Dataset Information

Chemical proteomics reveal the inventory of pyrroloquinoline quinone binding proteins in bacteria

ABSTRACT: Pyrroloquinoline quinone (PQQ) 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 so far unknown classes of PQQ-binding proteins. We designed and synthesized a structurally diverse suite of five PQQ probes equipped with a diazirine photocrosslinker and an alkyne handle for target identification. The fidelity of the probes was first evaluated for two well-characterized bacterial PQQ-dependent enzymes, demonstrating not only probe binding but also reconstitution of catalytic activity. We then commenced with proteome profiling of Escherichia coli and Pseudomonas putida cells and unraveled a distinct set of putative PQQ-binding proteins. Recombinant expression of selected hits including several chaperones validated PQQ binding. Notably, in some cases, PQQ 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 PQQ probes to further unravel the complement of cofactor-binding proteins in whole cells. It also provides a basis for future mechanistic studies on PQQ functions beyond redox catalysis.

INSTRUMENT(S):

ORGANISM(S): Escherichia Coli

SUBMITTER: Tao Wang

LAB HEAD: Stephan A. Sieber

PROVIDER: PXD075565 | Pride | 2026-04-27

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	E_coli_K12_PQQ_PQQ4_competition.zip	Other
	E_coli_K12_PQQ_PQQ4_competition_report.tsv	Tabular
	checksum.txt	Txt
	uniprot-download_true_format_fasta_query__28proteome_3AUP000000625_2-2023.05.02-11.18.14.75.fasta	Fasta

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Publications

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

Wang Tao T Mühlhofer Rahel R Lei E E Ding Wei W Klein Andreas S AS Zeymer Cathleen C Sieber Stephan A SA

Journal of the American Chemical Society 20260402 14

Pyrroloquinoline quinone (PQQ) 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 PQQ-binding proteins. We designed and synthesized a structurally diverse suite of five PQQ probes equipped with a diazirine photo-cross-linker and ...[more]

PMID: 41925033

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Project description:Unprecedented bacterial targets are urgently needed for the development of novel antibiotics to overcome the current resistance crisis. Challenges include the limited uptake of compounds as well as prioritizing proteins for their druggability and functional relevance. Especially, the wealth of uncharacterized proteins represents an untapped source for novel targets. However, tools to decipher their function are largely lacking. We here utilize the systematic mining of pyridoxal phosphate dependent enzymes (PLP DEs) in bacteria to focus on a target class, which is known to bind ligands, accesses PLP via active transport from the media and is involved in crucial metabolic processes. For this, we systematically exploited the chemical space of the pyridoxal (PL) scaffold and obtained eight PL probes bearing modifications at various ring positions. These probes were subsequently tested for phosphorylation by cognate kinases and labelling of PLP DEs in clinically relevant Gram-positive (Staphylococcus aureus) as well as Gram-negative (Escherichia coli and Pseudomonas aeruginosa) strains. Overall, the coverage of this diverse set of probes along with refined labelling conditions not only exceeded the performance of a previous probe generation, it also provided a detailed map of binding preferences of certain structural motifs. Although originally conducted in mutant cells devoid of PLP de novo biosynthesis, we here demonstrate efficient PLP DE labelling also in a wild type strain. Overall, the profiling revealed several putative PLP DEs with unknown function, of which we exemplarily characterized five via in-depth enzymatic assays. Finally, we screened a panel of putative PLP binders for antibiotic activity and unravelled the targets of hit molecules via competitive profiling with our probes. Here, an uncharacterized enzyme, essential for bacterial growth, was assigned as PLP dependent cysteine desulfurase and confirmed to be inhibited by the marketed drug phenelzine. Our approach provides a basis for deciphering novel PLP DEs as essential antibiotic targets along with corresponding ways to decipher small molecule inhibitors.