The Experts below are selected from a list of 192 Experts worldwide ranked by ideXlab platform
Renate Scheibe - One of the best experts on this subject based on the ideXlab platform.
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Transcriptional regulation of NADP-dependent Malate Dehydrogenase: comparative genetics and identification of DNA-binding proteins.
Journal of molecular evolution, 2007Co-Authors: Steffen Hameister, Jan E. Backhausen, Simone Holtgrefe, Vera Linke, Beril Becker, Inga Strodtkötter, Renate ScheibeAbstract:The transcriptional regulation of NADP-Malate Dehydrogenase (NADP-MDH) was analyzed in Arabidopsis ecotypes and other Brassicaceae. The amount of transcript increased twofold after transfer into low temperature (12 degrees C) or high light (750 microE) in all species. Analysis of the genomic DNA reveals that the NADP-MDH gene (At5g58330 in A. thaliana) in Brassicaceae is located between two other genes (At5g58320 and At5g58340 in Arabidopsis), both encoded on the opposite DNA strand. No promoter elements were identified in 5' direction of the NADP-MDH gene, and the expression of NADP-MDH was not affected in knock-out plants carrying a DNA insert in the 5' region. A yeast-one hybrid approach yielded only three DNA-binding proteins for the 500-bp fragment located upstream of the ATG sequence, but 34 proteins for its coding region. However, in Chlamydomonas and in some Poaceae, which do not possess any genes within the 1200 bp upstream region, typical promoter elements were identified. Alignments of genomic DNA reveal that, in contrast to Poaceae, the introns are highly conserved within Brassicaceae. We conclude that in Brassicaceae the majority of regulatory elements are located within the coding region. The NADP-MDH gene of both families evolved from a common precursor, similar to the gene in Chlamydomonas. Changes in the selection pressure allowed the insertion of At5g58340 into the promoter region of a common ancestor. When the demand for transcriptional regulation increased, At5g58340 disappeared in Poaceae, and a promoter developed in the 5' region. In contrast, Brassicaceae maintained At5g58340 and shifted all regulatory elements into the coding region of NADP-MDH.
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Transcriptional Regulation of NADP-Dependent Malate Dehydrogenase: Comparative Genetics and Identification of DNA-Binding Proteins
Journal of Molecular Evolution, 2007Co-Authors: Steffen Hameister, Jan E. Backhausen, Simone Holtgrefe, Vera Linke, Beril Becker, Inga Strodtkötter, Renate ScheibeAbstract:The transcriptional regulation of NADP-Malate Dehydrogenase (NADP-MDH) was analyzed in Arabidopsis ecotypes and other Brassicaceae. The amount of transcript increased twofold after transfer into low temperature (12°C) or high light (750 μE) in all species. Analysis of the genomic DNA reveals that the NADP-MDH gene (At5g58330 in A. thaliana ) in Brassicaceae is located between two other genes (At5g58320 and At5g58340 in Arabidopsis), both encoded on the opposite DNA strand. No promoter elements were identified in 5′ direction of the NADP-MDH gene, and the expression of NADP-MDH was not affected in knock-out plants carrying a DNA insert in the 5′ region. A yeast-one hybrid approach yielded only three DNA-binding proteins for the 500-bp fragment located upstream of the ATG sequence, but 34 proteins for its coding region. However, in Chlamydomonas and in some Poaceae, which do not possess any genes within the 1200 bp upstream region, typical promoter elements were identified. Alignments of genomic DNA reveal that, in contrast to Poaceae, the introns are highly conserved within Brassicaceae. We conclude that in Brassicaceae the majority of regulatory elements are located within the coding region. The NADP-MDH gene of both families evolved from a common precursor, similar to the gene in Chlamydomonas. Changes in the selection pressure allowed the insertion of At5g58340 into the promoter region of a common ancestor. When the demand for transcriptional regulation increased, At5g58340 disappeared in Poaceae, and a promoter developed in the 5′ region. In contrast, Brassicaceae maintained At5g58340 and shifted all regulatory elements into the coding region of NADP-MDH.
