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Publication Abstract from PubMed

N-Methylation of the peptide backbone confers pharmacologically beneficial characteristics to peptides that include greater membrane permeability and resistance to proteolytic degradation. The borosin family of ribosomally synthesized and post-translationally modified peptides offer a post-translational route to install amide backbone alpha-N-methylations. Previous work has elucidated the substrate scope and engineering potential of two examples of type I borosins, which feature autocatalytic precursors that encode N-methyltransferases that methylate their own C-termini in trans. We recently reported the first discrete N-methyltransferase and precursor peptide from Shewanella oneidensis MR-1, a minimally iterative, type IV borosin that allowed the first detailed kinetic analyses of borosin N-methyltransferases. Herein, we characterize the substrate scope and resilient regiospecificity of this discrete N-methyltransferase by comparison of relative rates and methylation patterns of over 40 precursor peptide variants along with structure analyses of nine enzyme-substrate complexes. Sequences critical to methylation are identified and demonstrated in assaying minimal peptide substrates and non-native peptide sequences for assessment of secondary structure requirements and engineering potential. This work grants understanding towards the mechanism of substrate recognition and iterative activity by discrete borosin N-methyltransferases.

alpha-N-Methyltransferase regiospecificity is mediated by proximal, redundant enzyme-substrate interactions.,Crone KK, Labonte JW, Elias MH, Freeman MF Protein Sci. 2025 Feb;34(2):e70021. doi: 10.1002/pro.70021. PMID:39840790^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.