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Properties of the endogenous components of the thioredoxin system in the psychrophilic eubacterium Pseudoalteromonas haloplanktis TAC 125

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Abstract

The endogenous components of the thioredoxin system in the Antarctic eubacterium Pseudoalteromonas haloplanktis have been purified and characterised. The temperature dependence of the activities sustained by thioredoxin (PhTrx) and thioredoxin reductase (PhTrxR) pointed to their adaptation in the cold growth environment. PhTrxR was purified as a flavoenzyme and its activity was significantly enhanced in the presence of molar concentration of monovalent cations. The energetics of the partial reactions leading to the whole electron transfer from NADPH to the target protein substrate in the reconstituted thioredoxin system was also investigated. While the initial electron transfer from NADPH to PhTrxR was energetically favoured, the final passage to the heterologous protein substrate enhanced the energetic barrier of the whole process. The energy of activation of the heat inactivation process essentially reflected the psychrophilic origin of PhTrxR. Vice versa, PhTrx possessed an exceptional heat resistance (half-life, 4.4 h at 95 °C), ranking this protein among the most thermostable enzymes reported so far in psychrophiles. PhTrxR was covalently modified by glutathione, mainly by its oxidised or nitrosylated forms. A mutagenic analysis realised on three non catalytic cysteines of the flavoenzyme allowed the identification of C303 as the target for the S-glutathionylation reaction.

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Abbreviations

Ap:

Aeropyrum pernix

Ec :

Escherichia coli

Ph :

Pseudoalteromonas haloplanktis

DTNB:

5,5′-dithiobis-2-nitrobenzoic acid

DTT:

Dithiothreitol

GSH:

Reduced glutathione

GSSG:

Oxidised glutathione

GSNO:

Nitrosylated glutathione

PMSF:

Phenylmethanesulphonyl fluoride

TNB:

2-nitro-5-thiobenzoate

Trx:

Thioredoxin

TrxR:

Thioredoxin reductase

Trx-S2 and Trx-(SH)2 :

Oxidised and reduced form of Trx, respectively

k in :

Heat inactivation rate constant

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Acknowledgments

This work was supported by grants from MIUR, PRIN 2009 (Rome) awarded to MM, EDV, GR.

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Authors and Affiliations

  1. Dipartimento di Scienze e Tecnologie dell’Ambiente e del Territorio, Università del Molise, Contrada Fonte Lappone, 86090, Pesche, IS, Italy

    Patrizia Falasca, Giovanna Evangelista & Gennaro Raimo

  2. Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, Via S. Pansini 5, 80131, Naples, Italy

    Roberta Cotugno, Salvatore Marco, Mariorosario Masullo, Emmanuele De Vendittis & Gennaro Raimo

  3. Dipartimento di Studi delle Istituzioni e dei Sistemi Territoriali, Università di Napoli “Parthenope”, Via Medina 40, 80133, Naples, Italy

    Mariorosario Masullo

Authors
  1. Patrizia Falasca
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  2. Giovanna Evangelista
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  3. Roberta Cotugno
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  4. Salvatore Marco
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  5. Mariorosario Masullo
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  6. Emmanuele De Vendittis
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  7. Gennaro Raimo
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Corresponding authors

Correspondence to Emmanuele De Vendittis or Gennaro Raimo.

Additional information

Communicated by F. Robb.

Authors P. Falasca and G. Evangelista equally contributed to this work.

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Falasca, P., Evangelista, G., Cotugno, R. et al. Properties of the endogenous components of the thioredoxin system in the psychrophilic eubacterium Pseudoalteromonas haloplanktis TAC 125. Extremophiles 16, 539–552 (2012). https://doi.org/10.1007/s00792-012-0453-0

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