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Biswarup Mukhopadhyay - One of the best experts on this subject based on the ideXlab platform.
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structure of an archaeal type Phosphoenolpyruvate Carboxylase sensitive to inhibition by aspartate
Proteins, 2011Co-Authors: Lakshmi Dharmarajan, Jessica L Kraszewski, Biswarup Mukhopadhyay, Pete W DuntenAbstract:The crystal structure of an archaeal-type Phosphoenolpyruvate Carboxylase from Clostridium perfringens has been determined based on X-ray data extending to 3 Ǻ. The asymmetric unit of the structure includes two tetramers (each a dimer-of-dimers) of the enzyme. The precipitant, malonate, employed for the crystallization is itself a weak inhibitor of Phosphoenolpyruvate Carboxylase and a malonate molecule is seen in the active-site in the crystal structure. The allosteric binding sites for aspartate (an inhibitor) and glucose-6-phosphate (an activator) observed in the Escherichia coli and Zea mays Phosphoenolpyruvate Carboxylase structures, respectively, are not conserved in the C. perfringens structure. Aspartate inhibits the C. perfringens enzyme competitively with respect to the substrate, Mg++.Phosphoenolpyruvate. A mechanism for inhibition is proposed based on the structure and sequence comparisons with other archaeal-type Phosphoenolpyruvate Carboxylases with differing sensitivity to inhibition by aspartate.
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Structure of an archaeal‐type Phosphoenolpyruvate Carboxylase sensitive to inhibition by aspartate
Proteins, 2011Co-Authors: Lakshmi Dharmarajan, Jessica L Kraszewski, Biswarup Mukhopadhyay, Pete W DuntenAbstract:The crystal structure of an archaeal-type Phosphoenolpyruvate Carboxylase from Clostridium perfringens has been determined based on X-ray data extending to 3 Ǻ. The asymmetric unit of the structure includes two tetramers (each a dimer-of-dimers) of the enzyme. The precipitant, malonate, employed for the crystallization is itself a weak inhibitor of Phosphoenolpyruvate Carboxylase and a malonate molecule is seen in the active-site in the crystal structure. The allosteric binding sites for aspartate (an inhibitor) and glucose-6-phosphate (an activator) observed in the Escherichia coli and Zea mays Phosphoenolpyruvate Carboxylase structures, respectively, are not conserved in the C. perfringens structure. Aspartate inhibits the C. perfringens enzyme competitively with respect to the substrate, Mg++.Phosphoenolpyruvate. A mechanism for inhibition is proposed based on the structure and sequence comparisons with other archaeal-type Phosphoenolpyruvate Carboxylases with differing sensitivity to inhibition by aspartate.
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Structure of an archaeal‐type Phosphoenolpyruvate Carboxylase sensitive to inhibition by aspartate
Proteins, 2011Co-Authors: Lakshmi Dharmarajan, Jessica L Kraszewski, Biswarup Mukhopadhyay, Pete W DuntenAbstract:The crystal structure of an archaeal-type Phosphoenolpyruvate Carboxylase from Clostridium perfringens has been determined based on X-ray data extending to 3 Ǻ. The asymmetric unit of the structure includes two tetramers (each a dimer-of-dimers) of the enzyme. The precipitant, malonate, employed for the crystallization is itself a weak inhibitor of Phosphoenolpyruvate Carboxylase and a malonate molecule is seen in the active-site in the crystal structure. The allosteric binding sites for aspartate (an inhibitor) and glucose-6-phosphate (an activator) observed in the Escherichia coli and Zea mays Phosphoenolpyruvate Carboxylase structures, respectively, are not conserved in the C. perfringens structure. Aspartate inhibits the C. perfringens enzyme competitively with respect to the substrate, Mg++.Phosphoenolpyruvate. A mechanism for inhibition is proposed based on the structure and sequence comparisons with other archaeal-type Phosphoenolpyruvate Carboxylases with differing sensitivity to inhibition by aspartate.
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The Phosphoenolpyruvate Carboxylase from Methanothermobacter thermautotrophicus has a novel structure.
