Regulation of chloroplast phosphoribulokinase by the ferredoxin/thioredoxin system

A newly found form of chloroplast phosphoribulokinase (designated the “regulatory form”) required reduced thioredoxin for activity. A second form of the enzyme (the “nonregulatory form”) was not appreciably affected by thioredoxin. The thioredoxin required for activation of the regulatory enzyme could be reduced (i) photochemically by chloroplast membranes that were supplemented with ferredoxin and ferredoxin-thioredoxin reductase or (ii) chemically in the dark with the sulfhydryl reagent dithiothreitol. Following activation by reduced thioredoxin, phosphoribulokinase was deactivated by the soluble chloroplast oxidants dehydroascorbate and oxidized glutathione. The results suggest that the regulatory form of phosphoribulokinase resembles fructose 1,6-bisphosphatase in its mode of regulation by the ferredoxin/thioredoxin system.     

Activation of Chloroplast NADP-linked Glyceraldehyde-3-Phosphate Dehydrogenase by the Ferredoxin/Thioredoxin System

NADP-glyceraldehyde-3-P dehydrogenase of spinach (Spinacia oleracea) chloroplasts was activated by thioredoxin that was reduced either photochemically with ferredoxin and ferredoxin-thioredoxin reductase or chemically with dithiothreitol. The activation process that was observed with the soluble protein fraction from chloroplasts and with the purified regulatory form of the enzyme was slow relative to the rate of catalysis. The NAD-linked glyceraldehyde-3-P dehydrogenase activity that is also present in chloroplasts and in the purified enzyme preparation was not affected by reduced thioredoxin.

When activated by dithiothreitol-reduced thioredoxin, the regulatory form of NADP-glyceraldehyde-3-P dehydrogenase was partly deactivated by oxidized glutathione. The enzyme activated by photochemically reduced thioredoxin was not appreciably affected by oxidized glutathione. The results suggest that although it resembles other regulatory enzymes in its requirements for light-dependent activation by the ferredoxin/thioredoxin system, NADP-glyceraldehyde-3-P dehydrogenase differs in its mode of deactivation and in its capacity for activation by enzyme effectors independently of thioredoxin.

     

Chloroplast phenylalanine ammonia-lyase from spinach leaves

Phenylalanine ammonia-lyase (PAL) from spinach (Spinacia oleracea L.) leaves was resolved into three forms by diethyl-aminoethyl(DEAE)-cellulose chromatography. Two forms were found in isolated chloroplasts, and the third form (the major component) was located outside of the chloroplasts. One of the chloroplast forms of the enzyme (designated the regulatory form) was activated by reduced thioredoxin. Neither the other chloroplast form nor the extra-chloroplast form showed a response to thioredoxin. After further purification by hydroxyapatite column chromatography and gel filtration, the regulatory form of chloroplast PAL was stimulated approximately 3-fold by thioredoxin reduced either photochemically by chloroplast membranes, via ferredoxin and ferredoxin-thioredoxin reductase, or chemically by dithiothreitol. Once activated, the enzyme required an added oxidant for deactivation. Physiological oxidants-oxidized glutathione (GSSG) and dehydroascorbate-as well as nonphysiological oxidants-sodium tetrathionate and diamide-were effective in deactivation. The results indicate that chloroplast PAL is regulated by light via the ferredoxin/thioredoxin system in a manner similar to that described for regulatory enzymes of CO₂ assimilation. The extra-chloroplast form of the enzyme, by contrast, appears to be regulated by light via the earlier-described phytochrome-linked system.     

Isolation and characterization of different forms of thioredoxins from the green alga Acetabularia mediterranea: identification of an NADP/thioredoxin system in the extrachloroplastic fraction.

A procedure has been developed for the simultaneous purification to apparent homogeneity of chloroplast thioredoxins f and m, and nonchloroplast thioredoxin h, from the green alga Acetabularia mediterranea. In the chloroplast fraction, three thioredoxins were isolated: one f type thioredoxin (Mr 13.4 kDa) and two m type thioredoxin forms (Mr of 12.9 and 13.8 kDa). A Western blot analysis of crude and purified chloroplast thioredoxin preparations revealed that Acetabularia thioredoxin m was immunologically related to its higher-plant counterparts whereas thioredoxin f was not. In the nonchloroplast fraction, a single form of thioredoxin h (Mr 13.4 kDa) and its associated enzyme NADP-thioredoxin reductase (NTR) were evidenced. Acetabularia NTR was partially purified and shown to be an holoenzyme composed of two 33.0-kDa subunits as is the case for other plant and bacterial NTRs. Similarity was confirmed by immunological tests: the algal enzyme was recognized by antibodies to spinach and Escherichia coli NTRs. Acetabularia thioredoxin h seemed to be more distant from higher-plant type h thioredoxins as recognition by antibodies to thioredoxin h from spinach and wheat was weak. The algal thioredoxin h was also slightly active with spinach and E. coli NTRs. These results suggest that in green algae as in the green tissues of higher plants the NADP and chloroplast thioredoxin systems are present simultaneously, and might play an important regulatory role in their respective cellular compartments.     

