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

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.