Dataset Information

Customizing functionalized cofactor mimics to study the human pyridoxal 5'-phosphate-binding proteome

ABSTRACT: Here we apply functionalized cofactor derivatives to study vitamin B6-dependent enzymes in human cells. We are able to cover for a large fraction of the pyridoxal 5'-phosphate-binding proteome (PLP-ome) and validate our results exemplarily for two PLP-DEs. One of those, named pyridoxal 5'-phosphate binding protein (PLPBP) is characterized further including studies with chemical cross-linking combined with mass spectrometry. We proceed with an in situ PLP-DE target screen applying B6-targeting compounds revealing interesting insights into selectivity. Finally, we broaden our analysis to multiple human cell lines revealing an altered PLP-DE target profile.

INSTRUMENT(S): Orbitrap Fusion ETD, Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Anja Fux

LAB HEAD: Stephan Axel Sieber

PROVIDER: PXD014771 | Pride | 2019-08-27

REPOSITORIES: Pride

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Customizing Functionalized Cofactor Mimics to Study the Human Pyridoxal 5'-Phosphate-Binding Proteome.

Fux Anja A Pfanzelt Martin M Kirsch Volker C VC Hoegl Annabelle A Sieber Stephan A SA

Cell chemical biology 20190822 10

Pyridoxal 5'-phosphate (PLP) is a versatile cofactor that catalyzes a plethora of chemical transformations within a cell. Although many human PLP-dependent enzymes (PLP-DEs) with crucial physiological and pathological roles are known, a global method enabling their cellular profiling is lacking. Here, we demonstrate the utility of a cofactor probe for the identification of human PLP-binding proteins in living cells. Striking selectivity of human pyridoxal kinase led to a customized labeling stra ...[more]

PMID: 31447350

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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.

			Action	DRS
	20180609_HeLa_PL1_1.raw	Raw
	20180609_HeLa_PL1_2.raw	Raw
	20180609_HeLa_PL1_3.raw	Raw
	20180609_HeLa_PL1_4.raw	Raw
	20180609_HeLa_PN1.raw	Raw