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1.
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. 相似文献
2.
Thioredoxin from Anabaena sp. has been purified 800-fold with an assay based on the reduction of insulin disulfides by NADPH and the heterologous calf thymus thioredoxin reductase. The final material was homogeneous on polyacrylamide gel electrophoresis and had a molecular weight of 12,000; the NH2-terminal residue was serine and the COOH-terminal was leucine. Anabaena thioredoxin-(SH)2 is a hydrogen donor for the adenosylcobalamin-dependent anabaena ribonucleotide reductase and is equally active with the iron-containing ribonucleotide reductase from Escherichia coli. Anabaena thioredoxin-S2 is a good substrate for E. coli thioredoxin reductase. We have compared the structure of Anabaena and E. coli thioredoxins. Clear structural differences between the proteins, compatible with the large evolutionary distance between the organisms, were seen with respect to total amino acid composition, isoelectric point, tryptic peptide maps, and a low immunochemical cross-reactivity. However, both thioredoxins contain a single oxidation-reduction active disulfide bridge with the amino acid sequence: Cys-Gly-Pro-Cys-Lys. The tryptophan fluorescence emission of Anabaena thioredoxin-S2 increases more than 3-fold on reduction to thioredoxin-(SH)2. This behavior is identical with that of E. coli thioredoxin, suggesting a very similar overall folding of homologous molecules. 相似文献
3.
目的:利用基因工程的方法原核表达无标签的重组人硫氧还蛋白(rhTrx)并对其进行大规模表达、纯化和鉴定.方法:从人胚胎肾HEK293细胞中提取总RNA,反转录合成cDNA,经PCR扩增、酶切后连入pET-22b(+)载体构建重组质粒,重组质粒转化大肠杆菌BL21( DE3)感受态细胞,IPTG诱导表达,经两步离子交换层析纯化重组蛋白,采用SDS-PAGE、Western blotting、HPLC、MALDI-TOF-MS及经典的胰岛素二硫键还原法对重组蛋白进行鉴定.结果:构建成功了rhTrx基因表达载体;实现了rhTrx在原核细胞中的可溶性表达;纯化出的蛋白经SDS-PAGE和Western blotting分析证实为rhTrx;HPLC和MALDI-TOF-MS分析表明,纯化出的目的蛋白纯度大于95%;胰岛素二硫键还原法证实纯化出的rhTrx具有生物学活性.结论:成功构建了rhTrx的原核表达体系,建立了rhTrx的纯化和鉴定方法,为其进一步的理论研究和生产开发提供了有效基础数据. 相似文献
4.
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.
相似文献
5.
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. 相似文献
6.
Mariam Sahrawy Valérie Hecht Javier Lopez-Jaramillo Ana Chueca Yvette Chartier Yves Meyer 《Journal of molecular evolution》1996,42(4):422-431
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. 相似文献
7.
Thioredoxin-thioredoxin reductase system of Streptomyces clavuligerus: sequences, expression, and organization of the genes. 总被引:4,自引:2,他引:2
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G Cohen M Yanko M Mislovati A Argaman R Schreiber Y Av-Gay Y Aharonowitz 《Journal of bacteriology》1993,175(16):5159-5167
The genes that encode thioredoxin and thioredoxin reductase of Streptomyces clavuligerus were cloned, and their DNA sequences were determined. Previously, we showed that S. clavuligerus possesses a disulfide reductase with broad substrate specificity that biochemically resembles the thioredoxin oxidoreductase system and may play a role in the biosynthesis of beta-lactam antibiotics. It consists consists of two components, a 70-kDa NADPH-dependent flavoprotein disulfide reductase with two identical subunits and a 12-kDa heat-stable protein general disulfide reductant. In this study, we found, by comparative analysis of their predicted amino acid sequences, that the 35-kDa protein is in fact thioredoxin reductase; it shares 48.7% amino acid sequence identity with Escherichia coli thioredoxin reductase, the 12-kDa protein is thioredoxin, and it shares 28 to 56% amino acid sequence identity with other thioredoxins. The streptomycete thioredoxin reductase has the identical cysteine redox-active region--Cys-Ala-Thr-Cys--and essentially the same flavin adenine dinucleotide- and NADPH dinucleotide-binding sites as E. coli thioredoxin reductase and is partially able to accept E. coli thioredoxin as a substrate. The streptomycete thioredoxin has the same cysteine redox-active segment--Trp-Cys-Gly-Pro-Cys--that is present in virtually all eucaryotic and procaryotic thioredoxins. However, in vivo it is unable to donate electrons to E. coli methionine sulfoxide reductase and does not serve as a substrate in vitro for E. coli thioredoxin reductase. The S. clavuligerus thioredoxin (trxA) and thioredoxin reductase (trxB) genes are organized in a cluster. They are transcribed in the same direction and separated by 33 nucleotides. In contrast, the trxA and trxB genes of E. coli, the only other organism in which both genes have been characterized, are physically widely separated. 相似文献
8.
