Author: "Gilles Curien" - Searchworks@Jio Institute Digital Library Search Results

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1. The Arabidopsis leaf quantitative atlas: a cellular and subcellular mapping through unified data integration

Author: Dimitri Tolleter, Edward N. Smith, Clémence Dupont-Thibert, Clarisse Uwizeye, Denis Vile, Pauline Gloaguen, Denis Falconet, Giovanni Finazzi, Yves Vandenbrouck, and Gilles Curien
Subjects: Arabidopsis, curation, leaf anatomy, modelling, organelle, Plant culture, SB1-1110, Botany, QK1-989
Abstract: Quantitative analyses and models are required to connect a plant’s cellular organisation with its metabolism. However, quantitative data are often scattered over multiple studies, and finding such data and converting them into useful information is time-consuming. Consequently, there is a need to centralise the available data and to highlight the remaining knowledge gaps. Here, we present a step-by-step approach to manually extract quantitative data from various information sources, and to unify the data format. First, data from Arabidopsis leaf were collated, checked for consistency and correctness and curated by cross-checking sources. Second, quantitative data were combined by applying calculation rules. They were then integrated into a unique comprehensive, referenced, modifiable and reusable data compendium representing an Arabidopsis reference leaf. This atlas contains the metrics of the 15 cell types found in leaves at the cellular and subcellular levels.
Published: 2024
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2. Morphological bases of phytoplankton energy management and physiological responses unveiled by 3D subcellular imaging

Author: Clarisse Uwizeye, Johan Decelle, Pierre-Henri Jouneau, Serena Flori, Benoit Gallet, Jean-Baptiste Keck, Davide Dal Bo, Christine Moriscot, Claire Seydoux, Fabien Chevalier, Nicole L. Schieber, Rachel Templin, Guillaume Allorent, Florence Courtois, Gilles Curien, Yannick Schwab, Guy Schoehn, Samuel C. Zeeman, Denis Falconet, and Giovanni Finazzi
Subjects: Science
Abstract: Phytoplankton account for a large proportion of global primary production and comprise a number of phylogenetically distinct lineages. Here, Uwizeye et al. use FIB-SEM to study ultrastructural plasticity of 7 distinct taxa and describe how subcellular organisation is linked to energy metabolism.
Published: 2021
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3. Consequences of Mixotrophy on Cell Energetic Metabolism in Microchloropsis gaditana Revealed by Genetic Engineering and Metabolic Approaches

Author: Davide Dal Bo, Leonardo Magneschi, Mariette Bedhomme, Elodie Billey, Etienne Deragon, Mattia Storti, Mathilde Menneteau, Christelle Richard, Camille Rak, Morgane Lapeyre, Mehdi Lembrouk, Melissa Conte, Valérie Gros, Guillaume Tourcier, Cécile Giustini, Denis Falconet, Gilles Curien, Guillaume Allorent, Dimitris Petroutsos, Frédéric Laeuffer, Laurent Fourage, Juliette Jouhet, Eric Maréchal, Giovanni Finazzi, and Séverine Collin
Subjects: Microchloropsis gaditana, mixotrophy, photosynthesis, mitochondrial alternative oxidase, TALE nuclease, lipid metabolism, Plant culture, SB1-1110
Abstract: Algae belonging to the Microchloropsis genus are promising organisms for biotech purposes, being able to accumulate large amounts of lipid reserves. These organisms adapt to different trophic conditions, thriving in strict photoautotrophic conditions, as well as in the concomitant presence of light plus reduced external carbon as energy sources (mixotrophy). In this work, we investigated the mixotrophic responses of Microchloropsis gaditana (formerly Nannochloropsis gaditana). Using the Biolog growth test, in which cells are loaded into multiwell plates coated with different organic compounds, we could not find a suitable substrate for Microchloropsis mixotrophy. By contrast, addition of the Lysogeny broth (LB) to the inorganic growth medium had a benefit on growth, enhancing respiratory activity at the expense of photosynthetic performances. To further dissect the role of respiration in Microchloropsis mixotrophy, we focused on the mitochondrial alternative oxidase (AOX), a protein involved in energy management in other algae prospering in mixotrophy. Knocking-out the AOX1 gene by transcription activator-like effector nuclease (TALE-N) led to the loss of capacity to implement growth upon addition of LB supporting the hypothesis that the effect of this medium was related to a provision of reduced carbon. We conclude that mixotrophic growth in Microchloropsis is dominated by respiratory rather than by photosynthetic energetic metabolism and discuss the possible reasons for this behavior in relationship with fatty acid breakdown via β-oxidation in this oleaginous alga.
Published: 2021
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4. Crystal Structure of the Chloroplastic Oxoene Reductase ceQORH from Arabidopsis thaliana