Differential effect of calcium ions on the cytosolic and mitochondrial thioredoxin reductase

The effect of calcium ions has been studied on three different isoforms of thioredoxin reductase. The cytosolic (TrxR1), mitochondrial (TrxR2), and the Escherichia coli enzymes were examined and compared. In our condition, TrxR1 appears extremely sensitive to Ca2+ showing an IC50 of about 160 nM, while Ca2+ exerts only a weak inhibitory effect on the mitochondrial isoform. The thioredoxin reductase purified from E. coli is almost completely insensitive to calcium ions. Circular dichroism analysis of highly purified mitochondrial and cytosolic thioredoxin reductases reveals that Ca2+ induces conformational alterations that are particularly relevant only in the cytosolic isoform. These observations are discussed with reference to the physiological role and, in particular, to the regulatory functions of the thioredoxin system.     

Oxidation-reduction and activation properties of chloroplast fructose 1,6-bisphosphatase with mutated regulatory site.

The concentration of Mg(2+) required for optimal activity of chloroplast fructose 1,6-bisphosphatase (FBPase) decreases when a disulfide, located on a flexible loop containing three conserved cysteines, is reduced by the ferredoxin/thioredoxin system. Mutation of either one of two regulatory cysteines in this loop (Cys155 and Cys174 in spinach FBPase) produces an enzyme with a S(0.5) for Mg(2+) (0.6 mM) identical to that observed for the reduced WT enzyme and significantly lower than the S(0.5) of 12.2 mM of oxidized WT enzyme. E(m) for the regulatory disulfide in WT spinach FBPase is -305 mV at pH 7.0, with an E(m) vs pH dependence of -59 mV/pH unit, from pH 5.5 to 8.5. Aerobic storage of the C174S mutant produces a nonphysiological Cys155/Cys179 disulfide, rendering the enzyme partially dependent on activation by thioredoxin. Circular dichroism spectra and thiol titrations provide supporting evidence for the formation of nonphysiological disulfide bonds. Mutation of Cys179, the third conserved cysteine, produces FBPase that behaves very much like WT enzyme but which is more rapidly activated by thioredoxin f, perhaps because the E(m) of the regulatory disulfide in the mutant has been increased to -290 mV (isopotential with thioredoxin f). Structural changes in the regulatory loop lower S(0.5) for Mg(2+) to 3.2 mM for the oxidized C179S mutant. These results indicate that opening the regulatory disulfide bridge, either through reduction or mutation, produces structural changes that greatly decrease S(0.5) for Mg(2+) and that only two of the conserved cysteines play a physiological role in regulation of FBPase.     

Evidence for function of the ferredoxin/thioredoxin system in the reductive activation of target enzymes of isolated intact chloroplasts

Results obtained with isolated intact chloroplasts maintained aerobically under light and dark conditions confirm earlier findings with reconstituted enzyme assays and indicate that the ferredoxin/thioredoxin system functions as a light-mediated regulatory thiol chain. The results were obtained by application of a newly devised procedure in which a membrane-permeable thiol labeling reagent, monobromobimane (mBBr), reacts with sulfhydryl groups and renders the derivatized protein fluorescent. The mBBr-labeled protein in question is isolated individually from chloroplasts by immunoprecipitation and its thiol redox status is determined quantitatively by combining sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorescence measurements. The findings indicate that each member of the ferredoxin/thioredoxin system containing a catalytically active thiol group is reduced in isolated intact chloroplasts after a 2-min illumination. The extents of reduction were FTR, 38%; thioredoxin m, 75% (11-kDa form) and 87% (13-kDa form); thioredoxin f, 95%. Reduction of each of these components was negligible both in the dark and when chloroplasts were transferred from light to dark conditions. The target enzyme, NADP-malate dehydrogenase, also underwent net reduction in illuminated intact chloroplasts. Fructose-1,6-bisphosphatase showed increased mBBr labeling under these conditions, but due to interfering gamma globulin proteins it was not possible to determine whether this was a result of net reduction as is known to take place in reconstituted assays. Related experiments demonstrated that mBBr, as well as N-ethylmaleimide, stabilized photoactivated NADP-malate dehydrogenase and fructose-1,6-bisphosphatase so that they remained active in the dark. By contrast, phosphoribulokinase, another thioredoxin-linked enzyme, was immediately deactivated following mBBr addition. These latter results provide new information on the relation between the regulatory and active sites of these enzymes.     

