Abstract：
The anthranilic diamide insecticide chlorantraniliprole has been extensively applied to control Lepidoptera pests. However, its overuse leads to the development of resistance and accumulation of residue in the environment. Four P450s (CYP6CV5, CYP9A68, CYP321F3, and CYP324A12) were first found to be constitutively overexpressed in an SSB CAP-resistant strain. It is imperative to further elucidate the molecular mechanisms underlying P450s-mediated CAP resistance for mitigating its environmental contamination. Here, we heterologously expressed these four P450s in insect cells and evaluated their abilities to metabolize CAP. Western blotting and reduced CO difference spectrum tests showed that these four P450 proteins had been successfully expressed in Sf9 cells, which are indicative of active functional enzymes. The recombinant proteins CYP6CV5, CYP9A68, CYP321F3, and CYP324A12 exhibited a preference for metabolizing the fluorescent P450 model probe substrates EC, BFC, EFC, and EC with enzyme activities of 0.54, 0.67, 0.57, and 0.46 pmol/min/pmol P450, respectively. In vitro metabolism revealed distinct CAP metabolic rates (0.97, 0.86, 0.75, and 0.55 pmol/min/pmol P450) and efficiencies (0.45, 0.37, 0.30, and 0.17) of the four recombinant P450 enzymes, thereby elucidating different protein catalytic activities. Furthermore, molecular model docking confirmed metabolic differences and efficiencies of these P450s and unveiled the hydroxylation reaction in generating N-demethylation and methylphenyl hydroxylation during CAP metabolism. Our findings not only first provide new insights into the mechanisms of P450s-mediated metabolic resistance to CAP at the protein level in SSB but also demonstrate significant differences in the capacities of multiple P450s for insecticide degradation and facilitate the evaluation and mitigation of toxic risks associated with CAP application in the environment.
摘要：
邻氨基二酰胺类杀虫剂氯antraniliprole已广泛应用于防治鳞翅目害虫。然而,它的过度使用导致环境中的抗性发展和残留物积累。首先发现四个P450（CYP6CV5，CYP9A68，CYP321F3和CYP324A12）在SSBCAP抗性菌株中组成性过表达。必须进一步阐明P450介导的CAP抗性的分子机制，以减轻其环境污染。这里,我们在昆虫细胞中异源表达了这四个P450，并评估了它们代谢CAP的能力。Westernblotting和还原型CO差异谱试验表明,这4种P450蛋白已在Sf9细胞中成功表达,这表明有活性的功能酶。重组蛋白CYP6CV5，CYP9A68，CYP321F3和CYP324A12表现出对代谢荧光P450模型探针底物EC的偏好，BFC,EFC,和EC的酶活性分别为0.54、0.67、0.57和0.46pmol/min/pmolP450。体外代谢显示四种重组P450酶的不同CAP代谢率（0.97、0.86、0.75和0.55pmol/min/pmolP450）和效率（0.45、0.37、0.30和0.17），从而阐明不同的蛋白质催化活性。此外，分子模型对接证实了这些P450的代谢差异和效率，并揭示了在CAP代谢过程中产生N-去甲基化和甲基苯基羟基化的羟基化反应。我们的研究结果不仅为SSB中P450介导的对CAP的代谢抗性在蛋白质水平上的机制提供了新的见解，而且还证明了多个P450对杀虫剂降解的能力存在显着差异，并有助于评估和减轻与环境中CAP应用相关的毒性风险。