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Redox regulation of chloroplast enzymes in Galdieria sulphuraria in view of eukaryotic evolution
Plant & cell physiology, 2007Co-Authors: Christine Oesterhelt, Simone Holtgrefe, Susanne Klocke, Vera Linke, Andreas P. M. Weber, Renate ScheibeAbstract:Redox modulation is a general mechanism for enzyme regulation, particularly for the post-translational regulation of the Calvin cycle in chloroplasts of green plants. Although red algae and photosynthetic protists that harbor plastids of red algal origin contribute greatly to global carbon fixation, relatively little is known about post-translational regulation of chloroplast enzymes in this important group of photosynthetic eukaryotes. To address this question, we used biochemistry, phylogenetics and analysis of recently completed genome sequences. We studied the functionality of the chloroplast enzymes phosphoribulokinase (PRK, EC 2.7.1.19), NADP-dependent glyceraldehyde 3-phosphate Dehydrogenase (NADP-GAPDH, GapA, EC 1.2.1.13), fructose 1,6-bisphosphatase (FBPase, EC 3.1.3.11) and glucose 6-phosphate Dehydrogenase (G6PDH, EC 1.1.1.49), as well as NADP-Malate Dehydrogenase (NADP-MDH, EC 1.1.1.37) in the unicellular red alga Galdieria sulphuraria (Galdieri) Merola. Despite high sequence similarity of G. sulphuraria proteins to those of other photosynthetic organisms, we found a number of distinct differences. Both PRK and GAPDH co-eluted with CP12 in a high molecular weight complex in the presence of oxidized glutathione, although Galdieria CP12 lacks the two cysteines essential for the formation of the N-terminal peptide loop present in higher plants. However, PRK inactivation upon complex formation turned out to be incomplete. G6PDH was redox modulated, but remained in its tetrameric form; FBPase was poorly redox regulated, despite conservation of the two redox-active cysteines. No indication for the presence of plastidic NADP-MDH (and other components of the Malate valve) was found.
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Adaptation of tobacco plants to elevated CO2: influence of leaf age on changes in physiology, redox states and NADP-Malate Dehydrogenase activity
Journal of Experimental Botany, 1999Co-Authors: Jan E. Backhausen, Renate ScheibeAbstract:Transgenic tobacco plants (Nicotiana tabacum L. cv. Xanthi) with altered chloroplast NADP-Malate Dehydrogenase (NADP-MDH) content were grown under ambient or under doubled atmospheric CO 2 in order to analyse the effect of elevated CO 2 on the redox state of the chloroplasts. Since large differences exist between the individual leaves of tobacco plants, gas exchange characteristics, enzyme capacities and metabolite contents were measured separately for each leaf of the plants. Large variations between leaves of different age were found in nearly every parameter analysed, and the differences between younger and older leaves were, in most cases, larger than the differences between comparable leaves at ambient or elevated CO 2 . For all parameters (chlorophyll fluorescence, P700 reduction, NADP-MDH activation) that are indicative for the redox situation in the electron transport chains and in the chloroplast stroma, more oxidized values were determined under elevated CO 2 . The increased redox state of ferredoxin, observed at ambient conditions in the NADP-MDH-under-expressing plants, disappeared under elevated CO 2 . It was concluded that the reduced rate of photorespiration under elevated CO 2 decreases the amount of excess electrons. Interestingly, this lowered not only the activation state of NADP-MDH, but also the expression of the enzyme in the wild-type plants. The results are discussed with respect to a possible interaction between stromal reduction state and gene expression.
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transgenic potato plants with altered expression levels of chloroplast NADP Malate Dehydrogenase interactions between photosynthetic electron transport and Malate metabolism in leaves and in isolated intact chloroplasts
Planta, 1998Co-Authors: Jan E. Backhausen, Andrea Emmerlich, Simone Holtgrefe, Peter Horton, Gabi Nast, Jennifer J M Rogers, Bernd Mullerrober, Renate ScheibeAbstract:The contribution of the Malate valve in the regulation of steady-state photosynthesis was studied in transgenic potato (Solanum tuberosum L. cv Desiree) plants with altered expression of plastidic NADP-dependent Malate Dehydrogenase (NADP-MDH; EC 1.1.1.82). Mutant plants were obtained after transformation with the homologous Nmdh gene in antisense orientation, or with the Nmdh gene from pea (Pisum sativum L.) in sense orientation. A total number of nine stable sense and antisense lines with 10% or 30%, and 400% of wild-type NADP-MDH capacity were selected. Intact chloroplasts were isolated from leaves of wild-type and mutant plants. In chloroplasts from sense transformants the increased enzyme amount was activated as in wild-type chloroplasts, but increased rates of oxaloacetate-dependent Malate formation were only measured upon partial uncoupling. In contrast, chloroplasts from antisense transformants produced only little Malate upon oxaloacetate addition. Measurements with intact leaves during steady-state photosynthesis yielded no differences in gas-exchange parameters and chlorophyll fluorescence. The leaf Malate content was unchanged in NADP-MDH underexpressors, but twice as high in overexpressing plants. The altered NADP-MDH expression clearly influences the redox state of ferredoxin, especially in low light. Furthermore, the Malate valve can successfully compete for electrons with cyclic electron flow, but the conditions under which this occurs are quite artificial.
