Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.1.1.37 (malate dehydrogenase)
4,591 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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 11500. 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") subunit 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.
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PMID:The ferredoxin-thioredoxin system of a green alga, Chlamydomonas reinhardtii: identification and characterization of thioredoxins and ferredoxin-thioredoxin reductase components. 1153 75

The activation pathway of the chloroplastic NADP-dependent malate dehydrogenase (MDH) by reduced thioredoxin has been examined using a method based on the mechanism of thiol/disulfide interchanges, i.e. the transient formation of a mixed disulfide between the target and the reductant. This disulfide can be stabilized when each of the partners is mutated in the less reactive cysteine of the disulfide/dithiol pair. As NADP-MDH has two regulatory disulfides per monomer, four different single cysteine mutants were examined, two for the C-terminal bridge and two for the N-terminal bridge. The results clearly show that the nucleophilic attack of thioredoxin on the C-terminal bridge proceeds through the formation of a disulfide with the most external Cys377. The results are less clear-cut for the N-terminal cysteines and suggest that the Cys24-Cys207 disulfide bridge previously proposed to be an intermediary step in MDH activation can form only when the C-terminal disulfide is reduced.
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PMID:Sites of interaction of thioredoxin with sorghum NADP-malate dehydrogenase. 1157 37

The function of a gene closely linked to nitrate assimilation loci from Chlamydomonas reinhardtii has been investigated. Gene expression analysis shows that its mRNA accumulation is modulated by light, carbon source and adaptation to light/dark cyclic conditions of growth. A full-length cDNA was isolated for the light-regulated transcript, and sequence characterization indicates that it encodes the NADP-malate dehydrogenase from C. reinhardtii (NADP-MDH;Cr). The primary structure of NADP-MDH;Cr is closely related to plant, mossfern and algal NADP-malate dehydrogenases, and shares structural determinants for chloroplast targeting, cofactor binding and catalysis. Sequence conservation extends to the carboxy end of the protein, where plant and mossfern enzymes have two cysteines and an acidic C-terminus with a critical role for regulation of NADP-MDH activity by the thioredoxin/ferredoxin system. Accordingly, incubation with DTT activates NADP-MDH enzyme in cell-free extracts from C. reinhardtii. Like NADP-malate dehydrogenases from two other green algae, the N-terminal extension of NADP-MDH;Cr lacks two thiol residues whose reduction constitutes the rate-limiting step in the activation reaction of plant enzymes. Homology-based 3D modelling of NADP-MDH;Cr, the first structure predicted for NADP-malate dehydrogenase from a lower eukaryote, evidences close positioning of two new cysteines in an accessible region of the protein surface. These results suggest that the algal enzyme has a different arrangement of regulatory disulfide bridges, which might involve the existence of new mechanisms that control functioning of the malate valve, the main system to export reducing power from the chloroplast of plant cells.
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PMID:NADP-malate dehydrogenase from Chlamydomonas: prediction of new structural determinants for redox regulation by homology modelling. 1185 23

A cDNA coding for a thioredoxin h has been isolated from a xylem/phloem poplar cDNA library by RACE-PCR. The nucleotide sequence called popTrx-h2 is homologous to other thioredoxins h isolated from plants but differs from the other thioredoxins h by presenting a 30 amino acid long N-terminus extension. A variant of this cDNA lacking the N-terminal extension was also generated by PCR. Both cDNAs have been introduced into an expression plasmid (pET-3d) and the recombinant proteins have been expressed to a high level and purified from Escherichia coli cells. Protein sequencing showed that a part of the N-terminal extension was cleaved in the E. coli cells, with the first 19 amino acids missing, suggesting the presence of a putative cleavage site in the N-terminal extension of popTrx-h2. Both recombinant proteins display unusual catalytic properties compared to other thioredoxins h characterized so far, i.e. a weak reduction by Arabidopsis thaliana NADPH-dependent thioredoxin reductase, and a weak activation of the chloroplastic NADP-malate dehydrogenase, a non-physiological target enzyme. Northern blot experiments indicate that the transcripts of popTrx-h2 are present in leaves and roots, albeit at a lower level compared to the earlier characterized popTrx-h1.
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PMID:Isolation and characterization of an extended thioredoxin h from poplar. 1190 63

Avoidance of over-reduction of the chloroplast ferredoxin pool is of paramount importance for plants in avoiding oxidative stress. The redox state of this pool can be controlled through regulation of the thylakoid electron transport chain. A model is presented for regulation of this chain via a thiol reduction mechanism, possibly involving a thioredoxin. It is shown in isolated thylakoids that electron transport is inhibited by the thiol reducing agent dithiothreitol. The kinetics of this reduction are rapid and readily reversible. The midpoint redox potential is -365 mV at pH 7.7, with a pH dependency of about -90 mV/pH. At physiological pH values, this places the potential of the species titrated between that of ferredoxin and NADPH and thus in the right potential range to be regulating the redox poise of the ferredoxin pool. This is also close to the potential of NADPH-malate dehydrogenase, an enzyme known to be regulated by thioredoxin. Regulation of electron transport by thioredoxin provides a mechanistic link between the regulation of photosynthesis and gene expression by sugars and the redox regulation of gene expression mediated through the plastoquinone pool.
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PMID:Thiol regulation of the thylakoid electron transport chain--a missing link in the regulation of photosynthesis? 1262 70

