Dataset Information

Mining the cellular inventory of pyridoxal phosphate-dependent enzymes with functionalized cofactor mimics

ABSTRACT: Pyridoxal phosphate (PLP) is an enzyme cofactor required for the chemical transformation of biological amines in numerous essential cellular processes. PLP-dependent enzymes (PLP-DEs) are ubiquitous and evolutionarily diverse, making their classification based on sequence homology challenging. This calls for a more direct approach for identifying new PLP-DEs and uncovering novel roles of PLP within the cell. We present a chemical proteomic method for reporting on PLP-DEs using functionalized cofactor probes. PL-analogs designed to be taken up by cells and metabolized in situ were synthesized. The probes are phosphorylated to functional cofactor surrogates by cellular PL kinases and bind to PLP-DEs via a reversible aldimine bond, which can be fixed by NaBH4-reduction. Conjugation to a reporter tag permits the identification of PLP-DEs using quantitative, label-free mass spectrometry via confidence ranking and profile searches. We label a significant portion of the PLP-ome in Staphylococcus aureus (41%) and annotate uncharacterized proteins as novel PLP-DEs. In applying our method, we were able to study structural tolerance within PLP-DE active sites and to screen for off-targets of the PLP-DE inhibitor D-cycloserine. Our cofactor profiling method represents a robust way to mine the cellular inventory of PLP-DEs which can be used for genome annotation, comparative profiling and drug development.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Staphylococcus Aureus

SUBMITTER: Nina Bach

LAB HEAD: Prof. Stephan A. Sieber

PROVIDER: PXD006483 | Pride | 2018-10-17

REPOSITORIES: Pride

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			Action	DRS
	171026_AH-2-162_100uM_D.raw	Raw
	171026_AH-2-162_100uM_E.raw	Raw
	171026_AH-2-162_100uM_F.raw	Raw
	171026_AH-2-162_DMSO_D.raw	Raw
	171026_AH-2-162_DMSO_E.raw	Raw

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Mining the cellular inventory of pyridoxal phosphate-dependent enzymes with functionalized cofactor mimics.

Hoegl Annabelle A Nodwell Matthew B MB Kirsch Volker C VC Bach Nina C NC Pfanzelt Martin M Stahl Matthias M Schneider Sabine S Sieber Stephan A SA

Nature chemistry 20181008 12

Pyridoxal phosphate (PLP) is an enzyme cofactor required for the chemical transformation of biological amines in many central cellular processes. PLP-dependent enzymes (PLP-DEs) are ubiquitous and evolutionarily diverse, making their classification based on sequence homology challenging. Here we present a chemical proteomic method for reporting on PLP-DEs using functionalized cofactor probes. We synthesized pyridoxal analogues modified at the 2'-position, which are taken up by cells and metaboli ...[more]

PMID: 30297752

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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.