An NADP/thioredoxin system in leaves: purification and characterization of NADP-thioredoxin reductase and thioredoxin h from spinach

An NADP/thioredoxin system, consisting of NADPH, NADP-thioredoxin reductase (NTR), and its thioredoxin, thioredoxin h, has been previously described for heterotrophic plant tissues, i.e., wheat seeds and cultured carrot cells. Until now there was no evidence for this system in green leaves. Here, we report the identification of protein components of the NADP/thioredoxin system in leaves of several species. Thioredoxin h and NTR, which were both recovered in the extrachloroplastic fraction, were purified to apparent homogeneity from spinach leaves. This represents the first time that NTR has been characterized from a plant source. Similar to that from bacterial and mammalian sources, spinach leaf NTR was a flavoprotein (Mr 68,000) composed of two subunits of identical molecular mass (Mr 33,000) that resembled Escherichia coli NTR immunologically. Spinach thioredoxin h existed in two forms (Mr of 13,500 and 12,000) and was highly specific for plant NTR. Thioredoxin h and NTR partially purified from spinach roots showed properties similar to their counterparts from leaves. Spinach cytosolic thioredoxin h differed from chloroplast thioredoxin m or f from the same source but was similar to thioredoxin h from wheat seed in immunological properties.     

The oxygen evolving enhancer protein 1 (OEE) of photosystem II in green algae exhibits thioredoxin activity

A thioredoxin-like chloroplast protein of the fructosebisphosphatase-stimulating f-type, but with an unusually high molecular mass of 28 kDa has previously been identified and purified to homogeneity in a fractionation scheme for resolution of the acid- and heat-stable, regular-size (12kDa) thioredoxins of the unicellular green algae, Scenedesmus obliquus. An apparently analogous protein of 26 kDa was described in a cyanobacterium, Anabaena sp., but no such large thioredoxin species f exists in the thioredoxin profiles of higher plants. The structure of the 28 kDa protein, which had been envisaged to represent a precursor, or fusion product of the two more specialized, common chloroplast thioredoxins f and m has now been determined by amino acid sequencing. Although it exhibits virtually all the properties and enzyme-modulating activities of a thioredoxin proper this algal protein, surprisingly, does not belong to the thioredoxin family of small redox proteins but is identical with OEE (oxygen evolving enhancer) protein 1, an auxiliary component of the photosystem II manganese cluster. Extracts of Chlorella vulgaris and Chlamydomonas reinhardtii also contain heat-stable protein fractions of 23-26 kDa capable of specifically stimulating chloroplast fructosebisphosphatase in vitro. In contrast, OEE protein 1 from spinach is not able to modulate FbPase or NADP malate dehydrogenase from spinach chloroplasts. A dual function of the OEE protein in algal photosynthesis is envisaged.     

A novel NADPH thioredoxin reductase, localized in the chloroplast, which deficiency causes hypersensitivity to abiotic stress in Arabidopsis thaliana

Plants contain three thioredoxin systems. Chloroplast thioredoxins are reduced by ferredoxin-thioredoxin reductase, whereas the cytosolic and mitochondrial thioredoxins are reduced by NADPH thioredoxin reductase (NTR). There is high similarity among NTRs from plants, lower eukaryotes, and bacteria, which are different from mammal NTR. Here we describe the OsNTRC gene from rice encoding a novel NTR with a thioredoxin-like domain at the C terminus, hence, a putative NTR/thioredoxin system in a single polypeptide. Orthologous genes were found in other plants and cyanobacteria, but not in bacteria, yeast, or mammals. Full-length OsNTRC and constructs with truncated NTR and thioredoxin domains were expressed in Escherichia coli as His-tagged polypeptides, and a polyclonal antibody specifically cross-reacting with the OsNTRC enzyme was raised. An in vitro activity assay showed that OsNTRC is a bifunctional enzyme with both NTR and thioredoxin activity but is not an NTR/thioredoxin system. Although the OsNTRC gene was expressed in roots and shoots of rice seedlings, the protein was exclusively found in shoots and mature leaves. Moreover, fractionation experiments showed that OsNTRC is localized to the chloroplast. An Arabidopsis NTRC knock-out mutant showed growth inhibition and hypersensitivity to methyl viologen, drought, and salt stress. These results suggest that the NTRC gene is involved in plant protection against oxidative stress.     

