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The unique serine/threonine phosphatase from the minimal bacterium Mycoplasma synoviae: biochemical characterization and metal dependence

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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 Mn2+ ions’ dependence. Thermal stability analysis demonstrated the enzyme stability at mild temperatures and the influence of Mn2+ 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.

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Abbreviations

ESI-MS:

Electrospray ionization-mass spectrometry

HAD:

Haloacid dehalogenase

MALDI-TOF/TOF:

Matrix-assisted laser desorption/ionization-time-of-flight

Mw:

Molecular weight

FCP/SCP:

[TFIIF(transcription initiation factor IIF)-associating component of CTD (C-terminal domain) phosphatase/small CTD phosphatase]

WT:

Wild type

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Acknowledgments

This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa Científica de Tecnológica do Estado de Santa Catarina (FAPESC), Financiadora de Estudos e Projetos (FINEP), and Instituto Nacional de Biologia Estrutural e Bioimagem (INBEB). We acknowledge the technical staff of CEBIME for assistance. We thank Maria Lucia Bianconi (UFRJ), Douglas Norberto, and Leonardo Rosado for helpful discussions about ITC data, and Catia Silene Klein for the M. synoviae strain 53 cells.

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Authors and Affiliations

  1. Departamento de Bioquímica-CCB, Centro de Biologia Molecular Estrutural, Universidade Federal de Santa Catarina, Florianópolis, SC, 88040-900, Brazil

    Angela C. O. Menegatti, Javier Vernal & Hernán Terenzi

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  1. Angela C. O. Menegatti
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  2. Javier Vernal
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  3. Hernán Terenzi
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Correspondence to Hernán Terenzi.

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Menegatti, A.C.O., Vernal, J. & Terenzi, H. The unique serine/threonine phosphatase from the minimal bacterium Mycoplasma synoviae: biochemical characterization and metal dependence. J Biol Inorg Chem 20, 61–75 (2015). https://doi.org/10.1007/s00775-014-1209-3

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  • DOI: https://doi.org/10.1007/s00775-014-1209-3

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