Author: Sarah Mas y mas, Gilles Curien, Cécile Giustini, Norbert Rolland, Jean-Luc Ferrer, and David Cobessi
Subjects: chloroplast envelope quinone oxidoreductase homolog, oxylipins, γ-ketols, α, β-unsaturated carbonyls, X-ray crystallography, Plant culture, SB1-1110
Abstract: Enzymatic and non-enzymatic peroxidation of polyunsaturated fatty acids give rise to accumulation of aldehydes, ketones, and α,β-unsaturated carbonyls of various lengths, known as oxylipins. Oxylipins with α,β-unsaturated carbonyls are reactive electrophile species and are toxic. Cells have evolved several mechanisms to scavenge reactive electrophile oxylipins and decrease their reactivity such as by coupling with glutathione, or by reduction using NAD(P)H-dependent reductases and dehydrogenases of various substrate specificities. Plant cell chloroplasts produce reactive electrophile oxylipins named γ-ketols downstream of enzymatic lipid peroxidation. The chloroplast envelope quinone oxidoreductase homolog (ceQORH) from Arabidopsis thaliana was previously shown to reduce the reactive double bond of γ-ketols. In marked difference with its cytosolic homolog alkenal reductase (AtAER) that displays a high activity toward the ketodiene 13-oxo-9(Z),11(E)-octadecadienoic acid (13-KODE) and the ketotriene 13-oxo-9(Z), 11(E), 15(Z)-octadecatrienoic acid (13-KOTE), ceQORH binds, but does not reduce, 13-KODE and 13-KOTE. Crystal structures of apo-ceQORH and ceQORH bound to 13-KOTE or to NADP+ and 13-KOTE have been solved showing a large ligand binding site, also observed in the structure of the cytosolic alkenal/one reductase. Positioning of the α,β-unsaturated carbonyl of 13-KOTE in ceQORH-NADP+-13-KOTE, far away from the NADP+ nicotinamide ring, provides a rational for the absence of activity with the ketodienes and ketotrienes. ceQORH is a monomeric enzyme in solution whereas other enzymes from the quinone oxidoreductase family are stable dimers and a structural explanation of this difference is proposed. A possible in vivo role of ketodienes and ketotrienes binding to ceQORH is also discussed.
Published: 2017
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5. E. coli chromosomal-driven expression of NADK2 from A. thaliana: A preferable alternative to plasmid-driven expression for challenging proteins

Author: Matthieu Goussé, Elisa Dell’Aglio, Gilles Curien, Stéphanie Borland, Sébastien Renoud, Caroline Ranquet, Alexia Chandor-Proust, BGene Genetics, Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), Light Photosynthesis & Metabolism (Photosynthesis), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and start-up Bgene
Subjects: Arabidopsis, Escherichia coli, E. coli, Expression system, T7-based expression, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Chromosomal insertion, NAD, Recombinant Proteins, NAD kinase, Biotechnology, Plasmids
Abstract: International audience; The expression and purification of large recombinant proteins or protein complexes is problematic for somebiotechnology laboratories. Indeed, it is often difficult to obtain enough active proteins to perform biologicalcharacterization or reach commercialization, when large proteins or protein complexes are expressed in E. colivia the popular T7-based plasmid-driven expression system. There is also an industrial demand to decrease our dependence on plasmid-driven expression, because of itsdrawbacks, such as: i) the common use of antibiotics to maintain the plasmid, ii) the issue of plasmid copynumber, and iii) the risk of overloading the expression system. Despite all these issues, alternative solutions, suchas gene integration in the bacterial chromosome, are rarely employed and their advantages are still a matter ofdebate.Plant plastidial NAD kinases (NADK; ATP:NAD 2′-phosphotransferase, EC 2.7.1.23) are a classic example ofproteins with high molecular weight, that are difficult to express and purify with traditional T7-based tech-nology. We therefore compared plasmid-driven and chromosomal-driven expression of the Arabidopsis thalianaNADK2 protein, using a proprietary counter-selection tool, COLIBELT®, that allows scar-free and marker-freechromosomal modifications.Here we show that chromosomal-driven expression allowed recovery of more active NADK2 protein thanclassic T7 expression systems, as well as better production, thus confirming that expression from one singlechromosomal copy is preferable to plasmid-driven expression and might be appealing for both basic and appliedresearch.
Published: 2022

6. Mixotrophic growth of the extremophile Galdieria sulphuraria reveals the flexibility of its carbon assimilation metabolism

