Multinuclear Non-Heme Iron-Dependent Oxidative Enzymes: Landscape of Their Substrates, Partner Proteins

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. IMPACT STATEMENT. The multinuclear non-heme iron-dependent oxidative (MNIO) enzymes belong to an emerging superfamily of modification enzymes that catalyze various oxidation reactions on ribosomally synthesized post-translationally modified peptides (RiPP) precursors. The largest families of MNIO-modified RiPPs, called bufferins, are involved in copper homeostasis. In this work we performed extensive _in silico_ analyses to explore the diversity of RiPP precursors genetically associated with MNIO enzymes and identified major families. Some precursors are much larger than typical RiPP precursors, indicating that MNIO enzymes may also modify proteins. We defined subtypes of MNIO enzymes’ partner proteins dedicated to specific families of precursors. Our analyses of the biosynthetic gene clusters unveiled functions beyond copper homeostasis, likely for the response to other metal stresses. The global landscape of MNIO-modified RiPP precursors will be a basis for investigations into new RiPP families and will undoubtedly lead to the discovery of new modifications and new functions.