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

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. This calls for a more direct approach for identifying new PLP-DEs and uncovering novel roles of PLP within the cell. We present a chemical proteomic method for reporting on PLP-DEs using functionalized cofactor probes. PL-analogs designed to be taken up by cells and metabolized in situ were synthesized. The probes are phosphorylated to functional cofactor surrogates by cellular PL kinases and bind to PLP-DEs via a reversible aldimine bond, which can be fixed by NaBH4-reduction. Conjugation to a reporter tag permits the identification of PLP-DEs using quantitative, label-free mass spectrometry via confidence ranking and profile searches. We label a significant portion of the PLP-ome in Staphylococcus aureus (41%) and annotate uncharacterized proteins as novel PLP-DEs. In applying our method, we were able to study structural tolerance within PLP-DE active sites and to screen for off-targets of the PLP-DE inhibitor D-cycloserine. Our cofactor profiling method represents a robust way to mine the cellular inventory of PLP-DEs which can be used for genome annotation, comparative profiling and drug development.