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Mining the cellular inventory of pyridoxal phosphate-dependent enzymes with functionalized cofactor mimics.


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.

SUBMITTER: Hoegl A 

PROVIDER: S-EPMC6252082 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Mining the cellular inventory of pyridoxal phosphate-dependent enzymes with functionalized cofactor mimics.

Hoegl Annabelle A   Nodwell Matthew B MB   Kirsch Volker C VC   Bach Nina C NC   Pfanzelt Martin M   Stahl Matthias M   Schneider Sabine S   Sieber Stephan A SA  

Nature chemistry 20181008 12


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 metaboli  ...[more]

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