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

The 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.

Sites of interaction of thioredoxin with sorghum NADP-malate dehydrogenase.

The activation pathway of the chloroplastic NADP-dependent malate dehydrogenase (MDH) by reduced thioredoxin has been examined using a method based on the mechanism of thiol/disulfide interchanges, i.e. the transient formation of a mixed disulfide between the target and the reductant. This disulfide can be stabilized when each of the partners is mutated in the less reactive cysteine of the disulfide/dithiol pair. As NADP-MDH has two regulatory disulfides per monomer, four different single cysteine mutants were examined, two for the C-terminal bridge and two for the N-terminal bridge. The results clearly show that the nucleophilic attack of thioredoxin on the C-terminal bridge proceeds through the formation of a disulfide with the most external Cys377. The results are less clear-cut for the N-terminal cysteines and suggest that the Cys24-Cys207 disulfide bridge previously proposed to be an intermediary step in MDH activation can form only when the C-terminal disulfide is reduced.

Heterologous complementation of yeast reveals a new putative function for chloroplast m-type thioredoxin.

In the chloroplast of higher plants, two types of thioredoxins (TRX), namely TRX m which shows high similarity to prokaryotic thioredoxins and TRX f which is more closely related to eukaryotic thioredoxins, have been found and biochemically characterized, but little is known about their physiological specificity with respect to their target(s). Here, we tested, in vivo, the ability of organelle-specific TRX from Arabidopsis thaliana to compensate for TRX deficiency of a Saccharomyces cerevisiae mutant strain. Seven plant organellar TRX (four of the m type, two of the f type and a newly discovered TRX x of prokaryotic type) were expressed in yeast in a putative mature form. None of these heterologous TRX were able to restore growth on sulphate or methionine sulphoxide of the mutant cells. When we tested their ability to rescue the oxidant-hypersensitive phenotype of the TRX-deficient strain, we found that TRX m and TRX x, but not TRX f, affected the tolerance to oxidative stress induced by either hydrogen peroxide or an alkyl hydroperoxide. Athm1, Athm2, Athm4 and Athx induced hydrogen peroxide tolerance like the endogenous yeast thioredoxins. Unexpectedly, Athm3 had a hypersensitizing effect towards oxidative stress. The presence of functional heterologous TRX was checked in the recombinant clones tested, supporting distinct abilities for organelle-specific plant TRX to compensate for TRX deficiency in yeast. We propose a new function for the prokaryotic-type chloroplastic TRX as an anti-oxidant and provide in vivo evidence for different roles of chloroplastic TRX isoforms.

The role of active site arginines of sorghum NADP-malate dehydrogenase in thioredoxin-dependent activation and activity.

The activation of sorghum NADP-malate dehydrogenase is initiated by thiol/disulfide interchanges with reduced thioredoxin followed by the release of the C-terminal autoinhibitory extension and a structural modification shaping the active site into a high efficiency and high affinity for oxaloacetate conformation. In the present study, the role of the active site arginines in the activation and catalysis was investigated by site-directed mutagenesis and arginyl-specific chemical derivatization using butanedione. Sequence and mass spectrometry analysis were used to identify the chemically modified groups. Taken together, our data reveal the involvement of Arg-134 and Arg-204 in oxaloacetate coordination, suggest an indirect role for Arg-140 in substrate binding and catalysis, and clearly confirm that Arg-87 is implicated in cofactor binding. In contrast with NAD-malate dehydrogenase, no lactate dehydrogenase activity could be promoted by the R134Q mutation. The decreased susceptibility of the activation of the R204K mutant to NADP and its increased sensitivity to the histidine-specific reagent diethylpyrocarbonate indicated that Arg-204 is involved in the locking of the active site. These results are discussed in relation with the recently published NADP-MDH three-dimensional structures and the previously established three-dimensional structures of NAD-malate dehydrogenase and lactate dehydrogenase.

Mechanism of activation of the chloroplast ATP synthase. A kinetic study of the thiol modulation of isolated ATPase and membrane-bound ATP synthase from spinach by Eschericia coli thioredoxin.

