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1.
2.
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   

3.
Uncovered in studies on photosynthesis 35 years ago, redox regulation has been extended to all types of living cells. We understand a great deal about the occurrence, function, and mechanism of action of this mode of regulation, but we know little about its origin and its evolution. To help fill this gap, we have taken advantage of available genome sequences that make it possible to trace the phylogenetic roots of members of the system that was originally described for chloroplasts—ferredoxin, ferredoxin:thioredoxin reductase (FTR), and thioredoxin as well as target enzymes. The results suggest that: (1) the catalytic subunit, FTRc, originated in deeply rooted microaerophilic, chemoautotrophic bacteria where it appears to function in regulating CO2 fixation by the reverse citric acid cycle; (2) FTRc was incorporated into oxygenic photosynthetic organisms without significant structural change except for addition of a variable subunit (FTRv) seemingly to protect the Fe–S cluster against oxygen; (3) new Trxs and target enzymes were systematically added as evolution proceeded from bacteria through the different types of oxygenic photosynthetic organisms; (4) an oxygenic type of regulation preceded classical light–dark regulation in the regulation of enzymes of CO2 fixation by the Calvin–Benson cycle; (5) FTR is not universally present in oxygenic photosynthetic organisms, and in certain early representatives is seemingly functionally replaced by NADP-thioredoxin reductase; and (6) FTRc underwent structural diversification to meet the ecological needs of a variety of bacteria and archaea.  相似文献   

4.
Thioredoxin functions in nearly all organisms as the major thiol-disulfide oxidoreductase within the cytosol. Its prime purpose is to maintain cysteine-containing proteins in the reduced state by converting intramolecular disulfide bonds into dithiols in a disulfide exchange reaction. Thioredoxin has been reported to contribute to a wide variety of physiological functions by interacting with specific sets of substrates in different cell types. To investigate the function of the essential thioredoxin A (TrxA) in the low-GC Gram-positive bacterium Bacillus subtilis, we purified wild-type TrxA and three mutant TrxA proteins that lack either one or both of the two cysteine residues in the CxxC active site. The pure proteins were used for substrate-binding studies known as “mixed disulfide fishing” in which covalent disulfide-bonded reaction intermediates can be visualized. An unprecedented finding is that both active-site cysteine residues can form mixed disulfides with substrate proteins when the other active-site cysteine is absent, but only the N-terminal active-site cysteine forms stable interactions. A second novelty is that both single-cysteine mutant TrxA proteins form stable homodimers due to thiol oxidation of the remaining active-site cysteine residue. To investigate whether these dimers resemble mixed enzyme-substrate disulfides, the structure of the most abundant dimer, C32S, was characterized by X-ray crystallography. This yielded a high-resolution (1.5Å) X-ray crystallographic structure of a thioredoxin homodimer from a low-GC Gram-positive bacterium. The C32S TrxA dimer can be regarded as a mixed disulfide reaction intermediate of thioredoxin, which reveals the diversity of thioredoxin/substrate-binding modes.  相似文献   

5.
Thioredoxin reductases control the redox state of thioredoxins (Trxs)—ubiquitous proteins that regulate a spectrum of enzymes by dithiol–disulfide exchange reactions. In most organisms, Trx is reduced by NADPH via a thioredoxin reductase flavoenzyme (NTR), but in oxygenic photosynthetic organisms, this function can also be performed by an iron-sulfur ferredoxin (Fdx)-dependent thioredoxin reductase (FTR) that links light to metabolic regulation. We have recently found that some cyanobacteria, such as the thylakoid-less Gloeobacter and the ocean-dwelling green oxyphotobacterium Prochlorococcus, lack NTR and FTR but contain a thioredoxin reductase flavoenzyme (formerly tentatively called deeply-rooted thioredoxin reductase or DTR), whose electron donor remained undefined. Here, we demonstrate that Fdx functions in this capacity and report the crystallographic structure of the transient complex between the plant-type Fdx1 and the thioredoxin reductase flavoenzyme from Gloeobacter violaceus. Thereby, our data demonstrate that this cyanobacterial enzyme belongs to the Fdx flavin-thioredoxin reductase (FFTR) family, originally described in the anaerobic bacterium Clostridium pasteurianum. Accordingly, the enzyme hitherto termed DTR is renamed FFTR. Our experiments further show that the redox-sensitive peptide CP12 is modulated in vitro by the FFTR/Trx system, demonstrating that FFTR functionally substitutes for FTR in light-linked enzyme regulation in Gloeobacter. Altogether, we demonstrate the FFTR is spread within the cyanobacteria phylum and propose that, by substituting for FTR, it connects the reduction of target proteins to photosynthesis. Besides, the results indicate that FFTR acquisition constitutes a mechanism of evolutionary adaptation in marine phytoplankton such as Prochlorococcus that live in low-iron environments.  相似文献   