Mutational analysis of human thioredoxin reductase 1. Effects on p53-mediated gene expression and interferon and retinoic acid-induced cell death

The interferon (IFN)-beta and all-trans-retinoic acid combination suppresses tumor growth by inducing apoptosis in several tumor cell lines. A genetic technique permitted the isolation of human thioredoxin reductase (TR) as a critical regulator of IFN/all-trans-retinoic acid-induced cell death. Our recent studies have shown that TR1:thioredoxin 1-regulated cell death is effected in part through the activation of p53-dependent responses. To understand its death regulatory function, we have performed a mutational analysis of TR. Human TR1 has three major structural domains, the FAD binding domain, the NADPH binding domain, and an interface domain (ID). Here, we show that the deletion of the C-terminal interface domain results in a constitutive activation of TR-dependent death responses and promotes p53-dependent gene expression. TR mutant without the ID still retains its dependence on thioredoxin for promoting these responses. Thus, our data suggest that TR-ID acts as a regulatory domain.     

The C-terminal extension of glyceraldehyde-3-phosphate dehydrogenase subunit B acts as an autoinhibitory domain regulated by thioredoxins and nicotinamide adenine dinucleotide

The regulatory isoform of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a light-activated enzyme constituted by subunits GapA and GapB. The NADPH-dependent activity of regulatory GAPDH from spinach chloroplasts was affected by the redox potential (E(m,7.9), -353 +/- 11 mV) through the action of thioredoxin f. The redox dependence of recombinant GapB (E(m,7.9), -347 +/- 9 mV) was similar to native GAPDH, whereas GapA was essentially redox-insensitive. GapB mutants having one or two C-terminal cysteines mutated into serines (C358S, C349S, C349S/C358S) were less redox-sensitive than GapB. Different mutants with other cysteines substituted by serines (C18S, C274S, C285S) still showed strong redox regulation. Fully active GapB was a tetramer of B-subunits, and, when incubated with NAD, it associated to a high molecular weight oligomer showing low NADPH-dependent activity. The C-terminal GapB mutants (C358S, C349S, C349S/C358S) were active tetramers unable to aggregate to higher oligomers in the presence of NAD, whereas other mutants (C18S, C274S, C285S) again behaved like GapB. We conclude that a regulatory disulfide, between Cys-349 and Cys-358 of the C-terminal extension of GapB, does form in the presence of oxidized thioredoxin. This covalent modification is required for the NAD-dependent association into higher oligomers and inhibition of the NADPH-activity. By leading to GAPDH autoinhibition, thioredoxin and NAD may thus concur to the dark inactivation of the enzyme in vivo.     

NADPH and oxidized thioredoxin mediate redox interconversion of calf-liver and Escherichia coli thioredoxin reductase

The activity of pure calf-liver and Escherichia coli thioredoxin reductases decreased drastically in the presence of NADPH or NADH, while NADP⁺, NAD⁺ and oxidized E. coli thioredoxin activated both enzymes significantly, particularly the bacterial one. The loss of activity under reducing conditions was time-dependent, thus suggesting an inactivation process: in the presence of 0.24 mM NADPH the half-lives for the E. coli and calf-liver enzymes were 13.5 and 2 min, respectively. Oxidized E. coli thioredoxin fully protected both enzymes from inactivation, and also promoted their complete reactivation after only 30 min incubation at 30° C. Lower but significant protection and reactivation was also observed with NADP⁺ and NAD⁺. EDTA protected thioredoxin reductase from NADPH inactivation to a great degree, thus indicating the participation of metals in the process; EGTA did not protect the enzyme from redox inactivation. Thioredoxin reductase was extensively inactivated by NADPH under aerobic and anaerobic conditions, thus excluding the participation of O₂ or oxygen active species in redox inactivation. The loss of thioredoxin reductase activity promoted by NADPH was much faster and complete in the presence of NAD⁺ glycohydrolase, thus suggesting that inactivation was related to full reduction of the redox-active disulfide. Those results indicate that thioredoxin reductase activity can be modulated in bacteria and mammals by the redox status of NADP(H) and thioredoxin pools, in a similar way to glutathione reductase. This would considerably expand the regulatory potential of the thioredoxin-thioredoxin reductase system with the enzyme being self-regulated by its own substrate, a regulatory protein.Abbreviations DTNB 5,5-dithiobis(2-nitrobenzoate) - EGTA Ethylenglycoltetraacetic Acid - TNB 5-thio-2-nitrobenzoate - Trx Thioredoxin - Trx(SH)2 Reduced Thioredoxin - Trx-S2 Oxidized Thioredoxin     

