Habrych Malgorzata, Rodriguez Sonia, Stewart Jon D
Department of Chemistry, University of Florida, Gainesville 32611, USA.
Biotechnol Prog. 2002 Mar-Apr;18(2):257-61. doi: 10.1021/bp0101841.
An NADPH-dependent enzyme that reduces ethyl 2-methylacetoacetate stereoselectively to ethyl (2R)-methyl-(3S)-hydroxybutanoate was purified 730-fold from Escherichia coli. The N-terminal amino acid sequence data obtained from the purified reductase were used to search the E. coli genome, and a single match was found at the start of the yqhE open reading frame. The YqhE protein had been identified previously by Yum et al. as a 2,5-diketo-D-gluconate reductase on the basis of sequence similarity to other bacterial homologues [Yum, D.-Y.; Lee, B.-Y.; Pan, J.-G. Appl.Environ. Microbiol. 1999, 65, 3341-3346]; however, it had not been examined for beta-keto ester reductions. Our results thus link a key enzyme in the microbial production of ascorbate with stereoselective beta-keto ester reductions, two important fields in biocatalysis. The purified YqhE reductase accepts ethyl acetoacetate and a variety of 2-substituted derivatives, and its sequence is similar to other aldose reductase superfamily members that also reduce alpha-substituted beta-keto esters to syn-(2R,3S) alcohols.
一种依赖烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的酶可将2-甲基乙酰乙酸乙酯立体选择性地还原为（2R）-甲基-（3S）-羟基丁酸乙酯，该酶从大肠杆菌中纯化出来，纯化倍数达730倍。从纯化的还原酶获得的N端氨基酸序列数据用于搜索大肠杆菌基因组，在yqhE开放阅读框起始处发现了唯一匹配。Yum等人先前已根据与其他细菌同源物的序列相似性将YqhE蛋白鉴定为2,5-二酮-D-葡萄糖酸还原酶[Yum, D.-Y.; Lee, B.-Y.; Pan, J.-G. Appl.Environ. Microbiol. 1999, 65, 3341-3346]；然而，尚未对其进行β-酮酯还原的研究。因此，我们的结果将微生物生产抗坏血酸中的一种关键酶与立体选择性β-酮酯还原联系起来，这是生物催化中的两个重要领域。纯化的YqhE还原酶可接受乙酰乙酸乙酯和多种2-取代衍生物，其序列与其他醛糖还原酶超家族成员相似，这些成员也将α-取代的β-酮酯还原为顺式-（2R,3S）醇。