Author: Andreas P.M. Weber, Phillip Westhoff, Marianne Tardif, Denis Falconet, Erika Guglielmino, Dagmar Lyska, Benoit Gallet, Giovanni Finazzi, Gilles Curien, Clément Hallopeau, Michele Carone, Davide Dal Bo, Claire Remacle, Johan Decelle, Sabine Brugière, Myriam Ferro, Janina Janetzko, Light Photosynthesis & Metabolism (Photosynthesis), Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), Cluster of Excellence on Plant Sciences (CEPLAS), Heinrich Heine Universität Düsseldorf = Heinrich Heine University [Düsseldorf]-Max Planck Institute for Plant Breeding Research (MPIPZ)-Universität zu Köln = University of Cologne, Etude de la dynamique des protéomes (EDyP), BioSanté (UMR BioSanté), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Photosymbiose, Institut de biologie structurale (IBS - UMR 5075), LIPID, Université de Liège, ARC grant (DARKMET proposal) for Concerted Research Actions (17/21-08), financed by the French Community of Belgium (Wallonia-Brussels Federation)., Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany´s Excellence Strategy – EXC-2048/1 – project ID 390686111, ANR-17-CE05-0029,MoMix,Modélisation de la Mixotrophie chez l'algue extrêmophile Galdieria sulphuraria(2017), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), ANR-10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), European Project: 833184, ChloroMito, Martin-Laffon, Jacqueline, Modélisation de la Mixotrophie chez l'algue extrêmophile Galdieria sulphuraria - - MoMix2017 - ANR-17-CE05-0029 - AAPG2017 - VALID, Grenoble Alliance for Integrated Structural Cell Biology - - GRAL2010 - ANR-10-LABX-0049 - LABX - VALID, CBH-EUR-GS - - CBH-EUR-GS2017 - ANR-17-EURE-0003 - EURE - VALID, Infrastructure Française de Protéomique - - ProFI2010 - ANR-10-INBS-0008 - INBS - VALID, Chloroplast and Mitochondria interactions for microalgal acclimation - ChloroMito - 833184 - INCOMING, Universität zu Köln-Heinrich Heine Universität Düsseldorf = Heinrich Heine University [Düsseldorf]-Max Planck Institute for Plant Breeding Research (MPIPZ), and InBios/Phytosystems Research Unit, University of Liege
Subjects: Proteomics, 0106 biological sciences, 0301 basic medicine, photorespiration, Physiology, Heterotroph, Plant Science, Photosynthesis, 7. Clean energy, 01 natural sciences, Galdieria sulphuraria, Extremophiles, 03 medical and health sciences, mixotrophy, Total inorganic carbon, Botany, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, red algae, photosynthesis, biology, Chemistry, Research, RuBisCO, Heterotrophic Processes, Carbon Dioxide, Full Papers, 15. Life on land, Carbon, 030104 developmental biology, Rhodophyta, biology.protein, Photorespiration, Energy source, Mixotroph, 010606 plant biology & botany
Abstract: Summary Galdieria sulphuraria is a cosmopolitan microalga found in volcanic hot springs and calderas. It grows at low pH in photoautotrophic (use of light as a source of energy) or heterotrophic (respiration as a source of energy) conditions, using an unusually broad range of organic carbon sources. Previous data suggested that G. sulphuraria cannot grow mixotrophically (simultaneously exploiting light and organic carbon as energy sources), its photosynthetic machinery being repressed by organic carbon.Here, we show that G. sulphuraria SAG21.92 thrives in photoautotrophy, heterotrophy and mixotrophy. By comparing growth, biomass production, photosynthetic and respiratory performances in these three trophic modes, we show that addition of organic carbon to cultures (mixotrophy) relieves inorganic carbon limitation of photosynthesis thanks to increased CO2 supply through respiration. This synergistic effect is lost when inorganic carbon limitation is artificially overcome by saturating photosynthesis with added external CO2.Proteomic and metabolic profiling corroborates this conclusion suggesting that mixotrophy is an opportunistic mechanism to increase intracellular CO2 concentration under physiological conditions, boosting photosynthesis by enhancing the carboxylation activity of Ribulose‐1,5‐bisphosphate carboxylase‐oxygenase (Rubisco) and decreasing photorespiration.We discuss possible implications of these findings for the ecological success of Galdieria in extreme environments and for biotechnological applications.
Published: 2021

7. Identification of the Arabidopsis Calmodulin-Dependent NAD+ Kinase That Sustains the Elicitor-Induced Oxidative Burst

Author: Michel Matringe, Giovanni Finazzi, Guillaume Decros, Yohann Couté, Alexandra Kraut, Christian Mazars, Alex Costa, Gilles Curien, Cécile Giustini, Yves Gibon, Elisa Dell’Aglio, Light Photosynthesis & Metabolism (Photosynthesis), Physiologie cellulaire et végétale (LPCV), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Etude de la dynamique des protéomes (EDyP), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Department of Biosciences. University of Milan, Biologie du fruit et pathologie (BFP), Université Bordeaux Segalen - Bordeaux 2-Institut National de la Recherche Agronomique (INRA)-Université Sciences et Technologies - Bordeaux 1, Laboratoire de Recherche en Sciences Végétales (LRSV), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, HFSP grant RGP0052/2015, Transition Grant 2015/2017 – Horizon 2020 Linea 1A, ANR–10–LABEX–04 ,GRAL,Labex, ANR-10-INBS-08-01/10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Etude de la dynamique des protéomes (EDyP ), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Université Sciences et Technologies - Bordeaux 1-Université Bordeaux Segalen - Bordeaux 2-Institut National de la Recherche Agronomique (INRA), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), ANR-10-LABX-0004,CeMEB,Mediterranean Center for Environment and Biodiversity(2010), ANR-10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Università degli Studi di Milano = University of Milan (UNIMI), Université Bordeaux Segalen - Bordeaux 2-Institut National de la Recherche Agronomique (INRA)-Université Sciences et Technologies - Bordeaux 1 (UB), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)
Subjects: 0106 biological sciences, chemistry.chemical_classification, biology, Calmodulin, Physiology, Chemistry, food and beverages, Plant Science, biology.organism_classification, 01 natural sciences, Cofactor, Elicitor, Enzyme, Biochemistry, Arabidopsis, Genetics, biology.protein, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, Arabidopsis thaliana, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, NAD+ kinase, Kinase activity, 010606 plant biology & botany
Abstract: NADP(H) is an essential cofactor of multiple metabolic processes in all living organisms, and in plants, NADP(H) is required as the substrate of Ca2+-dependent NADPH oxidases, which catalyze a reactive oxygen species burst in response to various stimuli. While NADP+ production in plants has long been known to involve a calmodulin (CaM)/Ca2+-dependent NAD+ kinase, the nature of the enzyme catalyzing this activity has remained enigmatic, as has its role in plant physiology. Here, we used proteomic, biochemical, molecular, and in vivo analyses to identify an Arabidopsis (Arabidopsis thaliana) protein that catalyzes NADP+ production exclusively in the presence of CaM/Ca2+. This enzyme, which we named NAD kinase-CaM dependent (NADKc), has a CaM-binding peptide located in its N-terminal region and displays peculiar biochemical properties as well as different domain organization compared with known plant NAD+ kinases. In response to a pathogen elicitor, the activity of NADKc, which is associated with the mitochondrial periphery, contributes to an increase in the cellular NADP+ concentration and to the amplification of the elicitor-induced oxidative burst. Based on a phylogenetic analysis and enzymatic assays, we propose that the CaM/Ca2+-dependent NAD+ kinase activity found in photosynthetic organisms is carried out by NADKc-related proteins. Thus, NADKc represents the missing link between Ca2+ signaling, metabolism, and the oxidative burst.
Published: 2019

