Cloning, homology modeling, and reaction mechanism analysis of a novel cis-epoxysuccinate hydrolase from Klebsiella sp.

被引：9

作者：

Cheng, Yongqing ^{[1
]}

Pan, Haifeng ^{[1
,2
]}

Bao, Wenna ^{[2
]}

Sun, Weirong ^{[2
]}

Xie, Zhipeng ^{[1
,2
]}

Zhang, Jianguo ^{[1
,2
]}

Zhao, Yuhua ^{[3
]}

机构：

[1] Zhejiang Univ, Coll Life Sci, Inst Biochem, Hangzhou 310058, Zhejiang, Peoples R China

[2] Hangzhou Bioking Biochem Engn Co Ltd, Hangzhou 311106, Zhejiang, Peoples R China

[3] Zhejiang Univ, Inst Microbiol, Coll Life Sci, Hangzhou 310058, Zhejiang, Peoples R China

来源：

BIOTECHNOLOGY LETTERS | 2014年 / 36卷 / 12期

基金：

中国国家自然科学基金;

关键词：

Catalytic mechanism; cis-epoxysuccinate hydrolase; Epoxide hydrolase; Homology modeling; Klebsiella sp; L(+)-Tartaric acid; CRYSTAL-STRUCTURES; L-2-HALOACID DEHALOGENASE; EPOXIDE HYDROLASE; ACID;

D O I：

10.1007/s10529-014-1638-7

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

The gene encoding a novel cis-epoxysuccinate hydrolase, which hydrolyzes cis-epoxysuccinate to l (+)-tartaric acid, was cloned from Klebsiella sp. BK-58 and expressed in Escherichia coli. The ORF was 825 bp encoding a mature protein of 274 amino acids with a molecular mass of 30.1 kDa. Multiple sequence alignment showed that the enzyme belonged to the haloacid dehalogenase-like super family. Homology modeling and site-directed mutagenesis were performed to investigate the structural characteristics of the enzyme. Its overall structure consisted of a core domain formed by six-stranded parallel beta-sheets flanked by seven alpha-helices and a subdomain that had a four helix bundle structure. Residues D48, T52, R85, N165, K195, Y201, A219, H221, and D224 were catalytically important forming the active pocket between the two domains. An O-18-labeling study suggested that the catalytic reaction of the enzyme proceeded through a two-step mechanism.

引用

页码：2537 / 2544

页数：8

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