Journal of Bacteriology, 2004Co-Authors: Hiten M. Patel, Jessica L Kraszewski, Biswarup MukhopadhyayAbstract:In Methanothermobacter thermautotrophicus, oxaloacetate synthesis is a major and essential CO2-fixation reaction. This methanogenic archaeon possesses two oxaloacetate-synthesizing enzymes, pyruvate Carboxylase and Phosphoenolpyruvate Carboxylase. The Phosphoenolpyruvate Carboxylase from this organism was purified to homogeneity. The subunit size of this homotetrameric protein was 55 kDa, which is about half that of all known bacterial and eukaryotic Phosphoenolpyruvate Carboxylases (PPCs). The NH2-terminal sequence identified this enzyme as the product of MTH943, an open reading frame with no assigned function in the genome sequence. A BLAST search did not show an obvious sequence similarity between MTH943 and known PPCs, which are generally well conserved. This is the first report of a new type of Phosphoenolpyruvate Carboxylase that we call PpcA (“A” for “archaeal”). Homologs to PpcA were present in most archaeal genomic sequences, but only in three bacterial (Clostridium perfringens, Oenococcus oeni, and Leuconostoc mesenteroides) and no eukaryotic genomes. PpcA was the only recognizable oxaloacetate-producing enzyme in Methanopyrus kandleri, a hydrothermal vent organism. Each PpcA-containing organism lacked a PPC homolog. The activity of M. thermautotrophicus PpcA was not influenced by acetyl coenzyme A and was about 50 times less sensitive to aspartate than the Escherichia coli PPC. The catalytic core (including His138, Arg587, and Gly883) of the E. coli PPC was partly conserved in PpcA, but three of four aspartate-binding residues (Lys773, Arg832, and Asn881) were not. PPCs probably evolved from PpcA through a process that added allosteric sites to the enzyme. The reverse is also equally possible.
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the Phosphoenolpyruvate Carboxylase from methanothermobacter thermautotrophicus has a novel structure
Journal of Bacteriology, 2004Co-Authors: Hiten M. Patel, Jessica L Kraszewski, Biswarup MukhopadhyayAbstract:The synthesis of oxaloacetate (OAA) is a major and essential CO2-fixation reaction in the methanarchaea (10, 11, 15, 16, 50, 52, 58, 60). These organisms possess an incomplete tricarboxylic acid (TCA) cycle which is used to generate intermediates (OAA and α-ketoglutarate [α-KG]) and a carrier (succinate) for the biosynthesis of amino acids and tetrapyrroles (10, 11, 15, 16, 50, 52, 58, 60). The organisms belonging to the orders of Methanococcales, Methanobacteriales, and Methanomicrobiales, which primarily use hydrogen as an energy source (2), employ a reductive sequence starting at OAA and terminating at α-KG (10, 11, 15, 16, 50, 52, 60). Methanosarcina species, which predominantly depend on acetotrophic or methylotrophic methanogenesis for energy generation (2), use an oxidative branch of the TCA cycle that initiates with OAA and acetate and terminates at α-KG (52, 58). Hence, OAA synthesis is a central anabolic process in methanarchaea. Thus far, pyruvate Carboxylase (PYC) (39, 41, 42, 50) and Phosphoenolpyruvate Carboxylase (PPC) (14, 31, 47, 60) have been found to be capable of fulfilling this requirement, as follows: PYC is ubiquitous in the methanogens (39, 41, 42, 50), and the primary structure, kinetic characteristics, and expression patterns of the methanogen PYCs have been investigated (39, 41, 42, 50). Methanothermobacter thermautotrophicus (formerly known as Methanobacterium thermoautotrophicum strain ΔH) (2, 57, 61) and Methanothermus sociabilis also possess Phosphoenolpyruvate Carboxylase (14, 31, 47, 60), but very little of such information is available on these enzymes. Although the Methanothermus sociabilis PPC was purified to homogeneity, the primary structure and the encoding gene of this protein were not identified (31, 47). A search of the genome databases with the amino acid sequences of known PPCs as queries did not detect a homolog of this enzyme in the archaea, even though such homologs were readily found in bacteria and eukaryotes. For these reasons and the probability that the methanarchaeal PPCs have a novel structure, we purified the enzyme from M. thermautotrophicus. Our results show that the PPC from M. thermautotrophicus has a novel structure and that most archaea possess a homolog of this enzyme.
Jean Vidal - One of the best experts on this subject based on the ideXlab platform.