6.
In response to changing light quantity and quality, photosynthetic organisms perform state transitions, a process which optimizes photosynthetic yield and mitigates photo-damage. The serine/threonine-protein kinase STN7 phosphorylates the light-harvesting complex of photosystem II (PSII; light-harvesting complex II), which then migrates from PSII to photosystem I (PSI), thereby rebalancing the light excitation energy between the photosystems and restoring the redox poise of the photosynthetic electron transport chain. Two conserved cysteines forming intra- or intermolecular disulfide bonds in the lumenal domain (LD) of STN7 are essential for the kinase activity although it is still unknown how activation of the kinase is regulated. In this study, we show lumen thiol oxidoreductase 1 (LTO1) is co-expressed with STN7 in Arabidopsis (Arabidopsis thaliana) and interacts with the LD of STN7 in vitro and in vivo. LTO1 contains thioredoxin (TRX)-like and vitamin K epoxide reductase domains which are related to the disulfide-bond formation system in bacteria. We further show that the TRX-like domain of LTO1 is able to oxidize the conserved lumenal cysteines of STN7 in vitro. In addition, loss of LTO1 affects the kinase activity of STN7 in Arabidopsis. Based on these results, we propose that LTO1 helps to maintain STN7 in an oxidized active state in state 2 through redox interactions between the lumenal cysteines of STN7 and LTO1.  相似文献   

7.
The ferredoxin:thioredoxin reductase is an essential enzyme of the light dependent regulatory system in oxygenic photosynthesis. It is composed of two dissimilar subunits and contains a 4Fe-4S cluster and a redox-active disulfide bridge. Artificial electron donors of redox potentials below –300 mV are capable of reducing the disulfide bridge. Based on our results we speculate that a group of more negative potential than the disulfide bridge is the first acceptor of the electrons in FTR. The chemical reduction of FTR has been used successfully for the detection of the enzyme during its purification.Abbreviation FBPase fructose 1,6-bisphosphatase - FTR ferredoxin:thioredoxin reductase - MV methyl viologen Dedicated to Prof. D.I. Arnon.  相似文献   

8.
Cyanobacteria perform oxygenic photosynthesis, which makes them unique among the prokaryotes, and this feature together with their abundance and worldwide distribution renders them a central ecological role. Cyanobacteria and chloroplasts of plants and algae are believed to share a common ancestor and the modern chloroplast would thus be the remnant of an endosymbiosis between a eukaryotic cell and an ancestral oxygenic photosynthetic prokaryote. Chloroplast metabolic processes are coordinated with those of the other cellular compartments and are strictly controlled by means of regulatory systems that commonly involve redox reactions. Disulphide/dithiol exchange catalysed by thioredoxin is a fundamental example of such regulation and represents the molecular mechanism for light-dependent redox control of an ever-increasing number of chloroplast enzymatic activities. In contrast to chloroplast thioredoxins, the functions of the cyanobacterial thioredoxins have long remained elusive, despite their common origin. The sequenced genomes of several cyanobacterial species together with novel experimental approaches involving proteomics have provided new tools for re-examining the roles of the thioredoxin systems in these organisms. Thus, each cyanobacterial genome encodes between one and eight thioredoxins and all components necessary for the reduction of thioredoxins. Screening for thioredoxin target proteins in cyanobacteria indicates that assimilation and storage of nutrients, as well as some central metabolic pathways, are regulated by mechanisms involving disulphide/dithiol exchange, which could be catalysed by thioredoxins or related thiol-containing proteins.  相似文献   

9.
A potential role in disulfide bond formation in the intracellular proteins of thermophilic organisms has recently been ascribed to a new family of protein disulfide oxidoreductases (PDOs). We report on the characterization of SsPDO, isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. SsPDO was cloned and expressed in Escherichia coli. We revealed that SsPDO is the substrate of a thioredoxin reductase in S. solfataricus (K(M) 0.3 microm) and not thioredoxins (TrxA1 and TrxA2). SsPDO/S. solfataricus thioredoxin reductase constitute a new thioredoxin system in aerobic thermophilic archaea. While redox (reductase, oxidative and isomerase) activities of SsPDO point to its central role in the biochemistry of cytoplasmic disulfide bonds, chaperone activities also on an endogenous substrate suggest a potential role in the stabilization of intracellular proteins. Northern and western analysis have been performed in order to analyze the response to the oxidative stress.  相似文献   

10.
Reduction of purothionin by the wheat seed thioredoxin system   总被引:1,自引:1,他引:0       下载免费PDF全文
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   

