Thioredoxins are ubiquitous proteins which catalyse the reduction of disulphide bridges on target proteins. The catalytic mechanism proceeds via a mixed disulphide intermediate whose breakdown should be enhanced by the involvement of a conserved buried residue, Asp-30, as a base catalyst towards residue Cys-39. We report here the crystal structure of wild-type and D30A mutant thioredoxin h from Chlamydomonas reinhardtii, which constitutes the first crystal structure of a cytosolic thioredoxin isolated from a eukaryotic plant organism. The role of residue Asp-30 in catalysis has been revisited since the distance between the carboxylate OD1 of Asp-30 and the sulphur SG of Cys-39 is too great to support the hypothesis of direct proton transfer. A careful analysis of all available crystal structures reveals that the relative positioning of residues Asp-30 and Cys-39 as well as hydrophobic contacts in the vicinity of residue Asp-30 do not allow a conformational change sufficient to bring the two residues close enough for a direct proton transfer. This suggests that protonation/deprotonation of Cys-39 should be mediated by a water molecule. Molecular-dynamics simulations, carried out either in vacuo or in water, as well as proton-inventory experiments, support this hypothesis. The results are discussed with respect to biochemical and structural data.
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October 2001
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Research Article|
September 24 2001
Crystal structure of the wild-type and D30A mutant thioredoxin h of Chlamydomonas reinhardtii and implications for the catalytic mechanism
Valeria MENCHISE;
Valeria MENCHISE
∗Laboratoire de Cristallographie et Modélisation des Matériaux Minéraux et Biologiques, Groupe Biocristallographie, ESA 7036, Université Henri Poincaré-Nancy I, BP 239, 54506Vandoeuvre-lès-Nancy Cedex, France
†Centro di Studio di Biocristallografia del CNR, Dipartimento di Chimica, Via Mezzocannone 4, 80134 Napoli, Italy
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Catherine CORBIER;
Catherine CORBIER
1
∗Laboratoire de Cristallographie et Modélisation des Matériaux Minéraux et Biologiques, Groupe Biocristallographie, ESA 7036, Université Henri Poincaré-Nancy I, BP 239, 54506Vandoeuvre-lès-Nancy Cedex, France
1To whom correspondence should be addressed (e-mail corbier@lcm3b.u-nancy.fr).
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Claude DIDIERJEAN;
Claude DIDIERJEAN
∗Laboratoire de Cristallographie et Modélisation des Matériaux Minéraux et Biologiques, Groupe Biocristallographie, ESA 7036, Université Henri Poincaré-Nancy I, BP 239, 54506Vandoeuvre-lès-Nancy Cedex, France
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Michele SAVIANO;
Michele SAVIANO
†Centro di Studio di Biocristallografia del CNR, Dipartimento di Chimica, Via Mezzocannone 4, 80134 Napoli, Italy
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Ettore BENEDETTI;
Ettore BENEDETTI
†Centro di Studio di Biocristallografia del CNR, Dipartimento di Chimica, Via Mezzocannone 4, 80134 Napoli, Italy
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Jean-Pierre JACQUOT;
Jean-Pierre JACQUOT
‡Interactions Arbres Microorganismes, UMR 1136 INRA, Université Henri Poincaré-Nancy I, BP 239, 54506Vandoeuvre-lès-Nancy Cedex, France
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André AUBRY
André AUBRY
∗Laboratoire de Cristallographie et Modélisation des Matériaux Minéraux et Biologiques, Groupe Biocristallographie, ESA 7036, Université Henri Poincaré-Nancy I, BP 239, 54506Vandoeuvre-lès-Nancy Cedex, France
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Publisher: Portland Press Ltd
Received:
October 23 2000
Revision Received:
June 13 2001
Accepted:
July 30 2001
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2001
2001
Biochem J (2001) 359 (1): 65–75.
Article history
Received:
October 23 2000
Revision Received:
June 13 2001
Accepted:
July 30 2001
Citation
Valeria MENCHISE, Catherine CORBIER, Claude DIDIERJEAN, Michele SAVIANO, Ettore BENEDETTI, Jean-Pierre JACQUOT, André AUBRY; Crystal structure of the wild-type and D30A mutant thioredoxin h of Chlamydomonas reinhardtii and implications for the catalytic mechanism. Biochem J 1 October 2001; 359 (1): 65–75. doi: https://doi.org/10.1042/bj3590065
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