An NADPH‐Dependent Ketoreductase Catalyses the Tetracyclic to Pentacyclic Skeletal Rearrangement in Chartreusin Biosynthesis

Abstract

Redox tailoring enzymes play key roles in generating structural complexity and diversity in type II polyketides. In chartreusin biosynthesis, the early 13 C-labeling experiments and bioinformatic analysis suggest the unusual aglycone is originated from a tetracyclic anthracyclic polyketide. Here, we demonstrated that the carbon skeleton rearrangement from a linear anthracyclic polyketide to an angular pentacyclic biosynthetic intermediate requires two redox enzymes. The flavin-dependent monooxygenase ChaZ catalyses a Baeyer-Villiger oxidation on resomycin C to form a seven-membered lactone. Subsequently, a ketoreductase ChaE rearranges the carbon skeleton and affords the a-pyrone containing pentacyclic intermediate in an NADPH-dependent manner via tandem reactions including the reduction of the lactone carbonyl group, Aldol-type reaction, followed by a spontaneous glactone ring formation, oxidation and aromatization. Our work reveals an unprecedented function of a ketoreductase that contributes to generate structural complexity of aromatic polyketide.

Alteration of the Catalytic Reaction Trajectory of a Vicinal Oxygen Chelate Enzyme by Directed Evolution

“…We therefore hypothesized that the CaB‐to‐CaA conversion could be realized via a β‐keto acid‐like decarboxylation process [43] . To address this assumption, 17‐hydroxy‐Chq (Table S8, Figures S28–S29) (isolated from the Δ chaL strain of S. chartreusis NA02069) [27] was transformed at 0 °C by ChaP D49L/Y92S to give a doubly decarboxylated product (named chatrudiol) via a singly decarboxylated transient compound, 17‐hydroxy‐CaA, which culminated 30 min after initializing the test but disappeared (fully decarboxylated) in about two hours (Figure 3B; Figures S8, S30, and S31). Correlating with the CaA's inertness to CVs, this observed time‐course showed that ChaP D49L/Y92S catalyzes the two‐round decarboxylation of the o‐ hydroxybenzoic acid motifs of the anhydride‐derived diacid (Figure 3A).…”

“…ChaP was found to catalyze the oxidative rearrangement step involved in the formation of chartreusin (Cha) from its benzoquinone precursor, chartrequinone (Chq) (Figures S15, S16; Table S4), via an α‐keto acid intermediate in exposure to flavin‐activated dioxygen. This is attributed to the C 15 –C 16 bond fissure of Chq (Scheme 1, carbon numbering deduced from the nascent (linear) polyketide precursor) [6, 27] . In addition to Cha, isochartreusin (Ich) was afforded as a trace product in the biotransformation of Chq using pure ChaP expressed in Escherichia coli (Figures S13, S14 and S17, S18; Tables S3 and S5) [6] .…”

Baeyer–Villiger oxidation: a promising tool for the synthesis of natural products: a review

“…It is well-known for its potent biological activity against tumor cells. 187 Initially, Xu and colleagues investigated the role of flavin-dependent ChaZ in the gene cluster of cha but chaZ gene suppressed the synthesis of compound 227 . For better understanding, Jiao et al in 2021, presented the biosynthesis of natural compound chartreusin 227 by using flavin-dependent BVMO (ChaZ), redox enzymes, and NADPH-dependent ketoreductase ChaE.…”

“…For better understanding, Jiao et al in 2021, presented the biosynthesis of natural compound chartreusin 227 by using flavin-dependent BVMO (ChaZ), redox enzymes, and NADPH-dependent ketoreductase ChaE. 187 Schematic route demonstrated the synthesis of compound 224 from a cofactor-scaffold (acetyl-CoA + MeI-CoA) 223 in the presence of a framework of enzymes which was further converted into decaketide scaffold of tetracyclic intermediate 225 over a few steps. Consequently, scaffold, resomycin C 225 underwent ChaZ-catalyzed Baeyer–Villiger oxidation to furnish a pentacyclic intermediate 226 .…”