title: The unique serine/threonine phosphatase from the minimal bacterium Mycoplasma synoviae: biochemical characterization and metal dependence.
abstract: Serine/threonine protein phosphatases have been described in many pathogenic bacteria as essential enzymes involved in phosphorylation-dependent signal transduction pathways and frequently associated with the virulence of these organisms. An inspection of Mycoplasma synoviae genome revealed the presence of a gene (prpC) encoding a putative protein phosphatase of the protein phosphatase 2C (PP2C) subfamily. Here, we report a complete biochemical characterization of M. synoviae phosphatase (PrpC) and the particular role of metal ions in the structure-function relationship of this enzyme. PrpC amino acid sequence analysis revealed that all the residues involved in the dinuclear metal center and the putative third metal ion-coordinating residues, conserved in PP2C phosphatases, are present in PrpC. PrpC is a monomeric protein able to dephosphorylate phospho-substrates with Mn(2+) ions' dependence. Thermal stability analysis demonstrated the enzyme stability at mild temperatures and the influence of Mn(2+) ions in this property. Mass spectrometry analysis suggested that three metal ions bind to PrpC, two of which with an apparent high-affinity constant. Mutational analysis of the putative third metal-coordinating residues, Asp122 and Arg164, revealed that these variants exhibited a weaker binding of manganese ions, and that both mutations affected PrpC phosphatase activity. According to these results, PrpC is a metal-dependent protein phosphatase member with an improved stability in the holo form and with Asp122, possibly implicated in the third metal-binding site, essential to catalytic activity.
authors.affiliation: Departamento de Bioquímica-CCB, Centro de Biologia Molecular Estrutural, Universidade Federal de Santa Catarina, Florianópolis, SC, 88040-900, Brazil.
authors.forename: Angela C O
authors.initials: ACO
authors.lastname: Menegatti
authors.affiliation: Departamento de Bioquímica-CCB, Centro de Biologia Molecular Estrutural, Universidade Federal de Santa Catarina, Florianópolis, SC, 88040-900, Brazil.
authors.forename: Javier
authors.initials: J
authors.lastname: Vernal
authors.affiliation: Departamento de Bioquímica-CCB, Centro de Biologia Molecular Estrutural, Universidade Federal de Santa Catarina, Florianópolis, SC, 88040-900, Brazil. hernan.terenzi@ufsc.br.
authors.forename: Hernán
authors.initials: H
authors.lastname: Terenzi
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D004795:Enzyme Stability; D007700:Kinetics; D008345:Manganese / Q000737:chemistry; D008969:Molecular Sequence Data; D045805:Mycoplasma synoviae / Q000201:enzymology*; D010748:Phosphopeptides / Q000737:chemistry; D010749:Phosphoprotein Phosphatases / Q000737:chemistry*; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational
chemical_list: D001426:Bacterial Proteins; D010748:Phosphopeptides; D008345:Manganese; D010749:Phosphoprotein Phosphatases