Jan E. Backhausen - One of the best experts on this subject based on the ideXlab platform.
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Transcriptional regulation of NADP-dependent Malate Dehydrogenase: comparative genetics and identification of DNA-binding proteins.
Journal of molecular evolution, 2007Co-Authors: Steffen Hameister, Jan E. Backhausen, Simone Holtgrefe, Vera Linke, Beril Becker, Inga Strodtkötter, Renate ScheibeAbstract:The transcriptional regulation of NADP-Malate Dehydrogenase (NADP-MDH) was analyzed in Arabidopsis ecotypes and other Brassicaceae. The amount of transcript increased twofold after transfer into low temperature (12 degrees C) or high light (750 microE) in all species. Analysis of the genomic DNA reveals that the NADP-MDH gene (At5g58330 in A. thaliana) in Brassicaceae is located between two other genes (At5g58320 and At5g58340 in Arabidopsis), both encoded on the opposite DNA strand. No promoter elements were identified in 5' direction of the NADP-MDH gene, and the expression of NADP-MDH was not affected in knock-out plants carrying a DNA insert in the 5' region. A yeast-one hybrid approach yielded only three DNA-binding proteins for the 500-bp fragment located upstream of the ATG sequence, but 34 proteins for its coding region. However, in Chlamydomonas and in some Poaceae, which do not possess any genes within the 1200 bp upstream region, typical promoter elements were identified. Alignments of genomic DNA reveal that, in contrast to Poaceae, the introns are highly conserved within Brassicaceae. We conclude that in Brassicaceae the majority of regulatory elements are located within the coding region. The NADP-MDH gene of both families evolved from a common precursor, similar to the gene in Chlamydomonas. Changes in the selection pressure allowed the insertion of At5g58340 into the promoter region of a common ancestor. When the demand for transcriptional regulation increased, At5g58340 disappeared in Poaceae, and a promoter developed in the 5' region. In contrast, Brassicaceae maintained At5g58340 and shifted all regulatory elements into the coding region of NADP-MDH.
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Transcriptional Regulation of NADP-Dependent Malate Dehydrogenase: Comparative Genetics and Identification of DNA-Binding Proteins
Journal of Molecular Evolution, 2007Co-Authors: Steffen Hameister, Jan E. Backhausen, Simone Holtgrefe, Vera Linke, Beril Becker, Inga Strodtkötter, Renate ScheibeAbstract:The transcriptional regulation of NADP-Malate Dehydrogenase (NADP-MDH) was analyzed in Arabidopsis ecotypes and other Brassicaceae. The amount of transcript increased twofold after transfer into low temperature (12°C) or high light (750 μE) in all species. Analysis of the genomic DNA reveals that the NADP-MDH gene (At5g58330 in A. thaliana ) in Brassicaceae is located between two other genes (At5g58320 and At5g58340 in Arabidopsis), both encoded on the opposite DNA strand. No promoter elements were identified in 5′ direction of the NADP-MDH gene, and the expression of NADP-MDH was not affected in knock-out plants carrying a DNA insert in the 5′ region. A yeast-one hybrid approach yielded only three DNA-binding proteins for the 500-bp fragment located upstream of the ATG sequence, but 34 proteins for its coding region. However, in Chlamydomonas and in some Poaceae, which do not possess any genes within the 1200 bp upstream region, typical promoter elements were identified. Alignments of genomic DNA reveal that, in contrast to Poaceae, the introns are highly conserved within Brassicaceae. We conclude that in Brassicaceae the majority of regulatory elements are located within the coding region. The NADP-MDH gene of both families evolved from a common precursor, similar to the gene in Chlamydomonas. Changes in the selection pressure allowed the insertion of At5g58340 into the promoter region of a common ancestor. When the demand for transcriptional regulation increased, At5g58340 disappeared in Poaceae, and a promoter developed in the 5′ region. In contrast, Brassicaceae maintained At5g58340 and shifted all regulatory elements into the coding region of NADP-MDH.
