Specificity of the loading acyltransferase from avermectin modular polyketide synthase

Abstract:

[Background] Under normal conditions in vivo, loading acyltransferase from avermectin modular polyketide synthase (AveAT0) recruits 2-methylbutyryl-coenzyme A (CoA) or isobutyryl-CoA as starter units to synthesize avermectins of “a” series or “b” series, respectively. [Objective] We are aiming to explore the catalytic specificity of AveAT0 on 2-methylbutyry-SNAC and isobutyryl-SNAC, as substrate analogues of 2-methylbutyryl-CoA and isobutyryl-CoA and modify its favorability towards the former. [Methods] The protein sequences of several AT0s were compared and residues playing important roles in protein-substrate identification were uncovered. The mutation experiments were then conducted on AveAT0 and concentration of free sulfhydryl (SH) released from SNAC was obtained via Ellman test to characterize the substrate speci?city of mutated enzymes. [Results] The Km value of AveAT0 towards 2-methylbutyry-SNAC was 0.4 mmol/L, kcat value was 14.1 min–1 and kcat/Km value was 32.1 L/(mmol·min); the Km value of AveAT0 towards isobutyryl-SNAC was 0.8 mmol/L, kcat value was 6.4 min–1 and the kcat/Km value was 7.5 L/(mmol·min). The mutation sites were selected as V224M, Q149L and L121M. After trials in combined mutations, the specificity of a triple mutant AveAT0 V224M/Q149L/L121M for both substrates increased. The Km value of AveAT0 V224M/Q149L/L121M towards 2-methylbutyryl SNAC was 0.8 mmol/L, kcat value was 5.4 min–1, and kcat/Km value was 6.9 L/(mmol·min); the kcat/Km towards isobutyryl-SNAC was 0.1 L/(mmol·min). [Conclusion] This study found key residues in AveAT0 identification, and provided insight for the rational modification of avermectin polyketide synthase acyltransferase.