AhyBURP, a member of the USP subfamily of BURP-domain proteins, catalyzes intramolecular macrocyclization of its core peptide during the biosynthesis of monocyclic lyciumin I and bicyclic legumenin. The protein contains two type II copper centers, derived from a conserved stapled-disulfide and His motif, and is involved in the formation of C-C, C-N, and C-O bonds between aromatic amino acid side chains and unactivated carbons. X-ray crystallography of AhyBURP reveals a unique BURP-domain fold with a 4xCH motif, which is essential for copper coordination and catalytic activity. The study shows that AhyBURP requires dioxygen and radical involvement for macrocyclization, as evidenced by enzyme assays in anoxic conditions and isotopic labeling. The copper center is located in the BURP domain and is essential for the formation of the macrocyclic bonds. The study also demonstrates that AhyBURP catalyzes sequential bond formation, starting with G82-W86 crosslinking and then Q79-Y84 crosslinking. The reaction is dioxygen-dependent and involves a radical mechanism, as indicated by the presence of radical intermediates and the incorporation of deuterated amino acids. The study provides structural and biochemical data on the AhyBURP copper center and its role in the biosynthesis of RiPPs. The findings expand the understanding of enzymatic intramolecular modifications in RiPP biosynthesis and highlight the importance of the BURP-domain fold in the catalytic activity of AhyBURP. The study also reveals that AhyBURP is an intramolecular macrocyclase, with the core peptide localized near the copper center, suggesting an intramolecular modification mechanism. The results provide insights into the structural and functional characteristics of AhyBURP and its role in the biosynthesis of RiPPs.AhyBURP, a member of the USP subfamily of BURP-domain proteins, catalyzes intramolecular macrocyclization of its core peptide during the biosynthesis of monocyclic lyciumin I and bicyclic legumenin. The protein contains two type II copper centers, derived from a conserved stapled-disulfide and His motif, and is involved in the formation of C-C, C-N, and C-O bonds between aromatic amino acid side chains and unactivated carbons. X-ray crystallography of AhyBURP reveals a unique BURP-domain fold with a 4xCH motif, which is essential for copper coordination and catalytic activity. The study shows that AhyBURP requires dioxygen and radical involvement for macrocyclization, as evidenced by enzyme assays in anoxic conditions and isotopic labeling. The copper center is located in the BURP domain and is essential for the formation of the macrocyclic bonds. The study also demonstrates that AhyBURP catalyzes sequential bond formation, starting with G82-W86 crosslinking and then Q79-Y84 crosslinking. The reaction is dioxygen-dependent and involves a radical mechanism, as indicated by the presence of radical intermediates and the incorporation of deuterated amino acids. The study provides structural and biochemical data on the AhyBURP copper center and its role in the biosynthesis of RiPPs. The findings expand the understanding of enzymatic intramolecular modifications in RiPP biosynthesis and highlight the importance of the BURP-domain fold in the catalytic activity of AhyBURP. The study also reveals that AhyBURP is an intramolecular macrocyclase, with the core peptide localized near the copper center, suggesting an intramolecular modification mechanism. The results provide insights into the structural and functional characteristics of AhyBURP and its role in the biosynthesis of RiPPs.