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Adaptation of tobacco plants to elevated CO2: influence of leaf age on changes in physiology, redox states and NADP-Malate Dehydrogenase activity
Journal of Experimental Botany, 1999Co-Authors: Jan E. Backhausen, Renate ScheibeAbstract:Transgenic tobacco plants (Nicotiana tabacum L. cv. Xanthi) with altered chloroplast NADP-Malate Dehydrogenase (NADP-MDH) content were grown under ambient or under doubled atmospheric CO 2 in order to analyse the effect of elevated CO 2 on the redox state of the chloroplasts. Since large differences exist between the individual leaves of tobacco plants, gas exchange characteristics, enzyme capacities and metabolite contents were measured separately for each leaf of the plants. Large variations between leaves of different age were found in nearly every parameter analysed, and the differences between younger and older leaves were, in most cases, larger than the differences between comparable leaves at ambient or elevated CO 2 . For all parameters (chlorophyll fluorescence, P700 reduction, NADP-MDH activation) that are indicative for the redox situation in the electron transport chains and in the chloroplast stroma, more oxidized values were determined under elevated CO 2 . The increased redox state of ferredoxin, observed at ambient conditions in the NADP-MDH-under-expressing plants, disappeared under elevated CO 2 . It was concluded that the reduced rate of photorespiration under elevated CO 2 decreases the amount of excess electrons. Interestingly, this lowered not only the activation state of NADP-MDH, but also the expression of the enzyme in the wild-type plants. The results are discussed with respect to a possible interaction between stromal reduction state and gene expression.
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transgenic potato plants with altered expression levels of chloroplast NADP Malate Dehydrogenase interactions between photosynthetic electron transport and Malate metabolism in leaves and in isolated intact chloroplasts
Planta, 1998Co-Authors: Jan E. Backhausen, Andrea Emmerlich, Simone Holtgrefe, Peter Horton, Gabi Nast, Jennifer J M Rogers, Bernd Mullerrober, Renate ScheibeAbstract:The contribution of the Malate valve in the regulation of steady-state photosynthesis was studied in transgenic potato (Solanum tuberosum L. cv Desiree) plants with altered expression of plastidic NADP-dependent Malate Dehydrogenase (NADP-MDH; EC 1.1.1.82). Mutant plants were obtained after transformation with the homologous Nmdh gene in antisense orientation, or with the Nmdh gene from pea (Pisum sativum L.) in sense orientation. A total number of nine stable sense and antisense lines with 10% or 30%, and 400% of wild-type NADP-MDH capacity were selected. Intact chloroplasts were isolated from leaves of wild-type and mutant plants. In chloroplasts from sense transformants the increased enzyme amount was activated as in wild-type chloroplasts, but increased rates of oxaloacetate-dependent Malate formation were only measured upon partial uncoupling. In contrast, chloroplasts from antisense transformants produced only little Malate upon oxaloacetate addition. Measurements with intact leaves during steady-state photosynthesis yielded no differences in gas-exchange parameters and chlorophyll fluorescence. The leaf Malate content was unchanged in NADP-MDH underexpressors, but twice as high in overexpressing plants. The altered NADP-MDH expression clearly influences the redox state of ferredoxin, especially in low light. Furthermore, the Malate valve can successfully compete for electrons with cyclic electron flow, but the conditions under which this occurs are quite artificial.
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Transgenic Tobacco Plants Expressing Pea Chloroplast Nmdh cDNA in Sense and Antisense Orientation (Effects on NADP-Malate Dehydrogenase Level, Stability of Transformants, and Plant Growth).
Plant physiology, 1997Co-Authors: M. Faske, Renate Scheibe, Jan E. Backhausen, M. Sendker, M. Singer-bayrle, A. Von SchaewenAbstract:A full-length cDNA encoding light-activated chloroplast NADP-Malate Dehydrogenase (NADP-MDH) (EC 1.1.1.82) from pea (Pisum sativum L.) was introduced in the sense and antisense orientation into tobacco (Nicotiana tabacum L.). Transgenic plants with decreased or increased expression levels were obtained. Because of substantial age-dependent differences in individual leaves of a single plant, standardization of NADP-MDH levels was required first. Then, extent and stability of over- or under-expression of Nmdh, the gene encoding NADP-MDH, was characterized in the various transformants. Frequently, cosuppression effects were observed, indicating sufficient homology between the endogenous tobacco and the heterologous pea gene. Analysis of the T1 and T2 progeny of a series of independent transgenic lines revealed that NADP-MDH capacity ranged between 10% and [greater than or equal to]10-fold compared with the wild type. Under ambient conditions whole-plant development, growth period, and fertility were unaffected by NADP-MDH reduction to 20% of the wild-type level; below this threshold plant growth was retarded. A positive growth effect was registered in young plants with stably enhanced NADP-MDH levels within a defined developmental window.
Paulette Decottignies - One of the best experts on this subject based on the ideXlab platform.
