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Functional and structural characterization of protein disulfide oxidoreductase from Thermus thermophilus HB27

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Abstract

The paper reports the characterization of a protein disulfide oxidoreductase (PDO) from the thermophilic Gram negative bacterium Thermus thermophilus HB27, identified as TTC0486 by genome analysis and named TtPDO. PDO members are involved in the oxidative folding, redox balance and detoxification of peroxides in thermophilic prokaryotes. Ttpdo was cloned and expressed in E. coli and the recombinant purified protein was assayed for the dithiol-reductase activity using insulin as substrate and compared with other PDOs characterized so far. In the thermophilic archaeon Sulfolobus solfataricus PDOs work as thiol-reductases constituting a peculiar redox couple with Thioredoxin reductase (SsTr). To get insight into the role of TtPDO, a hybrid redox couple with SsTr, homologous to putative Trs of T. thermophilus, was assayed. The results showed that SsTr was able to reduce TtPDO in a concentration dependent manner with a calculated K M of 34.72 μM, suggesting the existence of a new redox system also in thermophilic bacteria. In addition, structural characterization of TtPDO by light scattering and circular dichroism revealed the monomeric structure and the high thermostability of the protein. The analysis of the genomic environment suggested a possible clustering of Ttpdo with TTC0487 and TTC0488 (tlpA). Accordingly, transcriptional analysis showed that Ttpdo is transcribed as polycistronic messenger. Primer extension analysis allowed the determination of its 5′end and the identification of the promoter region.

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Acknowledgments

We gratefully acknowledge the grants from MIUR for project FIRB no. RBRN07BMCT and Università Federico II di Napoli and “Compagnia di San Paolo” (Naples Laboratory) for “Programma F.A.R.O, IV° tornata”. We acknowledge Dr. Carmen Sarcinelli for her helpful technical assistance and the grant from POR CAMPANIA FSE 2007/2013 for PROGETTO CARINA CUP B25B09000080007.

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Authors and Affiliations

  1. Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone 16, 80134, Naples, Italy

    Emilia Pedone

  2. Dipartimento di Biologia, Università degli Studi di Napoli ‘Federico II’, Complesso Universitario Monte S. Angelo, Via Cinthia, 80126, Naples, Italy

    Gabriella Fiorentino, Patrizia Contursi, Simonetta Bartolucci & Danila Limauro

  3. Istituto di Cristallografia, CNR, Via Giovanni Amendola 122/O, 70126, Bari, Italy

    Luciano Pirone

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  1. Emilia Pedone
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  2. Gabriella Fiorentino
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  3. Luciano Pirone
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  4. Patrizia Contursi
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  5. Simonetta Bartolucci
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Correspondence to Danila Limauro.

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Communicated by F. Robb.

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Pedone, E., Fiorentino, G., Pirone, L. et al. Functional and structural characterization of protein disulfide oxidoreductase from Thermus thermophilus HB27. Extremophiles 18, 723–731 (2014). https://doi.org/10.1007/s00792-014-0652-y

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