Mirko Zaffagnini

Regeneration Mechanisms of Arabidopsis thaliana Methionine Sulfoxide Reductases B by Glutaredoxins and Thioredoxins

Methionine oxidation leads to the formation of S- and R-diastereomers of methionine sulfoxide (MetSO), which are reduced back to methionine by methionine sulfoxide reductases (MSRs) A and B, respectively. MSRBs are classified in two groups depending on the conservation of one or two redox-active Cys; 2-Cys MSRBs possess a catalytic Cys-reducing MetSO and a resolving Cys, allowing regeneration by thioredoxins. The second type, 1-Cys MSRBs, possess only the catalytic Cys. The biochemical mechanisms involved in activity regeneration of 1-Cys MSRBs remain largely elusive. In the present work we used recombinant plastidial Arabidopsis thaliana MSRB1 and MSRB2 as models for 1-Cys and 2-Cys MSRBs, respectively, to delineate the Trx- and glutaredoxin-dependent reduction mechanisms. Activity assays carried out using a series of cysteine mutants and various reductants combined with measurements of free thiols under distinct oxidation conditions and mass spectrometry experiments show that the 2-Cys MSRB2 is reduced by Trx through a dithiol-disulfide exchange involving both redox-active Cys of the two partners. Regarding 1-Cys MSRB1, oxidation of the enzyme after substrate reduction leads to the formation of a stable sulfenic acid on the catalytic Cys, which is subsequently glutathionylated. The deglutathionylation of MSRB1 is achieved by both mono- and dithiol glutaredoxins and involves only their N-terminal conserved catalytic Cys. This study proposes a detailed mechanism of the regeneration of 1-Cys MSRB activity by glutaredoxins, which likely constitute physiological reductants for this type of MSR.

The thioredoxin-independent isoform of chloroplastic glyceraldehyde-3-phosphate dehydrogenase is selectively regulated by glutathionylation

In animal cells, many proteins have been shown to undergo glutathionylation under conditions of oxidative stress. By contrast, very little is known about this post‐translational modification in plants. In the present work, we showed, using mass spectrometry, that the recombinant chloroplast A4‐glyceraldehyde‐3‐phosphate dehydrogenase (A4‐GAPDH) from Arabidopsis thaliana is glutathionylated with either oxidized glutathione or reduced glutathione and H2O2. The formation of a mixed disulfide between glutathione and A4‐GAPDH resulted in the inhibition of enzyme activity. A4‐GAPDH was also inhibited by oxidants such as H2O2. However, the effect of glutathionylation was reversed by reductants, whereas oxidation resulted in irreversible enzyme inactivation. On the other hand, the major isoform of photosynthetic GAPDH of higher plants (i.e. the AnBn‐GAPDH isozyme in either A2B2 or A8B8 conformation) was sensitive to oxidants but did not seem to undergo glutathionylation significantly. GAPDH catalysis is based on Cys149 forming a covalent intermediate with the substrate 1,3‐bisphosphoglycerate. In the presence of 1,3‐bisphosphoglycerate, A4‐GAPDH was fully protected from either oxidation or glutathionylation. Site‐directed mutagenesis of Cys153, the only cysteine located in close proximity to the GAPDH active‐site Cys149, did not affect enzyme inhibition by glutathionylation or oxidation. Catalytic Cys149 is thus suggested to be the target of both glutathionylation and thiol oxidation. Glutathionylation could be an important mechanism of regulation and protection of chloroplast A4‐GAPDH from irreversible oxidation under stress.

Pattern of Expression and Substrate Specificity of Chloroplast Ferredoxins from Chlamydomonas reinhardtii

Ferredoxin (Fd) is the major iron-containing protein in photosynthetic organisms and is central to reductive metabolism in the chloroplast. The Chlamydomonas reinhardtii genome encodes six plant type [Fe2S2] ferredoxins, products of PETF, FDX2–FDX6. We performed the functional analysis of these ferredoxins by localizing Fd, Fdx2, Fdx3, and Fdx6 to the chloroplast by using isoform-specific antibodies and monitoring the pattern of gene expression by iron and copper nutrition, nitrogen source, and hydrogen peroxide stress. In addition, we also measured the midpoint redox potentials of Fd and Fdx2 and determined the kinetic parameters of their reactions with several ferredoxin-interacting proteins, namely nitrite reductase, Fd:NADP+ oxidoreductase, and Fd:thioredoxin reductase. We found that each of the FDX genes is differently regulated in response to changes in nutrient supply. Moreover, we show that Fdx2 (Em = −321 mV), whose expression is regulated by nitrate, is a more efficient electron donor to nitrite reductase relative to Fd. Overall, the results suggest that each ferredoxin isoform has substrate specificity and that the presence of multiple ferredoxin isoforms allows for the allocation of reducing power to specific metabolic pathways in the chloroplast under various growth conditions.

Biochemical characterization of glutaredoxins from Chlamydomonas reinhardtii reveals the unique properties of a chloroplastic CGFS-type glutaredoxin.

