Characterization of thioredoxin y, a new type of thioredoxin identified in the genome of Chlamydomonas reinhardtii

AbstractThe sequencing of the Arabidopsis genome revealed a multiplicity of thioredoxins (TRX), ubiquitous protein disulfide oxido-reductases. We have analyzed the TRX family in the genome of the unicellular green alga Chlamydomonas reinhardtii and identified eight different thioredoxins for which we have cloned and sequenced the corresponding cDNAs. One of these TRXs represents a new type that we named TRX y. This most probably chloroplastic TRX is highly conserved in photosynthetic organisms. The biochemical characterization of the recombinant protein shows that it exhibits a thermal stability profile and specificity toward target enzymes completely different from those of TRXs characterized so far

The dimer contact area of sorghum NADP-malate dehydrogenase: role of aspartate 101 in dimer stability and catalytic activity

AbstractDuring thioredoxin-mediated activation of chloroplastic NADP-malate dehydrogenase, a homodimeric enzyme, the interaction between subunits is known to be loosened but maintained. A modeling of the 3D structure of the protein identified Asp-101 as being potentially involved in the association between subunits through an electrostatic interaction. Indeed, upon site-directed substitution of Asp-101 by an asparagine, the mutated enzyme behaved mainly as a monomer. The mutation strongly affected the catalytical efficiency of the enzyme. The now available 3D structure of the enzyme shows that Asp-101 is protruding at the dimer interface, interacting with Arg-268 of the neighbouring subunit

Structural basis of redox signaling in photosynthesis: structure and function of ferredoxin:thioredoxin reductase and target enzymes

Abstract The role of the ferredoxin:thioredoxin system in the reversible light activation of chloroplast enzymes by thioldisulfide interchange with thioredoxins is now well established. Recent fruitful collaboration between biochemists and structural biologists, reflected by the shared authorship of the paper, allowed to solve the structures of all of the components of the system, including several target enzymes, thus providing a structural basis for the elucidation of the activation mechanism at a molecular level. In the present Review, these structural data are analyzed in conjunction with the information that was obtained previously through biochemical and site-directed mutagenesis approaches. The unique 4Fe-4S cluster enzyme ferredoxin:thioredoxin reductase (FTR) uses photosynthetically reduced ferredoxin as an electron donor to reduce the disulfide bridge of different thioredoxin isoforms. Thioredoxins in turn reduce regulatory disulfides of various target enzymes. This process triggers conformational changes on these enzymes, allowing them to reach optimal activity. No common activation mechanism can be put forward for these enzymes, as every thioredoxin-regulated protein undergoes specific structural modifications. It is thus important to solve the structures of the individual target enzymes in order to fully understand the molecular mechanism of the redox regulation of each of them. Abbreviations: FBPase -fructose

Hydrogen sulphide enhances photosynthesis through promoting chloroplast biogenesis, photosynthetic enzyme expression, and thiol redox modification in Spinacia oleracea seedlings

Hydrogen sulphide (H2S) is emerging as a potential messenger molecule involved in modulation of physiological processes in animals and plants. In this report, the role of H2S in modulating photosynthesis of Spinacia oleracea seedlings was investigated. The main results are as follows. (i) NaHS, a donor of H2S, was found to increase the chlorophyll content in leaves. (ii) Seedlings treated with different concentrations of NaHS for 30 d exhibited a significant increase in seedling growth, soluble protein content, and photosynthesis in a dose-dependent manner, with 100 μM NaHS being the optimal concentration. (iii) The number of grana lamellae stacking into the functional chloroplasts was also markedly increased by treatment with the optimal NaHS concentration. (iv) The light saturation point (Lsp), maximum net photosynthetic rate (Pmax), carboxylation efficiency (CE), and maximal photochemical efficiency of photosystem II (Fv/Fm) reached their maximal values, whereas the light compensation point (Lcp) and dark respiration (Rd) decreased significantly under the optimal NaHS concentration. (v) The activity of ribulose-1,5-bisphosphate carboxylase (RuBISCO) and the protein expression of the RuBISCO large subunit (RuBISCO LSU) were also significantly enhanced by NaHS. (vi) The total thiol content, glutathione and cysteine levels, internal concentration of H2S, and O-acetylserine(thiol)lyase and L-cysteine desulphydrase activities were increased to some extent, suggesting that NaHS also induced the activity of thiol redox modification. (vii) Further studies using quantitative real-time PCR showed that the gene encoding the RuBISCO large subunit (RBCL), small subunit (RBCS), ferredoxin thioredoxin reductase (FTR), ferredoxin (FRX), thioredoxin m (TRX-m), thioredoxin f (TRX-f), NADP-malate dehydrogenase (NADP-MDH), and O-acetylserine(thiol)lyase (OAS) were up-regulated, but genes encoding serine acetyltransferase (SERAT), glycolate oxidase (GYX), and cytochrome oxidase (CCO) were down-regulated after exposure to the optimal concentration of H2S. These findings suggest that increases in RuBISCO activity and the function of thiol redox modification may underlie the amelioration of photosynthesis and that H2S plays an important role in plant photosynthesis regulation by modulating the expression of genes involved in photosynthesis and thiol redox modification

ETUDE DE LA MALATE DESHYDROGENASE A NADP DE SORGHO (IMPLICATION DE RESIDUS DU SITE ACTIF DANS L'ACTIVATION ET DANS LA SPECIFICITE POUR LE SUBSTRAT ET POUR LE COFACTEUR)

ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Etude de l'interaction entre les thiorédoxines et leurs enzymes cibles (application à la recherche de nouveaux partenaires chez l'algue verte Chlamydomonas reinhardtii)

Le processus d'activation de la MDH à NADP choroplastique par les TRX est étudié par formation d'un disulfure mixte entre l'enzyme cible et la TRX. Le rôle de la Cys207 de la MDH est examiné par sa capacité à former des disulfures avec une TRX mutée sur une des Cys de son site actif. Les deux mutants de TRX forment des disulfures mixtes avec une MDH dépourvue dc son pont N-terminal. Ils sont faiblement efficaces avec la MDH sauvage oxydée. Lorsque la Cys207 est mutée, aucun disulfure mixte n'est formé, montrant que cette Cys est la seule, parmi les Cys internes, à former des disulfures avec la TRX. Ces expériences confirment que la réduction du pont N-terminal entreîne un relâchement dans l'interaction entre les sous-unités, rendant la Cys207 plus accessible. Quatre MDH différentes mutées sur une des Cys des ponts disulfures régulateurs sont examinées. Les résultats montrent que l'attaque de la TRX sur le pont C-terminal se fait sur la Cys377...The activation pathway of chloroplastic NADP-MDH by thioredoxin was examined using the formation of a mixed disulfide between the target and the reductant. The role of the Cys207 of MDH was investigated through the examination of the ability of this residue to form mixed-disulfides with thioredoxin mutated at either of its two active-site cysteines. Both thioredoxin mutants proved efficient in forming disulfides with a MDH devoid of its N-terminal bridge. They were poorly efficient with the WT oxidized MDH. Upon mutation of Cys207, no mixed disulfide could be formed, showing that this cysteine is the only one, among the internal cysteines, which can form mixed disulfides with thioredoxin. These experiments confirm that the opening of the N-terminal disulfide loosens the interaction between subits making Cys207 more accessible...ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF