Chemical Proteomics Reveal the Inventory of Pyrroloquinoline Quinone Binding Proteins in Bacteria

Pyrroloquinoline quinone (<b>PQQ</b>) is a bacterial redox cofactor enabling enzyme catalysis in various sugar and alcohol dehydrogenases. However, its proposed additional role as a "longevity vitamin" lacks a clear molecular basis and is thus highly debated. Here, we applied chemical proteomics to identify previously unknown classes of <b>PQQ</b>-binding proteins. We designed and synthesized a structurally diverse suite of five <b>PQQ</b> probes equipped with a diazirine photo-cross-linker and an alkyne handle for target identification. The fidelity of the probes was first evaluated for two well-characterized bacterial <b>PQQ</b>-dependent enzymes, demonstrating not only probe binding but also the reconstitution of catalytic activity. We then commenced with proteome profiling of <i>Escherichia coli</i> and <i>Pseudomonas putida</i> cells and unraveled a distinct set of putative <b>PQQ</b>-binding proteins. Recombinant expression of selected hits, including several chaperones, validated <b>PQQ</b> binding. Notably, in some cases, <b>PQQ</b> even formed covalent adducts with selected lysine residues, for instance, in the AAA+ ATPase RuvB involved in DNA remodeling. Overall, our work highlights the utility of <b>PQQ</b> probes to further unravel the complement of cofactor-binding proteins in whole cells. It also provides a basis for future mechanistic studies of <b>PQQ</b> functions beyond redox catalysis.