Substrate preference of carbamate hydrolase CehA reveals its environmental behavior.

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.