Multinuclear non-heme iron dependent oxidative enzymes (MNIOs) involved in unusual peptide modifications

Abstract

Multinuclear non-heme iron dependent oxidative enzymes (MNIOs), formerly known as domain of unknown function 692 (DUF692), are involved in the post-translational modification of peptides during the biosynthesis of peptide-based natural products. These enzymes catalyze highly unusual and diverse chemical modifications. Several class-defining features of this large family (>14 000 members) are beginning to emerge. Structurally, the enzymes are characterized by a TIM-barrel fold and a set of conserved residues for a di- or tri-iron binding site. They use molecular oxygen to modify peptide substrates, often in a four-electron oxidation taking place at a cysteine residue. This review summarizes the current understanding of MNIOs. Four modifications are discussed in detail: oxazolone-thioamide formation, β-carbon excision, hydantoin-macrocycle formation, and 5-thiooxazole formation. Briefly discussed are two other reactions that do not take place on Cys residues.

Keywords: Dinuclear/trinuclear iron enzyme; Metalloenzyme; Metallophore; Natural product biosynthesis; RiPPs.

Figure 1.

Diverse post-translational modifications performed by members of the MNIO family. (A) Reactions catalyzed by MbnBC, TglHI, ChrHI, BufBC, MovX, and ApyHI. (B) Sequence similarity network (SSN) of MNIOs (DUF692), colored by the top ten phyla in which MNIOs are most frequently found. Percentages represent the distribution of MNIO-containing species across phyla. The SSN was generated by using the Enzyme Function Initiative-Enzyme Similarity Tool with over 14 000 members [15,16].

Figure 2.

Proposed reaction mechanisms of MNIOs. (A) Proposed mechanisms of the cysteine modifying MNIOs MbnB, TglH, ChrH, and BufB. The mechanisms depicted here are consistent with the current evidence and are drawn to show possible similarities across the family. For example, all four mechanisms involving cysteine are drawn with a thiolate-coordinating ferric iron being different from the ferrous iron that activates O₂. Other mechanisms can be drawn in which the same Fe coordinates Cys and activates O₂ [14]. The fate of the carbonyl carbons during the ChrH reaction is indicated with an asterisk; X = enzyme residue. (B) Proposed mechanisms of the asparagine/aspartate modifying MNIOs MovX and ApyH. B = enzyme base. We note that for all reactions shown, other mechanisms are possible, with several steps having both homolytic or heterolytic possibilities.

Figure 3.

Structural insights into MNIO catalysis and substrate binding. (A) Structure of the MbnABC complex from Rugamonas rubra (PDB:7FC0), showing the common TIM-barrel fold shared across MNIOs and the three bound irons (orange spheres). The dotted box highlights the contact between MbnA and the MbnC partner protein that is required for substrate binding [11]. (B) Active site structure of RrMbnB showing coordinating side chains (teal sticks) and water molecules (red spheres). The cysteine residue of the substrate MbnA coordinating with Fe1 is shown in pink. A water molecule occupies this coordination site in the absence of substrate.