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Effect of LiCl on Phosphoenolpyruvate Carboxylase kinase and the phosphorylation of Phosphoenolpyruvate Carboxylase in leaf disks and leaves of Sorghum vulgare
Planta, 2006Co-Authors: Jose Antonio Monreal, Jean Vidal, Cristina Echevarría, Francisco Javier López-baena, Sofía García-mauriñoAbstract:In the present work, the effect of LiCl on Phosphoenolpyruvate Carboxylase kinase (PEPCase-k), C_4 Phosphoenolpyruvate Carboxylase (PEPCase: EC 4.1.1.31) and its phosphorylation process has been investigated in illuminated leaf disks and leaves of the C_4 plant Sorghum vulgare . Although this salt induced severe damages to older leaves, it did not significantly alter the physiological parameters (photosynthesis, transpiration rate, intercellular CO_2 concentration) of young leaves. An immunological approach was used to demonstrate that the PEPCase-k protein accumulated rapidly in illuminated leaf tissues, consistent with the increase in its catalytic activity. In vivo, LiCl was shown to strongly enhance the light effect on PEPCase-k protein content, this process being dependent on protein synthesis. In marked contrast, the salt was found to inhibit the PEPCase-k activity in reconstituted assays and to decrease the C_4 PEPCase content and phosphorylation state in LiCl treated plants. Short-term (15 min) LiCl treatment increased IP_3 levels, PPCK gene expression, and PEPCase-k accumulation. Extending the treatment (1 h) markedly decreased IP_3 and PPCK gene expression, while PEPCase-k activity was kept high. The cytosolic protein synthesis inhibitor cycloheximide (CHX), which blocked the light-dependent up-regulation of the kinase in control plants, was found not to be active on this process in preilluminated, LiCl-treated leaves. This suggested that the salt causes the kinase turnover to be altered, presumably by decreasing degradation of the corresponding polypeptide. Taken together, these results establish PEPCase-k and PEPCase phosphorylation as lithium targets in higher plants and that this salt can provide a means to investigate further the organization and functioning of the cascade controlling the activity of both enzymes.
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Effect of LiCl on Phosphoenolpyruvate Carboxylase kinase and the phosphorylation of Phosphoenolpyruvate Carboxylase in leaf disks and leaves of Sorghum vulgare
Planta, 2006Co-Authors: Jose Antonio Monreal, Jean Vidal, Cristina Echevarría, Francisco Javier López-baena, Sofía García-mauriñoAbstract:In the present work, the effect of LiCl on Phosphoenolpyruvate Carboxylase kinase (PEPCase-k), C_4 Phosphoenolpyruvate Carboxylase (PEPCase: EC 4.1.1.31) and its phosphorylation process has been investigated in illuminated leaf disks and leaves of the C_4 plant Sorghum vulgare . Although this salt induced severe damages to older leaves, it did not significantly alter the physiological parameters (photosynthesis, transpiration rate, intercellular CO_2 concentration) of young leaves. An immunological approach was used to demonstrate that the PEPCase-k protein accumulated rapidly in illuminated leaf tissues, consistent with the increase in its catalytic activity. In vivo, LiCl was shown to strongly enhance the light effect on PEPCase-k protein content, this process being dependent on protein synthesis. In marked contrast, the salt was found to inhibit the PEPCase-k activity in reconstituted assays and to decrease the C_4 PEPCase content and phosphorylation state in LiCl treated plants. Short-term (15 min) LiCl treatment increased IP_3 levels, PPCK gene expression, and PEPCase-k accumulation. Extending the treatment (1 h) markedly decreased IP_3 and PPCK gene expression, while PEPCase-k activity was kept high. The cytosolic protein synthesis inhibitor cycloheximide (CHX), which blocked the light-dependent up-regulation of the kinase in control plants, was found not to be active on this process in preilluminated, LiCl-treated leaves. This suggested that the salt causes the kinase turnover to be altered, presumably by decreasing degradation of the corresponding polypeptide. Taken together, these results establish PEPCase-k and PEPCase phosphorylation as lithium targets in higher plants and that this salt can provide a means to investigate further the organization and functioning of the cascade controlling the activity of both enzymes.
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metabolite and post translational control of Phosphoenolpyruvate Carboxylase from leaves and mesophyll cell protoplasts of arabidopsis thaliana
Plant Science, 2005Co-Authors: A Goussetdupont, Jeannoel Pierre, Cristina Echevarría, B Lebouteiller, Jose Antonio Monreal, Michael Hodges, Jean VidalAbstract:Abstract The four Phosphoenolpyruvate Carboxylase encoding genes (1–4) of Arabidopsis thaliana were found to be expressed in rosette leaves, with PEPC2 mRNA being the major species. The enzyme activity was sensitive to feedback inhibition by malate, aspartate and glutamate and this effect was antagonized by glucose-6-phosphate and modulated after a dark–light transition. Taken together, the light/dark differences detected in pH response, malate sensitivity, Phosphoenolpyruvate Carboxylase kinase1 (PEPCk1) transcript content, and in vivo [ 32 P]-phosphate labelling indicates that PEPC is subjected to reversible, light-dependent phosphorylation on its N-terminal regulatory serine. The light-transduction pathway in isolated mesophyll cell protoplasts involves a PI-dependent phospholipase C (PI-PLC), the second messenger inositol 1,4,5 trisphosphate (IP 3 ), calcium fluxes via IP 3 -dependent tonoplast calcium channels, photosynthetic electron transport and PEPCk1 synthesis.
