Jiang Wankui, Liu Yali, Ke Zhijian, Zhang Lu, Zhang Mingliang, Zhou Yidong, Wang Hui, Wu Chenglong, Qiu Jiguo, Hong Qing
Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, Jiangsu 210095, PR China.
Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, Jiangsu 210095, PR China.
J Hazard Mater. 2021 Feb 5;403:123677. doi: 10.1016/j.jhazmat.2020.123677. Epub 2020 Aug 12.
The cehA gene is the earliest reported and most widely found carbaryl hydrolase gene. CehA detoxifies carbaryl and other carbamate pesticides via de-esterification. Currently, there is no systematic research available on substrate preference or the mechanism of CehA action in different hosts. In this study, we found that CehA from different hosts is highly conserved, with more than 99% amino acid sequence similarity, and that transposable elements exist in both the upstream and downstream regions of cehA. By introducing point mutations into the cehA gene of Sphingobium sp. CFD-1, we obtained and heterologously expressed all reported CehA(CehAS) encoding genes. Assays to determine enzymatic properties and substrate profiles of CehAS showed that each CehA has a significant substrate preference for different carbamate insecticides. Specifically, CehA152Phe/Leu determines the catalytic preference for bicyclic carbamate substrates (carbofuran, carbaryl), while CehA494Thr/Ala and 570Thr/Ile determine the preference for monocyclic carbamate substrates (isoprocarb, propoxur) and linear carbamate substrates (oxamyl, aldicarb), respectively. Considering the existence of transposable elements in the flanking regions of cehA, we speculate that the cehA hosts may have acquired the hydrolysis ability, as well as substrate preference for carbamate pesticides, through horizontal gene transfer and genetic copying errors.
cehA基因是最早报道且分布最为广泛的西维因水解酶基因。CehA通过去酯化作用使西维因及其他氨基甲酸酯类农药解毒。目前，关于CehA在不同宿主中的底物偏好或作用机制尚无系统研究。在本研究中，我们发现来自不同宿主的CehA高度保守，氨基酸序列相似性超过99%，并且在cehA的上游和下游区域均存在转座元件。通过对鞘氨醇单胞菌属CFD-1的cehA基因引入点突变，我们获得并异源表达了所有已报道的编码CehA（CehAS）的基因。对CehAS的酶学性质和底物谱分析表明，每种CehA对不同氨基甲酸酯类杀虫剂都有显著的底物偏好。具体而言，CehA152Phe/Leu决定了对双环氨基甲酸酯底物（克百威、西维因）的催化偏好，而CehA494Thr/Ala和570Thr/Ile分别决定了对单环氨基甲酸酯底物（异丙威、残杀威）和线性氨基甲酸酯底物（杀线威、涕灭威）的偏好。考虑到cehA侧翼区域存在转座元件，我们推测cehA宿主可能通过水平基因转移和基因复制错误获得了水解能力以及对氨基甲酸酯类农药的底物偏好。
