Key Amino Acid Associated with Acephate Detoxification by Cydia pomonella Carboxylesterase Based on Molecular Dynamics with Alanine Scanning and Site-Directed Mutagenesis
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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DOI：
10.1021/ci500159q
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发表时间：
2014-05-01
影响因子：
5.6
通讯作者：
Zhang, Ya Lin
中科院分区：
文献类型：
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作者：
Yang, Xue Qing;Liu, Ji Yuan;Zhang, Ya Lin
杀虫剂解毒羧酸酯酶（CE）基因CpCE-1从苹果蠹蛾中克隆。分子动力学(MD)模拟和计算丙氨酸扫描(CAS)表明CpCE-1中的Asn 232构成了近似的结合热点，其结合自由能差(Delta Delta G(bind))值为3.66 kcal/mol。 N232A的催化效率（kcat/km）急剧下降，半抑菌浓度（IC50）值增加230倍以上。体外代谢测定表明，乙酰甲胺磷可以被野生型CpCE-1代谢，而N232A突变不能代谢乙酰甲胺磷，这表明热点Asn 232是乙酰甲胺磷代谢的关键残基。突变检测表明，欧洲田间菌株中 Asn 232 替换发生频率较低。我们的MD、CAS、定点突变和代谢研究引入了一个新的氨基酸残基Asn 232，该氨基酸残基参与CpCE-1对乙酰甲胺磷的代谢，该方法在杀虫剂抗性机制研究和与特定生理生化功能相关的蛋白质中关键氨基酸的预测中是可靠的。
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 (Delta Delta G(bind)) 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.