The sequencing of the genome of Arabidopsis thaliana revealed that this plant contained numerous isoforms of thioredoxin (Trx), a protein involved in thiol-disulfide exchanges. On the basis of sequence comparison, seven putative chloroplastic Trxs have been identified, four belonging to the m-type, two belonging to the f-type, and one belonging to a new x-type. In the present work, these isoforms were produced and purified as recombinant proteins without their putative transit peptides. Their activities were tested with two known chloroplast thioredoxin targets: NADP-malate dehydrogenase and fructose-1,6-bisphosphatase and also with a chloroplastic 2-Cys peroxiredoxin. The study confirms the strict specificity of fructose-bisphosphatase for Trx f, reveals that some Trxs are unable to activate NADP-malate dehydrogenase, and shows that the new x-type is the most efficient substrate for peroxiredoxin while being inactive toward the two other targets. This suggests that this isoform might be specifically involved in resistance against oxidative stress. Three-dimensional modeling shows that one of the m-type Trxs, Trx m3, which has no activity with any of the three targets, exhibits a negatively charged surface surrounding the active site. A green fluorescent protein approach confirms the plastidial localization of these Trxs.
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PMID:The Arabidopsis plastidial thioredoxins: new functions and new insights into specificity. 1270 79

The PSI-D subunit of photosystem I is a hydrophilic subunit of about 18 kDa, which is exposed to the stroma and has an important function in the docking of ferredoxin to photosystem I. We have used an antisense approach to obtain Arabidopsis thaliana plants with only 5-60% of PSI-D. No plants were recovered completely lacking PSI-D, suggesting that PSI-D is essential for a functional PSI in plants. Plants with reduced amounts of PSI-D showed a similar decrease in all other subunits of PSI including the light harvesting complex, suggesting that in the absence of PSI-D, PSI cannot be properly assembled and becomes degraded. Plants with reduced amounts of PSI-D became light-stressed even in low light although they exhibited high non-photochemical quenching (NPQ). The high NPQ was generated by upregulating the level of violaxanthin de-epoxidase and PsbS, which are both essential components of NPQ. Interestingly, the lack of PSI-D affected the redox state of thioredoxin. During the normal light cycle thioredoxin became increasingly oxidized, which was observed as decreasing malate dehydrogenase activity over a 4-h light period. This result shows that photosynthesis was close to normal the first 15 min, but after 2-4 h photoinhibition dominated as the stroma progressively became less reduced. The change in the thiol disulfide redox state might be fatal for the PSI-D-less plants, because reduction of thioredoxin is one of the main switches for the initiation of CO2 assimilation and photoprotection upon light exposure.
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PMID:Arabidopsis thaliana plants lacking the PSI-D subunit of photosystem I suffer severe photoinhibition, have unstable photosystem I complexes, and altered redox homeostasis in the chloroplast stroma. 1279 67

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.
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PMID:The oxygen evolving enhancer protein 1 (OEE) of photosystem II in green algae exhibits thioredoxin activity. 1502 27

Seeds of many plant species are green during embryogenesis. To directly assess the influence of light on the physiological status of green oilseeds in planta, Brassica napus and soybean (Glycine max) seeds were rapidly dissected from plants growing in the light or dark. The activation state of malate dehydrogenase, which reflects reduced thioredoxin and NADP/NADPH ratios, was found to be as high in seeds exposed to light as in leaves and to decrease in the dark. Rubisco was highly activated (carbamylated) in both light and dark, most likely reflecting high seed CO(2) concentrations. Activities of Rubisco and phosphoribulokinase were sufficient to account for significant refixation of CO(2) produced during B. napus oil biosynthesis. To determine the influence of light on oil synthesis in planta, siliques on intact plants in full sunlight or detached siliques fed (3)H(2)O were partly covered with aluminum foil. Seeds from light and dark sections were analyzed, and fatty acid accumulation was found to be higher in seeds exposed to light than seeds from dark sections. The spectrum of light filtering through silique walls and the pigment composition of developing B. napus embryos were determined. In addition to a low chlorophyll a/b ratio, the carotenoid pigments of seeds can provide additional capture of the green light that filters through siliques. Together, these results demonstrate that even the low level of light reaching seeds plays a substantial role in activating light-regulated enzymes, increasing fatty acid synthesis, and potentially powering refixation of CO(2).
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PMID:The capacity of green oilseeds to utilize photosynthesis to drive biosynthetic processes. 1534 83

The determinants of the thioredoxin (TRX)-dependent redox regulation of the chloroplastic NADP-malate dehydrogenase (NADP-MDH) from the eukaryotic green alga Chlamydomonas reinhardtii have been investigated using site-directed mutagenesis. The results indicate that a single C-terminal disulfide is responsible for this regulation. The redox midpoint potential of this disulfide is less negative than that of the higher plant enzyme. The regulation is of an all-or-nothing type, lacking the fine-tuning provided by the second N-terminal disulfide found only in NADP-MDH from higher plants. The decreased stability of specific cysteine/alanine mutants is consistent with the presence of a structural disulfide formed by two cysteine residues that are not involved in regulation of activity. Measurements of the ability of C. reinhardtii thioredoxin f (TRX f) to activate wild-type and site-directed mutants of sorghum (Sorghum vulgare) NADP-MDH suggest that the algal TRX f has a redox midpoint potential that is less negative than most those of higher plant TRXs f. These results are discussed from an evolutionary point of view.
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PMID:NADP-malate dehydrogenase from unicellular green alga Chlamydomonas reinhardtii. A first step toward redox regulation? 1557 63


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