An f-type thioredoxin fromArabidopsis thaliana Leaves

Thioredoxin, a small redox protein with an active site disulfide/dithiol, is ubiquitous in bacteria, plants, and animals and functions as a reducing agent and modulator of enzyme activity. A thioredoxin has been purified to electrophoretic homogeneity from the leaves ofArabidopsis thaliana using procedures such as DE-52 ion exchange chromatography, Sephadex G-50 gel filtration, Q-Sepharose ion exchange chromatography, and DEAE-Sephadex A-25 chromatography. The purified thioredoxin was determined to be a single band on SDS-PAGE, and its molecular weight was estimated to be 21 KDa, which was much larger than those of most other known thioredoxins. It was proved to be an f-type thioredoxin, since it could activate fructose-l,6-bisphosphatase, but it could not activate NADP⁺-malate dehydrogenase. As a protein disulfide reductase, it could reduce the disulfide bonds contained in insulin. As a substrate, it showed a Km value of 20.2 μM onEscherichia coli thioredoxin reductase, and it had an optimal pH of 8.0. The molecular weight of the purified f-type thioredoxin is not consistent with those of the five divergent h-type thioredoxins already identified by cDNA cloning. The purified f-type thioredoxin is the first example isolated fromA. thaliana.     

Involvement of thioredoxin y2 in the preservation of leaf methionine sulfoxide reductase capacity and growth under high light

Methionine (Met) in proteins can be oxidized to two diastereoisomers of methionine sulfoxide, Met‐S‐O and Met‐R‐O, which are reduced back to Met by two types of methionine sulfoxide reductases (MSRs), A and B, respectively. MSRs are generally supplied with reducing power by thioredoxins. Plants are characterized by a large number of thioredoxin isoforms, but those providing electrons to MSRs in vivo are not known. Three MSR isoforms, MSRA4, MSRB1 and MSRB2, are present in Arabidopsis thaliana chloroplasts. Under conditions of high light and long photoperiod, plants knockdown for each plastidial MSR type or for both display reduced growth. In contrast, overexpression of plastidial MSRBs is not associated with beneficial effects in terms of growth under high light. To identify the physiological reductants for plastidial MSRs, we analyzed a series of mutants deficient for thioredoxins f, m, x or y. We show that mutant lines lacking both thioredoxins y1 and y2 or only thioredoxin y2 specifically display a significantly reduced leaf MSR capacity (–25%) and growth characteristics under high light, related to those of plants lacking plastidial MSRs. We propose that thioredoxin y2 plays a physiological function in protein repair mechanisms as an electron donor to plastidial MSRs in photosynthetic organs.     

Compact reduced thioredoxin structure from the thermophilic bacteria Thermus thermophilus

The X-ray crystallographic structure of a thioredoxin from Thermus thermophilus was solved to 1.8 A resolution by molecular replacement. The crystals' space group was C2 with cell dimensions of a = 40.91, b = 95.44, c = 56.68 A, beta =91.41 degrees, with two molecules in the asymmetric unit. Unlike the reported thioredoxin structures, the biological unit of T. thermophilus thioredoxin is a dimer both in solution and in the crystal. The fold conforms to the "thioredoxin fold" that is common over a class of nine protein families including thioredoxin; however, the folded portion of this protein is much more compact than other thioredoxins previously solved by X-ray crystallography being reduced by one alpha-helix and one beta-strand. As with other thioredoxins, the active site is highly conserved even though the variation in sequence can be quite large. The T. thermophilus thioredoxin has some variability at the active site, especially compared with previously solved structures from bacterial sources.     

Thioredoxins in<Emphasis Type="Italic">Arabidopsis</Emphasis> and other plants

Regulation of disulfide dithiol exchange has become increasingly important in our knowledge of plant life. Initially discovered as regulators of light-dependent malate biosynthesis in the chloroplast, plant thioredoxins are now implicated in a large panel of reactions related to metabolism, defense and development. In this review we describe the numerous thioredoxin types encoded by the Arabidopsis genome, and provide evidence that they are present in all higher plants. Some results suggest cross-talk between thioredoxins and glutaredoxins, the second family of disulfide dithiol reductase. The development of proteomics in plants revealed an unexpectedly large number of putative target proteins for thioredoxins and glutaredoxins. Nevertheless, we are far from a clear understanding of the actual function of each thioredoxin in planta. Although hampered by functional redundancies between genes, genetic approaches are probably unavoidable to define which thioredoxin interacts with which target protein and evaluate the physiological consequences.     