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The unique Phosphoenolpyruvate Carboxylase kinase
Plant Physiology and Biochemistry, 2003Co-Authors: Cristina Echevarría, Jean VidalAbstract:Abstract This paper deals with the contribution of P. Gadal’s group to the study of the Phosphoenolpyruvate Carboxylase protein kinase. It traces the important steps from the discovery up to the present time leading to characterize a new protein kinase which is specific for plants and displays original properties.
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reversible phosphorylation in the regulation of photosynthetic Phosphoenolpyruvate Carboxylase in c4 plants
2001Co-Authors: Jean Vidal, Sylvie Coursol, Jeannoel PierreAbstract:C4 species have a specific isoform of Phosphoenolpyruvate Carboxylase (PEPC) that catalyzes primary CO2 fixation in the C4 photosynthesis pathway. It has long been known that the enzyme in the cytosol of the mesophyll cells is subject to allosteric control by opposing photosynthesis-related metabolites. The discovery of a phosphorylation process acting on C4 PEPC, via a complex light-signal transduction cascade, has revitalized interest in this enzyme and the ensuing wealth of data has highlighted one of a few signaling cascades known so far in the regulation of plant metabolism. The cascade depends upon a cross-talk between the two neighboring photosynthetic cell types, involves classical second messengers like pH, Inosital-1,4 5-trisphosphate (Ins(1,4,5) P3) and calcium, and upregulates the activity of a Ca2+-independent, C4 PEPC-specific protein-serine/threonine kinase, which finally phosphorylates PEPC. The final activity of C4 PEPC and the resulting carbon flux to bundle sheath cells are dependent on the mutual interaction between metabolite and covalent control mechanisms acting on this enzyme.
Bernard R. Glick - One of the best experts on this subject based on the ideXlab platform.
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The synthesis of Phosphoenolpyruvate Carboxylase in imbibing sorghum seeds
Biochemistry and cell biology = Biochimie et biologie cellulaire, 1991Co-Authors: Eli Khayat, Erwin B. Dumbroff, Bernard R. GlickAbstract:When sorghum seeds were imbibed either in the light or in the dark, the presence of newly synthesized Phosphoenolpyruvate Carboxylase (PEPC) could be detected immunologically after approximately 6 h. In addition, both PEPC mRNA and enzyme activity were detected in extracts of dry seeds prior to imbibition. By contrast, ribulose-1,5-bisphosphate Carboxylase mRNA, protein, and activity, as well as chlorophyll, were not detected even after 24 h of imbibition. These observations suggest that the nonphotosynthetic form of PEPC is synthesized during seed development and may play an important role in the germinative process.Key words: Phosphoenolpyruvate Carboxylase, sorghum seeds, germination, ribulose-1,5-bisphosphate Carboxylase.
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The synthesis of Phosphoenolpyruvate Carboxylase in imbibing sorghum seeds.
Biochemistry and Cell Biology, 1991Co-Authors: Eli Khayat, Erwin B. Dumbroff, Bernard R. GlickAbstract:When sorghum seeds were imbibed either in the light or in the dark, the presence of newly synthesized Phosphoenolpyruvate Carboxylase (PEPC) could be detected immunologically after approximately 6 h. In addition, both PEPC mRNA and enzyme activity were detected in extracts of dry seeds prior to imbibition. By contrast, ribulose-1,5-bisphosphate Carboxylase mRNA, protein, and activity, as well as chlorophyll, were not detected even after 24 h of imbibition. These observations suggest that the nonphotosynthetic form of PEPC is synthesized during seed development and may play an important role in the germinative process.Key words: Phosphoenolpyruvate Carboxylase, sorghum seeds, germination, ribulose-1,5-bisphosphate Carboxylase.
Pierre Gadal - One of the best experts on this subject based on the ideXlab platform.