11.
The Streptococcus genus includes the pathogenic species Streptococcus mutans, the main responsible of dental caries, and the safe microorganism Streptococcus thermophilus, used for the manufacture of dairy products. These facultative anaerobes control the levels of reactive oxygen species (ROS) and indeed, both S. mutans and S. thermophilus possess a cambialistic superoxide dismutase, the key enzyme for a preventive action against ROS. To evaluate the properties of a crucial mechanism for repairing ROS damages, the molecular and functional characterization of the thioredoxin system in these streptococci was investigated. The putative genes encoding its protein components in S. mutans and S. thermophilus were analysed and the corresponding recombinant proteins were purified. A single thioredoxin reductase was obtained from either S. mutans (SmTrxB) or S. thermophilus (StTrxB1), whereas two thioredoxins were prepared from either S. mutans (SmTrxA and SmTrxH1) or S. thermophilus (StTrxA1 and StTrxA2). Both SmTrxB and StTrxB1 reduced the synthetic substrate DTNB in the presence of NADPH, whereas only SmTrxA and StTrxA1 accelerated the insulin reduction in the presence of DTT. To reconstitute an in vitro streptococcal thioredoxin system, the combined activity of the thioredoxin components was tested through the insulin precipitation in the absence of DTT. The assay functions with a combination of SmTrxB or StTrxB1 with either SmTrxA or StTrxA1. These results suggest that the streptococcal members of the thioredoxin system display a direct functional interaction between them and that these protein components are interchangeable within the Streptococcus genus. In conclusion, our data prove the existence of a functioning thioredoxin system even in these microaerophiles.  相似文献   

12.
Trotter EW  Grant CM 《EMBO reports》2003,4(2):184-188
Our studies in yeast show that there is an essential requirement for either an active thioredoxin or an active glutathione (GSH)–glutaredoxin system for cell viability. Glutathione reductase (Glr1) and thioredoxin reductase (Trr1) are key regulatory enzymes that determine the redox state of the GSH–glutaredoxin and thioredoxin systems, respectively. Here we show that Trr1 is required during normal cell growth, whereas there is no apparent requirement for Glr1. Analysis of the redox state of thioredoxins and glutaredoxins in glr1 and trr1 mutants reveals that thioredoxins are maintained independently of the glutathione system. In contrast, there is a strong correlation between the redox state of glutaredoxins and the oxidation state of the GSSG/2GSH redox couple. We suggest that independent redox regulation of thioredoxins enables cells to survive in conditions under which the GSH–glutaredoxin system is oxidized.  相似文献   

13.
Intracellular redox homeostasis is crucial for many cellular functions but accurate measurements of cellular compartment-specific redox states remain technically challenging. To better characterize redox control in the nucleus, we targeted a yellow fluorescent protein-based redox sensor (rxYFP) to the nucleus of the yeast Saccharomyces cerevisiae. Parallel analyses of the redox state of nucleus-rxYFP and cytosol-rxYFP allowed us to monitor distinctively dynamic glutathione (GSH) redox changes within these two compartments under a given condition. We observed that the nuclear GSH redox environment is highly reducing and similar to the cytosol under steady-state conditions. Furthermore, these sensors are able to detect redox variations specific for their respective compartments in glutathione reductase (Glr1) and thioredoxin pathway (Trr1, Trx1, Trx2) mutants that have altered subcellular redox environments. Our mutant redox data provide in vivo evidence that glutathione and the thioredoxin redox systems have distinct but overlapping functions in controlling subcellular redox environments. We also monitored the dynamic response of nucleus-rxYFP and cytosol-rxYFP to GSH depletion and to exogenous low and high doses of H2O2 bursts. These observations indicate a rapid and almost simultaneous oxidation of both nucleus-rxYFP and cytosol-rxYFP, highlighting the robustness of the rxYFP sensors in measuring real-time compartmental redox changes. Taken together, our data suggest that the highly reduced yeast nuclear and cytosolic redox states are maintained independently to some extent and under distinct but subtle redox regulation. Nucleus- and cytosol-rxYFP register compartment-specific localized redox fluctuations that may involve exchange of reduced and/or oxidized glutathione between these two compartments. Finally, we confirmed that GSH depletion has profound effects on mitochondrial genome stability but little effect on nuclear genome stability, thereby emphasizing that the critical requirement for GSH during growth is linked to a mitochondria-dependent process.  相似文献   