NMR structures of thioredoxin m from the green alga Chlamydomonas reinhardtii

Chloroplast thioredoxin m from the green alga Chlamydomomas reinhardtii is very efficiently reduced in vitro and in vivo in the presence of photoreduced ferredoxin and a ferredoxin dependent ferredoxin-thioredoxin reductase. Once reduced, 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 thioredoxin 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 relaxation 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 dehydrogenase. The specific catalytic mechanism that takes place in the active site of thioredoxins is also discussed 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 cysteines of the redox active site may insulate each other from solvent attack by specific packing of invariable hydrophobic amino acids.     

Genomic organization and identification of a novel alternative splicing variant of mouse mitochondrial thioredoxin reductase (TrxR2) gene

Eukaryotic mitochondria are equipped with a complete thioredoxin system, composed of thioredoxin and thioredoxin reductase, which has been implicated in the protection against the reactive oxygen intermdiates generated during the respiratory process in this organelle. Like its cytosolic counterpart, mammalian mitochondrial thioredoxin reductase is a homodimeric selenoprotein. We report here the genomic organization of the mouse mitochondrial thioredoxin gene (TrxR2) that spans 53 kb and consists of 18 exons ranging from 20 to 210 bp. All splicing sites conformed to the GT/AG rule with the exon-intron boundaries located exactly at the same position as the human TrxR2 gene, the only mammalian mitochondrial thioredoxin reductase gene whose genomic structure has been elucidated to date. In addition, we have identified a novel mRNA splicing variant lacking intron 14 resulting in a protein subunit with a shorter interface domain. This new splicing variant provides a framework for further analysis of this important enzyme as its predicted homodimeric conformation can now be expanded to a putative heterodimeric structure as well as a small subunit homodimer with the obvious implications at the regulatory level.     

Target proteins of the cytosolic thioredoxins in Arabidopsis thaliana

Possible target proteins of cytosolic thioredoxin in higher plants have been investigated in the cell lysate of dark-grown Arabidopsis thaliana whole tissues. We immobilized a mutant of cytosolic thioredoxin, in which an internal cysteine at the active site was substituted with serine, on CNBr activated resin, and used the resin for the thioredoxin-affinity chromatography. By using this resin, the target proteins for thioredoxin in the higher plant cytosol were efficiently acquired. The obtained proteins were separated by two-dimensional gel electrophoresis and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Thus we have identified proteins of the anti-oxidative stress system proteins (ascorbate peroxidase, germin-like protein, and monomeric type II peroxiredoxin), proteins involved in protein biosynthesis (elongation factor-2 and eukaryotic translation initiation factor 4A), proteins involved in protein degradation (the regulatory subunit of 26S proteasome), and several metabolic enzymes (alcohol dehydrogenase, fructose 1,6-bis phosphate aldolase-like protein, cytosolic glyceraldehyde 3-phosphate dehydrogenase, cytosolic malate dehydrogenase, and vitamin B(12)-independent methionine synthase) together with some chloroplast proteins (chaperonin 60-alpha and 60-beta, heat shock protein 70, and glutamine synthase). The results in this study and recent proteomics studies on the target proteins of chloroplast thioredoxin indicate the versatility and the physiological significance of thioredoxin as reductant in plant cell.     

The ferredoxin/thioredoxin system: a key element in the regulatory function of light in photosynthesis

In addition to its well-established function in supplying the energy for carbon dioxide assimilation, light plays a regulatory role in photosynthesis. The ferredoxin/thioredoxin system is a major mechanism whereby light functions in this capacity. Here, light absorbed by chlorophyll is converted via ferredoxin into a reductant messenger, reduced thioredoxin, that interacts with key target enzymes, thereby changing their catalytic activities. In this way, the green plant achieves maximum efficiency of its photosynthetic (light) and heterotrophic (dark) capabilities.     

