Single amino acid variations drive functional divergence of cytochrome P450s in Helicoverpa species
Single amino acid variations drive functional divergence of cytochrome P450s in Helicoverpa species
复制标题
单一氨基酸变异导致 Helicoverpa 物种细胞色素 P450 的功能分化
DOI：
10.1016/j.ibmb.2022.103796
复制
发表时间：
2022
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;Shuo Sun;Yujun Zhang;Yingshi He;Minghong Du;Andrias O. O'Reilly;Shuwen Wu;Yihua Yang;Yidong Wu
基因功能的差异是进化的标志，但评估一个物种或物种之间的这种差异需要等位基因和同源基因功能改变的信息。本研究探讨了来自棉铃虫的两个旁系同源物CYP 6AE 19和CYP 6AE 20以及来自两个近缘种（玉米棉铃虫和棉铃虫）的两个直系同源物CYP 6 B8和CYP 6 B7的功能差异。armigera）;尽管在每对酶中存在高的序列同一性，但每对酶中的后一个P450已经丧失了对植物化感物质黄毒素的代谢能力。通过交换每对P450内的不同底物识别位点（SRS）和氨基酸来产生多个嵌合和单/双位点突变体。Sf 9细胞中的异源表达和体外代谢研究表明，SRS 4的交换交换了CYP 6AE 19和CYP 6AE 20的活性，随后的定点突变证明CYP 6AE 20 V318 M取代导致对黄毒素的功能获得。同时，发现SRS 6中的单个氨基酸取代（L489 P）在CYP 6 B直向同源物之间交换活性。CYP 6AE旁系同源物和所有已报道的昆虫黄毒素代谢P450的序列比对表明，M318和P489分别是CYP 6AE旁系同源物和CYP 6 B直系同源物的催化活性所必需的，但是不同亚家族中的P450对相同底物可能具有不同的机制。我们的研究结果表明，一个单一的氨基酸取代可以足以改变底物代谢和这种功能的分歧所造成的自然突变将有助于进一步我们了解的过程中的自然选择的P450基因和它们在昆虫寄主植物的相互作用。·两对P450旁系同源物和直系同源物在黄毒素代谢中显示出显著差异。·SRS 4中的单个氨基酸取代（V318 M）导致CYP 6AE 20的功能获得。·SRS 6中的单个氨基酸取代（L489 P）在CYP 6 B7和CYP 6 B8之间交换活性。· M318和P489分别是CYP 6AE旁系同源物和CYP 6 B直系同源物的催化活性所必需的。不同亚家族中的P450对同一底物可能具有不同的机制。
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. • 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.