Glutaredoxins (GRXs) are small ubiquitous disulfide oxidoreductases known to use GSH as electron donor. In photosynthetic organisms, little is known about the biochemical properties of GRXs despite the existence of ∼30 different isoforms in higher plants. We report here the biochemical characterization of Chlamydomonas GRX1 and GRX3, the major cytosolic and chloroplastic isoforms, respectively. Glutaredoxins are classified on the basis of the amino acid sequence of the active site. GRX1 is a typical CPYC-type GRX, which is reduced by GSH and exhibits disulfide reductase, dehydroascorbate reductase, and deglutathionylation activities. In contrast, GRX3 exhibits unique properties. This chloroplastic CGFS-type GRX is not reduced by GSH and has an atypically low redox potential (–323 ± 4 mV at pH 7.9). Remarkably, GRX3 can be reduced in the light by photoreduced ferredoxin and ferredoxin-thioredoxin reductase. Both GRXs proved to be very efficient catalysts of A4-glyceraldehyde-3-phosphate dehydrogenase deglutathionylation, whereas cytosolic and chloroplastic thioredoxins were inefficient. Glutathionylated A4-glyceraldehyde-3-phosphate dehydrogenase is the first physiological substrate identified for a CGFS-type GRX.

Prompt and Easy Activation by Specific Thioredoxins of Calvin Cycle Enzymes of Arabidopsis thaliana Associated in the GAPDH/CP12/PRK Supramolecular Complex

The Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) can form under oxidizing conditions a supramolecular complex with the regulatory protein CP12. Both GAPDH and PRK activities are inhibited within the complex, but they can be fully restored by reduced thioredoxins (TRXs). We have investigated the interactions of eight different chloroplast thioredoxin isoforms (TRX f1, m1, m2, m3, m4, y1, y2, x) with GAPDH (A(4), B(4), and B(8) isoforms), PRK and CP12 (isoform 2), all from Arabidopsis thaliana. In the complex, both A(4)-GAPDH and PRK were promptly activated by TRX f1, or more slowly by TRXs m1 and m2, but all other TRXs were ineffective. Free PRK was regulated by TRX f1, m1, or m2, while B(4)- and B(8)-GAPDH were absolutely specific for TRX f1. Interestingly, reductive activation of PRK caged in the complex was much faster than reductive activation of free oxidized PRK, and activation of A(4)-GAPDH in the complex was much faster (and less demanding in terms of reducing potential) than activation of free oxidized B(4)- or B(8)-GAPDH. It is proposed that CP12-assembled supramolecular complex may represent a reservoir of inhibited enzymes ready to be released in fully active conformation following reduction and dissociation of the complex by TRXs upon the shift from dark to low light. On the contrary, autonomous redox-modulation of GAPDH (B-containing isoforms) would be more suited to conditions of very active photosynthesis.

In vivo targets of S-thiolation in Chlamydomonas reinhardtii.

Glutathionylation is the major form of S-thiolation in cells. This reversible redox post-translational modification consists of the formation of a mixed disulfide between a free thiol on a protein and a molecule of glutathione. This recently described modification, which is considered to occur under oxidative stress, can protect cysteine residues from irreversible oxidation, and alter positively or negatively the activity of diverse proteins. This modification and its targets have been mainly studied in non-photosynthetic organisms so far. We report here the first proteomic approach performed in vivo on photosynthetically competent cells, using the eukaryotic unicellular green alga Chlamydomonas reinhardtii with radiolabeled [35S]cysteine to label the glutathione pool and diamide as oxidant. This method allowed the identification of 25 targets, mainly chloroplastic, involved in various metabolic processes. Several targets are related to photosynthesis, such as the Calvin cycle enzymes phosphoglycerate kinase and ribose-5-phosphate isomerase. A number of targets, such as chaperones and peroxiredoxins, are related to stress responses. The glutathionylation of HSP70B, chloroplastic 2-Cys peroxiredoxin and isocitrate lyase was confirmed in vitro on purified proteins and the targeted residues were identified.

Thioredoxin-dependent regulation of photosynthetic glyceraldehyde-3-phosphate dehydrogenase: autonomous vs. CP12-dependent mechanisms

