Abstract
Fungal enzymes containing domain of unknown function (DUF) 3328 have been implicated in the oxidative maturation of ribosomally synthesized and post-translationally modified peptides (RiPPs). We report here the functional characterization of one such enzyme, AprY, involved in the biosynthesis of RiPP asperipin-2a. Biochemical reconstitution of AprY showed the enzyme catalyzes two consecutive C-O cross-linking reactions between tyrosines and beta-carbons of residues in the core hexapeptide. The side-chain macrocyclization activities of AprY are copper- and oxygen-dependent and do not require a leader peptide sequence.