期刊
NATURE CHEMISTRY
卷 10, 期 12, 页码 1234-1245出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/s41557-018-0144-2
关键词
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资金
- European Research Council (ERC)
- European Union's Horizon 2020 research and innovation programme [725085]
- Deutscher Akademischer Austausch Dienst (DAAD)
- Studienstiftung des Deutschen Volkes
- European Research Council (ERC) [725085] Funding Source: European Research Council (ERC)
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.
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