate pathway, or alternatively it can be reduced to glycolate by constitutive glyoxylate reductase activity. At present no information is available on this latter enzyme in Escherichia coli, although similar enzymes, classified as 2-hydroxyacid dehydrogenases, have been characterized in other organisms. A BLAST search using as the query sequence the hydroxypyruvate/glyoxylate reductase from Cucumis sativus identified as an orthologue the yiaE gene of E. coli encoding a ketoaldonate reductase. Use of this sequence in a subsequent BLAST search yielded the ycdW gene as a good candidate to encode glyoxylate reductase in this bacterium. Cloning and overexpression of the ycdW gene showed that its product displayed a high NADPH-linked glyoxylate reductase activity, and also catalysed the reduction of hydroxypyruvate with a lower efficiency. Disruption of the ycdW gene by a chloramphenicol acetyltransferase ('CAT') cassette did not totally abolish the glyoxylate reductase activity, indicating that another enzyme accomplished this function. The similarity with YiaE led us to test whether this protein was responsible for the remaining glyoxylate reductase activity. Purification of YcdW and YiaE proteins permitted their kinetic characterization and comparison. Analysis of the catalytic power (k(cat)/K(m)) disclosed a higher ratio of YcdW for glyoxylate and of YiaE for hydroxypyruvate.
乙醛酸是由乙酸、尿囊素或乙醇酸形成的微生物中心代谢的重要中间体。根据生理条件，乙醛酸通过苹果酸合酶活性和D-甘油酸途径的联合作用被纳入中心代谢，或者它可以通过组成型乙醛酸还原酶活性被还原为乙醇酸。目前在大肠杆菌中没有关于后一种酶的信息，尽管在其他生物体中已经鉴定出了类似的酶，归类为2-羟基酸脱氢酶。使用来自黄瓜的羟基丙酮酸/乙醛酸还原酶作为查询序列进行BLAST搜索，鉴定出大肠杆菌的yiaE基因是一种酮醛酸还原酶的直系同源物。在随后的BLAST搜索中使用该序列，得到ycdW基因是该细菌中编码乙醛酸还原酶的一个良好候选基因。ycdW基因的克隆和过表达表明其产物具有高NADPH连接的乙醛酸还原酶活性，并且还以较低效率催化羟基丙酮酸的还原。用氯霉素乙酰转移酶（“CAT”）盒破坏ycdW基因并没有完全消除乙醛酸还原酶活性，这表明另一种酶完成了该功能。与YiaE的相似性使我们测试该蛋白质是否负责剩余的乙醛酸还原酶活性。YcdW和YiaE蛋白的纯化允许对它们进行动力学表征和比较。催化能力（k(cat)/K(m)）分析表明，YcdW对乙醛酸的比例较高，而YiaE对羟基丙酮酸的比例较高。