Isolation and characterization of thioredoxin f from the filamentous cyanobacterium, Anabaena sp. 7119 总被引:2,自引:0,他引:2
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. 相似文献
9.
The genes encoding thioredoxin and thioredoxin reductase of Clostridium litorale were cloned and sequenced. The thioredoxin reductase gene (trxB) encoded a protein of 33.9 kDa, and the deduced amino acid sequence showed 44% identity to the corresponding protein from
Escherichia coli. The gene encoding thioredoxin (trxA) was located immediately downstream of trxB. TrxA and TrxB were each encoded by two gene copies, both copies presumably located on the chromosome. Like other thioredoxins
from anaerobic, amino-acid-degrading bacteria investigated to date by N-terminal amino acid sequencing, thioredoxin from C. litorale exhibited characteristic deviations from the consensus sequence, e.g., GCVPC instead of WCGPC at the redox-active center.
Using heterologous enzyme assays, neither thioredoxin nor thioredoxin reductase were interchangeable with the corresponding
proteins of the thioredoxin system from E. coli. To elucidate the molecular basis of that incompatibility, Gly-31 in C. litorale thioredoxin was substituted with Trp (the W in the consensus sequence) by site-directed mutagenesis. The mutant protein was
expressed in E. coli and was purified to homogeneity. Enzyme assays using the G31W thioredoxin revealed that Gly-31 was not responsible for the
observed incompatibility with the E. coli thioredoxin reductase, but it was essential for activity of the thioredoxin system in C. litorale.
Received: 19 September 1996 / Accepted: 21 May 1997 相似文献
10.
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。 相似文献
11.
Heather C. Huppe Frédéric de Lamotte-Guéry Jean-Pierre Jacquot Bob B. Buchanan 《Planta》1990,180(3):341-351
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 (Mrs) 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
- Mr
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. 相似文献
12.
We purified to homogeneity and characterized a heat stable thioredoxin which catalyzes thiol/disulfide exchange reaction,
for the first time from dromedary pancreas. The purification involved heat and acidic treatment (90 °C; pH 2.5), precipitation
by ammonium sulphate and ethanol, respectively followed by sequential column chromatography reverse HPLC column, and it resulted
in an apparently pure protein after a 217-fold purification with a final yield of 55% of the initial thioredoxin activity.
The thioredoxin preparation obtained was homogeneous as judged by polyacrylamide gel electrophoresis and the presence of valine
as the only NHt-terminal amino acid. MALDI-TOF mass spectrometry revealed that the protein has a molecular mass of 11,302.9 Da.
The first 40 amino-acid residues at the N-terminal extremity of purified DrTrx was determined by automatic Edman degradation
and showed a high sequence homology with known Thioredoxin. It contained he tetrapeptide-Cys-Gly-Pro-Cys-, which constitutes
the active site of mammalian thioredoxins. DrTrx activity was compatible with the presence of organic solvents and the maximum
activity appeared at pH 7.5 using the insulin precipitation assay. Thioredoxin stability in the presence of organic solvents,
as well as in acidic and alkaline pHs and at high temperatures makes it a good candidate for its application in pharmaceutical
and food industry. 相似文献
13.
Enzyme Regulation in Crassulacean Acid Metabolism Photosynthesis : Studies on the Ferredoxin/Thioredoxin System of KalanchoE daigremontiana
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Cell-free preparations of the Crassulacean acid metabolism (CAM) plant, Kalanchoë daigremontiana, were analyzed for thioredoxins and ferredoxin-thioredoxin reductase. Three distinct forms of thioredoxin were identified in Kalanchoë leaves, two of which specifically activated fructose 1,6-bisphosphatase (designated f1 and f2) and a third which activated NADP-malate dehydrogenase (thioredoxin m). The apparent molecular weight of both forms of thioredoxin f was 11,000 and that of thioredoxin m was 10,000. In parallel studies, ferredoxin and ferredoxin-thioredoxin reductase were purified from Kalanchoë leaf preparations. Kalanchoë ferredoxin-thioredoxin reductase was similar to that of C3 and C4 plants in molecular weight (31,000) and immunological cross-reactivity. Kalanchoë ferredoxin-thioredoxin reductase exhibited an affinity for ferredoxin as demonstrated by its binding to an immobilized ferredoxin affinity column. The purified components of the Kalanchoë ferredoxin-thioredoxin system could be recombined to function in the photoregulation of chloroplast enzymes. The data suggest that the ferredoxin/thioredoxin system plays a role in enzyme regulation of all higher plants irrespective of whether they show C3, C4, or CAM photosynthesis. 相似文献
14.