Contrasting evolutionary histories of chloroplast thioredoxins f and m

Fourteen thioredoxin sequences were used to construct a minimal phylogenetic tree by using parsimony. The bacterial thioredoxins clustered into three groups: one containing the photosynthetic purple bacteria, Escherichia and Corynebacterium; a second containing the photosynthetic green bacterium, Chlorobium; and a third containing cyanobacteria. These groupings are similar to those generated from earlier 16s RNA analyses. Animal thioredoxins formed a fourth group. The two thioredoxins of chloroplasts (f and m) showed contrasting phylogenetic patterns. As predicted from prior studies, spinach chloroplast thioredoxin m grouped with its counterparts from cyanobacteria and eukaryotic algae, but, unexpectedly, thioredoxin f grouped with the animal thioredoxins. The results indicate that, during evolution, thioredoxin m of contemporary photosynthetic eukaryotic cells was derived from a prokaryotic symbiont, whereas thioredoxin f descended from an ancestral eukaryote common to plants and animals. The findings illustrate the potential of thioredoxin as a phylogenetic marker and suggest a relationship between the animal and f-type thioredoxins.      

Characterization and primary structure of a second thioredoxin from the green alga, Chlamydomonas reinhardtii

A second thioredoxin, Ch1, distinct from the one recently reported [Decottignies, P., Schmitter, J.M., Jacquot, J. P., Dutka, S., Picaud, A. & Gadal, P. (1990) Arch, Biochem. Biophys. 280, 112-121] has been purified from the green alga, Chlamydomonas reinhardtii, and its functional and structural properties investigated. Its activity in various enzymatic assays has been compared with the activities of different plant thioredoxins (Ch2 from C. reinhardtii and spinach m and f). Ch1 cannot serve as a substrate for Escherichia coli thioredoxin reductase, but can be reduced by spinach ferredoxin-thioredoxin reductase. It is less efficient than its spinach counterpart in the activation of corn leaf NADP-dependent malate dehydrogenase by light or dithiothreitol, and it only activates spinach fructose-1,6-bisphosphatase at very high concentrations. The complete primary structure of C. reinhardtii thioredoxin Ch1 was determined by automated Edman degradation of the intact protein and of peptides derived from trypsin, chymotrypsin and Staphylococcus aureus V8 protease digestions. When needed, peptide masses were verified by plasma desorption mass spectrometry. Ch1 consists of a polypeptide of 111 amino acids (11634 Da) and contains the well-conserved active site sequence Trp-Cys-Gly-Pro-Cys. Compared to thioredoxins from other sources, the algal thioredoxin Ch1 displays few sequence similarities with all the thioredoxins sequenced so far. Preliminary evidence indicates that Ch1 may be an h-type thioredoxin.     

Enzyme regulation in C4 photosynthesis: purification, properties, and activities of thioredoxins from C4 and C3 plants

Procedures are described for the purification to homogeneity of chloroplast thioredoxins f and m from leaves of corn (Zea mays, a C4 plant) and spinach (Spinacea oleracea, a C3 plant). The C3 and C4f thioredoxins were similar immunologically and biochemically, but differed in certain of their physiochemical properties. The f thioredoxins from the two species were capable of activating both NADP-malate dehydrogenase (EC 1.1.1.37) and fructose-1,6-bisphosphatase (EC 3.1.3.11) when tested in standard thioredoxin assays. Relative to its spinach counterpart, corn thioredoxin f showed a greater molecular mass (15.0-16.0 kDa vs 10.5 kDa), lower isoelectric point (ca. 5.2 vs 6.0), and lower ability to form a stable noncovalent complex with its target fructose bisphosphatase enzyme. The C3 and C4 m thioredoxins were similar in their specificity (ability to activate NADP-malate dehydrogenase, and not fructose-1,6-bisphosphatase) and isoelectric points (ca. 4.8), but differed slightly in molecular mass (13.0 kDa for spinach vs 13.5 kDa for corn) and substantially in their immunological properties. Results obtained in conjunction with these studies demonstrated that the thioredoxin m-linked activation of NADP-malate dehydrogenase in selectively enhanced by the presence of halide ions (e.g., chloride) and by an organic solvent (e.g., 2-propanol). The results suggest that in vivo NADP-malate dehydrogenase interacts with thylakoid membranes and is regulated to a greater extent by thioredoxin m than thioredoxin f.     

