Genome mining for natural products made by multinuclear iron-dependent oxidation enzymes (MNIOs).

Multinuclear non-heme iron-dependent oxidative enzymes (MNIOs) are a family of diiron/triiron enzymes that install post-translational modifications (PTMs) on ribosomally produced peptides. These modifications include oxazolone-thioamide formation, carbon excision, thiooxazole formation, α-keto acid formation, and N-Cα bond cleavage, demonstrating the high functional diversity of MNIOs. Many MNIOs function together with a partner protein that helps recruit the substrate peptide. This review outlines experimental methods for the expression and purification of a representative MNIO (TglH) and its peptide substrate (TglA), as well as the characterization of the resulting PTM using various spectroscopic methods and isotope labeling. These protocols can be applied to study other MNIO-encoding pathways, with case-by-case adaptations and differences highlighted. Continued genome mining of MNIOs is likely to reveal more novel enzymatic functions, advancing our understanding of their catalytic mechanisms and their roles in natural product biosynthesis.