Peptide Arginases from Cryptic Pathways Install Ornithine Residues in Uncharacterized Members of Orphan RiPP Families

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are remarkable natural products with interesting chemical structures and potent bioactivities. RiPP pathways are abundant in all domains of life and harbor a large biosynthetic potential in the form of post-translationally acting enzymes. A relatively small number of RiPP biosynthetic gene clusters encode peptide arginases, a recently discovered maturase family capable of hydrolyzing arginine residues of RiPP core peptides to ornithines. In this study, members of the peptide arginase family (FlmR and OhkR), which are associated with uncharacterized precursors from orphan RiPP families, are identified. In vivo and in vitro activity of FlmR and OhkR with the five associated precursor peptides (FlmA1-3 and OhkA1-2) is demonstrated and kinetic studies to biochemically characterize the enzymes are performed. Furthermore, in silico structural analysis with AlphaFold 3 is used to predict precursor-arginase complexes, providing insights into how peptide arginases could bind their precursor substrates. In the case of OhkA-OhkR complexes, this analysis also allows a hypothesis as to which of the arginine residues of the core peptide is modified first, which is confirmed experimentally. This detailed biochemical and structural enzyme characterization is a prerequisite for the application of peptide arginases in peptide-based drug discovery platforms.