Substrate Specificity of Acyltransferase Domains for Efficient Transfer of Acyl Groups.

Acyltransferase domains (ATs) of polyketide synthases (PKSs) are critical for loading of acyl groups on acyl carrier protein domains (A) via - and -acylation reactions， to produce structurally diverse polyketides. However， the interaction specificity between ATs and unusual acyl units is rarely documented. In YN06， we found that AT4 [an AT in the fourth module of tacrolimus (FK506) PKS] transferred both allylmalonyl (allmal) and emthylmalonyl (ethmal) units to ACPs， which was supposed responsible for the production of both FK506 and its analog FK520， respectively. Mutations of five residues in AT4 (Q119A， L185I-V186D-V187T， and F203L) caused decreased efficiency of allmal transfer， but a higher ratio of ethmal transfer， supposedly due to less nucleophilic attacks between Ser599 in the active site of AT4 and the carbonyl carbon in the allmal unit， as observed from molecular dynamics simulations. Furthermore， reverse mutations of these five residues in ethmal-specific ATs to the corresponding residues of AT4 increased its binding affinity to allmal-CoA. Among these residues， Val187 of AT4 mainly contributed to allmal recognition， and V187K mutant produced less FK520 than wild type. Our findings thus suggested that five critical residues within AT4 were important for AT functionality in polyketide extension and potentially for targeting biosynthesis by generating desirable products and eliminating undesirable analogs.