Phosphohistidine (pHis) is a reversible protein post-translational modification (PTM) that is currently poorly understood. The P-N bond in pHis is heat and acid-sensitive, making it more challenging to study than the canonical phosphoamino acids pSer, pThr, and pTyr. As advancements in the development of tools to study pHis have been made, the roles of pHis in cells are slowly being revealed. To date, a handful of enzymes responsible for controlling this modification have been identified, including the histidine kinases NME1 and NME2, as well as the phosphohistidine phosphatases PHPT1, LHPP, and PGAM5. These tools have also identified the substrates of these enzymes, granting new insights into previously unknown regulatory mechanisms. Here, we discuss the cellular function of pHis and how it is regulated on known pHis-containing proteins, as well as cellular mechanisms that regulate the activity of the pHis kinases and phosphatases themselves. We further discuss the role of the pHis kinases and phosphatases as potential tumor promoters or suppressors. Finally, we give an overview of various tools and methods currently used to study pHis biology. Given their breadth of functions, unraveling the role of pHis in mammalian systems promises radical new insights into existing and unexplored areas of cell biology.

Two resorufin-based substrates for protein tyrosine phosphatase (PTP) activity have been synthesized. These substrates provide sensitive fluorogenic readouts of PTP activity in vitro and in living cells at both acidic and neutral pH. In addition, the presence of the pathogenic bacteria Staphylococcus aureus was detected visually using a colorimetric readout.

Although the importance of protein histidine phosphorylation in mammals has been a subject of increasing interest, few chemical probes are available for monitoring and manipulating PHP activity. Here, we present an optimized and validated protocol for assaying the activity of PHPT1 using the fluorogenic substrate DiFMUP. The kinetic parameters of our optimized assay are significantly improved as compared with other PHPT1 assays in the literature, with a kcat of 0.39 ± 0.02 s-1, a Km of 220 ± 30 μM, and a kcat/Km of 1800 ± 200 M-1 s-1. In addition, the assay is significantly more sensitive as a result of using a fluorescent probe, requiring only 109 nM enzyme as compared with 2.4 µM as required by previously published assays. In the process of assay optimization, we discovered that PHPT1 is sensitive to a reducing environment and inhibited by transition metal ions, with one apparent Cu(II) binding site with IC50 value of 500 ± 20 μM and two apparent Zn(II) binding sites with IC50 values of 25 ± 1 μM and 490 ± 20 μM.