The mechanism of thiol modulation of the chloroplast ATP synthase by Escherichia coli thioredoxin was investigated in the isolated ATPase subcomplex and in the ATP synthase complex reconstituted in bacteriorhodopsin proteoliposomes. Thiol modulation was resolved kinetically by continuously monitoring ATP hydrolysis by the isolated subcomplex and ATP synthesis by proteoliposomes. The binding rate constant of reduced thioredoxin to the oxidized ATPase subcomplex devoid of its epsilon subunit could be determined. It did not depend on the catalytic turnover. Reciprocically, the catalytic turnover did not seem to depend on thioredoxin binding. Thiol modulation by Trx of the epsilon-bearing ATPase subcomplex was slow and favored the release of epsilon. The rate constant of thioredoxin binding to the membrane-bound ATP synthase increased with the protonmotive force. It was lower in the presence of ADP than in its absence, revealing a specific effect of the ATP synthase turnover on thioredoxin-gamma subunit interaction. These findings, and more especially the comparisons between the isolated ATPase subcomplex and the ATP synthase complex, can be interpreted in the frame of the rotational catalysis hypothesis. Finally, thiol modulation changed the catalytic properties of the ATP synthase, the kinetics of which became non-Michaelian. This questions the common view about the nature of changes induced by ATP synthase thiol modulation.

Inhibition of the thioredoxin-dependent activation of the NADP-malate dehydrogenase and cofactor specificity.

The chloroplastic NADP-malate dehydrogenase is activated by reduction of its N- and C-terminal disulfides by reduced thioredoxin. The activation is inhibited by NADP(+), the oxidized form of the cofactor. Previous studies suggested that the C-terminal disulfide was involved in this process. Recent structural data pointed toward a possible direct interaction between the C terminus of the oxidized enzyme and the cofactor. In the present study, the relationship between the cofactor specificity for catalysis and for inhibition of activation has been investigated by changing the cofactor specificity of the enzyme by substitution of selected residues of the cofactor-binding site. An NAD-specific thiol-regulated MDH was engineered. Its activation was inhibited by NAD(+) but no longer by NADP(+). These results demonstrate that the oxidized cofactor is bound at the same site as the reduced cofactor and support the idea of a direct interaction between the negatively charged C-terminal end of the enzyme and the positively charged nicotinamide ring of the cofactor, in agreement with the structural data. The structural requirements for cofactor specificity are modeled and discussed.

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

During 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.

Oxidation-reduction properties of the regulatory disulfides of sorghum chloroplast nicotinamide adenine dinucleotide phosphate-malate dehydrogenase.

Oxidation-reduction midpoint potentials (E(m)) have been measured for the thioredoxin-dependent, reductive activation of sorghum nicotinamide adenine dinucleotide phosphate- (NADP-) dependent malate dehydrogenase (MDH) in the wild-type enzyme and in a number of site-specific mutants. The E(m) value associated with activation of the wild-type enzyme, -330 mV at pH 7.0, can be attributed to the E(m) of the C365/C377 disulfide present in the C-terminal region of the enzyme. The C24/C29 disulfide, located in the N-terminal region of the enzyme and the only other disulfide present in oxidized, wild-type MDH, has a E(m) value of -280 mV at pH 7.0. A third regulatory disulfide, C24/C207, that is absent in the oxidized enzyme but is thought to be formed during the activation process, has an E(m) value at pH 7.0 of -310 mV. E(m) vs pH profiles suggest pK(a) values for the more acidic cysteine involved in the formation of each of these disulfides of 8.5 for C24/C29; 8.1 for C24/C207; and 8.7 for C365/C377. The results of this study show that the N-terminal disulfide formed between C24 and C29 has a more positive E(m) value than the two other disulfides and is thus is likely to be the "preregulatory disulfide" postulated to function in activating the enzyme.

Redox signalling in the chloroplast: structure of oxidized pea fructose-1,6-bisphosphate phosphatase.

Sunlight provides the energy source for the assimilation of carbon dioxide by photosynthesis, but it also provides regulatory signals that switch on specific sets of enzymes involved in the alternation of light and dark metabolisms in chloroplasts. Capture of photons by chlorophyll pigments triggers redox cascades that ultimately activate target enzymes via the reduction of regulatory disulfide bridges by thioredoxins. Here we report the structure of the oxidized, low-activity form of chloroplastic fructose-1, 6-bisphosphate phosphatase (FBPase), one of the four enzymes of the Calvin cycle whose activity is redox-regulated by light. The regulation is of allosteric nature, with a disulfide bridge promoting the disruption of the catalytic site across a distance of 20 A. Unexpectedly, regulation of plant FBPases by thiol-disulfide interchange differs in every respect from the regulation of mammalian gluconeogenic FBPases by AMP. We also report a second crystal form of oxidized FBPase whose tetrameric structure departs markedly from D(2) symmetry, a rare event in oligomeric structures, and the structure of a constitutively active mutant that is unable to form the regulatory disulfide bridge. Altogether, these structures provide a structural basis for redox regulation in the chloroplast.

