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论文类型：期刊论文

发表时间：2017-10-01

发表刊物：FEBS LETTERS

收录刊物：Scopus、SCIE、PubMed

卷号：591

期号：20

页面范围：3402-3413

ISSN号：1873-3468

关键字：meso-2; 3-butanediol dehydrogenase; rational design; short-chain dehydrogenase/reductase

摘要：Meso-2,3-butanediol dehydrogenase (meso-2,3-BDH) catalyzes NAD(+)-dependent conversion of meso-2,3-butanediol to acetoin, a crucial external energy storage molecule in fermentive bacteria. In this study, the active tunnel of meso-2,3-BDH was identified. The two short helixes positioned away from the 4-helix possibly expose the hydrophobic ligand-binding cavity, gating the exit of product and cofactor from the activity pocket. Further MM/GBSA-binding free energy analysis shows that Phe212 and Asn146 function as the key product-release sites. Site-directed mutagenesis experiments targeted to the sites show that the k(cat) of Phe212Tyr is enhanced up to (4-8)-fold. The original activity of Asn146Gln is retained, but the activity of Asn146Ala mutation is lost. These results could provide helpful guidance on rational design of short-chain dehydrogenases/reductases.