Mining the cellular inventory of pyridoxal phosphate-dependent enzymes with functionalized cofactor mimics
| Autor: | Sabine Schneider, Nina C. Bach, Matthew B. Nodwell, Martin Pfanzelt, Stephan A. Sieber, Volker C. Kirsch, Matthias Stahl, Annabelle Hoegl |
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| Jazyk: | angličtina |
| Předmět: |
0301 basic medicine
Models Molecular Aldimine General Chemical Engineering Coenzymes chemical and pharmacologic phenomena 010402 general chemistry Ornithine Decarboxylase 01 natural sciences Cofactor Article 03 medical and health sciences chemistry.chemical_compound immune system diseases Pyridoxal phosphate Phosphorylation Pyridoxal Transaminases chemistry.chemical_classification Glycine Hydroxymethyltransferase biology Molecular Structure Kinase Alanine Racemase General Chemistry 0104 chemical sciences nervous system diseases Kinetics 030104 developmental biology Enzyme chemistry Biochemistry Pyridoxal Phosphate Proteome biology.protein Dopa Decarboxylase lipids (amino acids peptides and proteins) |
| Zdroj: | Nature Chemistry Nature chemistry |
| ISSN: | 1755-4349 1755-4330 |
| DOI: | 10.1038/s41557-018-0144-2 |
| Popis: | 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 metabolized in situ. These pyridoxal analogues are phosphorylated to functional cofactor surrogates by cellular pyridoxal kinases and bind to PLP-DEs via an aldimine bond which can be rendered irreversible by NaBH4 reduction. Conjugation to a reporter tag enables the subsequent identification of PLP-DEs using quantitative, label-free mass spectrometry. Using these probes we accessed a significant portion of the Staphylococcus aureus PLP-DE proteome (73%) and annotate uncharacterized proteins as novel PLP-DEs. We also show that this approach can be used to study structural tolerance within PLP-DE active sites and to screen for off-targets of the PLP-DE inhibitor D-cycloserine. |
| Databáze: | OpenAIRE |
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