Direct NMR observation of the thioredoxin-mediated reduction of the chloroplast NADP-malate dehydrogenase provides a structural basis for the relief of autoinhibition.

The chloroplastic NADP-dependent malate dehydrogenase (NADP-MDH) catalyzing the reduction of oxaloacetate into L-malate is regulated by light. Its activation results from the thioredoxin-mediated reduction of two disulfides, located, respectively, in N- and C-terminal sequence extensions typical of all NADP-dependent light-regulated forms. Site-directed mutagenesis studies and the resolution of the three-dimensional structure of the oxidized (inactive) Sorghum vulgare enzyme showed that the C-terminal Cys(365)-Cys(377) disulfide constrains the C-terminal extension to fold into the active site where it acts as an internal inhibitor. In the present study, two-dimensional proton NMR spectra of an engineered NADP-MDH rendered monomeric by a 33-amino acid deletion at the N terminus (38 kDa) revealed that a 15-amino acid-long C-terminal peptide (Ala(375) to C-terminal Val(389)) acquired an increased mobility upon reduction, allowing its direct sequence-specific NMR assignment. The location of the flexible peptide in the sequence suggests that the first part of the C-terminal peptide is still folded near the core of the enzyme, so that cysteines 365 and 377 remain in proximity to allow for an efficient reoxidation/inactivation of the enzyme.

The complex regulation of ferredoxin/thioredoxin-related genes by light and the circadian clock.

The biochemical properties of the ferredoxin/thioredoxin transduction pathway regulating the activity of key carbon-fixation enzymes through post-translational modifications are well characterized but little is known about the regulation of the different genes. In the present study, we investigated in Chlamydomonas reinhardtii the regulation of the expression of ferredoxin, thioredoxin m, ferredoxin-NADP reductase, phosphoribulokinase, as well as that of cytosolic thioredoxin h, the function of which is still largely unknown. The effects of light, the circadian clock and active cell division were investigated by northern blotting. The five genes were found to be regulated by light and the circadian clock but with different kinetics and amplitudes. This leads for the first time to the proposal that an extra-chloroplastic thioredoxin is possibly implicated in light and/or circadian-related processes. An interplay between several light-transduction pathways in controlling the expression of the genes is suggested by the expression studies and the theoretical analysis of the promoters.

Heavy-metal regulation of thioredoxin gene expression in chlamydomonas reinhardtii

Heavy metals are highly toxic compounds for cells. In this report we demonstrate that the expression of Chlamydomonas reinhardtii thioredoxins (TRX) m and h is induced by heavy metals. Upon exposure of the cells to Cd and Hg, a strong accumulation of both messengers was observed. Western-blot experiments revealed that among these two TRXs, only TRX h polypeptides accumulated in response to the toxic cations. A biochemical analysis indicated that heavy metals inhibit TRX activity, presumably by binding at the level of their active site. Sequence analysis of the C. reinhardtii TRX h promoter revealed the presence of cis-acting elements related to cadmium induction. The origins and purposes of this regulation are discussed. Our data suggest, for the first time to our knowledge, a possible implication of TRXs in defense mechanisms against heavy metals.

Regulation of chloroplast enzyme activities by thioredoxins: activation or relief from inhibition?

Studies on redox signaling and light regulation of chloroplast enzymes have highlighted the importance of the ferredoxin-thioredoxin thiol-disulfide interchange cascade. Recent research has focused on the intramolecular mechanism by which the reduction status of a chloroplast enzyme affects its catalytic properties, and site-directed mutagenesis has been used to identify the regulatory cysteines involved. For some of the thiol-regulated enzymes, structure-function studies have revealed that the complex conformational changes that occur might be associated with disulfide isomerization and auto-inhibition. Transgenic approaches indicate that this regulation constitutes a rapid means to adjust enzyme activity to metabolic needs.

Structural basis for light activation of a chloroplast enzyme: the structure of sorghum NADP-malate dehydrogenase in its oxidized form.

