Key Amino Acid Associated with Acephate Detoxification by Cydia pomonella Carboxylesterase Based on Molecular Dynamics with Alanine Scanning and Site-Directed Mutagenesis
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- 作者：Xue Qing Yang ; Ji Yuan Liu ; Xian Chun Li ; Mao Hua Chen ; Ya Lin Zhang
- 刊名：Journal of Chemical Information and Modeling
- 出版年：2014
- 出版时间：May 27, 2014
- 年：2014
- 卷：54
- 期：5
- 页码：1356-1370
- 全文大小：751K
- 年卷期：v.54,no.5(May 27, 2014)
- ISSN：1549-960X
文摘
Insecticide-detoxifying carboxylesterase (CE) gene CpCE-1 was cloned from Cydia pomonella. Molecular dynamics (MD) simulation and computational alanine scanning (CAS) indicate that Asn 232 in CpCE-1 constitutes an approximate binding hot-spot with a binding free energy difference (螖螖Gbind) value of 3.66 kcal/mol. The catalytic efficiency (kcat/km) of N232A declined dramatically, and the half inhibitory concentrations (IC50) value increased by more than 230-fold. Metabolism assay in vitro reveals that the acephate could be metabolized by wild CpCE-1, whereas N232A mutation is unable to metabolize the acephate, which suggests that the hot-spot Asn 232 is a crucial residue for acephate metabolism. Mutation detection suggests that low frequency of Asn 232 replacement occurred in Europe field strains. Our MD, CAS, site-directed mutagenesis, and metabolism studies introduce a new amino acid residue Asn 232 involved in the metabolism of the acephate with CpCE-1, and this method is reliable in insecticide resistance mechanism research and prediction of key amino acids in a protein which is associated with specific physiological and biochemical functions.