Multinuclear non-heme iron dependent oxidative enzymes: Landscape of their substrates, partner proteins and biosynthetic gene clusters
Authors: Antoine R. , Leprevost L. , Jünger S. , Zirah S. , Lippens G. , Li Y. , Dubiley S. , Jacob-Dubuisson F. 
Published: January 2025

AbstractEnzymes of themultinuclearnon-heme iron-dependentoxidase (MNIO) superfamily catalyze various modification reactions on the precursors ofribosomally synthesized,post-translationally modifiedpeptides (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 extensivein silicostudies. Analyses of MNIO-coding biosynthetic gene clusters (BGCs) identified all major groups of putative precursors 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 Terrabacteria, 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 specific associations between subgroups of MNIO enzymes, families of precursors, and types of partner proteins, indicating some specificity in these systems. This study depicts the global landscape of potential MNIO-dependent natural products by unveiling the major 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 statementThemultinuclear non-heme iron-dependentoxidative (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 extensivein silicoanalyses to explore the diversity of RiPP precursors genetically associated with MNIO enzymes and identified all 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 and 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.