Isolation and characterization of thioredoxin f from the filamentous cyanobacterium, Anabaena sp. 7119

Two thioredoxin fractions had previously been reported to occur in Anabaena 7119 by Buchanan and co-workers (Yee, B. C., dela Torre, A., Crawford, N. A., Lara, C., Carlson, D. E., and Buchanan, B. B. (1981) Arch. Microbiol. 130, 14-18). These proteins were detected by their ability to activate spinach fructose-1,6-bisphosphatase (Fru-P2-ase). The partially purified proteins resembled similar thioredoxins found in spinach chloroplasts and were designated thioredoxin f (Tf) for the fraction most effective in activating spinach Fru-P2-ase and thioredoxin m (Tm) for the fraction most effective in activating spinach NADPH-malate dehydrogenase. Using the assay system of Yee and co-workers, we were able to separate and purify to homogeneity two thioredoxin fractions from Anabaena extracts. Tm corresponded to the thioredoxin fraction we had isolated and studied previously (Gleason, F. K., and Holmgren, A. (1981) J. Biol. Chem. 256, 8301-8309). The other fraction, Tf, was characterized further. Unlike the thioredoxins found in higher plants, the cyanobacterial thioredoxins do not appear to be related. Anabaena thioredoxin f has a Mr = 25,500 as compared to the more usual Mr = 12,000 for Tm. From a comparison of the amino acid composition, Tf is not obviously a dimer or otherwise related to Tm. Tf has one active center cystine disulfide. Anabaena Tf activates spinach Fru-P2-ase very efficiently but has very little activity with spinach malate dehydrogenase. Anabaena Tf, unlike Tm, does not reduce the homologous ribonucleotide reductase. Anabaena Tf also does not activate a partially purified preparation of Anabaena Fru-P2-ase. We conclude that the cyanobacterial Tf is a unique protein with no structural or functional properties in common with other thioredoxins.     

Purification, characterization, and complete amino acid sequence of a thioredoxin from a green alga, Chlamydomonas reinhardtii

Two thioredoxins (named Ch1 and Ch2 in reference to their elution pattern on an anion-exchange column) have been purified to homogeneity from the green alga, Chlamydomonas reinhardtii. In this paper, we described the properties and the sequence of the most abundant form, Ch2. Its activity in various enzymatic assays has been compared with those of Escherichia coli and spinach thioredoxins. C. reinhardtii thioredoxin Ch2 can serve as a substrate for E. coli thioredoxin reductase with a lower efficiency when compared to the homologous system. In the presence of dithiothreitol (DTT), the protein is able to catalyze the reduction of porcine insulin. Thioredoxin Ch2 is as efficient as its spinach counterpart in the DTT or light activation of corn NADP-malate dehydrogenase, but it only activates spinach fructose-1, 6-bisphosphatase at very high concentrations. The complete primary structure of the C. reinhardtii thioredoxin Ch2 was determined by automated Edman degradation of the intact protein and of peptides derived from trypsin, chymotrypsin, clostripain, and SV8 protease digestions. It consists of a polypeptide of 106 amino acids (MW 11,808) and contains the well-conserved active site sequence Trp-Cys-Gly-Pro-Cys. The sequence of the algal thioredoxin Ch2 has been compared to that of thioredoxins from other sources and has the greatest similarity (67%) with the thioredoxin from Anabaena 7119.     

