title: Polyketide intermediate mimics as probes for revealing cryptic stereochemistry of ketoreductase domains.
abstract: 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 K(M) values (2.4 ± 0.8 and 7.8 ± 2.7 mM, respectively), indicating molecular recognition of intermediate-like substrates. Due to an abundance of the nonreducable enol-tautomer, the k(cat) values were attenuated by as much as 15-336 fold relative to known substrates. This study reveals the high stereoselectivity of PikKR2 in the face of gross substrate permutation, highlighting the utility of a chemical probe-based approach in the study of polyketide ketoreductases.
authors.affiliation: Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota , Minneapolis, Minnesota 55455, United States.
authors.forename: Yang
authors.initials: Y
authors.lastname: Li
authors.forename: William D
authors.initials: WD
authors.lastname: Fiers
authors.forename: Steffen M
authors.initials: SM
authors.lastname: Bernard
authors.forename: Janet L
authors.initials: JL
authors.lastname: Smith
authors.forename: Courtney C
authors.initials: CC
authors.lastname: Aldrich
authors.forename: Robert A
authors.initials: RA
authors.lastname: Fecik
mesh_terms: D000595:Amino Acid Sequence; D040761:Biomimetic Materials / Q000138:chemical synthesis / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D008958:Models, Molecular; D015335:Molecular Probes / Q000138:chemical synthesis / Q000737:chemistry*; D008969:Molecular Sequence Data; D009249:NADP / Q000737:chemistry; D048630:Polyketide Synthases / Q000737:chemistry* / Q000235:genetics; D061065:Polyketides / Q000138:chemical synthesis / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013237:Stereoisomerism; D013302:Streptomyces / Q000737:chemistry* / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D015335:Molecular Probes; D061065:Polyketides; D011994:Recombinant Proteins; D009249:NADP; D048630:Polyketide Synthases