Tailored Cofactor Traps for the Detection of Hemithioacetal-Forming Pyridoxal Kinases.
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Issue Date
2020-12-03
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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 importance of a cysteine, embedded within a distant flexible lid region, was recently demonstrated. 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 yet available to study the relevance of this lid residue. Here, we introduce pyridoxal probes, each equipped with an electrophilic trapping group in place of the aldehyde to target PLK reactive lid cysteines as a mimic of hemithioacetal formation. The addition of alkyne handles placed at two different positions within the pyridoxal structure facilitates enrichment of PLKs from living cells. Interestingly, depending on the position, the probes displayed a preference for either Gram-positive or Gram-negative PLK enrichment. 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, qualifying them as chemical tools for mining further diverse proteomes for this important enzyme class.Citation
ACS Chem Biol. 2020 Dec 18;15(12):3227-3234. doi: 10.1021/acschembio.0c00787. Epub 2020 Dec 3.Affiliation
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.Publisher
American Society for Chemistry (ACS)Journal
ACS chemical biologyPubMed ID
33269909Type
ArticleLanguage
enEISSN
1554-8937ae974a485f413a2113503eed53cd6c53
10.1021/acschembio.0c00787
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- Creative Commons
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