Insights from Reactions of Phylogenetically Related Thioesterases with Semi-Synthetic Substrates

Polyketides are a diverse class of natural products with a wide range of therapeutic uses, serving as antibiotics, anticancer agents, and immunosuppressants. A major subset of these molecules is synthesized by Type I Assembly-Line Polyketide Synthases (T1ALPKSs), large enzyme complexes that catalyze their stepwise construction. Understanding the selectivity and scope of T1ALPKS domains could greatly expand the diversity of products generated by these synthases and their components. This study explores the reactivity and selectivity of the terminal domains of T1ALPKSs, known as thioesterases (TEs). A panel of 25 wild-type and 10 mutant (Ser->Cys) thioesterases was reacted with semi-synthetic substrates. The selectivity with which these TEs form 12- and/or 14-membered macrolactones and a pair of diastereomeric 14-membered macrolactones was quantified. The results indicate that the selectivity with which wild-type TEs react with unnatural substrates correlates with their parent T1ALPKSs, enabling the assignment of TEs into groups. Additionally, our findings reveal that mutant (Ser->Cys) TEs often exhibit expanded scopes compared to their wild-type counterparts. We demonstrated the synthetic applications of TEs with kinetic resolutions and preparative-scale reactions. This study provides a framework for organizing TEs and predicting their reactivity and selectivity, facilitating future research on structure-activity relationships and supporting directed evolution studies.