Active site analysis of <Emphasis Type="Italic">cis</Emphasis>-epoxysuccinate hydrolase from <Emphasis Type="Italic">Nocardia tartaricans</Emphasis> using homology modeling and site-directed mutagenesis |
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Authors: | Vinayagam?Vasu Jayaraman?Kumaresan Manoharan?Ganesh?Babu Email author" target="_blank">Sankaranarayanan?MeenakshisundaramEmail author |
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Institution: | (1) Centre for Biotechnology, Anna University, Guindy, Chennai, 600 025, India; |
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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. |
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