title: Structural and activity characterization of human PHPT1 after oxidative modification.
abstract: 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 (H2O2). 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 H2O2 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 H2O2-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.
authors.affiliation: Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, Tampa, FL 33620, USA.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Martin
authors.affiliation: Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, Tampa, FL 33620, USA.
authors.forename: Priyanka
authors.initials: P
authors.lastname: Dutta
authors.affiliation: Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, Tampa, FL 33620, USA.
authors.forename: Shikha
authors.initials: S
authors.lastname: Mahajan
authors.affiliation: Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, Tampa, FL 33620, USA.
authors.forename: Sameer
authors.initials: S
authors.lastname: Varma
authors.affiliation: Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, Tampa, FL 33620, USA.
authors.forename: Stanley M
authors.initials: SM
authors.lastname: Stevens
mesh_terms: D020134:Catalytic Domain / Q000187:drug effects; D006639:Histidine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008715:Methionine / Q000187:drug effects; D056004:Molecular Dynamics Simulation; D016877:Oxidants / Q000494:pharmacology*; D010084:Oxidation-Reduction; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D016877:Oxidants; D011994:Recombinant Proteins; D006639:Histidine; D008715:Methionine; D006861:Hydrogen Peroxide; C504019:PHPT1 protein, human; D010744:Phosphoric Monoester Hydrolases; C005436:phosphohistidine