A Standalone Flavoprotein Monooxygenase Directs Divergent Tropolone Skeletal Editing through Baeyer–Villiger-Triggered Rearrangement

Abstract Late-stage oxidative remodeling of tropolone cores underpins the remarkable scaffold diversity in fungal meroterpenoid biosynthesis. However, the mechanistic basis by which a single enzymatic catalyst diverts a common substrate toward carbon-excising ring contraction versus carbon-retentive skeletal rearrangement remains elusive. Here, via comparative transcriptomics and targeted gene deletion, we identified a standalone host-encoded class A flavoprotein monooxygenase, ANIA_01821 (AN1821), as indispensable for tropolone ring remodeling in Aspergillus nidulans. A functionally homologous enzyme, AO090103000276 (AO276), from Aspergillus oryzae was further characterized as a tractable biocatalyst for in vitro studies. Reconstitution assays using purified AO276 revealed that this single flavoprotein monooxygenase alone converts the bistropolone meroterpenoid eupenifeldin into both ring-contracted phenolic products and carbon-retentive bridged polycycles, establishing a unified enzymatic origin for two structurally distinct natural-product scaffolds. Structure-guided mutagenesis uncovered a precisely organized electrostatic network that is critical for flavin-dependent oxygenation. Complementary well-tempered metadynamics simulations and quantum-chemical calculations support a Baeyer–Villiger branching mechanism, wherein the AO276 active site may preferentially stabilize alternative migration-competent conformations of a putative Criegee intermediate. Each conformation aligns a distinct C–C bond in an antiperiplanar geometry relative to the O–O bond; restricted conformational interconversion within the active site could preserve the initial reactive geometry and bias divergent carbon migration. Collectively, our findings identify conformational preorganization as a plausible contributor to regiodivergent enzymatic Baeyer–Villiger catalysis, offering a rational strategy for steering oxidative skeletal remodeling in complex natural product molecules.