Mining the cellular inventory of pyridoxal phosphate-dependent enzymes with functionalized cofactor mimics.
Nat Chem
; 10(12): 1234-1245, 2018 12.
Article
en En
| MEDLINE
| ID: mdl-30297752
ABSTRACT
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.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Ornitina Descarboxilasa
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Fosfato de Piridoxal
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Glicina Hidroximetiltransferasa
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Coenzimas
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Alanina Racemasa
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Dopa-Decarboxilasa
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Transaminasas
Idioma:
En
Revista:
Nat Chem
Asunto de la revista:
QUIMICA
Año:
2018
Tipo del documento:
Article
País de afiliación:
Alemania