title: Active site analysis of cis-epoxysuccinate hydrolase from Nocardia tartaricans using homology modeling and site-directed mutagenesis.
abstract: Cis-epoxysuccinate hydrolase (CESH, EC 3.3.2.3) from Nocardia tartaricans is known to catalyze the opening of an epoxide ring of cis-epoxysuccinate (CES), thereby converting it to corresponding vicinal diol, L(+)-tartaric acid. An attempt has been made to build a 3D homology model of CESH to investigate the structure-function relationship, and also to understand the mechanism of the enzymatic reaction. Using a combination of molecular-docking simulation and multiple sequence alignment, a set of putative residues that are involved in the CESH catalysis has been identified. Functional roles of these putative active-site residues were further evaluated by site-directed mutagenesis. Interestingly, the mutants D18A, D18E, Q20E, T22A, R55E, N134D, K164A, H190A, H190N, H190Q, D193A, and D193E resulted in complete loss of activity, whereas the mutants Y58F, T133A, S189A, and Y192D retained partial enzyme activity. Furthermore, the active-site residues responsible for the opening of CES were analyzed, and the mechanism underlying the catalytic triad involved in L(+)-tartaric acid biosynthesis was proposed.
authors: Centre for Biotechnology, Anna University, Guindy, Chennai, India.
authors: Vinayagam
authors: V
authors: Vasu
authors: Jayaraman
authors: J
authors: Kumaresan
authors: Manoharan Ganesh
authors: MG
authors: Babu
authors: Sankaranarayanan
authors: S
authors: Meenakshisundaram
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D020134:Catalytic Domain; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009615:Nocardia / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D006867:Hydrolases; C492296:cis-epoxysuccinate hydrolase, Nocardia tartaricans