8. Morphological bases of phytoplankton energy management and physiological responses unveiled by 3D subcellular imaging

Author: Guy Schoehn, Clarisse Uwizeye, Gilles Curien, Rachel Templin, Samuel C. Zeeman, Yannick Schwab, Davide Dal Bo, Denis Falconet, Fabien Chevalier, Serena Flori, Christine Moriscot, Florence Courtois, Claire Seydoux, Jean-Baptiste Keck, Benoit Gallet, Johan Decelle, Nicole L. Schieber, Pierre-Henri Jouneau, Giovanni Finazzi, Guillaume Allorent, LIPID, Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), Photosymbiose, Modélisation et Exploration des Matériaux (MEM), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Light Photosynthesis & Metabolism (Photosynthesis), Institut de biologie structurale (IBS - UMR 5075), Calcul des Variations, Géométrie, Image (CVGI), Laboratoire Jean Kuntzmann (LJK), Institut National de Recherche en Informatique et en Automatique (Inria)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP ), Université Grenoble Alpes (UGA)-Institut National de Recherche en Informatique et en Automatique (Inria)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP ), Université Grenoble Alpes (UGA), Cell Biology and Cell Biophysics Unit, European Molecular Biology Laboratory, European Molecular Biology Laboratory [Grenoble] (EMBL), Department of Biology [ETH Zürich] (D-BIOL), Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich), Défi X-Life grant from CNRS, CEA DRFimpulsion FIB-Bioprogram, Auvergne-Rhône-Alpes Region, Fondation Recherche Medicale (FRM), FEDER and the GIS-Infrastructures en Biologie Santé et Agronomie (IBiSA), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), ANR-17-CE05-0029,MoMix,Modélisation de la Mixotrophie chez l'algue extrêmophile Galdieria sulphuraria(2017), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-15-IDEX-0002,UGA,IDEX UGA(2015), European Project: 833184, ChloroMito, and European Project: 654248,H2020,H2020-INFRADEV-1-2014-1,CORBEL(2015)
Subjects: 0106 biological sciences, 0301 basic medicine, Cell biology, Light, Bioenergetics, MESH: Mitochondria, Acclimatization, Science, General Physics and Astronomy, MESH: Acclimatization, MESH: Imaging, Three-Dimensional, Cellular imaging, Photosynthesis, 01 natural sciences, Article, General Biochemistry, Genetics and Molecular Biology, Pyrenoid, MESH: Microalgae, 03 medical and health sciences, Imaging, Three-Dimensional, Phytoplankton, Microalgae, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Plastids, Plastid, Cellular microbiology, Trophic level, Biomass (ecology), Multidisciplinary, biology, Ecology, MESH: Energy Metabolism, fungi, MESH: Plastids, General Chemistry, biology.organism_classification, MESH: Light, Mitochondria, 030104 developmental biology, Diatom, MESH: Subcellular Fractions, Energy Metabolism, Subcellular Fractions, MESH: Phytoplankton, 010606 plant biology & botany
Abstract: Eukaryotic phytoplankton have a small global biomass but play major roles in primary production and climate. Despite improved understanding of phytoplankton diversity and evolution, we largely ignore the cellular bases of their environmental plasticity. By comparative 3D morphometric analysis across seven distant phytoplankton taxa, we observe constant volume occupancy by the main organelles and preserved volumetric ratios between plastids and mitochondria. We hypothesise that phytoplankton subcellular topology is modulated by energy-management constraints. Consistent with this, shifting the diatom Phaeodactylum from low to high light enhances photosynthesis and respiration, increases cell-volume occupancy by mitochondria and the plastid CO2-fixing pyrenoid, and boosts plastid-mitochondria contacts. Changes in organelle architectures and interactions also accompany Nannochloropsis acclimation to different trophic lifestyles, along with respiratory and photosynthetic responses. By revealing evolutionarily-conserved topologies of energy-managing organelles, and their role in phytoplankton acclimation, this work deciphers phytoplankton responses at subcellular scales., Nature Communications, 12 (1), ISSN:2041-1723
Published: 2021