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Light‐activation of NADP‐Malate Dehydrogenase: A highly controlled process for an optimized function
Physiologia Plantarum, 2008Co-Authors: Myroslawa Miginiac-maslow, Jean-pierre Jacquot, E Ruelland, Aymeric Goyer, Emmanuelle Issakidis-bourguet, Kenth Johansson, I. Schepens, M. Lemaire-chamley, P. Le Maréchal, Paulette DecottigniesAbstract:The chloroplastic nicotinamide adenine dinucleotide phosphate-Malate Dehydrogenase (NADP-MDH) (EC 1.1.1.82), a key enzyme of photosynthetic carbon assimilation of the C4 NADP-malic enzyme type plants, is strictly regulated by light through the ferredoxin-thioredoxin system. It is inactive in the dark, in the oxidized form, and activated in the light by the reduction of specific regulatory disulfides. A site-directed mutagenesis approach allowed localization of the regulatory disulfides in the N- and C-terminal sequence extensions conserved in all the light-regulated chloroplastic Malate Dehydrogenases. These extensions do not exist in the permanently active NAD-dependent MDHs (EC 1.1.1.37). Biochemical characterization of the mutants and elimination of negative charges at the C-terminus provided evidence for auto-inhibition of the oxidized enzyme by its C-terminal end through interaction with the active site and showed that the more compact structure of the oxidized dimer was linked to the presence of the N-terminal disulfide. The recently published 3-dimensional structures of the oxidized enzyme confirmed the location of the regulatory disulfides and fully support the auto-inhibition hypothesis. Indeed, the C-terminus is trapped inside the active site, interacting with active-site residues, and the N-termini are inserted at the dimer contact area where they are bound by hydrophobic interactions with both subunits. The physiological function of such complex regulation is discussed.
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light activation of NADP Malate Dehydrogenase a highly controlled process for an optimized function
Physiologia Plantarum, 2000Co-Authors: Myroslawa Miginiacmaslow, Jean-pierre Jacquot, Emmanuelle Issakidisbourguet, E Ruelland, Aymeric Goyer, Kenth Johansson, I. Schepens, M Lemairechamley, Le P Marechal, Paulette DecottigniesAbstract:The chloroplastic nicotinamide adenine dinucleotide phosphate-Malate Dehydrogenase (NADP-MDH) (EC 1.1.1.82), a key enzyme of photosynthetic carbon assimilation of the C4 NADP-malic enzyme type plants, is strictly regulated by light through the ferredoxin-thioredoxin system. It is inactive in the dark, in the oxidized form, and activated in the light by the reduction of specific regulatory disulfides. A site-directed mutagenesis approach allowed localization of the regulatory disulfides in the N- and C-terminal sequence extensions conserved in all the light-regulated chloroplastic Malate Dehydrogenases. These extensions do not exist in the permanently active NAD-dependent MDHs (EC 1.1.1.37). Biochemical characterization of the mutants and elimination of negative charges at the C-terminus provided evidence for auto-inhibition of the oxidized enzyme by its C-terminal end through interaction with the active site and showed that the more compact structure of the oxidized dimer was linked to the presence of the N-terminal disulfide. The recently published 3-dimensional structures of the oxidized enzyme confirmed the location of the regulatory disulfides and fully support the auto-inhibition hypothesis. Indeed, the C-terminus is trapped inside the active site, interacting with active-site residues, and the N-termini are inserted at the dimer contact area where they are bound by hydrophobic interactions with both subunits. The physiological function of such complex regulation is discussed.
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Mechanism of Auto-Inhibition of NADP-Malate Dehydrogenase by Its C-Terminal Extension
Photosynthesis: Mechanisms and Effects, 1998Co-Authors: Eric Ruelland, Paulette Decottignies, N. Djukic, Myroslawa Miginiac-maslowAbstract:NADP-Malate Dehydrogenase (NADP-MDH: EC. 1.1.1.82) is a chloroplastic enzyme activated in the light by the ferredoxin/thioredoxin system. Its totally inactive oxidized form contains two disulfide bridges per subunit, located in specific sequence extensions, one at the N-terminus [1, 2] and the other at the C-terminus [3]. The C-terminal extension shields the access to the active site [4]. Upon reduction, the N-terminal bridge is isomerized and the newly created disulfide is reduced. During this reduction process, the active site undergoes a conformational change towards a high catalytic efficiency conformation [5]. The reduction of the C-terminal bridge leads to a displacement of the C-terminal extension, uncovering the access to the active site. Up to now, the molecular mechanism of the shielding of the active site by the oxidized C-terminal extension is poorly understood. The extension might act as a lid, as suggested by proteolysis experiments [6], or it might enter the active site and bind to specific active-site residues, as suggested by molecular modeling [7]. In this regard, it can be noted that the C-terminal end of the protein bears two negative charges: the C-terminal carboxyl group and the lateral carboxyl group of the penultimate glutamate residue and thus can mimick the dicarboxylic substrate of the enzyme.
