Oxidative Peptide Backbone Cleavage by a HEXXH Enzyme during RiPP Biosynthesis

Ribosomally synthesized and post-translationally modified peptides (RiPPs) rely on a diverse array of enzymes to tailor peptide backbones and side chains. In this study, we characterized enzymes from two different biosynthetic gene clusters (BGCs) from Pseudomonas strains (<i>pfl</i> and <i>pos</i>) that catalyze new transformations in RiPP biosynthesis. Two α-ketoglutarate-dependent HEXXH enzymes, PflC and PosC, perform hydroxylation of multiple consecutive glutamine residues and selectively recognize a C-terminal ARMD tetrapeptide to trigger oxidative backbone cleavage that generates an amide terminus. Mutational analysis pinpoints the first position of this motif as a critical determinant. Notably, PflC displays proteolytic activity in the absence of the leader peptide, indicating that leader peptide-enzyme interactions modulate the observed reaction selectivity. The biosynthetic gene clusters also encode a unique MNIO-nitroreductase fusion enzyme that installs a rare <i>Z</i>-dehydrophenylalanine and hydroxylates an Asp residue. Collectively, this work expands both the catalytic repertoire and structural diversity accessible through bacterial RiPP biosynthesis.