Plant thioredoxin h: an animal-like thioredoxin occurring in multiple cell compartments

Thioredoxin h has been purified to electrophoretic homogeneity from spinach roots using a procedure devised for leaves. The root thioredoxin (h2 form) differed from chloroplast and animal thioredoxins in showing an atypical active site (Cys-Ala-Pro-Cys) but otherwise resembled animal thioredoxin in structure. Sequence data for a total of 72 residues of spinach root thioredoxin h2 (about 69% of the primary structure) showed 43-44% identity with rabbit and rat thioredoxin. Analysis of cell fractions from the endosperm of germinating castor beans revealed that thioredoxin h occurs in the cytosol, endoplasmic reticulum, and mitochondria. The present findings demonstrate a similarity between plant thioredoxin h and animal thioredoxins in structure and intracellular location and raise the question of whether these proteins have similar functions.     

Chlamydomonas reinhardtii thioredoxins: structure of the genes coding for the chloroplastic m and cytosolic h isoforms; expression in Escherichia coli of the recombinant proteins,purification and biochemical properties

Based on known amino acid sequences, probes have been generated by PCR and used for the subsequent isolation of cDNAs and genes coding for two thioredoxins (m and h) of Chlamydomonas reinhardtii. Thioredoxin m, a chloroplastic protein, is encoded as a preprotein of 140 amino acids (15 101 Da) containing a transit peptide of 34 amino acids with a very high content of Ala and Arg residues. The sequence for thioredoxin h codes for a 113 amino acid protein with a molecular mass of 11817 Da and no signal sequence. The thioredoxin m gene contains a single intron and seems to be more archaic in structure than the thioredoxin h gene, which is split into 4 exons. The cDNA sequences encoding C. reinhardtii thioredoxins m and h have been integrated into the pET-3d expression vector, which permits efficient production of proteins in Escherichia coli cells. A high expression level of recombinant thioredoxins was obtained (up to 50 mg/l culture). This has allowed us to study the biochemical/biophysical properties of the two recombinant proteins. Interestingly, while the m-type thioredoxin was found to have characteristics very close to the ones of prokaryotic thioredoxins, the h-type thioredoxin was quite different with respect to its kinetic behaviour and, most strikingly, its heat denaturation properties.Abbreviations DTT dithiothreitol - FBPase Fructose 1,6-biphosphate phosphatase - FTR ferredoxin-thioredoxin reductase - IPTG isopropyl thiogalactoside - NADP-MDH NADPH-dependent malate dehydrogenase - NMR nuclear magnetic resonance - NTR NADPH-dependent thioredoxin reductase Dedicated to the memory of Claude Crétin     

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.     

Intron position as an evolutionary marker of thioredoxins and thioredoxin domains

In contrast to prokaryotes, which typically possess one thioredoxin gene per genome, three different thioredoxin types have been described in higher plants. All are encoded by nuclear genes, but thioredoxins m and f are chloroplastic while thioredoxins h have no transit peptide and are probably cytoplasmic. We have cloned and sequencedArabidopsis thaliana genomic fragments encoding the five previously described thioredoxins h, as well as a sixth gene encoding a new thioredoxin h. In spite of the high divergence of the sequences, five of them possess two introns at positions identical to the previously sequenced tobacco thioredoxin h gene, while a single one has only the first intron. The recently published sequence ofChlamydomonas thioredoxin h shows three introns, two at the same positions as in higher plants. This strongly suggests a common origin for all cytoplasmic thioredoxins of plants and green algae. In addition, we have cloned and sequenced pea DNA genomic fragments encoding thioredoxins m and f. The thioredoxin m sequence shows only one intron between the regions encoding the transit peptide and the mature protein, supporting the prokaryotic origin of this sequence and suggesting that its association with the transit peptide has been facilitated by exon shuffling. In contrast, the thioredoxin f sequence shows two introns, one at the same position as an intron in various plant and animal thioredoxins and the second at the same position as an intron in thioredoxin domains of disulfide isomerases. This strongly supports the hypothesis of a eukaryotic origin for chloroplastic thioredoxin f.     

