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.

磷酸吡哆醛 (PLP) 是许多中枢细胞过程中生物胺化学转化所需的酶辅因子。PLP 依赖性酶 (PLP-DE) 普遍存在且进化多样化，这使得它们基于序列同源性的分类具有挑战性。在这里，我们提出了一种使用功能化辅因子探针报告 PLP-DE 的化学蛋白质组学方法。我们合成了在 2'-位修饰的吡哆醛类似物，它们被细胞吸收并原位代谢。这些吡哆醛类似物被细胞吡哆醛激酶磷酸化为功能性辅助因子替代物，并通过醛亚胺键与 PLP-DE 结合，NaBH _{4可使其不可逆}减少。与报告标记结合后，可以使用定量、无标记质谱法对 PLP-DE 进行后续鉴定。使用这些探针，我们访问了大部分金黄色葡萄球菌PLP-DE 蛋白质组 (73%)，并将未表征的蛋白质注释为新的 PLP-DE。我们还表明，这种方法可用于研究 PLP-DE 活性位点内的结构耐受性，并筛选 PLP-DE 抑制剂d-环丝氨酸的脱靶。