A Penta-Thiooxazole RiPP Confers Periplasmic Defense to Oxidative Stress in Pseudomonas aeruginosa
Multinuclear non-heme iron-dependent oxidases (MNIOs) constitute an emerging family of post-translational modifying enzymes that catalyze atypical modifications. Despite their widespread distribution across bacteria, only a small number of MNIO family members have been biochemically characterized, and the catalytic capabilities, product structures, and physiological functions of the vast majority remain unexplored. Here, we report the discovery and characterization of aen gene cluster, a previously uncharacterized MNIO-associated gene cluster in the opportunistic pathogen Pseudomonas aeruginosa PAO1. We demonstrate that the AenBC enzyme complex catalyzes four-electron oxidation of all five cysteine residues in the precursor peptide AenA, yielding a mature product, aeruginin, whose cysteine-derived modifications are most consistent with five 5-thiooxazole motifs. Under competitive metal incubation conditions, HR-MS analysis revealed that aeruginin preferentially associates with Fe3+ rather than copper. Functional analyses reveal that deletion of aenA results in a highly specific growth defect under chlorite stress, accompanied by significant upregulation of the periplasmic disulfide isomerase DsbG. Together, these findings expand the known structural diversity of MNIO-derived RiPPs and suggest a potential role for aeruginin in chlorite responsive periplasmic stress adaptation.