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

Vinayagam Vasu, Jayaraman Kumaresan, Manoharan Ganesh Babu, Sankaranarayanan Meenakshisundaram*

*Corresponding author for this work

Research output: Contribution to journalArticleResearchpeer-review

10 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)2377-2386
Number of pages10
JournalApplied Microbiology and Biotechnology
Volume93
Issue number6
DOIs
Publication statusPublished - Mar 2012
Externally publishedYes

Keywords

  • Active site
  • Cis-epoxysuccinate hydrolase
  • Homology modeling
  • Nocardia tartaricans
  • Site-directed mutagenesis

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