9. ChloroKB, a cell metabolism reconstruction of the model plant Arabidopsis thaliana

Author: Pauline Gloaguen, Yves Vandenbrouck, Gilles Curien, Jacques Joyard, Light Photosynthesis & Metabolism (Photosynthesis), Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Martin-Laffon, Jacqueline
Subjects: 0106 biological sciences, Chloroplasts, [SDV]Life Sciences [q-bio], Arabidopsis, Metabolic network, Computational biology, MESH: Arabidopsis Proteins, 01 natural sciences, Mesophyll Cell, Chloroplast, Modelling, [SDV.BV.BOT] Life Sciences [q-bio]/Vegetal Biology/Botanics, Knowledge base, Plant cell, 03 medical and health sciences, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Arabidopsis thaliana, MESH: Arabidopsis, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Plant metabolism, 030304 developmental biology, 2. Zero hunger, 0303 health sciences, MESH: Chloroplasts, biology, Arabidopsis Proteins, food and beverages, General Medicine, [SDV.BV.BOT]Life Sciences [q-bio]/Vegetal Biology/Botanics, biology.organism_classification, Cell metabolism, Metabolism, MESH: Metabolic Networks and Pathways, Metabolic Networks and Pathways, 010606 plant biology & botany
Abstract: Can we understand how plant cell metabolism really works? An integrated large-scale modelling of plant metabolism predictive model would make possible to analyse the impact of disturbances in environmental conditions on cellular functioning and diversity of plant-made molecules of interest. ChloroKB, a Web application initially developed for exploration of Arabidopsis chloroplast metabolic network now covers Arabidopsis mesophyll cell metabolism. Interconnected metabolic maps show subcellular compartments, metabolites, proteins, complexes, reactions, and transport. Data in ChloroKB have been structured to allow for mathematical modelling and will be used as a reference for modelling work dedicated to a particular issue., Peut-on comprendre comment fonctionne réellement le métabolisme des cellules végétales ? Un modèle prédictif intégré à grande échelle du métabolisme des plantes permettrait d’analyser l’impact des perturbations des conditions environnementales sur le fonctionnement cellulaire et la diversité des molécules d’intérêt fabriquées par les plantes. ChloroKB, une application Web initialement développée pour l’exploration du réseau métabolique du chloroplaste d’Arabidopsis, couvre désormais le métabolisme des cellules du mésophylle d’Arabidopsis. Des cartes métaboliques interconnectées décrivent les compartiments subcellulaires, les métabolites, les protéines, les complexes, les réactions et le transport. Les données de ChloroKB ont été structurées pour permettre la modélisation mathématique et seront utilisées comme référence pour les travaux de modélisation consacrés à une question particulière.
Published: 2021

10. In-cell quantitative structural imaging of phytoplankton using 3D electron microscopy

Author: Nicole L. Schieber, Gilles Curien, Rachel Templin, Johan Decelle, Jean-Baptiste Keck, Samuel C. Zeeman, Denis Falconet, Pierre-Henri Jouneau, Christine Moriscot, Guy Schoehn, Fabien Chevalier, Giovanni Finazzi, Clarisse Uwizeye, Benoit Gallet, Yannick Schwab, LIPID, Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), Light Photosynthesis & Metabolism (Photosynthesis), Modélisation et Exploration des Matériaux (MEM), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Institut de biologie structurale (IBS - UMR 5075), Calcul des Variations, Géométrie, Image (CVGI), Laboratoire Jean Kuntzmann (LJK), Institut National de Recherche en Informatique et en Automatique (Inria)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP ), Université Grenoble Alpes (UGA)-Institut National de Recherche en Informatique et en Automatique (Inria)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP ), Université Grenoble Alpes (UGA), Integrated Structural Biology Grenoble (ISBG - UMS 3518 ), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-European Molecular Biology Laboratory [Grenoble] (EMBL)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), European Molecular Biology Laboratory (EMBL), Department of Biology [ETH Zürich] (D-BIOL), Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich), This project received funding from the European Research Council: ERC Chloro-mito (grant no. 833184). Research was also supported by a Défi X-Life grant from CNRS, funds from the CEA DRF impulsion FIB-Bio program, the LabEx GRAL (ANR-10-LABX-49-01), financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003) and the ANR ‘Momix’ (Projet-ANR-17-CE05-0029). This project also received funds from the European Union’s Horizon 2020 research and innovation programme CORBEL under the grant agreement No 654248. This work used the platforms of the Grenoble Instruct-ERIC centre (ISBG, UMS 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-05-02) and GRAL. The electron microscope facility is supported by the Auvergne-Rhône-Alpes Region, the Fondation Recherche Medicale (FRM), the funds FEDER and the GIS-Infrastructures en Biologie Santé et Agronomie (IBISA). J.D. was supported by ATIP-Avenir program. C.U. is supported by a joint UGA-ETH Zurich PhD grant in the framework of the 'Investissements d’avenir' programme (ANR-15-IDEX-02). The authors thank the Roscoff Culture Collection that provided phytoplankton strains and Noan Le Bescot (Ternog Design) for help in the conception and realisation of the figures of this article., ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), ANR-17-CE05-0029,MoMix,Modélisation de la Mixotrophie chez l'algue extrêmophile Galdieria sulphuraria(2017), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-15-IDEX-0002,UGA,IDEX UGA(2015), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), and Integrated Structural Biology Grenoble (ISBG)
Subjects: 0106 biological sciences, Abiotic component, 0303 health sciences, Biomass (ecology), Biotic component, Chemistry, [SDV]Life Sciences [q-bio], fungi, Genomics, 01 natural sciences, Chromatin, 03 medical and health sciences, Phytoplankton, Organelle, Biophysics, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, 030304 developmental biology, 010606 plant biology & botany, Biomineralization
Abstract: Phytoplankton is a minor fraction of the global biomass playing a major role in primary production and climate. Despite improved understanding of phytoplankton diversity and genomics, we lack nanoscale subcellular imaging approaches to understand their physiology and cell biology. Here, we present a complete Focused Ion Beam - Scanning Electron Microscopy (FIB-SEM) workflow (from sample preparation to image processing) to generate nanometric 3D phytoplankton models. Tomograms of entire cells, representatives of six ecologically-successful phytoplankton unicellular eukaryotes, were used for quantitative morphometric analysis. Besides lineage-specific cellular architectures, we observed common features related to cellular energy management: i) conserved cell-volume fractions occupied by the different organelles; ii) consistent plastid-mitochondria interactions, iii) constant volumetric ratios in these energy-producing organelles. We revealed detailed subcellular features related to chromatin organization and to biomineralization. Overall, this approach opens new perspectives to study phytoplankton acclimation responses to abiotic and biotic factors at a relevant biological scale.
Published: 2020

