The study investigates the intramolecular macrocyclization of AhyBURP, a representative of the Unknown Seed Protein (USP)-type BURP-domain subfamily, during the biosynthesis of monocyclic lycumin I and bicyclic legumenin in plants. X-ray crystallography reveals that AhyBURP has a unique BURP-domain fold with two type II copper centers derived from a conserved stapled-disulfide and His motif. The macrocyclization of lycumin I and legumenin involves dioxygen and radical involvement, as demonstrated by enzyme assays in anoxic conditions and isotopic labeling. The study expands the understanding of enzymatic intramolecular modifications in ribosomally synthesized and post-translationally modified peptides (RiPPs) beyond catalytic moiety and chromophore biogenesis. The findings highlight the importance of the core peptide and the copper center in the catalytic mechanism, providing insights into the evolution and function of BURP-domain proteins in plant RiPP biosynthesis.The study investigates the intramolecular macrocyclization of AhyBURP, a representative of the Unknown Seed Protein (USP)-type BURP-domain subfamily, during the biosynthesis of monocyclic lycumin I and bicyclic legumenin in plants. X-ray crystallography reveals that AhyBURP has a unique BURP-domain fold with two type II copper centers derived from a conserved stapled-disulfide and His motif. The macrocyclization of lycumin I and legumenin involves dioxygen and radical involvement, as demonstrated by enzyme assays in anoxic conditions and isotopic labeling. The study expands the understanding of enzymatic intramolecular modifications in ribosomally synthesized and post-translationally modified peptides (RiPPs) beyond catalytic moiety and chromophore biogenesis. The findings highlight the importance of the core peptide and the copper center in the catalytic mechanism, providing insights into the evolution and function of BURP-domain proteins in plant RiPP biosynthesis.