We report the structure of a fungal enzyme that activates the amide bond, allowing nucleophilic chemistry.

Backbone N-methylations impart several favorable characteristics to peptides including increased proteolytic stability and membrane permeability. Nonetheless, amide bond N-methylations incorporated as post-translational modifications are scarce in nature, and were first demonstrated in 2017 for a single set of fungal metabolites. Here we expand on our previous discovery of iterative, autocatalytic α-N-methylating precursor proteins in the borosin family of ribosomally encoded peptide natural products. We identify over fifty putative pathways in a variety of ascomycete and basidiomycete fungi, and functionally validate nearly a dozen new self-α-N-methylating catalysts. Significant differences in precursor size, architecture, and core peptide properties subdivide this new peptide family into three discrete structural types. Lastly, using targeted genomics, we link the biosynthetic origins of the potent antineoplastic gymnopeptides to the borosin natural product family. This work highlights the metabolic potential of fungi for ribosomally synthesized peptide natural products. 7Gymnopeptide B possesses a β-hairpin-like structure containing cis amide bonds between residues Val7-Ala8 and Thr15-Val16. 37 In proteins, β-hairpins are often surfaceexposed motifs involved in protein-protein interactions, and are frequently found in antibodies and cytokine receptors. Consequently, β-hairpins can also be found in a wide variety of peptide natural products that include gramicidin S, ωconotoxin, defensins, cyclotides, and many antimicrobial peptides. 42 Interestingly, the type-IV-like β-turn at Val7-Ala8 in the gymnopeptides usually requiring proline at the i+3 position is replaced by an α-N-methylated amino acid, a property that has been observed in model synthetic peptides. 43 Thus, borosin peptides, with their exclusive properties of genetically templated residues resulting in α-N-methylated amino acids, can survey a wide variety of β-hairpin motifs and other structures otherwise inaccessible by peptides and proteins produced by the ribosome. CONCLUSIONThis work outlines the biosynthetic landscape of the α-Nmethylated borosin RiPP family of natural products. Through genome mining and heterologous expression, over 50 putative gene clusters encoded in basidiomycete and ascomycete fungi were identified. Through catalytic validation of over 10 autocatalytic borosin precursors, two additional borosin precursor structural types were discovered, with type II precursors defined by multiple core sequences and type III characterized by extraordinarily long catalytic leaders and highly repetitive acidic core sequences. Lastly, our evidence advocates that the antineoplastic gymnopeptides are biosynthesized via a borosin pathway. Basidiomycetes appear to be particularly robust hosts for borosin natural products, as 25 species out of several hundred sequenced genomes were found to encode one or more borosin pathways. With over 30,000 basidiomycete species, 60,000 ascomycetes, and five million total fungi currently estimated to exist on...

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.