title: Reversible oxidation controls the activity and oligomeric state of the mammalian phosphoglycolate phosphatase AUM.
abstract: Redox-dependent switches of enzyme activity are emerging as important fine-tuning mechanisms in cell signaling. For example, protein tyrosine phosphatases employ a conserved cysteine residue for catalysis, which also renders them highly susceptible to reversible inactivation by oxidation. In contrast, haloacid dehalogenase (HAD)-type phosphatases perform catalysis via a phosphoaspartyltransferase reaction. The potential regulation of HAD phosphatases by reversible oxidation has not yet been explored. Here, we investigate the redox-sensitivity of the HAD-type phosphoglycolate phosphatase PGP, also known as AUM or glycerol-3-phosphate phosphatase. We show that recombinant, purified murine PGP is inhibited by oxidation and re-activated by reduction. We identify three reactive cysteine residues in the catalytic core domain of PGP (Cys35, Cys104 and Cys243) that mediate the reversible inhibition of PGP activity and the associated, redox-dependent conformational changes. Structural analysis suggests that Cys35 oxidation weakens van-der-Waals interactions with Thr67, a conserved catalytic residue required for substrate coordination. Cys104 and Cys243 form a redox-dependent disulfide bridge between the PGP catalytic core and cap domains, which may impair the open/close-dynamics of the catalytic cycle. In addition, we demonstrate that Cys297 in the PGP cap domain is essential for redox-dependent PGP oligomerization, and that PGP oxidation/oligomerization occurs in response to stimulation of cells with EGF. Finally, employing a modified cysteinyl-labeling assay, we show that cysteines of cellular PGP are transiently oxidized to sulfenic acids. Taken together, our findings establish that PGP, an aspartate-dependent HAD phosphatase, is transiently inactivated by reversible oxidation in cells.
authors.affiliation: Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Strasse 9, D-97078 Würzburg, Germany; Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Josef-Schneider-Strasse 2, D-97080 Würzburg, Germany.
authors.forename: Annegrit
authors.initials: A
authors.lastname: Seifried
authors.affiliation: Université de Montréal, Department of Medicine and Department of Biochemistry, Montréal, QC, Canada H3C 3J7; Montreal Heart Institute, 5000 Rue Bélanger, Montréal, QC, Canada H1T 1C8.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Bergeron
authors.affiliation: Université de Montréal, Department of Medicine and Department of Biochemistry, Montréal, QC, Canada H3C 3J7; Montreal Heart Institute, 5000 Rue Bélanger, Montréal, QC, Canada H1T 1C8; Colleges of Nanoscale Science & Engineering, SUNY Polytechnic Institute, 257 Fuller Road, Albany, NY 12203, USA.
authors.forename: Benoit
authors.initials: B
authors.lastname: Boivin
authors.affiliation: Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Strasse 9, D-97078 Würzburg, Germany; Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Josef-Schneider-Strasse 2, D-97080 Würzburg, Germany. Electronic address: antje.gohla@uni-wuerzburg.de.
authors.forename: Antje
authors.initials: A
authors.lastname: Gohla
mesh_terms: D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000378:metabolism; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000235:genetics*; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D004220:Disulfides; D017382:Reactive Oxygen Species; D001224:Aspartic Acid; D006861:Hydrogen Peroxide; D006867:Hydrolases; C022408:phosphoglycolate phosphatase; D010744:Phosphoric Monoester Hydrolases; C034507:2-haloacid dehalogenase; D003545:Cysteine
keywords: AUM; Cysteinyl-labeling assay; EGF; Glycerol-3-phosphate phosphatase; Haloacid dehalogenase-type phosphatase; Phosphoglycolate phosphatase; Reactive oxygen species; Reversible cysteine oxidation