Macrocyclization of backbone <i>N</i> -methylated peptides by a prolyl oligopeptidase with a distinctive substrate recognition mechanism

Macrocyclization and multiple backbone <i>N</i>-methylations can significantly improve the pharmacological properties of peptides. Since chemical synthesis of such compounds is often challenging, enzyme-based production platforms are an interesting option. Here, we characterized OphP, a serine peptidase involved in the cyclization of omphalotins, a group of ribosomally produced dodecapeptides with multiple backbone <i>N</i>-methylations. OphP displays robust peptidase and macrocyclase activity towards multiply α-<i>N</i>-methylated peptides of various lengths and composition derived from the omphalotin precursor protein OphMA. In addition, OphP processes, with lower efficiency, peptides unrelated to OphMA, containing a ^MeGly, ^MeAla or Pro residue at the P1 site. Structural analysis reveals that OphP adopts a canonical prolyl oligopeptidase fold but, unlike other enzymes of this enzyme family, recognizes its substrates by their hydrophobic and multiply backbone <i>N</i>-methylated core rather than by the follower peptide. The activity of OphP could be harnessed for the enzymatic production of therapeutic peptides.