Pyridoxal phosphate (PLP) is an enzyme cofactor required for the chemical transformation of biological amines in numerous essential 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 (PL)-analogues modified at the 2’-position which are taken up by cells and metabolized in situ. These PL-analogues are phosphorylated to functional cofactor surrogates by cellular PL 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.
Hoegl, A., Nodwell, M.B., Kirsch, V.C., Bach, N.C., Pfanzelt, M., Stahl, M., Schneider, S., Sieber, S.A., "Mining the cellular inventory of pyridoxal phosphate-dependent enzymes with functionalized cofactor mimics", Nat. Chem. 2018, accepted
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