Multinuclear non-heme iron dependent oxidative enzymes: Landscape of their substrates, partner proteins and biosynthetic gene clusters

Proteins of the multinuclear non-heme iron-dependent oxidative (MNIO) enzyme superfamily catalyze various modification reactions on the precursors of ribosomally synthesized, post-translationally modified peptides (RiPPs). We recently identified two large families of MNIO-modified RiPPs called bufferins, which enhance bacterial growth under copper stress by chelating the excess metal ions. Here, we explored the diversity of potential MNIO substrates by performing extensive in silico studies. Analyses of MNIO-coding biosynthetic gene clusters (BGCs) identified various groups of putative precursors most of which are characterized by specific Cys-containing motifs, throughout the eubacterial phylogenetic tree. The precursors of most MNIO-modified RiPPs harbor N-terminal Sec-dependent signal peptides, a rare feature among bacterial RiPPs. Some precursors are very long relative to those of typical RiPPs, indicating that MNIO enzymes could modify both peptide and protein substrates. We also identified a distinct family of integral membrane proteins with large predicted extra-cytoplasmic domains mostly found in Actinomycetota, frequently but not systematically associated with MNIOs. Most MNIO BGCs harbor genes coding for DUF2063 domain-containing proteins or structurally related proteins, serving as partners of the enzymes for precursor modification. We uncovered a correlation between the presence or the absence of Sec signal peptides in the precursors and the types of partner proteins of the MNIO enzymes. This study depicts the global landscape of potential MNIO-dependent natural products by unveiling groups of peptides and proteins genetically associated with MNIOs. It reveals a treasure trove of potential new RiPP precursors which likely represent a widespread bacterial strategy to deal with copper stress, and most likely other stresses, in natural environments.