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J. Biol. Chem., Vol. 283, Issue 34, 23062-23072, August 22, 2008

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An Atypical Catalytic Mechanism Involving Three Cysteines of Thioredoxin^*

Cha San Koh¹, Nicolas Navrot^¶||, Claude Didierjean, Nicolas Rouhier^¶, Masakazu Hirasawa^**, David B. Knaff^**, Gunnar Wingsle^||, Razip Samian, Jean-Pierre Jacquot^¶, Catherine Corbier, and Eric Gelhaye^¶2

From the LCM3B, Equipe Biocristallographie, UMR 7036 CNRS-Université Henri Poincaré, ^¶Unité Mixte de recherche INRA-UHP 1136, Interactions Arbres/Micro-organismes, URAFPA, Equipe PB2P, Faculté des Sciences et Techniques, Nancy Université, BP 239, 54506 Vandoeuvre Cedex France, the ^**Department of Chemistry and Biochemistry, and Center for Biotechnology and Genomics, Texas Tech University, Lubbock, Texas 79409-1061, the School of Biology, Universiti Sains Malaysia, Minden, 11800 USM, Penang, Malaysia, and the ^||Department of Forest Genetics and Plant Physiology, Faculty of Forestry, Swedish University of Agricultural Sciences, SE-901 83 Umea, Sweden

Unlike other thioredoxins h characterized so far, a poplar thioredoxin of the h type, PtTrxh4, is reduced by glutathione and glutaredoxin (Grx) but not NADPH:thioredoxin reductase (NTR). PtTrxh4 contains three cysteines: one localized in an N-terminal extension (Cys⁴) and two (Cys⁵⁸ and Cys⁶¹) in the classical thioredoxin active site (⁵⁷WCGPC⁶¹). The property of a mutant in which Cys⁵⁸ was replaced by serine demonstrates that it is responsible for the initial nucleophilic attack during the catalytic cycle. The observation that the C4S mutant is inactive in the presence of Grx but fully active when dithiothreitol is used as a reductant indicates that Cys⁴ is required for the regeneration of PtTrxh4 by Grx. Biochemical and x-ray crystallographic studies indicate that two intramolecular disulfide bonds involving Cys⁵⁸ can be formed, linking it to either Cys⁶¹ or Cys⁴. We propose thus a four-step disulfide cascade mechanism involving the transient glutathionylation of Cys⁴ to convert this atypical thioredoxin h back to its active reduced form.

Received for publication, March 17, 2008 , and in revised form, June 13, 2008.

^* This work was supported in part by Grant D-0710 (to D. B. K.) from the Robert A. Welch Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (code 3D21 and 3D22) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ Recipient of the ASTS (Academic Staff Training Scheme) fellowship from the Universiti Sains Malaysia.

² To whom correspondence should be addressed: Unité Mixte de recherche INRA-UHP 1136, Interactions Arbres/Micro-organismes, Faculté des Sciences et Techniques, Nancy Université, BP 239, 54506 Vandoeuvre Cedex France. Tel.: 33-0-3-83-68-42-28; E-mail: gelhaye{at}lcb.uhp-nancy.fr.

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