Polyketide IntermediateMimics as Probes for RevealingCryptic Stereochemistry of Ketoreductase Domains

摘要

Among natural product families, polyketides have shown the most promise for combinatorial biosynthesis of natural product-like libraries. Though recent research in the area has provided many mechanistic revelations, a basic-level understanding of kinetic and substrate tolerability is still needed before the full potential of combinatorial biosynthesis can be realized. We have developed a novel set of chemical probes for the study of ketoreductase domains of polyketide synthases. This chemical tool-based approach was validated using the ketoreductase of pikromycin module 2 (PikKR2) as a model system. Triketide substrate mimics >12 and >13 were designed to increase stability (incorporating a nonhydrolyzable thioether linkage) and minimize nonessential functionality (truncating the phosphopantetheinyl arm). PikKR2 reduction product identities as well as steady-state kinetic parameters were determined by a combination of LC-MS/MS analysis of synthetic standards and a NADPH consumption assay. The d-hydroxyl product is consistent with bioinformatic analysis and results from a complementary biochemical and molecular biological approach.When compared to widely employed substrates in previous studies, diketide >63 and trans-decalone >64, substrates >12 and >13 showed 2–10 fold lower KM values (2.4 ± 0.8 and 7.8 ± 2.7mM, respectively), indicating molecular recognition of intermediate-likesubstrates. Due to an abundance of the nonreducable enol-tautomer,the kcat values were attenuated by asmuch as 15–336 fold relative to known substrates. This studyreveals the high stereoselectivity of PikKR2 in the face of grosssubstrate permutation, highlighting the utility of a chemical probe-basedapproach in the study of polyketide ketoreductases.