Chemical-proteomic tools to monitor pyridoxal phosphorylation and its function as an enzyme cofactor in disease-related pathways
Final Report Abstract
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 Grampositive 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. Furthermore, we developed a chemical probe for the in situ labeling of PLP-dependent enzymes based on the mechanism-based inhibition of vigabatrin. The synthesis was successfully accomplished, however, the probe turned out not to crosslink its dedicated target. We thus enhanced the electrophilicity by a new chemical design and introduction of a halogen leaving group. Again the synthesis was efficiently performed but labeling could still not be observed suggesting that further factors such as sterics need to be considered in follow-up studies.
Publications
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"Tailored cofactor traps for the in situ detection of hemithioacetal-forming pyridoxal kinases", ACS Chem Bio, 2020
Hübner, I., Dienemann J.-N., Friederich, J., Sieber, S.A.