Isomers in thioredoxins of spinach chloroplasts

We have developed a method for the concomitant purification of several components of the ferredoxin/thioredoxin system of spinach chloroplasts. By applying this method to spinach-leaf extract or spinach-chloroplast extract we separated and purified three thioredoxins indigenous to chloroplasts. The three thioredoxins, when reduced, will activate certain chloroplast enzymes such as fructose-1,6-bisphosphatase and NADP-dependent malate dehydrogenase. Fructose-1,6-bisphosphatase is activated by thioredoxin f exclusively. Malate dehydrogenase is activated by thioredoxin mb and thioredoxin mc in a similar way, and it is also activated by thioredoxin f but with different kinetics. All three thioredoxins have very similar relative molecular masses of about 12,000 but distinct isoelectric points of 6.1 (thioredoxin f), 5.2 (thioredoxin mb) and 5.0 (thioredoxin mc). The amino acid composition as well as the C-terminal and N-terminal sequences have been determined for each thioredoxin. Thioredoxin f exhibits clear differences in amino acid composition and terminal sequences when compared with the m-type thioredoxins. Thioredoxin mb and thioredoxin mc, however, are very similar, the only difference being an additional lysine residue at the N-terminus of thioredoxin mb. Amino acid analyses, terminal sequences, immunological tests and the activation properties of the thioredoxins support our conclusion that thioredoxins mb and mc are N-terminal redundant isomers coming from one gene whereas thioredoxin f is a different protein coded by a different gene.     

Crystal structures of oxidized and reduced forms of human mitochondrial thioredoxin 2

Mammalian thioredoxin 2 is a mitochondrial isoform of highly evolutionary conserved thioredoxins. Thioredoxins are small ubiquitous protein-disulfide oxidoreductases implicated in a large variety of biological functions. In mammals, thioredoxin 2 is encoded by a nuclear gene and is targeted to mitochondria by a N-terminal mitochondrial presequence. Recently, mitochondrial thioredoxin 2 was shown to interact with components of the mitochondrial respiratory chain and to play a role in the control of mitochondrial membrane potential, regulating mitochondrial apoptosis signaling pathway. Here we report the first crystal structures of a mammalian mitochondrial thioredoxin 2. Crystal forms of reduced and oxidized human thioredoxin 2 are described at 2.0 and 1.8 A resolution. Though the folding is rather similar to that of human cytosolic/nuclear thioredoxin 1, important differences are observed during the transition between the oxidized and the reduced states of human thioredoxin 2, compared with human thioredoxin 1. In spite of the absence of the Cys residue implicated in dimer formation in human thioredoxin 1, dimerization still occurs in the crystal structure of human thioredoxin 2, mainly mediated by hydrophobic contacts, and the dimers are associated to form two-dimensional polymers. Interestingly, the structure of human thioredoxin 2 reveals possible interaction domains with human peroxiredoxin 5, a substrate protein of human thioredoxin 2 in mitochondria.     

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.     

重组玉米f型和m型硫氧还蛋白的功能确定及其靶向蛋白的捕获

RT-PCR从玉米幼叶总RNA中克隆f型和m型硫氧还蛋白（Thioredoxin, Trx）的编码基因,分别将两种类型Trx活性中心的第二个保守Cys残基定点突变成Ser残基和Ala残基。在大肠杆菌分别重组表达和纯化了含组氨酸标签的Trx及其突变体蛋白,SDS-PAGE显示纯化的蛋白显示一条主带,蛋白分子量分别估计为f型Trx为18kDa,m型Trx为14kDa;纯化的含有SUMO标签融合Trx,用SUMO专一性SUMO水解酶Ulp除去SUMO,等点聚焦电泳显示m型和f型Trx的等电点分别为4.6和5.9。m型Trx比f型Trx有更强的还原胰岛素能力,而突变体蛋白几乎没有还原能力。用Cys残基专一性标记化合物AMS标记Trx,显示野生型Trx有氧化还原态,而突变体蛋白仅有还原态。SDS-PAGE电泳显示固定化的f型Trx突变体比m型Trx突变体捕获的玉米幼叶靶蛋白更具有多样性。     

Interaction of thioredoxins with target proteins: role of particular structural elements and electrostatic properties of thioredoxins in their interplay with 2-oxoacid dehydrogenase complexes

