title: Cloning, homology modeling, and reaction mechanism analysis of a novel cis-epoxysuccinate hydrolase from Klebsiella sp.
abstract: 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 β-sheets flanked by seven α-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 (18)O-labeling study suggested that the catalytic reaction of the enzyme proceeded through a two-step mechanism.
authors.affiliation: Institute of Biochemistry, College of Life Science, Zhejiang University, Hangzhou, 310058, Zhejiang, China.
authors.forename: Yongqing
authors.initials: Y
authors.lastname: Cheng
authors.forename: Haifeng
authors.initials: H
authors.lastname: Pan
authors.forename: Wenna
authors.initials: W
authors.lastname: Bao
authors.forename: Weirong
authors.initials: W
authors.lastname: Sun
authors.forename: Zhipeng
authors.initials: Z
authors.lastname: Xie
authors.forename: Jianguo
authors.initials: J
authors.lastname: Zhang
authors.forename: Yuhua
authors.initials: Y
authors.lastname: Zhao
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004852:Epoxy Compounds / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007709:Klebsiella / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013386:Succinates / Q000378:metabolism; D013644:Tartrates / Q000378:metabolism
chemical_list: D004852:Epoxy Compounds; D011994:Recombinant Proteins; D013386:Succinates; D013644:Tartrates; D006867:Hydrolases; C029768:tartaric acid