14.
The Glutaredoxin Family in Oxygenic Photosynthetic Organisms   总被引:12,自引:0,他引:12  
Glutaredoxins (GRXs) are small redox proteins of the thioredoxin (TRX) superfamily. Compared to TRXs, much less information on the GRX family is available, especially in photosynthetic organisms since GRXs have been mainly studied in E. coli, yeast and mammal cells. The analysis of the TRX family in oxygenic photosynthetic organisms revealed an unsuspected multiplicity of TRXs but it is not known if the same situation holds for GRXs. Despite the availability of genome sequences from different oxygenic photosynthetic organisms, the number of GRXs and the different groups present in these organisms are still undescribed. This paper presents a comparative analysis of the GRX families present in Arabidopsis, Chlamydomonas and Synechocystis which were found to contain 30, 6 and 3 GRX genes, respectively. The putative subcellular localization of each GRX and its relative expression level, based on EST data, have been investigated. This analysis reveals the presence of three major classes of GRXs, the CPYC type, the CGFS type and a previously undescribed type, called the CC type that appears specific to higher plants. These data are discussed in view of recent results suggesting a complex cross-regulation between the TRX and GRX systems.  相似文献   

15.
Cameron JC  Pakrasi HB 《Plant physiology》2010,154(4):1672-1685
Glutathione, a nonribosomal thiol tripeptide, has been shown to be critical for many processes in plants. Much less is known about the roles of glutathione in cyanobacteria, oxygenic photosynthetic prokaryotes that are the evolutionary precursor of the chloroplast. An understanding of glutathione metabolism in cyanobacteria is expected to provide novel insight into the evolution of the elaborate and extensive pathways that utilize glutathione in photosynthetic organisms. To investigate the function of glutathione in cyanobacteria, we generated deletion mutants of glutamate-cysteine ligase (gshA) and glutathione synthetase (gshB) in Synechocystis sp. PCC 6803. Complete segregation of the ΔgshA mutation was not achieved, suggesting that GshA activity is essential for growth. In contrast, fully segregated ΔgshB mutants were isolated and characterized. The ΔgshB strain lacks reduced glutathione (GSH) but instead accumulates the precursor compound γ-glutamylcysteine (γ-EC). The ΔgshB strain grows slower than the wild-type strain under favorable conditions and exhibits extremely reduced growth or death when subjected to conditions promoting oxidative stress. Furthermore, we analyzed thiol contents in the wild type and the ΔgshB mutant after subjecting the strains to multiple environmental and redox perturbations. We found that conditions promoting growth stimulate glutathione biosynthesis. We also determined that cellular GSH and γ-EC content decline following exposure to dark and blue light and during photoheterotrophic growth. Moreover, a rapid depletion of GSH and γ-EC is observed in the wild type and the ΔgshB strain, respectively, when cells are starved for nitrate or sulfate.  相似文献   

16.
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   

17.
The photosystem two (PSII) complex found in oxygenic photosynthetic organisms is susceptible to damage by UV-B irradiation and undergoes repair in vivo to maintain activity. Until now there has been little information on the identity of the enzymes involved in repair. In the present study we have investigated the involvement of the FtsH and Deg protease families in the degradation of UV-B-damaged PSII reaction center subunits, D1 and D2, in the cyanobacterium Synechocystis 6803. PSII activity in a ΔFtsH (slr0228) strain, with an inactivated slr0228 gene, showed increased sensitivity to UV-B radiation and impaired recovery of activity in visible light after UV-B exposure. In contrast, in ΔDeg-G cells, in which all the three deg genes were inactivated, the damage and recovery kinetics were the same as in the WT. Immunoblotting showed that the loss of both the D1 and D2 proteins was retarded in ΔFtsH (slr0228) during UV-B exposure, and the extent of their restoration during the recovery period was decreased relative to the WT. However, in the ΔDeg-G cells the damage and recovery kinetics of D1 and D2 were the same as in the WT. These data demonstrate a key role of FtsH (slr0228), but not the Deg proteases, for the repair of PS II during and following UV-B radiation at the step of degrading both of the UV-B damaged D1 and D2 reaction center subunits.  相似文献   

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Ferredoxin (Fd) is a small [2Fe‐2S] cluster‐containing protein found in all organisms performing oxygenic photosynthesis. Fd is the first soluble acceptor of electrons on the stromal side of the chloroplast electron transport chain, and as such is pivotal to determining the distribution of these electrons to different metabolic reactions. In chloroplasts, the principle sink for electrons is in the production of NADPH, which is mostly consumed during the assimilation of CO2. In addition to this primary function in photosynthesis, Fds are also involved in a number of other essential metabolic reactions, including biosynthesis of chlorophyll, phytochrome and fatty acids, several steps in the assimilation of sulphur and nitrogen, as well as redox signalling and maintenance of redox balance via the thioredoxin system and Halliwell‐Asada cycle. This makes Fds crucial determinants of the electron transfer between the thylakoid membrane and a variety of soluble enzymes dependent on these electrons. In this article, we will first describe the current knowledge on the structure and function of the various Fd isoforms present in chloroplasts of higher plants and then discuss the processes involved in oxidation of Fd, introducing the corresponding enzymes and discussing what is known about their relative interaction with Fd.  相似文献   

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