The thioredoxin action upon the 2-oxoacid dehydrogenase complexes is investigated by using different thioredoxins, both wild-type and mutated. The attacking cysteine residue of thioredoxin is established to be essential for the thioredoxin-dependent activation of the complexes. Mutation of the buried cysteine residue to serine is not crucial for the activation, but prevents inhibition of the complexes, exhibited by the Clamydomonas reinhardtii thioredoxin m disulfide. Site-directed mutagenesis of D26, W31, F/W12, and Y/A70 (the Escherichia coli thioredoxin numbering is employed for all the thioredoxins studied) indicates that both the active site and remote residues of thioredoxin are involved in its interplay with the 2-oxoacid dehydrogenase complexes. Sequences of 11 thioredoxin species tested biochemically are aligned. The thioredoxin residues at the contact between the alpha3/3(10) and alpha1 helices, the length of the alpha1 helix and the charges in the alpha2-beta3 and beta4-beta5 linkers are found to correlate with the protein influence on the 2-oxoacid dehydrogenase complexes (the secondary structural elements of thioredoxin are defined according to Eklund H et al., 1991, Proteins 11:13-28). The distribution of the charges on the surface of the thioredoxin molecules is analyzed. The analysis reveals the species specific polarization of the thioredoxin active site surroundings, which corresponds to the efficiency of the thioredoxin interplay with the 2-oxoacid dehydrogenase systems. The most effective mitochondrial thioredoxin is characterized by the strongest polarization of this area and the highest value of the electrostatic dipole vector of the molecule. Not only the magnitude, but also the orientation of the dipole vector show correlation with the thioredoxin action. The dipole direction is found to be significantly influenced by the charges of the residues 13/14, 51, and 83/85, which distinguish the activating and inhibiting thioredoxin disulfides.     

AtCXXS: atypical members of the Arabidopsis thaliana thioredoxin h family with a remarkably high disulfide isomerase activity

The Arabidopsis thaliana thioredoxin subgroup h III is composed of four members and includes the two monocysteinic (CXXS) thioredoxins encoded by the genome. We show that AtCXXS1 is the ortholog of monocysteinic thioredoxins present in all higher plants. In contrast, unicellular algae and the moss Physcomitrella patens do not encode monocysteinic thioredoxin. AtCXXS2, the second monocysteinic thioredoxin of Arabidopsis has no ortholog in any other higher plants. It probably appeared recently by duplications of a dicysteinic thioredoxin of the same subgroup h III. Both monocysteinic thioredoxins show a low disulfide reductase activity in vitro but are very efficient as disulfide isomerases in RNAse refolding tests. The possible interactions of these proteins with the glutathione glutaredoxin pathway are discussed on the basis of recent papers.     

The Arabidopsis thaliana genome encodes at least four thioredoxins m and a new prokaryotic-like thioredoxin

Screening of cDNA libraries at low stringency and complete sequencing of EST clones with homology to thioredoxins allowed us to characterize five new prokaryotic type Arabidopsis thaliana thioredoxins. All present N-terminal extensions with characteristics of transit peptides. Four are clustered in a phylogenetic tree with the chloroplastic thioredoxin m from red and green algae and higher plants, and their transit peptides have typical characteristics of chloroplastic transit peptides. One is clearly divergent and defines a new prokaryotic thioredoxin type that we have named thioredoxin x. Its transit peptide sequence presents characteristics of both chloroplastic and mitochondrial transit peptides. The five corresponding genes are expressed at different levels, but mostly in green tissues and in in-vitro cultivated cells.     

Evolution of redoxin genes in the green lineage

The availability of the Arabidopsis genome revealed the complexity of the gene families implicated in dithiol disulfide exchanges. Most non-green organisms present less dithiol oxidoreductase genes. The availability of the almost complete genome sequence of rice now allows a systematic search for thioredoxins, glutaredoxins and their reducers. This shows that all redoxin families previously defined for Arabidopsis have members in the rice genome and that all the deduced rice redoxins fall within these families. This establishes that the redoxin classification applies both to dicots and monocots. Nevertheless, within each redoxin type the number of members is not the same in these two higher plants and it is not always possible to define orthologues between rice and Arabidopsis. The sequencing of two unicellular algae (Chlamydomonas and Ostreococcus) genomes are almost finished. This allowed us to follow the origin of the different gene families in the green lineage. It appears that most thioredoxin and glutaredoxin types, their chloroplastic, mitochondrial and cytosolic reducers are always present in these unicellular organisms. Nevertheless, striking differences appear in comparison to higher plant redoxins. Some thioredoxin types are not present in these algal genomes including thioredoxins o, clot and glutaredoxins CCxC. Numerous redoxins, including the cytosolic thioredoxins, do not fit with the corresponding higher plant classification. In addition both algae present a NADPH-dependent thioredoxin reductase with a selenocysteine which is highly similar to the animal thioredoxin reductases, a type of thioredoxin reductase not present in higher plants. An erratum to this article can be found at