Mechanism of installing multiple oxazolone/thioamide pairs on a ubiquitous family of copper-chelating proteins in oxidative stress defense

ABSTRACT: Ribosomally synthesized and post‑translationally modified peptides (RiPPs) constitute a structurally diverse class of natural products with wide‑ranging bioactivities. Multinuclear non‑heme iron‑dependent oxidases (MNIOs) have recently emerged as important enzymes expanding the types of post-translational modifications (PTM) of RiPPs. Here we characterize two MNIO‑modified small proteins from Pseudomonas protegens Pf‑5, designated pseudoprobactin 1 and 2. Mass spectrometry and NMR spectroscopy revealed that their precursor proteins, PbnA1 and PbnA2, contain an N-terminal signal peptide and an intramolecular disulfide bond between the N-terminal and C-terminal cysteines. The remaining cysteine residues undergo MNIO-mediated modification into oxazolone/thioamide pairs, catalyzed by the PbnB1C1/PbnB2C2 enzyme complex. While the signal peptide and disulfide bond are dispensable for modification, the residues immediately downstream of the cysteines in the precursor proteins critically influence PbnB1C1/PbnB2C2 activity, with PbnB2C2 exhibiting a specific order in modifying multiple cysteine residues. Functional assays demonstrate that pseudoprobactin 1 and 2 bind copper with high specificity and enhance Pf-5 survival under chlorite-induced oxidative stress. Combined with our bioinformatic analysis, these findings establish oxazolone/thioamide as a prevalent MNIO-mediated modification, define the substrate recognition determinants, and reveal a previously unrecognized role for MNIO-modified small proteins in bacterial oxidative stress defense.

INSTRUMENT(S):

ORGANISM(S): Pseudomonas Protegens Pf-5 Bacteria

TISSUE(S): Cell Culture

SUBMITTER: hanzhong feng  

LAB HEAD: Yong-Xing He

PROVIDER: PXD066785 | Pride | 2026-07-29

REPOSITORIES: Pride