Key Laboratory of Plant Protection Resources & Pest Management of Ministry of Education and §Key Laboratory of Crop Pest Integrated Pest Management on the Loess Plateau of Ministry of Agriculture, Northwest A & F University , Yangling 712100, Shaanxi, China.
J Chem Inf Model. 2014 May 27;54(5):1356-70. doi: 10.1021/ci500159q. Epub 2014 May 1.
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 (ΔΔGbind) 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.
从苹果蠹蛾（Cydia pomonella）中克隆了杀虫剂解毒羧酸酯酶（CE）基因 CpCE-1。分子动力学（MD）模拟和计算丙氨酸扫描（CAS）表明，CpCE-1 中的天冬酰胺 232 构成了一个近似结合热点，其结合自由能差异（ΔΔGbind）值为 3.66kcal/mol。N232A 的催化效率（kcat/km）显著下降，半抑制浓度（IC50）值增加了 230 多倍。体外代谢测定表明，野生型 CpCE-1 可以代谢乙酰甲胺磷，而 N232A 突变则无法代谢乙酰甲胺磷，这表明热点天冬酰胺 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 N232 替换的频率较低。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天冬酰胺 232，它与 CpCE-1 一起参与了乙酰甲胺磷的代谢，这种方法可用于研究杀虫剂抗性机制和预测与特定生理生化功能相关的蛋白质中的关键氨基酸。
