title: High yield recombinant expression, characterization and homology modeling of two types of cis-epoxysuccinic acid hydrolases.
abstract: The cis-epoxysuccinate hydrolases (CESHs), members of epoxide hydrolase, catalyze cis-epoxysuccinic acid hydrolysis to form D: (-)-tartaric acid or L: (+)-tartaric acid which are important chemicals with broad scientific and industrial applications. Two types of CESHs (CESH[D: ] and CESH[L: ], producing D: (-)- and L: (+)-tartaric acids, respectively) have been reported with low yield and complicated purification procedure in previous studies. In this paper, the two CESHs were overexpressed in Escherichia coli using codon-optimized genes. High protein yields by one-step purifications were obtained for both recombinant enzymes. The optimal pH and temperature were measured for both recombinant CESHs, and the properties of recombinant enzymes were similar to native enzymes. Kinetics parameters measured by Lineweaver-Burk plot indicates both enzymes exhibited similar affinity to cis-epoxysuccinic acid, but CESH[L: ] showed much higher catalytic efficiency than CESH[D: ], suggesting that the two CESHs have different catalytic mechanisms. The structures of both CESHs constructed by homology modeling indicated that CESH[L: ] and CESH[D: ] have different structural folds and potential active site residues. CESH[L: ] adopted a typical α/β-hydrolase fold with a cap domain and a core domain, whereas CESH[D: ] possessed a unique TIM barrel fold composed of 8 α-helices and 8 β-strands, and 2 extra short α-helices exist on the top and bottom of the barrel, respectively. A divalent metal ion, preferred to be zinc, was found in CESH[D: ], and the ion was proved to be crucial to the enzymatic activity. These results provide structural insight into the different catalytic mechanisms of the two CESHs.
authors.affiliation: Shandong Provincial Key Laboratory of Energy Genetics, Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao, 266101, China.
authors.forename: Gu-Zhen
authors.initials: GZ
authors.lastname: Cui
authors.forename: Shan
authors.initials: S
authors.lastname: Wang
authors.forename: Yifei
authors.initials: Y
authors.lastname: Li
authors.forename: Yi-Jun
authors.initials: YJ
authors.lastname: Tian
authors.forename: Yingang
authors.initials: Y
authors.lastname: Feng
authors.forename: Qiu
authors.initials: Q
authors.lastname: Cui
mesh_terms: D001884:Bordetella / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D004851:Epoxide Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004852:Epoxy Compounds / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008958:Models, Molecular; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012240:Rhodococcus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D019802:Succinic Acid / Q000737:chemistry / Q000378:metabolism*; D013644:Tartrates / Q000378:metabolism
chemical_list: D004852:Epoxy Compounds; D011994:Recombinant Proteins; D013644:Tartrates; D019802:Succinic Acid; D004851:Epoxide Hydrolases; C029768:tartaric acid