Multinuclear iron enzymes expand RiPP chemical diversity to enable bacterial oxidative stress defense

RiPP structural complexity is significantly expanded by multinuclear non-heme iron-dependent oxidative enzymes (MNIOs). Here, we characterize pseudoprobactin 1 and 2, two MNIO-modified proteins from Pseudomonas protegens Pf-5. Using MS, NMR, and X-ray crystallography, we show that the PbnBC converts precursor cysteines into 5-thiooxazoles. While the precursors feature an N-terminal signal peptide and an intramolecular disulfide, both are dispensable for catalysis. Instead, residues downstream of the target cysteines are the primary determinants of substrate recognition. Furthermore, PbnB2C2 modifies multiple sites in a strictly ordered, stepwise manner. Functionally, pseudoprobactins coordinate Cu 2+ , enhancing bacterial fitness under chlorite-induced oxidative stress. This work establishes 5-thiooxazole as a widespread MNIO-mediated modification, defines its biosynthetic logic, and reveals a role for MNIO-modified proteins in bacterial oxidative stress defense.