Intermolecular Carbon–Carbon Naphthalene Coupling Involves a Radical Mechanism Driven by Cytochrome P450 in Alternaria alternata

ABSTRACT Dihydroxynaphthalene (DHN) derivatives are precursors of biologically important molecules, including melanin and perylenequinones (PQs). Biosynthesis of DHN‐derived metabolites requires precise spatial control of radical reactions to form specific C─C or C─O─C bonds. Investigating fungal PQ biosynthesis in Alternaria alternata , we identified a cytochrome P450 enzyme that catalyzes a highly regio‐ and stereospecific C─C radical coupling reaction. Conversely, a DHN‐converting P450 from Berkleasmium sp. utilizes a diradical mechanism yielding a C─O─C bridge. Comparing these enzymes revealed five sequence motifs that, with structural modeling, allowed the identification of a novel A. alternata enzyme converting 1,8‐DHN to a 2‐2' DHN dimer. We demonstrate that P450‐dependent PQ biosynthesis in A. alternata is fundamentally distinct from the laccase‐fasciclin‐mediated pathway in Cercospora beticola , indicating that PQ biosynthesis evolved via distinct, lineage‐specific pathways through convergent evolution. Finally, PQs contribute to microbial niche shaping on tomato fruits. Understanding these pathways provides a foundation for producing PQs for medical applications.