Single amino acid variations drive functional divergence of cytochrome P450s in Helicoverpa species

• Published in: Insect biochemistry and molecular biology Vol. 146; p. 103796
• Main Authors: Shi, Yu, Sun, Shuo, Zhang, Yujun, He, Yingshi, Du, Minghong, ÓReilly, Andrias O., Wu, Shuwen, Yang, Yihua, Wu, Yidong
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.07.2022
• Subjects: Cytochrome P450; Detoxification; Functional divergence; Molecular modelling; Xanthotoxin; Functional divergence; Molecular modelling; Cytochrome P450; Xanthotoxin; Detoxification
• ISSN: 0965-1748; 1879-0240
• DOI: 10.1016/j.ibmb.2022.103796

Divergence of gene function is a hallmark of evolution, but assessing such divergence in one species or between species requires information on functional alterations of the alleles and homologs. Here, we explore the functional divergence of two paralogs, CYP6AE19 and CYP6AE20, from Helicoverpa armigera, and two close orthologs, CYP6B8 and CYP6B7, from two related species (Helicoverpa zea and H. armigera); although there is high sequence identity within each pair of enzymes, the latter P450 of each pair has lost metabolic competence towards the plant allelochemical xanthotoxin. Multiple chimeric and single/double site mutants were created by exchanging the diverse substrate recognition sites (SRSs) and amino acids within each pair of P450s. Heterologous expression in Sf9 cells and in vitro metabolism studies showed that the exchange of SRS4 swapped the activity of CYP6AE19 and CYP6AE20, and subsequent site-directed mutagenesis demonstrated that the CYP6AE20 V318M substitution causes a gain-of-function towards xanthotoxin. Meanwhile, a single amino acid substitution (L489P) in SRS6 was found to swap activity between the CYP6B orthologs. Sequence alignments of CYP6AE paralogs and all reported insect xanthotoxin-metabolizing P450s suggest M318 and P489 are essential for the catalytic activities of CYP6AE paralogs and CYP6B orthologs, respectively, but P450s in different subfamilies may have different mechanisms towards the same substrate. Our findings demonstrate that a single amino acid substitution can suffice to alter substrate metabolism and this functional divergence resulting from natural mutations will help to further our understanding of the process of natural selection of P450 genes and their role in insect-host plant interactions. [Display omitted] •Two pairs of P450 paralogs and orthologs display significant differences in xanthotoxin metabolism.•Single amino acid substitution (V318M) in SRS4 causes a gain-of-function of CYP6AE20.•Single amino acid substitution (L489P) in SRS6 swaps activity between CYP6B7 and CYP6B8.•M318 and P489 are essential for the catalytic activities of CYP6AE paralogs and CYP6B orthologs, respectively.•P450s in different subfamilies may have different mechanisms towards the same substrate.