Engineering of a Peptide α-N-Methyltransferase to Methylate Non-Proteinogenic Amino Acids

Abstract

Introduction of α-N-methylated non-proteinogenic amino acids into peptides can improve their biological activities, membrane permeability and proteolytic stability. This is commonly achieved, in nature and in the lab, by assembling pre-methylated amino acids. The more appealing route of methylating amide bonds is challenging. Biology has evolved an α-N-automethylating enzyme, OphMA, which acts on the amide bonds of peptides fused to its C-terminus. Due to the ribosomal biosynthesis of its substrate, the activity of this enzyme towards peptides with non-proteinogenic amino acids has not been addressed. An engineered OphMA, intein-mediated protein ligation and solid-phase peptide synthesis have allowed us to demonstrate the methylation of amide bonds in the context of non-natural amides. This approach may have application in the biotechnological production of therapeutic peptides.

Keywords: RiPPs; cyclic peptide; non-proteinogenic amino acids; split intein; α-N-methylation.

Figure 1

OphMA‐ and split‐intein‐mediated peptide ligation. A) OphMA catalyzes nine processive backbone N‐methylations of its C‐terminal peptide which is converted to omphalotin A by OphP (macrocyclase).[ ²³ , ²⁴ , ²⁵ ] The leader peptide is shown green, the follower as gray circles. B) The scheme for one‐pot core peptide (blue) ligation to OphMA mediated by split intein Cfa^N and mutated Cfa^C (denoted Cfa^C‐AA) inserts a cysteine at the ligation site (see also Figure S1). C) Key to constructs. Native OphMA (numbers show the first residue of each region); OphMAΔC27 and OphMAΔC12 (27 or 12 C‐terminal residues deleted); OphMAΔC12‐G390_E391insCys as OphMAΔC12 but cysteine (yellow) inserted between 390 and 391; fOphMA2ΔC12 is the fused dimer with 12 C‐terminal residues deleted; fOphMA2ΔC12‐G390_E391InsCys is fOphMA2ΔC12 with cysteine insertion; fOphMA2ΔC27‐Cfa^N‐SUMO intein and sumo added to C‐terminus; fOphMA2ΔC12‐401UAA the post ligation protein.

Figure 2

OphMA catalyzes backbone N‐methylation of ligated synthetic peptides. A) MS analysis of C‐terminal peptide of the expressed OphMAΔC12 (i), OphMAΔC12‐G390_E391insCys (ii) and fOphMA2ΔC12‐G390_E391insCys (iii). B) Intact mass analysis of ligated products, OphMAΔC12‐401Val (left) and fOphMA2ΔC12‐401Val (right). C) MS analysis of C‐terminal peptide confirmed N‐methylation for OphMAΔC12‐401Val (iv) and fOphMA2ΔC12‐401Val (v). Both are ligated protein with cysteine insertion between Gly390 and Glu391, and incubated with SAM for three days. D) MS² spectrum confirms the N‐methylated site of fOphMA2ΔC12‐401Val.

Figure 3

Non‐proteinogenic amino acids N‐methylated by OphMA. The methylation was determined by MS² of the Glu‐C digested fOphMA2‐ligated protein. Values in the bracket represent relative percentage of mono‐methylation species with non‐proteinogenic site methylated and are calculated by integrating all methylated species observed. * Di‐methylation was also detected; ** indicates methylation of the residue but low abundance.