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bioRxiv · 10.1101/2022.07.21.500988

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

Matabaro, E.·Song, H.·Gherlone, F.·Sonderegger, L.·Giltrap, A.·Liver, S.·Gossert, A.·Künzler, M.·Naismith, J. H.
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Abstract

The enzyme OphP is essential for the biosynthesis of the macrocyclic peptide omphalotin A, a dodecamer with 9 backbone N-methylations produced by the wood-degrading fungus Omphalotus olearius. Heterologous expression of OphP and the peptide-precursor protein OphMA in yeast, yields omphalotin A. Thus, Oph P was hypothesized to have a dual function; catalyzing both endoproteolytic release of a peptide intermediate from OphMA, and macrocyclization of the multiply -N-methylated core peptide with concomitant release of a C-terminal follower peptide. In our in vitro activity assays, OphP showed robust endoproteolytic and macrocyclase activity on -N-methylated peptides but was unable to cleave OphMA. The enzyme had a strong preference for hydrophobic, highly -N-methylated peptides and an -N-methylated glycine residue at the P1 site. OphP adopts a canonical prolyl oligopeptidase (POP) fold with a predominantly hydrophobic substrate binding cleft, and a small and hydrophobic P1 binding pocket. We demonstrate that OphP is a POP-type macrocyclase with a specificity and a substrate route to the active site different from other members of the family. These results could be exploited for the biotechnological production of macrocyclic peptides with multiple backbone N-methylations, which are interesting due to their favorable pharmacological properties.

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Matabaro, E., Song, H., Gherlone, F., Sonderegger, L., Giltrap, A., Liver, S., Gossert, A., Künzler, M., Naismith, J. H.. 2022-07-23. Molecular basis for the enzymatic macrocyclization of multiply backbone N-methylated peptides. https://doi.org/10.1101/2022.07.21.500988

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