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Essential histidine at the active site of sorghum leaf NADP-dependent Malate Dehydrogenase.
The Journal of biological chemistry, 1994Co-Authors: M. Lemaire, Myroslawa Miginiac-maslow, Pierre Gadal, Jean-marie Schmitter, Emmanuelle Issakidis, Paulette DecottigniesAbstract:Chloroplastic NADP-dependent Malate Dehydrogenase (NADP-MDH) is a key enzyme in the photosynthetic CO2 fixation pathway of C4-plants. The presence of a histidine at its active site has been proposed, based on sequence alignment with nonchloroplastic NAD-dependent Malate Dehydrogenases. In order to investigate this hypothesis, the effect of diethylpyrocarbonate on the sorghum leaf enzyme has been tested. Diethylpyrocarbonate strongly inhibited NADP-MDH activity, its effect being dramatically decreased in the presence of substrates and reversed by hydroxylamine. When diethylpyrocarbonate-inactivated NADP-MDH was cleaved with trypsin, one peptide with increased absorbance at 240 nm was detected. Sequencing of this peptide and analysis by mass spectrometry demonstrated that histidine 229 was modified by diethylpyrocarbonate. This amino acid was changed to an alanine by site-directed mutagenesis, and the modified protein was produced in Escherichia coli. It was similar to the plant enzyme except that it was totally inactive. Taken together, these results indicate that His229 is an essential residue in the active site of sorghum NADP-MDH.
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Purification and characterization of pea thioredoxin f expressed in Escherichia coli
Plant Molecular Biology, 1994Co-Authors: Michael Hodges, Myroslawa Miginiac-maslow, Claude Crétin, Paulette Decottignies, Jean-pierre Jacquot, Loïc Lepiniec, Mariana Stein, Pierre GadalAbstract:The recently cloned cDNA for pea chloroplast thioredoxin f was used to produce, by PCR, a fragment coding for a protein lacking the transit peptide. This cDNA fragment was subcloned into a pET expression vector and used to transform E. coli cells. After induction with IPTG the transformed cells produce the protein, mainly in the soluble fraction of the broken cells. The recombinant thioredoxin f has been purified and used to raise antibodies and analysed for activity. The antibodies appear to be specific towards thioredoxin f and do not recognize other types of thioredoxin. The recombinant protein could activate two chloroplastic enzymes, namely NADP-dependent Malate Dehydrogenase (NADP-MDH) and fructose 1,6-bisphosphatase (FBPase), both using dithiothreitol as a chemical reductant and in a light-reconstituted/thylakoid assay. Recombinant pea thioredoxin f turned out to be an excellent catalyst for NADP-MDH activation, being the more efficient than a recombinant m-type thioredoxin of Chlamydomonas reinhardtii and the thioredoxin of E. coli . At the concentrations of thioredoxin used in the target enzyme activation assays only the recombinant thioredoxin f activated the FBPase.
Na Sui - One of the best experts on this subject based on the ideXlab platform.
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NADP-Malate Dehydrogenase of Sweet Sorghum Improves Salt Tolerance of Arabidopsis thaliana
Journal of agricultural and food chemistry, 2018Co-Authors: Yuanyuan Guo, Yushuang Song, Hongxiang Zheng, Yi Zhang, Jianrong Guo, Na SuiAbstract:Sweet sorghum is a C4 crop that shows high salt tolerance and high yield. NADP-Malate Dehydrogenase (NADP-ME) is a crucial enzyme of the C4 pathway. The regulatory mechanism of NADP-ME remains unclear. In this study, we isolated SbNADP-ME from sweet sorghum. The open reading frame of SbNADP-ME is 1911 bp and 637 amino acid residues. Quantitative real-time PCR analysis showed that SbNADP-ME transcription in sweet sorghum was enhanced by salt stress. The SbNADP-ME transcript level was highest under exposure to 150 mM NaCl. Arabidopsis overexpressing SbNADP-ME showed increased germination rate and root length under NaCl treatments. At the seedling stage, physiological photosynthesis parameters, chlorophyll content, PSII photochemical efficiency, and PSI oxidoreductive activity in the wild type decreased more severely than in the overexpression lines but less than in T-DNA insertion mutants under salt stress. Overexpression of SbNADP-ME in Arabidopsis may also increase osmotic adjustment and scavenging activi...