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Phosphoenolpyruvate Carboxylase: structure, regulation and evolution
Plant Science, 1994Co-Authors: Loïc Lepiniec, Raymond Chollet, Jean Vidal, Pierre Gadal, Claude CrétinAbstract:Abstract Plant Phosphoenolpyruvate Carboxylase (EC 4.1.1.31; PEPC) is encoded by a small multigene family in which the expression of each member is controlled individually by exogenous (light, environmental) and/or endogenous (hormonal and developmental) stimuli. The involvement of putative trans-actig factors and consensus cis-elements of promoters in the specific transcriptional regulation of the PEPC genes is discussed. At the post-translational level, the regulatory strategy of the plant enzyme is mainly to offset the negative effect of the feedback inhibitor, L-malate, the end-product of the oxaloacetate reduction. All plant PEPC-forms are under positive and negative allosteric control by metabolite effectors and possess a consensus phosphorylation site containing a target serine residue near their N-terminus (e.g. Ser8 in C4 PEPC from sorghum). In C4 and Crassulacean acid metabolism (CAM) plants, a complex signal-transduction chain activates a Ca2+-independent protein-serine kinase responsible for regulatory phosphorylation of PEPC. A more thorough understanding of the functional and regulatory properties of the bacterial and C4 enzymes has emerged by exploiting recombinant proteins and site-directed mutagenesis. In these newly opened areas, PEPC offers one of the best characterized paradigms of plant signaling. Finally, some emerging ideas on the evolution and phylogenetic relationships of the various PEPC isoforms are presented.
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Production inEscherichia coli of activeSorghum Phosphoenolpyruvate Carboxylase which can be phosphorylated
Plant Molecular Biology, 1991Co-Authors: Claude Crétin, Jean Vidal, Loïc Lepiniec, Naïma Bakrim, Eliane Kéryer, Simonetta Santi, Pierre GadalAbstract:Phosphoenolpyruvate Carboxylase (PEPC)-deficient mutants of Escherichia coli have been complemented with a plasmid bearing a full-length cDNA encoding the C4-type form of Sorghum leaf PEPC. Transformed cells grew on minimal medium. Two clones were selected which produce a functional and full-sized enzyme protein as determined by activity assays, immunochemical behavior and SDS-PAGE. In addition, regulatory phosphorylation of immunopurified recombinant PEPC was observed when the enzyme was incubated with a partially purified plant PEPC kinase. These results establish that E. coli cells produce a genuine, phosphate-free, higher-plant PEPC. Application of immunoadsorbtion chromatography to bacterial extracts makes it possible to prepare highly pure protein available for biochemical studies.
Min-xian Wu - One of the best experts on this subject based on the ideXlab platform.
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Inactivation of Maize Phosphoenolpyruvate Carboxylase by Urea
Plant Physiology, 1992Co-Authors: Randolph T. Wedding, Paul Dole, Thierry Chardot, Min-xian WuAbstract:Phosphoenolpyruvate Carboxylase purified from leaves of maize (Zea mays, L.) is sensitive to the presence of urea. Exposure to 2.5 m urea for 30 min completely inactivates the enzyme, whereas for a concentration of 1.5 m urea, about 1 h is required. Malate appears to have no effect on inactivation by urea of Phosphoenolpyruvate Carboxylase. However, the presence of 20 mm Phosphoenolpyruvate or 20 mm glucose-6-phosphate prevents significant inactivation by 1.5 m urea for at least 1 h. The inactivation by urea is reversible by dilution. The inhibition by urea and the protective effects of Phosphoenolpyruvate and glucose-6-phosphate are associated with changes in aggregation state.
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Inactivation of Maize Phosphoenolpyruvate Carboxylase by Urea
Plant Physiology, 1992Co-Authors: Randolph T. Wedding, Paul Dole, Thierry Chardot, Min-xian WuAbstract:Phosphoenolpyruvate Carboxylase purified from leaves of maize (Zea mays, L.) is sensitive to the presence of urea. Exposure to 2.5 m urea for 30 min completely inactivates the enzyme, whereas for a concentration of 1.5 m urea, about 1 h is required. Malate appears to have no effect on inactivation by urea of Phosphoenolpyruvate Carboxylase. However, the presence of 20 mm Phosphoenolpyruvate or 20 mm glucose-6-phosphate prevents significant inactivation by 1.5 m urea for at least 1 h. The inactivation by urea is reversible by dilution. The inhibition by urea and the protective effects of Phosphoenolpyruvate and glucose-6-phosphate are associated with changes in aggregation state.
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Inactivation of maize leaf Phosphoenolpyruvate Carboxylase by the binding to chloroplast membranes.
Plant Physiology, 1992Co-Authors: Min-xian Wu, Randolph T. WeddingAbstract:Phosphoenolpyruvate Carboxylase (PEPC) purified from maize (Zea mays L.) leaves associates with maize leaf chloroplast membrane in vitro. The binding of PEPC to the membrane results in enzyme inactivation. A protein isolated from a maize leaf chloroplast membrane preparation inactivated PEPC. Treatment with membrane preparation or with partially purified inactivating protein accelerates PEPC inactivation at low temperature (4°C). Interaction of PEPC with chloroplast membrane or inactivating protein may inactivate the enzyme by influencing dissociation of the enzyme active tetramer.