Some key chloroplast enzymes are activated by light via a ferredoxin-thioredoxin reduction system which reduces disulfide bridges in the enzymes. We describe for the first time the structural basis for the redox activation of a chloroplast enzyme, the NADP-dependent malate dehydrogenase (MDH) from Sorghum vulgare whose structure has been determined and refined at 2.4 A resolution. In addition to the normal structural components of MDHs, the enzyme exhibits extensions at both the N- and C-termini, each of which contains a regulatory disulfide bridge which must be reduced for activation. The N-terminal disulfide motif is inserted in a cleft between the two subunits of the dimer, thereby locking the domains in each subunit. The C-terminal disulfide keeps the C-terminal residues tight to the enzyme surface and blocks access to the active site. Reduction of the N-terminal disulfide would release the stopper between the domains and give the enzyme the necessary flexibility. Simultaneous reduction of the C-terminal disulfide would free the C-terminal residues from binding to the enzyme and make the active site accessible.

The internal Cys-207 of sorghum leaf NADP-malate dehydrogenase can form mixed disulphides with thioredoxin.

The role of the internal Cys-207 of sorghum NADP-malate dehydrogenase (NADP-MDH) in the activation of the enzyme has been investigated through the examination of the ability of this residue to form mixed disulphides with thioredoxin mutated at either of its two active-site cysteines. The h-type Chlamydomonas thioredoxin was used, because it has no additional cysteines in the primary sequence besides the active-site cysteines. Both thioredoxin mutants proved equally efficient in forming mixed disulphides with an NADP-MDH devoid of its N-terminal bridge either by truncation, or by mutation of its N-terminal cysteines. They were poorly efficient with the more compact WT oxidised NADP-MDH. Upon mutation of Cys-207, no mixed disulphide could be formed, showing that this cysteine is the only one, among the four internal cysteines, which can form mixed disulphides with thioredoxin. These experiments confirm that the opening of the N-terminal disulphide loosens the interaction between subunits, making Cys-207, located at the dimer contact area, more accessible.

The autoinhibition of sorghum NADP malate dehydrogenase is mediated by a C-terminal negative charge.

The chloroplastic NADP malate dehydrogenase is completely inactive in its oxidized form and is activated by thiol/disulfide interchange with reduced thioredoxin. To elucidate the molecular mechanism underlying the absence of activity of the oxidized enzyme, we used site-directed mutagenesis to delete or substitute the two most C-terminal residues (C-terminal Val, penultimate Glu, both bearing negative charges). We also combined these mutations with the elimination of one or both of the possible regulatory N-terminal disulfides by mutating the corresponding cysteines. Proteins mutated at the C-terminal residues had no activity in the oxidized form but were partially inhibited when pretreated with the histidine-specific reagent diethyl pyrocarbonate before activation, showing that the active site was partially accessible. Proteins missing both N-terminal regulatory disulfides reached almost full activity without activation upon elimination of the negative charge of the penultimate Glu. These results strongly support a model where the C-terminal extension is docked into the active site through a negatively charged residue, acting as an internal inhibitor. They show also that the reduction of both N-terminal bridges is necessary to release the C-terminal extension from the active site. This is the first report for a thiol-activated enzyme of a regulatory mechanism resembling the well known intrasteric inhibition of protein kinases.

The single mutation Trp35-->Ala in the 35-40 redox site of Chlamydomonas reinhardtii thioredoxin h affects its biochemical activity and the pH dependence of C36-C39 1H-13C NMR.

The role of the invariant Trp residue at the redox site of thioredoxins was investigated by site-directed mutagenesis of a Chlamydomonas reinhardtii thioredoxin h. Though being still redox active with NADPH-thioredoxin reductase and chemical substrates [dithiothreitol and 5,5'-dithio-bis(2-nitrobenzoic acid)] the Trp35-->Ala-mutated protein completely lost the capacity to activate the thiol-regulated NADPH-dependent malate dehydrogenase. However, it was able to activate a mutant malate dehydrogenase where only the most exposed disulfide was retained. The pH dependence of the redox-site Cys beta 1H/13C-NMR frequencies of the wild-type and mutated proteins, in both the reduced and oxidised states, were compared over the pH range 5.8-10. The mutation does not affect the conserved buried Asp30, which titrates with a pKa of 7.5 in the oxidised proteins in agreement with previous studies. However, for the reduced forms of the proteins, the pH dependence of resonances of both Cys was strongly affected by the mutation. In the case of the wild-type thioredoxin, two apparent pKa values were found around 7.0 and 9.5 and could be assigned to the titration of Cys36 and Cys39 thiol, respectively, similar to the case of Escherichia coli thioredoxin. For the mutated thioredoxin a single pKa was found around 8.3. This result can be interpreted as a single pKa of either Cys36 or Cys39 or both. While the mutation clearly affects ionisations, the measured redox potentials of the active-site Cys pair are not significantly affected by the Trp35-->Ala mutation. Possible roles of an aromatic side chain on the reactivity of the catalytic Cys residues in thioredoxins are proposed.

