Molecular basis for the enzymatic macrocyclization of multiply backbone N-methylated peptides

OphP is an essential enzyme for the biosynthesis of the macrocyclic peptide natural product omphalotin A. The enzyme has been proposed to proteolytically release a peptide intermediate from the precursor protein OphMA and to macrocyclize the multiply α-N-methylated core of this intermediate to produce omphalotin A. Our in vitro experiments demonstrate that OphP does not process OphMA but shows robust macrocyclase activity on pre-processed α-N-methylated peptides. We conclude that in vivo, an as yet, unidentified fungal protease liberates the peptide substrate from OphMA. OphP tolerates sequence promiscuity in the substrate but has a preference for highly α-N-methylated substrates and for N-methylated glycine residue at the catalytic site. The promiscuity of OphP is explained by structural analysis of co-complexes which reveals a predominantly hydrophobic substrate binding cleft with few interactions between substrate side chains and OphP. These structures also describe a small and hydrophobic P1 pocket that we propose rationalizes the preference for N-methylated glycine. Our data suggests a substrate entry path that is different from other members of the POP family. Since macrocyclic peptides with multiple backbone N-methylations such as omphalotin A possess potent biological activity, exploitation of enzymes to produce such compounds is highly desirable. ### Competing Interest Statement MK is an inventor on a patent application filed by ETH Zurich (no. WO2017174760A1, priority date: 7 April 2016). The authors declare no other competing interests