11. Identification of the Arabidopsis Calmodulin-Dependent NAD

Author: Elisa, Dell'Aglio, Cécile, Giustini, Alexandra, Kraut, Yohann, Couté, Alex, Costa, Guillaume, Decros, Yves, Gibon, Christian, Mazars, Michel, Matringe, Giovanni, Finazzi, and Gilles, Curien
Subjects: Research Report, Arabidopsis Proteins, Arabidopsis, food and beverages, Models, Biological, Mitochondria, Kinetics, Phosphotransferases (Alcohol Group Acceptor), Protein Domains, Seedlings, Calcium-Calmodulin-Dependent Protein Kinases, Calcium, Amino Acid Sequence, Photosynthesis, Peptides, Phylogeny, Flagellin, Protein Binding, Respiratory Burst
Abstract: NADP(H) is an essential cofactor of multiple metabolic processes in all living organisms, and in plants, NADP(H) is required as the substrate of Ca(2+)-dependent NADPH oxidases, which catalyze a reactive oxygen species burst in response to various stimuli. While NADP(+) production in plants has long been known to involve a calmodulin (CaM)/Ca(2+)-dependent NAD(+) kinase, the nature of the enzyme catalyzing this activity has remained enigmatic, as has its role in plant physiology. Here, we used proteomic, biochemical, molecular, and in vivo analyses to identify an Arabidopsis (Arabidopsis thaliana) protein that catalyzes NADP(+) production exclusively in the presence of CaM/Ca(2+). This enzyme, which we named NAD kinase-CaM dependent (NADKc), has a CaM-binding peptide located in its N-terminal region and displays peculiar biochemical properties as well as different domain organization compared with known plant NAD(+) kinases. In response to a pathogen elicitor, the activity of NADKc, which is associated with the mitochondrial periphery, contributes to an increase in the cellular NADP(+) concentration and to the amplification of the elicitor-induced oxidative burst. Based on a phylogenetic analysis and enzymatic assays, we propose that the CaM/Ca(2+)-dependent NAD(+) kinase activity found in photosynthetic organisms is carried out by NADKc-related proteins. Thus, NADKc represents the missing link between Ca(2+) signaling, metabolism, and the oxidative burst.
Published: 2019

12. Tyrosine metabolism: identification of a key residue in the acquisition of prephenate aminotransferase activity by 1β aspartate aminotransferase

Author: Gilles Curien, Cécile Giustini, Matthieu Graindorge, David Cobessi, Michel Matringe, Adeline Robin, Serge Crouzy, Physiologie cellulaire et végétale (LPCV), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Groupe modélisation et chimie théorique (MCT), Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), FRISBI ANR-10-INSB-05-02, GRAL within the Grenoble Partnership for Structural Biology (PSB) ANR-10-LABX-49-01, Institut National de la Recherche Agronomique (INRA), Centre National de la Recherche Scientifique (CNRS), French Atomic Energy Commission, University of Grenoble Alpes, University of Grenoble Alpes (Grenoble Alliance for Integrated Structural Cell Biology) ANR-10-LABEX-04, Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG)
Subjects: Models, Molecular, 0301 basic medicine, Chloroplasts, Cyclohexanecarboxylic Acids, Protein Conformation, Transamination, Amino Acid Motifs, Arabidopsis, Crystallography, X-Ray, Biochemistry, Substrate Specificity, Isoaspartate, 0302 clinical medicine, 1 aspartate aminotransferase, structure function, Arabidopsis thaliana, Transferase, Tyrosine, Conserved Sequence, chemistry.chemical_classification, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, molecular modeling Abreviation: AAT Aspartate aminotransferase, Thermus thermophilus, Recombinant Proteins, 1β aspartate/prephenate aminotransferase, Amino Acids, Dicarboxylic, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], 030220 oncology & carcinogenesis, congenital, hereditary, and neonatal diseases and abnormalities, Molecular Dynamics Simulation, 03 medical and health sciences, Species Specificity, Cyclohexenes, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, Amino Acid Sequence, Aspartate Aminotransferases, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Molecular Biology, Transaminases, Sequence Homology, Amino Acid, Arabidopsis Proteins, molecular modeling, Mutagenesis, Cell Biology, arogenate route, biology.organism_classification, [INFO.INFO-MO]Computer Science [cs]/Modeling and Simulation, tyrosine metabolism, 030104 developmental biology, Enzyme, prephenate, PAT Prephenate aminotransferase, Sequence Alignment, Sinorhizobium meliloti
Abstract: International audience; Alternative routes for the post-chorismate branch of the biosynthetic pathway leading to tyrosine exist, the 4-hydroxyphenylpyruvate or the arogenate route. The arogenate route involves the transamination of prephenate into arogenate. In a previous study, we found that, depending on the microorganisms possessing the arogenate route, three different aminotransferases evolved to perform prephenate transamination, that is, 1β aspartate aminotransferase (1β AAT), N-succinyl-l,l-diaminopimelate aminotransferase, and branched-chain aminotransferase. The present work aimed at identifying molecular determinant(s) of 1β AAT prephenate aminotransferase (PAT) activity. To that purpose, we conducted X-ray crystal structure analysis of two PAT competent 1β AAT from Arabidopsis thaliana and Rhizobium meliloti and one PAT incompetent 1β AAT from R. meliloti. This structural analysis supported by site-directed mutagenesis, modeling, and molecular dynamics simulations allowed us to identify a molecular determinant of PAT activity in the flexible N-terminal loop of 1β AAT. Our data reveal that a Lys/Arg/Gln residue in position 12 in the sequence (numbering according to Thermus thermophilus 1β AAT), present only in PAT competent enzymes, could interact with the 4-hydroxyl group of the prephenate substrate, and thus may have a central role in the acquisition of PAT activity by 1β AAT.
Published: 2019