Purification and characterization of the malate dehydrogenase from Streptomyces aureofaciens.

The malate dehydrogenase (MDH) from Streptomyces aureofaciens was purified to homogeneity and its physical and biochemical properties were studied. Its amino-terminal sequence perfectly matched the amino-terminal sequence of the MDH from Streptomyces atratus whose biochemical characteristics have never been determined. The molecular mass of the native enzyme, estimated by size-exclusion chromatography, was 70 kDa. The protein was a homodimer, with a 38-kDa subunit molecular mass. It showed a strong specificity for NADH and was much more efficient for the reduction of oxaloacetate than for the oxidation of malate, with a pH optimum of 8. Unlike MDHs from other sources, it was not inhibited by excess oxaloacetate. This first complete functional characterization of an MDH from Streptomyces shows that the enzyme is very similar in many respects to other bacterial MDHs with the notable exception of a lack of inhibition by excess substrate.

An internal cysteine is involved in the thioredoxin-dependent activation of sorghum leaf NADP-malate dehydrogenase.

The chloroplastic NADP-malate dehydrogenase is activated by thiol/disulfide interchange with reduced thioredoxins. Previous experiments showed that four cysteines located in specific N- and carboxyl-terminal extensions were implicated in this process, leading to a model where no internal cysteine was involved in activation. In the present study, the role of the conserved four internal cysteines was investigated. Surprisingly, the mutation of cysteine 207 into alanine yielded a protein with accelerated activation time course, whereas the mutations of the three other internal cysteines into alanines yielded proteins with unchanged activation kinetics. These results suggested that cysteine 207 might be linked in a disulfide bridge with one of the four external cysteines, most probably with one of the two amino-terminal cysteines whose mutation similarly accelerates the activation rate. To investigate this possibility, mutant malate dehydrogenases (MDHs) where a single amino-terminal cysteine was mutated in combination with the mutation of both carboxyl-terminal cysteines were produced and purified. The C29S/C365A/C377A mutant MDH still needed activation by reduced thioredoxin, while the C24S/C365A/C377A mutant MDH exhibited a thioredoxin-insensitive spontaneous activity, leading to the hypothesis that a Cys24-Cys207 disulfide bridge might be formed during the activation process. Indeed, an NADP-MDH where the cysteines 29, 207, 365, and 377 are mutated yielded a permanently active enzyme very similar to the previously created permanently active C24S/C29S/C365A/C377A mutant. A two-step activation model involving a thioredoxin-mediated disulfide isomerization at the amino terminus is proposed.

Cysteine-153 is required for redox regulation of pea chloroplast fructose-1,6-bisphosphatase.

Chloroplastic fructose-1,6-bisphosphatases are redox regulatory enzymes which are activated by the ferredoxin thioredoxin system via the reduction/isomerization of a critical disulfide bridge. All chloroplastic sequences contain seven cysteine residues, four of which are located in, or close to, an amino acid insertion region of approximately 17 amino acids. In order to gain more information on the nature of the regulatory site, five cysteine residues (Cys49, Cys153, Cys173, Cys178 and Cys190) have been modified individually into serine residues by site-directed mutagenesis. While mutations C173S and C178S strongly affected the redox regulatory properties of the enzyme, the most striking effect was observed with the C153S mutant which became permanently active and redox independent. On the other hand, the C190S mutant retained most of the properties of the wild-type enzyme (except that it could now also be partially activated by the NADPH/NTR/thioredoxin h system). Finally, the C49S mutant is essentially identical to the wild-type enzyme. These results are discussed in the light of recent crystallographic data obtained on spinach FBPase [Villeret et al. (1995) Biochemistry 34, 4299-4306].