Differential effects of chilling-induced photooxidation on the redox regulation of photosynthetic enzymes

Photosynthesis in plant species that are evolutionarily adapted for growth in warm climates is highly sensitive to illumination under cool conditions. Although it is well documented that illumination of these sensitive species under cool conditions results in the photosynthetic production of reactive oxygen molecules, the underlying mechanism for the inhibition of photosynthesis remains uncertain. Determinations of chloroplast fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase activity showed that the light-dependent, reductive activation of these key carbon reduction cycle enzymes was substantially inhibited in tomato (Lycopersicon esculentum) following illumination at 4 degrees C. However, other chloroplast enzymes also dependent on thioredoxin-mediated reductive activation were largely unaffected. We performed equilibrium redox titrations to investigate the thermodynamics of the thiol/disulfide exchange between thioredoxin f and the regulatory sulfhydryl groups of fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase, phosphoribulokinase, NADP-glyceraldehyde phosphate dehydrogenase, and the chloroplast ATPsynthase. We determined that the redox midpoint potentials for the regulatory sulfhydryl groups of the various enzymes spanned a broad range ( approximately 50 mV at pH 7. 9). The electron-sharing equilibria among thioredoxin f and its target enzymes largely explained the differential effects of photooxidation induced at low temperature on thioredoxin-mediated activation of chloroplast enzymes in tomato. These results not only provide a plausible mechanism for the low-temperature-induced inhibition of photosynthesis in this important group of plants, but also provide a quantitative basis to evaluate the influence of thioredoxin/target enzyme electron-sharing equilibria on the differential activation and deactivation kinetics of thioredoxin-regulated chloroplast enzymes.     

Kinetics of the modulation of chloroplastic fructose-1,6-bisphosphatase activity by thioredoxin fb

The inactivation of reduced chloroplast fructose-bisphosphatase by oxidized thioredoxin fb has been studied during the enzyme reaction along the principle of Tian and Tsou [Biochemistry (1982) 21, 1028-1032]. A minimum model for this process is presented and its kinetic and equilibrium parameters have been determined. Thioredoxin fb binding to the enzyme is fast relative to catalysis and product desorption. Under quasi-equilibrium conditions oxidized thioredoxin is a non-competitive inhibitor of the enzyme reaction and must bind to a regulatory 'thioredoxin site'. The slow deactivation is thermodynamically favoured, and as expected from binding data, slowed down by the presence of substrate, fructose bisphosphate. The desorption of thioredoxin fb from the enzyme is extremely slow and this small protein may be regarded as a 'regulatory' subunit of fructose-bisphosphatase.     

Crystal structures of two functionally different thioredoxins in spinach chloroplasts

Thioredoxins are small ubiquitous proteins which act as general protein disulfide reductases in living cells. Chloroplasts contain two distinct thioredoxins ( f and m) with different phylogenetic origin. Both act as enzyme regulatory proteins but have different specificities towards target enzymes. Thioredoxin f (Trx f), which shares only low sequence identity with thioredoxin m (Trx m) and with all other known thioredoxins, activates enzymes of the Calvin cycle and other photosynthetic processes. Trx m shows high sequence similarity with bacterial thioredoxins and activates other chloroplast enzymes. The here described structural studies of the two chloroplast thioredoxins were carried out in order to gain insight into the structure/function relationships of these proteins. Crystal structures were determined for oxidized, recombinant thioredoxin f (Trx f-L) and at the N terminus truncated form of it (Trx f-S), as well as for oxidized and reduced thioredoxin m (at 2.1 and 2.3 A resolution, respectively). Whereas thioredoxin f crystallized as a monomer, both truncated thioredoxin f and thioredoxin m crystallized as non-covalent dimers. The structures of thioredoxins f and m exhibit the typical thioredoxin fold consisting of a central twisted five-stranded beta-sheet surrounded by four alpha-helices. Thioredoxin f contains an additional alpha-helix at the N terminus and an exposed third cysteine close to the active site. The overall three-dimensional structures of the two chloroplast thioredoxins are quite similar. However, the two proteins have a significantly different surface topology and charge distribution around the active site. An interesting feature which might significantly contribute to the specificity of thioredoxin f is an inherent flexibility of its active site, which has expressed itself crystallographically in two different crystal forms.