Understanding andEngineering of C4 Indole PrenyltransferaseFgaPT2 by Theoretical Study and Mutation Experiments

Indole prenyltransferases (IPTs) play vital roles in the biosynthesis of abundant natural products with diverse biological activities. However, the underlying mechanisms of specific members are not fully understood. Herein, we investigated the detailed reaction mechanism of FgaPT2, a C4 IPT involved in the biosynthesis of important pharmaceutical ergot alkaloids, by employing multiscale calculations and experimental validation. Our study indicates that the C4-prenylation process in FgaPT2 is an unconventional associative reaction accompanied by a short-lived carbocation intermediate rather than a dissociative reaction. The tyrosine shield in FgaPT2 facilitates the prenylation step mainly through hydrogen bond interactions with the dimethylallyl diphosphate. The conserved E89 residue significantly lowers the energy barrier of the prenylation step through the electrostatic interaction. We also confirmed the Cope rearrangement process in the K174A mutant, which results in a reverse-prenylated C3 tricyclic product. By analyzing the MD trajectories, we propose a C6-prenylation mechanism and a prenyl shift reaction from C6 to C5, and finally to the C4 site, which was identified by QM calculation and QCT-MD simulation. Based on our newly proposed mechanism, the C5-prenylated product 5-dimethylallyltryptophan was successfully obtained in the T102N mutant through rational engineering. Our study expands the current understanding of the catalytic mechanism of IPTs and provides insights into the rational modification of IPTs to synthesize a wide variety of high-value prenylated indole products.