Structural Study Of Bemisia Tabaci Acetyl CoA Carboxylase In Complex With Spirotetramat

Acetyl CoA carboxylase(ACC)is a key enzyme in lipid metabolism of organisms.It catalyzes the conversion of Acetyl CoA to malonyl CoA and participates in the fatty acid synthesis.ACC is a target of a variety of drugs,including herbicides,insecticides,etc.Spirotetramat,a cyclic keto-enol insecticide,is widely used to control sucking pests such as white flies(Bemisia tabaci)and Aphids(Aphidoidea).It targets at the ACC and causes growth arrest.Recent studies showed that spirotetramat targets at the carboxyl transferase domain(CT)of ACC,and A2083V mutation of white fly ACC is related to spirotetramat resistance.However,the specific binding site of spirotetramat remains unknown.Therefore,we aim to determine the structures of whitefly ACC in complex with spirotetramat-enol.It may help reveal the potential resistant site,monitor the resistance level of the field pest populations and develop the next-generation pesticides.The coding region of ACC was cloned from B.tabaci and used for protein expression and purification.According to the mutation sites of the resistant B.tabaci population to spirotetramat,genotypes of B.tabaci from 5 provinces in China were determined,and no resistance individuals was found.Two recombinant vectors were constructed to express and purify the full-length ACC.Results showed that the expression level was low and the protein-purity and homogeneity are not good enough for Cryo-EM.Negative staining showed that the full-length ACC is present in aggregates.Then,seven recombinant vectors were constructed to express and purify the CT domain of ACC and high purity proteins can be generated through multiple purificaition steps.However,negative staining showed that the purified protein was poor in homogeneity and could not be usd for Cryo-EM.The crystal structure of the CT domain of Saccharomyces cerevisiae in complex with pinoxaden was selected as a template to generate a homology modeling.The spirotetramat-enol was docked into the CT domain of B.tabaci.Results showed that the A2083V substitution reported in the previous study is located in the binding pocket of spirotetramat.The A2083V substitution may result in steric hindrance between the valine residue and spirotetramat-enol,hinder the binding of spirotetramat to ACC,thus produces resistance.