Meyer Y Riondet C Constans L Abdelgawwad MR Reichheld JP Vignols F 《Photosynthesis research》2006,89(2-3):179-192
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 相似文献
15.
A kininogenase from bovine erythrocyte membranes has been purified 140-fold by affinity chromatography on pepstatin A-Agarose followed by ion exchange chromatography on CM Cellulose. The purified enzyme showed an apparent molecular weight of 31,000 daltons as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. ItspH optimum is 7.5, and it was totally inhibited by soybean trypsin inhibitor, phenylmethylsulfonylfluoride, aprotinin, pepstatin, and dithiotreitol, suggesting the presence of a disulfide bond(s) whose integrity is(are) essential for maintaining the native three-dimensional structure. The referred enzyme was able to release kinin from a substrate partially purified from rat plasma. The kininogenase was activated by Zn2+, Ca2+, and cysteine-HCl. 相似文献
16.
17.
Mariana Stein Jean-Pierre Jacquot Emmanuelle Jeannette Paulette Decottignies Michael Hodges Jean-Marc Lancelin Virginie Mittard Jean-Marie Schmitter Myroslawa Miginiac-Maslow 《Plant molecular biology》1995,28(3):487-503
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 相似文献
18.
Thioredoxin h, the thioredoxin characteristic of heterotrophic plant tissues, was purified to homogeneity from wheat endosperm (flour) and found to resemble its counterpart from carrot cell cultures. In the presence of NADPH, homogeneous thioredoxin h and partially purified wheat endosperm thioredoxin reductase (NADPH), (EC 1.6.4.5), purothionin promoted the activation of chloroplast fructose-1,6-bisphosphatase (EC 3.1.3.11). Under these conditions, NADPH provided the reducing equivalents for a series of thiol reactions in which (a) thioredoxin reductase reduced thioredoxin h thereby converting it from disulfide (S-S) to sulfhydryl (SH) form; (b) the sulfhydryl form of thioredoxin h reduced the disulfide form of purothionin—a 5 kilodalton seed storage protein with 4 S-S bridges; and (c) the sulfhydryl form of purothionin reductively activated fructose-1,6-bisphosphatase. The results show that, since thioredoxin h does not react effectively with fructose-1,6-bisphosphatase, the thioredoxin system can activate an enzyme through purothionin by secondary thiol redox control. In a related type reaction, purothionin, inhibited the activity of either Escherichia coli or calf thymus ribonucleotide reductase with reduced thioredoxin as hydrogen donor. The results suggest that purothionin competes with ribonucleotide reductase for reducing equivalents from thioredoxin. Thus, inhibition of deoxyribonucleotide synthesis should be considered a possible mechanism when examining the toxic effects of purothionin on mammalian cells in S-phase. 相似文献
19.
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. 相似文献
20.
Crawford NA Sutton CW Yee BC Johnson TC Carlson DC Buchanan BB 《Archives of microbiology》1984,139(2-3):124-129
Enzymes that are regulated by the ferredoxin/thioredoxin system in chloroplasts — fructose-1,6-bisphosphatase (FBPase), sedoheptulose-1,7-bisphosphatase purified from two different types of photosynthetic prokaryotes (cyanobacteria, purple sulfur bacteria) and tested for a response to thioredoxins. Each of the enzymes from the cyanobacterium Nostoc muscorum, an oxygenic organism known to contain the ferredoxin/thioredoxin system, was activated by thioredoxins that had been reduced either chemically by dithiothreitol or photochemically by reduced ferredoxin and ferredoxin-thioredoxin reductase. Like their chloroplast counterparts, N. muscorum FBPase and SBPase were activated preferentially by reduced thioredoxin f. SBPase was also partially activated by thioredoxin m. PRK, which was present in two regulatory forms in N. muscorum, was activated similarly by thioredoxins f and m. Despite sharing the capacity for regulation by thioredoxins, the cyanobacterial FBPase and SBPase target enzymes differed antigenically from their chloroplast counterparts. The corresponding enzymes from Chromatium vinosum, an anoxygenic photosynthetic purple bacterium found recently to contain the NADP/thioredoxin sytem, differed from both those of cyanobacteria and chloroplasts in showing no response to reduced thioredoxin. Instead, C. vinosum FBPase, SBPase, and PRK activities were regulated by a metabolite effector, 5-AMP. The evidence is in accord with the conclusion that thioredoxins function in regulating the reductive pentose phosphate cycle in oxygenic prokaryotes (cyanobacteria) that contain the ferredoxin/thioredoxin system, but not in anoxygenic prokaryotes (photosynthetic purple bacteria) that contain the NADP/thioredoxin system. In organisms of the latter type, enzyme effectors seem to play a dominant role in regulating photosynthetic carbon dioxide assimilation. 相似文献