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NADP-Malate Dehydrogenase of Sweet Sorghum Improves Salt Tolerance of Arabidopsis thaliana
2018Co-Authors: Yuanyuan Guo, Yushuang Song, Hongxiang Zheng, Yi Zhang, Jianrong Guo, Na SuiAbstract:Sweet sorghum is a C4 crop that shows high salt tolerance and high yield. NADP-Malate Dehydrogenase (NADP-ME) is a crucial enzyme of the C4 pathway. The regulatory mechanism of NADP-ME remains unclear. In this study, we isolated SbNADP-ME from sweet sorghum. The open reading frame of SbNADP-ME is 1911 bp and 637 amino acid residues. Quantitative real-time PCR analysis showed that SbNADP-ME transcription in sweet sorghum was enhanced by salt stress. The SbNADP-ME transcript level was highest under exposure to 150 mM NaCl. Arabidopsis overexpressing SbNADP-ME showed increased germination rate and root length under NaCl treatments. At the seedling stage, physiological photosynthesis parameters, chlorophyll content, PSII photochemical efficiency, and PSI oxidoreductive activity in the wild type decreased more severely than in the overexpression lines but less than in T-DNA insertion mutants under salt stress. Overexpression of SbNADP-ME in Arabidopsis may also increase osmotic adjustment and scavenging activity on DPPH and decrease membrane peroxidation. These results suggest that SbNADP-ME overexpression in Arabidopsis increases salt tolerance and alleviates PSII and PSI photoinhibition under salt stress by improving photosynthetic capacity
Myroslawa Miginiac-maslow - One of the best experts on this subject based on the ideXlab platform.
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Light‐activation of NADP‐Malate Dehydrogenase: A highly controlled process for an optimized function
Physiologia Plantarum, 2008Co-Authors: Myroslawa Miginiac-maslow, Jean-pierre Jacquot, E Ruelland, Aymeric Goyer, Emmanuelle Issakidis-bourguet, Kenth Johansson, I. Schepens, M. Lemaire-chamley, P. Le Maréchal, Paulette DecottigniesAbstract:The chloroplastic nicotinamide adenine dinucleotide phosphate-Malate Dehydrogenase (NADP-MDH) (EC 1.1.1.82), a key enzyme of photosynthetic carbon assimilation of the C4 NADP-malic enzyme type plants, is strictly regulated by light through the ferredoxin-thioredoxin system. It is inactive in the dark, in the oxidized form, and activated in the light by the reduction of specific regulatory disulfides. A site-directed mutagenesis approach allowed localization of the regulatory disulfides in the N- and C-terminal sequence extensions conserved in all the light-regulated chloroplastic Malate Dehydrogenases. These extensions do not exist in the permanently active NAD-dependent MDHs (EC 1.1.1.37). Biochemical characterization of the mutants and elimination of negative charges at the C-terminus provided evidence for auto-inhibition of the oxidized enzyme by its C-terminal end through interaction with the active site and showed that the more compact structure of the oxidized dimer was linked to the presence of the N-terminal disulfide. The recently published 3-dimensional structures of the oxidized enzyme confirmed the location of the regulatory disulfides and fully support the auto-inhibition hypothesis. Indeed, the C-terminus is trapped inside the active site, interacting with active-site residues, and the N-termini are inserted at the dimer contact area where they are bound by hydrophobic interactions with both subunits. The physiological function of such complex regulation is discussed.
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Intrasteric inhibition in redox signalling: light activation of NADP-Malate Dehydrogenase
Photosynthesis Research, 2002Co-Authors: Myroslawa Miginiac-maslow, Jean-marc LancelinAbstract:Chloroplast NADP-dependent Malate Dehydrogenase (NADP-MDH, EC 1.1.1.82) is inactive in the dark and activated in the light via a reduction of specific disulfides by thiol-disulfide interchange with thioredoxin, reduced by the photosynthetic electron transfer. Compared to the constitutively active NAD-dependent forms, NADP-MDH exhibits two regulatory disulfides per subunit, one located in an N-terminal extension and the other in a C-terminal extension. Convergent information gathered from biochemical, site-directed mutagenesis and structural approaches allowed to solve almost completely the activation mechanism. In the oxidized enzyme, the C-terminal extension is pulled back by the disulfide bridge toward the active-site cleft where the penultimate C-terminal glutamate interacts with one of the arginines involved in substrate binding, thus acting as an internal inhibitor obstructing the access of oxaloacetate. The N-terminal extensions are located at the subunit interface area and rigidify the overall structure of the dimer. Their reduction by reduced thioredoxin triggers a conformational change of the active site towards high-activity conformation, whereas the reduction of the C-terminal bridge expells the C-terminal end from the active site, thus opening the way for the substrate.
