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...

Borosins are ribosomally synthesized and post-translationally modified peptides (RiPPs) with α-N-methylations installed on the peptide backbone that impart unique properties like proteolytic stability to these natural products. The borosin RiPP family was initially reported only in fungi until our recent discovery and characterization of a Type IV split borosin system in the metal-respiring bacterium Shewanella oneidensis. Here, we used hidden Markov models and sequence similarity networks to identify over 1600 putative pathways that show split borosin biosynthetic gene clusters are widespread in bacteria. Noteworthy differences in precursor and α-N-methyltransferase open reading frame sizes, architectures, and core peptide properties allow further subdivision of the borosin family into six additional discrete structural types, of which five have been validated in this study.