Study On The Molecular Mechailism Of The Catalysis By Cjs-epoxysuccinate Hydrolase From Bordetella Sp.BK-52

D(-)-tartaric acid, mainly used in chemical and pharmacy industry, is an important chiral auxiliary reagent, resolving reagent, food additive and nutritional feed additive. Chemical resolution is the traditional method to produce D(-)-tartaric acid. However, owning to its economic and environmental friendliness, microbial transformation has become an inevitable development trend, namely the transformation cis-epoxysuccinic acid into D(-)-tartaric acid by the microorganism which contains cis-epoxysuccinate hydrolase (CESH).Genus Pseudomonas, Alcaligenes and Bordetella have been reported for the production of D(-)-tartaric acid, but only the CESH gene from Alcaligenes and Bordetella were reported, which is also restricted to the primary structure level. There is no report on its further study from molecular structure and reaction mechanism. Therefore, this study is first to reveal the3D structure and catalytic mechanisms of CESH from Bordetella sp. BK-52by means of multiple sequence alignment, homology modeling, docking study, site-directed mutagenesis, characterization enzymes, inductively coupled plasma mass spectrometry and isotopic labeling experiment. The results of this study were summarized as follows:1. It showed that the CESH belongs to prokaryotic protein of unknown function (DUF849) superfamily by means of multiple sequence alignment and secondary structure analysis.2. It showed that the CESH has a predicted canonical (β/a)8TIM barrel fold, and the active site is situated in the central β-barrel by means of homology modeling and docking study.3. It showed that the CESH is a Zn2-dependent enzyme with about1molar ratio of zinc to enzyme by means of metal chelating agent test and inductively coupled plasma mass spectrometry analysis.4. It showed that the following8amino acids play important roles in the catalysis of CESH, His47, His49, Arg51, Thr82, Tyrl38, Asn140, Trp164and Asp251by means of site-directed mutagenesis, circular dichroism spectra, enantioselectivity analysis and kinetic analysis of totally27conserved sites. The distances between the substrate and the active sites, and the distances between each active site were calculated according to the result of homology modeling and docking study.5. Zinc may act as Lewis acid in the CESH and it showed that His47and His49were identified as zinc binding ligands by means of inductively coupled plasma mass spectrometry analysis.6. H218O-labelling study suggested that an oxygen atom18O from a water molecule was directly transferred to the product and a Zn2+-dependent one-step mechanism was proposed for the CESH according to the results of homology modeling, docking study and site-directed mutagenesis.7. CESH belongs to epoxide hydrolase (EH), but the CESH was remarkably different from most of previously reported EHs, which have an a/β hydrolase fold, act via a two-step catalytic mechanism and require neither cofactors, prosthetic groups nor metal ions, revealing that it’s a novel EH.