title: Analysis of essential amino acid residues for catalytic activity of cis-epoxysuccinate hydrolase from Bordetella sp. BK-52.
abstract: cis-Epoxysuccinate hydrolase (CESH) from Bordetella sp. BK-52, an epoxide hydrolase (EH), catalyzes the stereospecific hydrolysis of cis-epoxysuccinate to D(-)-tartrate. The enzyme, which shows no homology to other reported EHs, belongs to the DUF849 superfamily of prokaryotic proteins, which have unknown function. Metal composition analysis revealed that the CESH is a Zn(2+)-dependent enzyme with an approximately 1:1 molar ratio of zinc to enzyme. The results of an (18)O-labeling study suggest that the enzyme catalyzes epoxide hydrolysis by means of a one-step mechanism. We evaluated the relationship between the structure and function of the enzyme by means of sequence alignment, modeling, substrate binding, and reaction kinetics studies. The CESH has a canonical (β/α)₈ TIM barrel fold, and we used site-directed mutagenesis to identify eight residues (H47, H49, R51, T82, Y138, N140, W164, and D251) as being localized to the active site or highly conserved. On the basis of these results and theoretical considerations, we identified H47 and H49 as zinc-binding ligands, and we propose that a zinc atom and R51, T82, Y138, N140, W164, and D251 are the catalytic residues and participate in substrate binding. In summary, the structure and catalytic mechanism of the CESH from Bordetella sp. BK-52 differ from those of classic EHs, which have an α/β hydrolase fold, act via a two-step catalytic mechanism, and do not require cofactors, prosthetic groups, or metal ions.
authors.affiliation: Institute of Biochemistry, College of Life Science, Zhejiang University, Hangzhou, 310058, China.
authors.forename: Wenna
authors.initials: W
authors.lastname: Bao
authors.forename: Haifeng
authors.initials: H
authors.lastname: Pan
authors.forename: Zhenhong
authors.initials: Z
authors.lastname: Zhang
authors.forename: Yongqing
authors.initials: Y
authors.lastname: Cheng
authors.forename: Zhipeng
authors.initials: Z
authors.lastname: Xie
authors.forename: Jianguo
authors.initials: J
authors.lastname: Zhang
authors.forename: Yongquan
authors.initials: Y
authors.lastname: Li
mesh_terms: D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism*; D001884:Bordetella / Q000201:enzymology*; D002384:Catalysis; D006867:Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics
chemical_list: D000596:Amino Acids; D006867:Hydrolases