Institute of Biochemistry, College of Life Science, Zhejiang University, Hangzhou, 310058, China.
Appl Microbiol Biotechnol. 2014 Feb;98(4):1641-9. doi: 10.1007/s00253-013-5019-2. Epub 2013 Jun 14.
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.
布鲁氏菌 BK-52 来源的顺式-环氧琥珀酸水解酶（CESH）是一种环氧化物水解酶（EH），能够立体专一性地水解顺式-环氧琥珀酸生成 D(-)-酒石酸。该酶与其他已报道的 EH 没有同源性，属于未知功能的 DUF849 超家族原核蛋白。金属组成分析表明，CESH 是一种依赖 Zn(2+)的酶，锌与酶的摩尔比约为 1:1。¹⁸O 标记研究的结果表明，该酶通过一步机制催化环氧化物水解。我们通过序列比对、建模、底物结合和反应动力学研究评估了该酶的结构与功能之间的关系。CESH 具有典型的（β/α）₈ TIM 桶折叠结构，我们通过定点突变鉴定了八个残基（H47、H49、R51、T82、Y138、N140、W164 和 D251）定位于活性位点或高度保守。基于这些结果和理论考虑，我们鉴定 H47 和 H49 为锌结合配体，并提出锌原子以及 R51、T82、Y138、N140、W164 和 D251 是催化残基，并参与底物结合。综上所述，布鲁氏菌 BK-52 来源的 CESH 的结构和催化机制与具有α/β水解酶折叠结构、通过两步催化机制发挥作用且不需要辅因子、辅酶或金属离子的经典 EH 不同。
