Biosynthesis of Peptidic Thiooxazole Metallophores Installed by Multinuclear Nonheme Iron Enzymes

Significant effort has been directed toward the characterization of nonheme iron enzymes owing to their breadth of unique reactivity. Through genome mining, we identified a conserved biosynthetic gene cluster within Pseudomonadota encoding one such family, the multinuclear nonheme iron-dependent oxidative enzymes (MNIO, formerly DUF692). Using a representative gene cluster from Fontimonas thermophila, we heterologously produced the post-translationally modified peptide fontiphorin, and detailed spectral analysis revealed MNIO-catalyzed installation of seven 5-thiooxazole (5TO) moieties. During our work, additional MNIO products were reported with conflicting structural assignments, so we investigated the related biosynthetic gene clusters from Haemophilus influenzae and Neisseria gonorrhoeae. Using alkylation-assisted HMBC correlations, we demonstrated that these products also contain 5TO, resulting in a revision of the structure of oxazolin. We further provide evidence supporting a role for 5TO-containing peptides in copper detoxification and recommend that this emerging class of Cu-associated peptidic thiooxazole metallophores be referred to as captophorins. To further explore the captophorins, we reconstituted fontiphorin biosynthesis in vitro and investigated its enzymatic requirements. Using cell-free production of single-site, double-site, and naturally occurring variants, we examined enzyme-substrate interactions to determine key sites governing catalysis by 5TO-forming MNIOs. Through our detailed spectroscopic approach for 5TO assignment and investigation of enzyme-substrate interactions, our work unifies tens of thousands of MNIOs in the biosynthesis of captophorins.