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.