Oxidative Electrocyclization Assembles 2,2‐Dimethyl‐2 <i>H</i> ‐pyran in Fungal Indole Alkaloids

Abstract Pericyclic reactions, such as cycloaddition and electrocyclization, are among the most useful approaches for constructing complex natural products with diverse biological activities. While enzymatic pericyclic reactions have been increasingly reported in recent years, there are only a few examples of enzyme‐mediated electrocyclization reactions. The 2,2‐dimethyl‐2 H ‐pyran (DMP) ring plays a significant role in constituting many bioactive fungal and plant natural products. In this study, we characterize the vsc biosynthetic gene cluster of the DMP‐containing natural product versicamide A ( 1 ) in Aspergillus versicolor DL0022. The biosynthetic route for DMP formation and the biochemical functions of the involved enzymes are fully elucidated. Specifically, we reveal that the berberine bridge‐like oxidoreductase VscB mediates a crucial oxidative process, followed by an electrocyclization to form a C─O bridge bond, thereby affording the key DMP ring. Structural simulation, molecular modeling, mutational analysis, and quantum chemical calculations elucidate the oxidative electrocyclization mechanism. The flavin adenine dinucleotide (FAD) cofactor of VscB/NotD' facilitates hydride abstraction at the C25 position of the substrate, while the key amino acid residue E529 acts as a general base to deprotonate the C6 hydroxyl group.