Characterization of the biosynthetic enzymes of the <i>Morus alba</i> benzofuran natural products

Moracins are a distinct class of benzofuran natural products, primarily found in Moraceae plants. Despite their varying biological activities and interesting chemical structures, little is known about their biosynthesis. Here, we elucidate the biosynthetic pathway of moracin M, a common intermediate in moracin biosynthesis. Using thermal proteome profiling, we identified a noncanonical laccase from Morus alba (MaLAC) that catalyzes an unprecedented oxidative cyclization of oxyresveratrol to moracin M. Distinct from other plant laccases, MaLAC is the first plant laccase shown to catalyze an intramolecular oxidative cyclization reaction in natural product biosynthesis. Mechanistic studies revealed a copper-mediated radical cyclization, providing guidance for engineering laccases to catalyze synthetically valuable intramolecular oxidative cyclization. The biosynthetic pathway for moracin M was heterologously constructed in Nicotiana benthamiana . This work not only provides important insights into moracin biosynthesis, laying the foundation for manufacturing bioactive moracins via metabolic engineering strategies, but also demonstrates the power of label-free chemoproteomics in elucidating unknown biosynthetic pathways of plant secondary metabolites.