13. Identification of the Calmodulin-dependent NAD+kinase sustaining the elicitor-induced oxidative burst in plants

Author: Alexandra Kraut, Elisa Dell’Aglio, Christian Mazars, Michel Matringe, Gilles Curien, Giovanni Finazzi, Cécile Giustini, and Yohann Couté
Subjects: NAD+ kinase activity, biology, Calmodulin, Chemistry, Kinase, ATPase, food and beverages, biology.organism_classification, Cofactor, Elicitor, Biochemistry, Arabidopsis, biology.protein, NAD+ kinase
Abstract: NADP(H) is an essential cofactor ofmultiple metabolic processes in all living organisms. While NADP+ production in plants has long been known to involve a Calmodulin (CaM)/Ca2+-dependent NAD+kinase, the nature of the enzyme catalyzing this activity has remained enigmatic, as well as its role in plant physiology. Here, we identify an Arabidopsis P-loop ATPase (Atlg04280) with a bacterial type II zeta toxin domain, that catalyzes NADP+production upon binding of CaM/Ca2+to a domain located in its N-terminal region. The encoded protein (NADKc-1) is associated with the mitochondria and amplifies the elicitor-induced oxidative burst in Arabidopsis leaves representing the missing link between calcium signalling and metabolism in the response to pathogen elicitor. By analysis of various plants and algae, we show that NADKc is well conserved in the plant lineage and present in basal plants. Our data allows proposing that the CaM-dependent NAD kinase activity is only found in photosynthetic species carrying NADKc-1 related proteins, which would represent the only proteins harboring CaM-dependent NAD kinase activity in plants and algae.
Published: 2019

14. Investigating mixotrophic metabolism in the model diatom Phaeodactylum tricornutum

Author: Julien Pagliardini, Juliette Jouhet, Adeline Le Monnier, Melissa Conte, Toshihiro Obata, Giovanni Finazzi, Antonio Emidio Fortunato, Angela Falciatore, Eric Maréchal, Alisdair R. Fernie, Mark G. Poolman, Davide Dal Bo, Valeria Villanova, Dimitris Petroutsos, Gilles Curien, Dipali Singh, Fermentalg, Physiologie cellulaire et végétale (LPCV), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Biologie Computationnelle et Quantitative = Laboratory of Computational and Quantitative Biology (LCQB), Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Department of Biological and Medical Sciences, Oxford Brookes University, Max-Planck-Institut für Molekulare Pflanzenphysiologie (MPI-MP), Max-Planck-Gesellschaft, Marie Curie Initial Training Network Accliphot (FP7-PEPOPLE-2012-ITN, 316427), Region Rhone-Alpes (Cible project), Programme Investissement d’Avenir Oceanomics, ANR-12-BIME-0005,DiaDomOil,Domestication des diatomées pour la production de biocarburants(2012), ANR-10- LABX-49-01,Labex GRAL,Labex GRAL, Physiologie cellulaire et végétale [2016-2019] (LPCV [2016-2019]), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), UMR 1417 PCV Laboratoire de Physiologie Cellulaire Végétale, Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut National de la Recherche Agronomique (INRA), Fermentalg SA, Department of Biological and Medical Sciences, Oxford Brookes University, Max Planck Institute of Molecular Plant Physiology (MPI-MP), Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Université Pierre et Marie Curie - Paris 6 (UPMC), ANR (DiaDomOil) [ANR-12BIME-0005], CEA Bioenergies programme, Programme Investissement d'Avenir Oceanmics, CNRS Defi, HFSP [HFSP0052], Marie Curie Initial Training Network CALIPSO (ITN) [GA 607607], ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), European Project: 316427, Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Université Grenoble Alpes (UGA), Université Pierre et Marie Curie (Paris 6), Villanova V., Fortunato A.E., Singh D., Bo D.D., Conte M., Obata T., Jouhet J., Fernie A.R., Marechal E., Falciatore A., Pagliardini J., Le Monnier A., Poolman M., Curien G., Petroutsos D., and Finazzi G.
Subjects: 0301 basic medicine, Glycerol, [SDV.OT]Life Sciences [q-bio]/Other [q-bio.OT], Light, Metabolic flux, Biology, Settore BIO/19 - Microbiologia Generale, Photosynthesis, Phaeodactylum tricornutum, General Biochemistry, Genetics and Molecular Biology, Glycerolipid, 03 medical and health sciences, Nutrient, mixotrophy, Botany, Microalgae, Settore BIO/04 - Fisiologia Vegetale, Metabolomics, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, photosynthèse, 14. Life underwater, Biomass, Transcriptomics, métabolisme, micro-algue, Diatoms, photosynthesis, Phototroph, marine diatoms, fungi, Carbon metabolism, Lipid metabolism, Articles, approche omique, biology.organism_classification, Carbon, Triacylglycerol biosynthesis, 030104 developmental biology, Diatom, Biomass production, Biochemistry, General Agricultural and Biological Sciences, Energy source, metabolism, Mixotroph, omics analyses
Abstract: Diatoms are prominent marine microalgae, interesting not only from an ecological point of view, but also for their possible use in biotechnology applications. They can be cultivated in phototrophic conditions, using sunlight as the sole energy source. Some diatoms, however, can also grow in a mixotrophic mode, wherein both light and external reduced carbon contribute to biomass accumulation. In this study, we investigated the consequences of mixotrophy on the growth and metabolism of the pennate diatom Phaeodactylum tricornutum , using glycerol as the source of reduced carbon. Transcriptomics, metabolomics, metabolic modelling and physiological data combine to indicate that glycerol affects the central-carbon, carbon-storage and lipid metabolism of the diatom. In particular, provision of glycerol mimics typical responses of nitrogen limitation on lipid metabolism at the level of triacylglycerol accumulation and fatty acid composition. The presence of glycerol, despite provoking features reminiscent of nutrient limitation, neither diminishes photosynthetic activity nor cell growth, revealing essential aspects of the metabolic flexibility of these microalgae and suggesting possible biotechnological applications of mixotrophy. This article is part of the themed issue ‘The peculiar carbon metabolism in diatoms'.
Published: 2017

