Substrate Specificity Of The Loading Acyltransferase From Avermectin Modular Polyketide Synthase

Avermectins are a family of macrocylic lactone anthelmintics with a wide range of activity against endoparasites and ectoparasites.Avermectins are extensively used in the field of animal health,agriculture,and human infections.Eight major compounds are produced by Streptomyces avermitilis.Under normal conditions in vivo,loading acyltransferase domain of avermectin modular polyketide synthase?AveAT0?recruits 2-methylbutyryl-coenzyme A?CoA?or isobutyryl-CoA as the starter unit to synthesize avermectins of“a”series or“b”series,respectively.In order to explore the substrate specificity of AveAT0,the protein sequences of several AT0s were compared and residues playing important roles in protein-substrate identification were covered.N-acetyl cysteamine thioesters?SNAC,where“S''indicates the thioester linkage?,2-methylbutyry-SNAC and isobutyryl-SNAC were used as surrogate substrates to assay the catalytic activities of AveAT0 and its mutants.The free SH group of released SNAC was monitored by Ellman?DTNB?assays.The K_m value of AveAT0 towards 2-methylbutyry-SNAC was 0.4 mM and towards isobutyryl-SNAC was 0.8 mM.The k_cat value of AveAT0 towards 2-methylbutyry-SNAC was 14.1 min^-1 and towards isobutyryl-SNAC was 6.4 min^-1.The k_cat/K_m value of AveAT0 towards 2-methylbutyry-SNAC was 32.1 mM^-11 min^-1and towards isobutyryl-SNAC was 7.5 mM^-11 min^-1.According to sequence alignment of AT0 and the structural characteristics of AveAT0,the mutation sites were selected as V224M,Q149L,121M,L158M.After trials in combined mutations,the specificity of a triple mutant AveAT0 V224M/Q149L/L121M for 2-methylbutyry-SNAC increased.The K_m value of AveAT0V224M/Q149L/L121M towards 2-methylbutyryl-SNAC was 0.8 mM and k_cat value was 5.4 min^-1.The k_cat/K_m value value of AveAT0V224M/Q149L/L121M towards 2-methylbutyryl SNAC was 6.9 mM^-11 min^-1and towards isobutyryl-SNAC was 0.1 mM^-11 min^-1.The specificity of AveAT0 V224M/Q149L/L121M for 2-methylbutyryl SNAC were increased than AveAT0.This study altered the substrate specificity of AveAT0 and provided insight for the rational modification of avermectin polyketide synthase.Also,the study had a potentiality to increase the proportion of“a”series avermectin,cut cost of purification.