PXD021791

PXD021791 is an original dataset announced via ProteomeXchange.

Title	Tailored cofactor traps for the in situ detection of hemithioacetal-forming pyridoxal kinases
Description	Pyridoxal kinases (PLK) are crucial enzymes for the biosynthesis of pyridoxal phosphate, an important cofactor in a plethora of enzymatic reactions. The evolution of these enzymes resulted in different catalytic designs. In addition to the active site, the relevance of a cysteine, embedded within a distant flexible lid region, was recently shown. This cysteine forms a hemithioacetal with the pyridoxal aldehyde and is essential for catalysis. Despite the prevalence of these enzymes in various organisms, no tools were available to assess the general relevance of the lid residue. We here introduce pyridoxal probes equipped with an electrophilic trapping group in place of the aldehyde which readily reacts with reactive lid cysteines as a mimic of hemithioacetal formation. This irreversible capture not only facilitates the readout of cysteine reactivity but also the enrichment of PLKs from living cells via alkyne handles placed at two different positions within the pyridoxal structure. Interestingly, depending on the position, the probes either displayed a preference for Gram-positive or Gram-negative PLK enrichment in living cells. By applying the cofactor traps, we were able to validate not only previously investigated Staphylococcus aureus and Enterococcus faecalis PLKs but also Escherichia coli and Pseudomonas aeruginosa PLKs unravelling a crucial role of the lid cysteine for catalysis. Overall, our tailored probes facilitated a reliable readout of lid cysteine containing PLKs and thereby qualify them as chemical tools for further mining diverse proteomes for this important enzyme class.
HostingRepository	PRIDE
AnnounceDate	2021-03-04
AnnouncementXML	Submission_2021-03-04_12:36:56.453.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Ines Hübner
SpeciesList	scientific name: Escherichia coli; NCBI TaxID: 562; scientific name: Enterococcus faecalis (Streptococcus faecalis); NCBI TaxID: 1351; scientific name: Staphylococcus aureus; NCBI TaxID: 1280;
ModificationList	acetylated residue; monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2020-10-02 04:15:47	ID requested
⏵ 1	2021-03-04 12:36:57	announced

H, ü, bner I, Dienemann JN, Friederich J, Schneider S, Sieber SA, Detection of Hemithioacetal-Forming Pyridoxal Kinases. ACS Chem Biol, 15(12):3227-3234(2020) [pubmed]

submitter keyword: Pyridoxal, Pyridoxal kinase, S. aureus, E. faecalis, E. coli, P. aeruginosa, LC-MS/MS

Stephan A. Sieber
contact affiliation	Department of Chemistry, Chair of Organic Chemistry II, Center For Functional Protein Assemblies (CPA), Technische Universität München, Lichtenbergstraße 4, 85748 Garching, Germany
contact email	stephan.sieber@tum.de
lab head
Ines Hübner
contact affiliation	Technical University of Munich
contact email	ines.huebner@tum.de
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD021791
     1. Label: PRIDE project
     2. Name: Tailored cofactor traps for the in situ detection of hemithioacetal-forming pyridoxal kinases