Tandem Enzymatic Oxygenations in Biosynthesis of Epoxyquinone Pharmacophore of Manumycin-type Metabolites

•Epoxyquinone pharmacophore was biosynthesized through tandem enzymatic oxygenations•Single-component monooxygenase (AsuE1) de-aromatizes 3,4-AHB moiety by hydroxylation•When flavin is scarce, flavin reductase (AsuE2) specifically elicits AsuE1 activity•FMN-dependent monooxygenase (AsuE3) catalyzes the formation of the epoxide moiety

SummaryMany natural products contain epoxyquinone pharmacophore with unknown biosynthetic mechanisms. Recent genetic analysis of the asukamycin biosynthetic gene cluster proposed enzyme candidates related to epoxyquinone formation for manumycin-type metabolites. Our biochemical studies reveal that 3-amino-4-hydroxyl benzoic acid (3,4-AHBA) precursor is activated and loaded on aryl carrier protein (AsuC12) by ATP-dependent adenylase (AsuA2). AsuE1 and AsuE3, both single-component flavin-dependent monooxygenases, catalyze the exquisite regio- and enantiospecific postpolyketide synthase (PKS) assembly oxygenations. AsuE1 installs a hydroxyl group on the 3,4-AHB ring to form a 4-hydroxyquinone moiety, which is epoxidized by AsuE3 to yield the epoxyquinone functionality. Despite being a single-component monooxygenase, AsuE1 activity is elicited by AsuE2, a pathway-specific flavin reductase. We further demonstrate that the epoxyquinone moiety is critical for anti-MRSA activity by analyzing the bioactivity of various manumycin-type metabolites produced through mutasynthesis.

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