Regulation of the Calvin–Benson cycle under varying light/dark conditions is a common property of oxygenic photosynthetic organisms and photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the targets of this complex regulatory system. In cyanobacteria and most algae, photosynthetic GAPDH is a homotetramer of GapA subunits which do not contain regulatory domains. In these organisms, dark-inhibition of the Calvin–Benson cycle involves the formation of a kinetically inhibited supramolecular complex between GAPDH, the regulatory peptide CP12 and phosphoribulokinase. Conditions prevailing in the dark, i.e. oxidation of thioredoxins and low NADP(H)/NAD(H) ratio promote aggregation. Although this regulatory system has been inherited in higher plants, these phototrophs contain in addition a second type of GAPDH subunits (GapB) resulting from the fusion of GapA with the C-terminal half of CP12. Heterotetrameric A2B2-GAPDH constitutes the major photosynthetic GAPDH isoform of higher plants chloroplasts and coexists with CP12 and A4-GAPDH. GapB subunits of A2B2-GAPDH have inherited from CP12 a regulatory domain (CTE for C-terminal extension) which makes the enzyme sensitive to thioredoxins and pyridine nucleotides, resembling the GAPDH/CP12/PRK system. The two systems are similar in other respects: oxidizing conditions and low NADP(H)/NAD(H) ratios promote aggregation of A2B2-GAPDH into strongly inactivated A8B8-GAPDH hexadecamers, and both CP12 and CTE specifically affect the NADPH-dependent activity of GAPDH. The alternative, lower activity with NADH is always unaffected. Based on the crystal structure of spinach A4-GAPDH and the analysis of site-specific mutants, a model of the autonomous (CP12-independent) regulatory mechanism of A2B2-GAPDH is proposed. Both CP12 and CTE seem to regulate different photosynthetic GAPDH isoforms according to a common and ancient molecular mechanism.

Structure-function relationship of the chloroplastic glutaredoxin S12 with an atypical WCSYS active site.

Glutaredoxins (Grxs) are efficient catalysts for the reduction of mixed disulfides in glutathionylated proteins, using glutathione or thioredoxin reductases for their regeneration. Using GFP fusion, we have shown that poplar GrxS12, which possesses a monothiol 28WCSYS32 active site, is localized in chloroplasts. In the presence of reduced glutathione, the recombinant protein is able to reduce in vitro substrates, such as hydroxyethyldisulfide and dehydroascorbate, and to regenerate the glutathionylated glyceraldehyde-3-phosphate dehydrogenase. Although the protein possesses two conserved cysteines, it is functioning through a monothiol mechanism, the conserved C terminus cysteine (Cys87) being dispensable, since the C87S variant is fully active in all activity assays. Biochemical and crystallographic studies revealed that Cys87 exhibits a certain reactivity, since its pKa is around 5.6. Coupled with thiol titration, fluorescence, and mass spectrometry analyses, the resolution of poplar GrxS12 x-ray crystal structure shows that the only oxidation state is a glutathionylated derivative of the active site cysteine (Cys29) and that the enzyme does not form inter- or intramolecular disulfides. Contrary to some plant Grxs, GrxS12 does not incorporate an iron-sulfur cluster in its wild-type form, but when the active site is mutated into YCSYS, it binds a [2Fe-2S] cluster, indicating that the single Trp residue prevents this incorporation.

Redox regulation of chloroplastic glucose‐6‐phosphate dehydrogenase: A new role for f‐type thioredoxin

Glucose‐6‐phosphate dehydrogenase (G6PDH) is the key enzyme of the oxidative pentose phosphate pathway supplying reducing power (as NADPH) in non‐photosynthesizing cells. We have examined in detail the redox regulation of the plastidial isoform predominantly present in Arabidopsis green tissues (AtG6PDH1) and found that its oxidative activation is strictly dependent on plastidial thioredoxins (Trxs) that show differential efficiencies. Light/dark modulation of AtG6PDH1 was reproduced in vitro in a reconstituted ferredoxin/Trx system using f‐type Trx allowing to propose a new function for this Trx isoform co‐ordinating both reductive (Calvin cycle) and oxidative pentose phosphate pathways.

Regulation of Photosynthetic GAPDH Dissected by Mutants

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of higher plants catalyzes an NADPH-consuming reaction, which is part of the Calvin cycle. This reaction is regulated by light via thioredoxins and metabolites, while a minor NADH-dependent activity is constant and constitutive. The major native isozyme is formed by A- and B-subunits in stoichiometric ratio (A2B2, A8B8), but tetramers of recombinant B-subunits (GapB) display similar regulatory features to A2B2-GAPDH. The C-terminal extension (CTE) of B-subunits is essential for thioredoxin-mediated regulation and NAD-induced aggregation to partially inactive oligomers (A8B8, B8). Deletion mutant B(minCTE) is redox insensitive and invariably tetrameric, and chimeric mutant A(plusCTE) acquired redox sensitivity and capacity to aggregate to very large oligomers in presence of NAD. Redox regulation principally affects the turnover number, without significantly changing the affinity for either 1,3-bisphosphoglycerate or NADPH. Mutant R77A of GapB, B(R77A), is down-regulated and mimics the behavior of oxidized GapB under any redox condition, whereas mutant B(E362Q) is constantly up-regulated, resembling reduced GapB. Despite their redox insensitivity, both B(R77A) and B(E362Q) mutants are notably prone to aggregate in presence of NAD. Based on structural data and current functional analysis, a model of GAPDH redox regulation is presented. Formation of a disulfide in the CTE induces a conformational change of the GAPDH with repositioning of the terminal amino acid Glu-362 in the proximity of Arg-77. The latter residue is thus distracted from binding the 2′-phosphate of NADP, with the final effect that the enzyme relaxes to a conformation leading to a slower NADPH-dependent catalytic activity.