Roles of the variable P450 substrate recognition sites SRS1 and SRS6 in esfenvalerate metabolism by CYP6AE subfamily enzymes in Helicoverpa armigera
Roles of the variable P450 substrate recognition sites SRS1 and SRS6 in esfenvalerate metabolism by CYP6AE subfamily enzymes in Helicoverpa armigera
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可变 P450 底物识别位点 SRS1 和 SRS6 在棉铃虫 CYP6AE 亚家族酶 Esfenvalerate 代谢中的作用
DOI：
10.1016/j.ibmb.2020.103486
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发表时间：
2020
影响因子：
3.8
通讯作者：
Yidong Wu
中科院分区：
文献类型：
--
作者：
Yu Shi;Andrias O. O’Reilly;Shuo Sun;Qiong Qu;Yihua Yang;Yidong Wu
聚簇CYP 6AE亚家族的棉铃虫P450具有高序列同一性，但在代谢异生物质，特别是右旋氰戊菊酯的能力方面存在显著差异。其中，CYP 6AE 17与CYP 6AE 18具有最高的序列同一性，但显示出高107倍的代谢效率。CYP 6AE 11对顺式氰戊菊酯最具活性，但CYP 6AE 20无活性，尽管酶具有54.8%的序列同一性。序列分析表明，CYP 6AE 17和CYP 6AE 18之间的SRS 1（底物识别位点）和SRS 6，以及CYP 6AE 11和CYP 6AE 20之间的SRS 1是所有6个SRSs中变异最大的。为了确定所观察到的催化差异的关键因素，我们在两对P450之间交换了这些SRS序列，并研究了所得杂交突变体或嵌合体的活性。体外代谢表明，CYP 6AE 17/18嵌合体对顺式氰戊菊酯的活性降低2倍和10倍，CYP 6AE 18/17嵌合体对顺式氰戊菊酯的活性增加6倍和10倍。同时，交换SRS 1后，CYP 6AE 11/20嵌合体错误折叠，但CYP 6AE 20/11嵌合体获得了对顺式氰戊菊酯的中等活性。分子模拟表明，SRS 1或SRS 6内的氨基酸变体改变了活性位点的形状和化学环境，这可能会影响配体结合相互作用。这些结果表明，由SSR序列多样性引起的蛋白质结构变异促进了昆虫化学防御的进化，并有助于昆虫对农药抗性的发展。
The cotton bollworm P450s of the clustered CYP6AE subfamily share high sequence identities but differ dramatically in their capacity to metabolize xenobiotics, especially esfenvalerate. Among them, CYP6AE17 has the highest sequence identity with CYP6AE18 but shows ∼7-fold higher metabolic efficiency. CYP6AE11 is most active towards esfenvalerate but CYP6AE20 is inactive even though the enzymes share 54.8% sequence identity. Sequence analysis revealed the SRS1 (Substrate Recognition Site) and SRS6 between CYP6AE17 and CYP6AE18, and SRS1 between CYP6AE11 and CYP6AE20 are the most variable among all six SRSs. In order to identify the key factors that underlie the observed catalytic difference, we exchanged these SRS sequences between two pairs of P450s and studied the activity of the resulting hybrid mutants or chimeras. In vitro metabolism showed that the CYP6AE17/18 chimeras had 2- and 10-fold decreased activities and the CYP6AE18/17 chimeras had 6- and 10-fold increased activities to esfenvalerate. Meanwhile, after exchanging SRS1 with each other, the CYP6AE11/20 chimera folded incorrectly but the CYP6AE20/11 chimera gained moderate activity to esfenvalerate. Molecular modelling showed that amino acids variants within SRS1 or SRS6 change the shape and chemical environment of the active sites, which may affect the ligand-binding interactions. These results indicate that the protein structure variation resulting from the sequence diversity of SRSs promotes the evolution of insect chemical defense and contributes to the development of insect resistance to pesticides.