Active site analysis of cis-epoxysuccinate hydrolase from Nocardia tartaricans using homology modeling and site-directed mutagenesis

• Published in: Applied microbiology and biotechnology 2012-03, Vol.93 (6), p.2377-2386
• Main Authors: Vasu, Vinayagam, Kumaresan, Jayaraman, Babu, Manoharan Ganesh, Meenakshisundaram, Sankaranarayanan
• Format: Article
• Language: English
• Subjects: Acids; Amino Acid Sequence; Analysis; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biomedical and Life Sciences; Biosynthesis; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Catalysis; Catalytic Domain; Catalytic oxidation; Cloning; DNA polymerase; E coli; Enzymatic activity; Enzyme kinetics; Enzymes; Gene expression; Gram-positive bacteria; Hydrolases; Hydrolases - chemistry; Hydrolases - genetics; Hydrolases - metabolism; Kinetics; Life Sciences; Microbial Genetics and Genomics; Microbiology; Microorganisms; Models, Molecular; Molecular Sequence Data; Mutagenesis; Mutagenesis, Site-Directed; Nocardia; Nocardia - chemistry; Nocardia - enzymology; Nocardia - genetics; Oxidation; Physiological aspects; Sequence Alignment; Studies; Thermal cycling
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-011-3548-0

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.