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.
Crystal structure of the wild-type and D30A mutant thioredoxin h of Chlamydomonas reinhardtii and implications for the catalytic mechanism
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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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