A Single Active Site Mutation in the Pikromycin Thioesterase Generates a More Effective Macrocyclization Catalyst

Macrolactonization
of natural product analogs presents a significant
challenge to both biosynthetic assembly and synthetic chemistry. In
the preceding paper, we identified a thioesterase
(TE) domain catalytic bottleneck processing unnatural substrates in
the pikromycin (Pik) system, preventing the formation of epimerized
macrolactones. Here, we perform molecular dynamics simulations showing
the epimerized hexaketide was accommodated within the Pik TE active
site; however, intrinsic conformational preferences of the substrate
resulted in predominately unproductive conformations, in agreement
with the observed hydrolysis. Accordingly, we engineered the stereoselective
Pik TE to yield a variant (TES148C) with improved reaction
kinetics and gain-of-function processing of an unnatural, epimerized
hexaketide. Quantum mechanical comparison of model TES148C and TEWT reaction coordinate diagrams revealed a change
in mechanism from a stepwise addition–elimination (TEWT) to a lower energy concerted acyl substitution (TES148C), accounting for the gain-of-function and improved reaction kinetics.
Finally, we introduced the S148C mutation into a polyketide synthase
module (PikAIII-TE) to impart increased substrate flexibility, enabling
the production of diastereomeric macrolactones.