Chemoproteomic identification of phosphohistidine acceptors: posttranslational activity regulation of a key glycolytic enzyme

Histidine phosphorylation, an unconventional and understudied posttranslational modification, often involves phosphohistidine (pHis) "acceptor" proteins, which bind to pHis residues and undergo phosphotransfer from pHis. While the roles of pHis acceptors are well-documented in bacterial cell signalling and metabolism, the presence and functions of additional pHis acceptors remain largely unknown. In this study, we introduce a chemoproteomic strategy leveraging a stable analogue of 3-pHis to identify 13 putative pHis acceptors in <i>Escherichia coli</i>. Among these, we identified phosphofructokinase-1 (PfkA), a central enzyme in glycolysis, as a pHis acceptor phosphorylated at His249 by phosphocarrier protein HPr (PtsH). This phosphorylation, modulated by carbon source availability, inhibited PfkA's kinase activity, while the pHis-specific phosphatase signal inhibitory factor X (SixA) reversed the effect, restoring the kinase function. Our findings reveal a novel regulatory mechanism in which histidine phosphorylation dynamically controls a key glycolytic enzyme, implicating a broader role for pHis in bacterial metabolism.