Structural and activity characterization of human PHPT1 after oxidative modification

Phosphohistidine phosphatase 1 (PHPT1), the only known phosphohistidine phosphatase in mammals, regulates phosphohistidine levels of several proteins including those involved in signaling, lipid metabolism, and potassium ion transport. While the high-resolution structure of human PHPT1 (hPHPT1) is available and residues important for substrate binding and catalytic activity have been reported, little is known about post-translational modifications that modulate hPHPT1 activity. Here we characterize the structural and functional impact of hPHPT1 oxidation upon exposure to a reactive oxygen species, hydrogen peroxide (H(2)O(2)). Specifically, liquid chromatography-tandem mass spectrometry was used to quantify site-specific oxidation of redox-sensitive residues of hPHPT1. Results from this study revealed that H(2)O(2) exposure induces selective oxidation of hPHPT1 at Met95, a residue within the substrate binding region. Explicit solvent molecular dynamics simulations, however, predict only a minor effect of Met95 oxidation in the structure and dynamics of the apo-state of the hPHPT1 catalytic site, suggesting that if Met95 oxidation alters hPHPT1 activity, then it will do so by altering the stability of an intermediate state. Employing a novel mass spectrometry-based assay, we determined that H(2)O(2)–induced oxidation does not impact hPHPT1 function negatively; a result contrary to the common conception that protein oxidation is typically a loss-of-function modification.