NMR Structures of Thioredoxin m from the Green Alga Chlamydomonas reinhardtii Jean-Marc Lancelin, 1 * Laure Guilhaudis, 2 Isabelle Krimm, 1 Martin J. Blackledge, 2 Dominique Marion, 2 and Jean-Pierre Jacquot 3 1 Laboratoire de RMN Biomole ´culaire associe ´ au CNRS, Universite ´ Claude Bernard-Lyon 1 and Ecole Supe ´rieure de Chimie, Physique et Electronique de Lyon, Villeurbanne, France 2 Institut de Biologie Structurale Jean-Pierre Ebel, CEA-CNRS, Grenoble, France 3 Laboratoire de Biologie Forestie `re associe ´a ` l’INRA, Biochimie et Biologie Mole ´culaire Ve ´ge ´tales, Universite ´ de Nancy I, Vandoeuvre Cedex, France ABSTRACT Chloroplast thioredoxin m from the green alga Chlamydomomas reinhardtii is very effi- ciently reduced in vitro and in vivo in the presence of photoreduced ferredoxin and a ferredoxin depen- dent ferredoxin-thioredoxin reductase. Once re- duced, thioredoxin m has the capability to quickly activate the NADP malate dehydrogenase (EC 1.1.1.82) a regulatory enzyme involved in an energy- dependent assimilation of carbon dioxide in C4 plants. This activation is the result of the reduction of two disulfide bridges by thioredoxin m, that are located at the N- and C-terminii of the NADP malate dehydrogenase. The molecular structure of thiore- doxin m was solved using NMR and compared to other known thioredoxins. Thioredoxin m belongs to the prokaryotic type of thioredoxin, which is divergent from the eukaryotic-type thioredoxins also represented in plants by the h (cytosolic) and f (chloroplastic) types of thioredoxins. The dynamics of the molecule have been assessed using 15 N relax- ation data and are found to correlate well with regions of disorder found in the calculated NMR ensemble. The results obtained provide a novel basis to interpret the thioredoxin dependence of the activation of chloroplast NADP-malate dehydroge- nase. The specific catalytic mechanism that takes place in the active site of thioredoxins is also dis- cussed on the basis of the recent new understanding and especially in the light of the dual general acid- base catalysis exerted on the two cysteines of the redox active site. It is proposed that the two cys- teines of the redox active site may insulate each other from solvent attack by specific packing of invariable hydrophobic amino acids. Proteins 2000; 41:334 –349. © 2000 Wiley-Liss, Inc. Key words: chloroplast thioredoxin; cysteine reac- tivity; dithiol redox control; enzymatic photoregulation; NADP malate dehydro- genase INTRODUCTION Plant thioredoxins are a multigenic family of redox proteins containing a disulfide bridge involved in several metabolic functions inside and outside the cells. 1,2 In chloroplasts, several light-regulated enzymes of the Calvin cycle were identified as dependent on reduced thioredoxins for their activation, which is triggered by the reduction of specific disulfide bridges. In bacteria, anaerobic photosyn- thetic prokaryotes and animal cells, thioredoxins are reduced by NADPH and a flavoenzyme (NADPH-thiore- doxin reductase), whereas in photosynthetic eukaryotes, the cytosolic NADPH dependent reduction system of thiore- doxin coexists with an alternate system located in the chloroplasts that consists of the photosynthetic electron chain, ferredoxin and a ferredoxin-thioredoxin reductase (FTR). 3 Chloroplast thioredoxins appear therefore as a disulfide redox link needed to activate the carbon fixation pathway during the light period and avoid unnecessary and energy-consuming futile cycles. Sequence analysis has revealed three different thiore- doxin groups in the plant cells. 1 The first group contains thioredoxins similar to the prokaryotic thioredoxins, such as the Escherichia coli thioredoxin which was the first, and to date the one for which the highest resolution 3-D structures are available. 4–7 These thioredoxins are nucleus encoded but localized in the chloroplasts and are reduced by FTR 3 ; they are efficient catalysts in vitro of the reduc- tion/activation of the chloroplast NADPH-malate dehydro- genase (NADPH-MDH) and have been accordingly called thioredoxins m. The second group of nucleus-encoded chloroplast thioredoxins also reduced by FTR, and called thioredoxins f, are very specific activators of the fructose- 1,6-bisphosphate phosphatase of the Calvin cycle. Finally, plants contain a third type of thioredoxins, the thioredox- Abbreviations: DQF-COSY, double quantum filtered correlation spectroscopy; FTR, ferredoxin thioredoxin reductase; MDH, NADP malate dehydrogenase (EC 1.1.1.82); HSQC, heteronuclear single quantum coherence; NOESY, nuclear Overhauser enhancement spec- troscopy; PDB, Protein Data Bank; rMD, restrained molecular dy- namic; Rmsd, root mean square deviation; SA, simulated annealing; TOCSY, total correlation spectroscopy. The Supplementary Material referred to in this article can be found at http://www.interscience.wiley.com/jpages/0887-3585/suppmat/ 41_3/v41_3.334.html *Correspondence to: Laboratoire de RMN Biomole ´culaire, UMR CNRS 5078, Universite ´Claude Bernard-Lyon 1, Ecole Supe ´rieure de Chimie Physique et Electronique de Lyon (CPE-Lyon), Ba ˆ timent 308G, F-69622 Villeurbanne, France. E-mail: lancelin@hikari.cpe.fr Received 13 April 2000; Accepted 28 June 2000 PROTEINS: Structure, Function, and Genetics 41:334 –349 (2000) © 2000 WILEY-LISS, INC.