Copper-Dependent Oxidase Catalyzes Aryl Cross-Coupling in the Biosynthesis of a Minimal Ribosomally Derived Natural Product

Ribosomally synthesized and post-translationally modified peptides (RiPPs) form a rapidly growing natural product family. Compared to bacterial RiPP biosynthesis, RiPPs from fungi remain underexplored in terms of both structural diversities and enzymology. Members of an enzyme family predicted to contain the domain of unknown function 3328 (DUF3328) were recently characterized as copper-dependent oxidases in fungal natural product biosynthesis. These enzymes catalyze diverse C(sp³)-H functionalization reactions, including halogenation, macrocyclization, hydroxylation, and desaturation. Bioinformatic and genetic analyses have shown that DUF3328 enzymes are associated with post-translational modifications of fungal RiPPs. Toward expanding the catalytic scope of DUF3328 enzymes, we targeted the biosynthesis of cyclic peptidyl natural products from fungi and characterized the enzyme TruY involved in the formation of aminopeptidase B inhibitor OF4949. Biochemical characterizations revealed that TruY catalyzes the copper-dependent C_β-hydroxylation of an asparagine residue in the concatemeric repeats, followed by a C(sp²)-O aryl coupling reaction between two flanking Tyr residues. The hydroxylated and cyclized concatemeric repeat is then proteolyzed to afford OF4949. The roles of fungal proteases Kex1p and Kex2p in processing the concatemeric repeats in RiPP precursor peptides are also demonstrated. Collectively, this work refines our understanding of the maturation steps involved in fungal RiPP biosynthesis.