Abstract
The methylation of amide nitrogen atoms can improve the stability, oral availability, and cell permeability of peptide therapeutics. Chemical N-methylation of peptides is challenging. Omphalotin A is a ribosomally synthesized, macrocylic dodecapeptide with nine backbone N-methylations. The fungal natural product is derived from the precursor protein, OphMA, harboring both the core peptide and a SAM-dependent peptide α-N-methyltransferase domain. OphMA forms a homodimer and its α-N-methyltransferase domain installs the methyl groups in trans on the hydrophobic core dodecapeptide and some additional C-terminal residues of the protomers. These post-translational backbone N-methylations occur in a processive manner from the N- to the C-terminus of the peptide substrate. We demonstrate that OphMA can methylate polar, aromatic, and charged residues when these are introduced into the core peptide. Some of these amino acids alter the efficiency and pattern of methylation. Proline, depending on its sequence context, can act as a tunable stop signal. Crystal structures of OphMA variants have allowed rationalization of these observations. Our results hint at the potential to control this fungal α-N-methyltransferase for biotechnological applications.
Highlights
Peptide macrocyclization offers a way to increase peptide stability, membrane permeability, and rigidity.[1]
Chemical modifications do likewise[2] and include exocyclic amide bonds,[3] D-amino acids,[4−7] tailoring of side chains,[4,8,9] and backbone (α-)N-methylation.[10−14] Backbone N-methylation is potent, because it alters the energetic landscape of backbone dihedral angles,[15] shifts the amide bond cis/trans equilibrium, disrupts hydrogen bonding,[12] reduces hydrophilicity of the amide backbone, and, in the macrocyclic peptide cyclosporine A, allows “shape shifting”
Omphalotin A was believed to be produced via nonribosomal peptide synthesis (NRPS)
Summary
Peptide macrocyclization offers a way to increase peptide stability, membrane permeability, and rigidity.[1]. Adenosylmethionine (SAM)-dependent peptide α-N-methyltransferase domain of the precursor protein (OphMA), a prolyl oligopeptidase (OphP), member of the structurally characterized POP family of macrocycle forming enzymes, an acetyl transferase (OphD), and at least two cytochrome P450 monooxygenases (OphB1 and OphB2) (Figure 1B).[24,25] OphMA consists of the N-terminal peptide α-N-methyltransferase domain (NMT, residues 1−251), a clasp domain (residues 252−378), and the C-terminal substrate peptide (residues 379−417).[26,27] The precise boundary between the clasp and C-terminal substrate peptide is somewhat arbitrary.
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