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

Area of Science:

• Natural Product Biosynthesis
• Enzymology
• Structural Biology

Background:

• Ribosomally synthesized and post-translationally modified peptides (RiPPs) are diverse natural products with significant bioactivities.
• RiPP biosynthesis involves specialized enzymes, including a recently identified class of peptide arginases.
• Peptide arginases modify RiPP core peptides by converting arginine to ornithine residues.

Purpose of the Study:

• To identify and characterize novel peptide arginases and their associated precursor peptides.
• To elucidate the biochemical and structural mechanisms of peptide arginase activity.
• To explore the potential of peptide arginases in peptide-based drug discovery.

Main Methods:

• Identification of peptide arginase family members (FlmR, OhkR) and associated precursors (FlmA1-3, OhkA1-2).
• In vivo and in vitro activity assays to demonstrate enzyme function.
• Kinetic studies for biochemical characterization.
• In silico structural analysis using AlphaFold 3 to predict enzyme-substrate complexes.

Main Results:

• FlmR and OhkR enzymes were shown to be active with their cognate precursor peptides.
• Biochemical characterization provided kinetic parameters for the enzymes.
• Structural predictions offered insights into substrate binding and modification specificity.
• Experimental validation confirmed the predicted modification order for OhkA-OhkR.

Conclusions:

• This study provides a detailed biochemical and structural characterization of novel peptide arginases.
• Understanding these enzymes is essential for harnessing their potential in synthetic biology and drug discovery.
• The findings pave the way for utilizing peptide arginases in developing new peptide-based therapeutics.