Thioredoxin system of the photosynthetic anaerobe Chromatium vinosum

Chromatium vinosum, an anaerobic photosynthetic purple sulfur bacterium, resembles aerobic bacterial cells in that it has an NADP-thioredoxin system composed of a single thioredoxin which is reduced by NADPH via NADP-thioredoxin reductase. Both protein components were purified to homogeneity, and some of their properties were determined. Chromatium vinosum thioredoxin was slightly larger than other bacterial thioredoxins (13 versus 12 kilodaltons) but was similar in its specificity (ability to activate chloroplast NADP-malate dehydrogenase more effectively than chloroplast fructose-1,6-bisphosphatase) and immunological properties. As in other bacteria, Chromatium vinosum NADP-thioredoxin reductase was an arsenite-sensitive flavoprotein composed of two 33.5-kilodalton subunits, that required thioredoxin for the NADPH-linked reduction of 5,5'-dithiobis(2-nitrobenzoic acid). Chromatium vinosum NADP-thioredoxin reductase very effectively reduced several different bacterial-type thioredoxins (Escherichia coli, Chlorobium thiosulfatophilum (this name has not been approved by the International Committee of Systematic Bacteriology), Rhizobium meliloti) but not others (Clostridium pasteurianum, spinach chloroplast thioredoxin m). The results show that Chromatium vinosum contains an NADP-thioredoxin system typical of evolutionarily more advanced microorganisms.     

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。     

Human thioredoxin reactivity-structure/function relationship

The reactivity of human thioredoxin (HTR) was tested in several reactions. HTR was as efficient as E. coli or plant and algal thioredoxins when assayed with E. coli ribonucleotide reductase or for the reduction of insulin. On the other hand, HTR was poorly reduced by NADPH and the E. coli flavoenzyme NADPH thioredoxin reductase as monitored in the DTNB reduction test. When reduced with dithiothreitol (DTT), HTR was much less efficient than thioredoxin m and thioredoxin f, the respective specific thioredoxins for the chloroplast enzymes NADP-malate dehydrogenase (NADP-MDH) and fructose 1,6 bisphosphatase (FBPase). Finally, HTR could be used in the photoactivation of NADP-MDH although less efficiently than thioredoxin m, proving nevertheless that it can be reduced by the iron sulfur enzyme ferredoxin thioredoxin reductase in the presence of photoreduced ferredoxin. Based on sequence comparisons, it was expected that HTR would display a reactivity similar to chloroplast thioredoxin f rather than to thioredoxin m. However the observed behavior of FTR did not exactly fit this prediction. The results are discussed in relation to the structural data available for the proteins.     

The ferredoxin-thioredoxin system of a green alga,Chlamydomonas reinhardtii

The components of the ferredoxin-thioredoxin (FT) system of Chlamydomonas reinhardtii have been purified and characterized. The system resembled that of higher plants in consisting of a ferredoxin-thioredoxin reductase (FTR) and two types of thioredoxin, a single f and two m species, m1 and m2. The Chlamydomonas m and f thioredoxins were antigenically similar to their higher-plant counterparts, but not to one another. The m thioredoxins were recognized by antibodies to both higher-plant m and bacterial thioredoxins, whereas the thioredoxin f was not. Chlamydomonas thioredoxin f reacted, although weakly, with the antibody to spinach thioredoxin f. The algal thioredoxin f differed from thioredoxins studied previously in behaving as a basic protein on ion-exchange columns. Purification revealed that the algal thioredoxins had molecular masses (M_rs) typical of thioredoxins from other sources, m1 and m2 being 10700 and f 11 500. Chlamydomonas FTR had two dissimilar subunits, a feature common to all FTRs studied thus far. One, the 13-kDa (similar) subunit, resembled its counterpart from other sources in both size and antigenicity. The other, 10-kDa (variable) sub-unit was not recognized by antibodies to any FTR tested. When combined with spinach, (Spinacia oleracea L.) thylakoid membranes, the components of the FT system functioned in the light activation of the standard target enzymes from chloroplasts, corn (Zea mays L.) NADP-malate dehydrogenase (EC 1.1.1.82) and spinach fructose 1,6-bisphosphatase (EC 3.1.3.11) as well as the chloroplast-type fructose 1,6-bisphosphatase from Chlamydomonas. Activity was greatest if ferredoxin and other components of the FT system were from Chlamydomonas. The capacity of the Chlamydomonas FT system to activate autologous FBPase indicates that light regulates the photosynthetic carbon metabolism of green algae as in other oxygenic photosynthetic organisms.Abbreviations DEAE diethylaminoethyl - ELISA enzyme-linked immunosorption assay - FBPase fructose 1,6-bisphosphatase - Fd ferredoxin - FPLC fast protein liquid chromatography - FTR ferredoxin-thioredoxin reductase - FT system ferredoxin-thioredoxin system - kDa kilodaltons - M_r relative molecular mass - NADP-MDH NADP-malate dehydrogenase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work was supported in part by a grant from the National Aeronautics and Space Administration. We would like to thank Don Carlson and Jacqueline Girard for their assistance with cell cultures.     

