α-N-Methyltransferase regiospecificity is mediated by proximal, redundant enzyme-substrate interactions.

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 α-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.