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Direct NMR observation of the thioredoxin-mediated reduction of the chloroplast NADP-Malate Dehydrogenase provides a structural basis for the relief of autoinhibition.
The Journal of biological chemistry, 1999Co-Authors: Isabelle Krimm, Myroslawa Miginiac-maslow, Aymeric Goyer, Emmanuelle Issakidis-bourguet, Jean-marc LancelinAbstract:The chloroplastic NADP-dependent Malate Dehydrogenase (NADP-MDH) catalyzing the reduction of oxaloacetate into L-Malate is regulated by light. Its activation results from the thioredoxin-mediated reduction of two disulfides, located, respectively, in N- and C-terminal sequence extensions typical of all NADP-dependent light-regulated forms. Site-directed mutagenesis studies and the resolution of the three-dimensional structure of the oxidized (inactive) Sorghum vulgare enzyme showed that the C-terminal Cys(365)-Cys(377) disulfide constrains the C-terminal extension to fold into the active site where it acts as an internal inhibitor. In the present study, two-dimensional proton NMR spectra of an engineered NADP-MDH rendered monomeric by a 33-amino acid deletion at the N terminus (38 kDa) revealed that a 15-amino acid-long C-terminal peptide (Ala(375) to C-terminal Val(389)) acquired an increased mobility upon reduction, allowing its direct sequence-specific NMR assignment. The location of the flexible peptide in the sequence suggests that the first part of the C-terminal peptide is still folded near the core of the enzyme, so that cysteines 365 and 377 remain in proximity to allow for an efficient reoxidation/inactivation of the enzyme.
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Mechanism of Auto-Inhibition of NADP-Malate Dehydrogenase by Its C-Terminal Extension
Photosynthesis: Mechanisms and Effects, 1998Co-Authors: Eric Ruelland, Paulette Decottignies, N. Djukic, Myroslawa Miginiac-maslowAbstract:NADP-Malate Dehydrogenase (NADP-MDH: EC. 1.1.1.82) is a chloroplastic enzyme activated in the light by the ferredoxin/thioredoxin system. Its totally inactive oxidized form contains two disulfide bridges per subunit, located in specific sequence extensions, one at the N-terminus [1, 2] and the other at the C-terminus [3]. The C-terminal extension shields the access to the active site [4]. Upon reduction, the N-terminal bridge is isomerized and the newly created disulfide is reduced. During this reduction process, the active site undergoes a conformational change towards a high catalytic efficiency conformation [5]. The reduction of the C-terminal bridge leads to a displacement of the C-terminal extension, uncovering the access to the active site. Up to now, the molecular mechanism of the shielding of the active site by the oxidized C-terminal extension is poorly understood. The extension might act as a lid, as suggested by proteolysis experiments [6], or it might enter the active site and bind to specific active-site residues, as suggested by molecular modeling [7]. In this regard, it can be noted that the C-terminal end of the protein bears two negative charges: the C-terminal carboxyl group and the lateral carboxyl group of the penultimate glutamate residue and thus can mimick the dicarboxylic substrate of the enzyme.
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Essential histidine at the active site of sorghum leaf NADP-dependent Malate Dehydrogenase.
The Journal of biological chemistry, 1994Co-Authors: M. Lemaire, Myroslawa Miginiac-maslow, Pierre Gadal, Jean-marie Schmitter, Emmanuelle Issakidis, Paulette DecottigniesAbstract:Chloroplastic NADP-dependent Malate Dehydrogenase (NADP-MDH) is a key enzyme in the photosynthetic CO2 fixation pathway of C4-plants. The presence of a histidine at its active site has been proposed, based on sequence alignment with nonchloroplastic NAD-dependent Malate Dehydrogenases. In order to investigate this hypothesis, the effect of diethylpyrocarbonate on the sorghum leaf enzyme has been tested. Diethylpyrocarbonate strongly inhibited NADP-MDH activity, its effect being dramatically decreased in the presence of substrates and reversed by hydroxylamine. When diethylpyrocarbonate-inactivated NADP-MDH was cleaved with trypsin, one peptide with increased absorbance at 240 nm was detected. Sequencing of this peptide and analysis by mass spectrometry demonstrated that histidine 229 was modified by diethylpyrocarbonate. This amino acid was changed to an alanine by site-directed mutagenesis, and the modified protein was produced in Escherichia coli. It was similar to the plant enzyme except that it was totally inactive. Taken together, these results indicate that His229 is an essential residue in the active site of sorghum NADP-MDH.