title: Key amino acid associated with acephate detoxification by Cydia pomonella carboxylesterase based on molecular dynamics with alanine scanning and site-directed mutagenesis.
abstract: 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.
authors.affiliation: 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.
authors.forename: Xue Qing
authors.initials: XQ
authors.lastname: Yang
authors.forename: Ji Yuan
authors.initials: JY
authors.lastname: Liu
authors.forename: Xian Chun
authors.initials: XC
authors.lastname: Li
authors.forename: Mao Hua
authors.initials: MH
authors.lastname: Chen
authors.forename: Ya Lin
authors.initials: YL
authors.lastname: Zhang
mesh_terms: D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D043182:Carboxylesterase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D007700:Kinetics; D007915:Lepidoptera / Q000201:enzymology*; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009946:Organothiophosphorus Compounds / Q000378:metabolism* / Q000633:toxicity; D063088:Phosphoramides / Q000378:metabolism* / Q000633:toxicity; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013816:Thermodynamics
chemical_list: D004791:Enzyme Inhibitors; D009946:Organothiophosphorus Compounds; D063088:Phosphoramides; C001969:acephate; D043182:Carboxylesterase; D000409:Alanine