Redox control of hydrogenase activity in the green alga Scenedesmus obliquus by thioredoxin and other thiols.

The activity of the NiFe-hydrogenase from the green alga Scenedesmus obliquus is inhibited by both algal thioredoxins f and I+II, and by Escherichia coli thioredoxin. The strongest inhibition was observed with homologous chloroplastic thioredoxin f (I50 = 21 nM) and E. coli thioredoxin (I50 = 83 nM). For the homologous cytoplasmic thioredoxins I+II an I50 of 667 nM was determined. Glutathione shows a similar but much less pronounced inhibitory effect whereas dithiothreitol had no effect. In addition to glucose-6-phosphate dehydrogenase, NiFe-hydrogenase is only the second enzyme known to be inhibited by reduced thioredoxin.     

Induction by different thioredoxins of ATPase activity in coupling factor 1 from spinach chloroplasts

ATPase activity of the coupling factor 1, CF1, isolated from spinach chloroplasts, was enhanced by reduction with dithiothreitol. Reduced thioredoxins from spinach chloroplasts, Escherichia coli and human lymphocytes replaced dithiothreitol as reductant and activator of the ATPase. CF1 must be in an oxidized activated state to be further activated by reduced thioredoxin. This state was obtained either by heating CF1 or removing the inhibitory intrinsic epsilon subunit from CF1. Efficiency and primary structure of the different thioredoxins were compared. The progressive addition of KCl during ATPase activation by reduced thioredoxin increases then decreases this process. We proposed that three basic amino acids corresponding to arginine 73 and lysines 82 and 96 in Escherichia coli thioredoxin play an important role in the anchorage of the thioredoxin to the negatively charged surface of the CF1 and are involved in the dual effect of KCl. The variations in the screening effect of the negative charges of the CF1 surface by K+ ions can indeed explain the changes in the anchorage of these 3 basic amino acids with concomitant variation in ATPase activity. Human thioredoxin must be 10 times more concentrated than Escherichia coli or spinach chloroplast thioredoxin to exhibit the same activation effect on the ATPase. This fact was related to the properties of a sequence equivalent to the part from amino acid 59 to 72 in Escherichia coli thioredoxin. This part which joins the two lobes of the thioredoxin is more hydrophilic and more negatively charged in human thioredoxin than in Escherichia coli or spinach chloroplast thioredoxin. Although ATPase activation was obtained at a very low concentration of the reduced spinach chloroplast thioredoxin, the thioredoxin formed only a loose complex with CF1.     

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.     

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.     

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.     

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.     

Activities of two dissimilar thioredoxins from the cyanobacterium Anabaena sp. strain PCC 7120.

Thioredoxin is a small redox protein that functions as a reducing agent and modulator of enzyme activity. A gene for an unusual thioredoxin was previously isolated from the cyanobacterium Anabaena sp. strain PCC 7120 and cloned and expressed in Escherichia coli. However, the protein could not be detected in Anabaena cells (J. Alam, S. Curtis, F. K. Gleason, M. Gerami-Nejad, and J. A. Fuchs, J. Bacteriol. 171:162-171, 1989). Polyclonal antibodies to the atypical thioredoxin were prepared, and the protein was detected by Western immunoblotting. It occurs at very low levels in extracts of Anabaena sp. and other cyanobacteria. No antibody cross-reaction was observed in extracts of eukaryotic algae, plants, or eubacteria. The anti-Anabaena thioredoxin antibodies did react with another unusual thioredoxin-glutaredoxin produced by bacteriophage T4. Like the T4 protein and other glutaredoxins, the unusual cyanobacterial thioredoxin can be reduced by glutathione. The Anabaena protein can also activate enzymes of carbon metabolism and has some functional similarity to spinach chloroplast thioredoxin f. However, it shows only 23% amino acid sequence identity to the spinach chloroplast protein and appears to be distantly related to other thioredoxins. The data indicate that cyanobacteria, like plant chloroplasts, have two dissimilar thioredoxins. One is related to the more common protein found in other prokaryotes, and the other is an unusual thioredoxin that can be reduced by glutathione and may function in glucose catabolism.