15. ChloroKB:a web-application for the integration of knowledge related to chloroplast metabolic network

Author: Daphné Seigneurin-Berny, Sylvain Bournais, Christophe Bruley, Claude Alban, Gilles Curien, Marianne Tardif, Marcel Kuntz, Stéphane Ravanel, Pauline Gloaguen, Michel Matringe, Myriam Ferro, Yves Vandenbrouck, Norbert Rolland, Etude de la dynamique des protéomes (EDyP ), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Physiologie cellulaire et végétale (LPCV), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire Biologie, Informatique et Mathématiques, Commissariat à l'énergie atomique et aux énergies alternatives (CEA), ANR (GRAL Labex, Grenoble Alliance for Integrated Structural Cell Biology) [ANR-10-LABEX-04], ANR-10-LABX-0004,CeMEB,Mediterranean Center for Environment and Biodiversity(2010), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'étude de la dynamique des protéomes (LEDyP), Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de Biosciences et de Biotechnologies de Grenoble (ex-IRTSV) (BIG), Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS), Etude de la dynamique des protéomes (EDyP), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and ANR–10–LABEX–04 ,GRAL,Labex
Subjects: 0301 basic medicine, Chloroplasts, Traceability, Arabidopsis thaliana, Physiology, Knowledge Bases, [SDV]Life Sciences [q-bio], Arabidopsis, Metabolic network, Context (language use), Plant Science, Computational biology, Bioinformatics, Chloroplast, Metabolic engineering, Knowledge base, 03 medical and health sciences, Software, Genetics, Web application, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, ComputingMilieux_MISCELLANEOUS, Internet, biology, business.industry, Plant, Breakthrough Technologies, biology.organism_classification, Biocuration, Biological network visualization, ComputingMethodologies_PATTERNRECOGNITION, 030104 developmental biology, Metabolism, Metabolic pathways, business, Metabolic Networks and Pathways, Subcellular Fractions
Abstract: International audience; Higher plants, as autotrophic organisms, are effective sources of molecules. They hold great promise for metabolic engineering, but the behavior of plant metabolism at the network level is still incompletely described. Although structural models (stoichiometry matrices) and pathway databases are extremely useful, they cannot describe the complexity of the metabolic context, and new tools are required to visually represent integrated biocurated knowledge for use by both humans and computers. Here, we describe ChloroKB, a Web application (http://chlorokb.fr/) for visual exploration and analysis of the Arabidopsis (Arabidopsis thaliana) metabolic network in the chloroplast and related cellular pathways. The network was manually reconstructed through extensive biocuration to provide transparent traceability of experimental data. Proteins and metabolites were placed in their biological context (spatial distribution within cells, connectivity in the network, participation in supramolecular complexes, and regulatory interactions) using CellDesigner software. The network contains 1,147 reviewed proteins (559 localized exclusively in plastids, 68 in at least one additional compartment, and 520 outside the plastid), 122 proteins awaiting biochemical/genetic characterization, and 228 proteins for which genes have not yet been identified. The visual presentation is intuitive and browsing is fluid, providing instant access to the graphical representation of integrated processes and to a wealth of refined qualitative and quantitative data. ChloroKB will be a significant support for structural and quantitative kinetic modeling, for biological reasoning, when comparing novel data with established knowledge, for computer analyses, and for educational purposes. ChloroKB will be enhanced by continuous updates following contributions from plant researchers.
Published: 2017

16. The chloroplast membrane associated ceQORH putative quinone oxidoreductase reduces long-chain, stress-related oxidized lipids

Author: Gilles Curien, Jean-Luc Montillet, Jean-Luc Ferrer, David Cobessi, Alexander N. Grechkin, Michel Matringe, Cécile Giustini, Norbert Rolland, Sarah Mas-y-Mas, Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Biologie végétale et microbiologie environnementale - UMR7265 (BVME), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire d'Ecophysiologie Moléculaire des Plantes (LEMP), Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Kazan Institute of Biochemistry and Biophysics, ANR-10- LABX-49-01,Labex GRAL,Labex GRAL, Physiologie cellulaire et végétale (LPCV), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Protéines de Protection des Végétaux (PPV), Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), French National Research Agency GRAL Labex ANR-10-LABX-49-01, Russian Academy of Sciences, Labex GRAL (Alliance Grenobloise pour la Biologie Structurale et Cellulaire integrees), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Direction de Recherche Fondamentale (CEA) (DRF (CEA))
Subjects: 0301 basic medicine, Chloroplasts, Membrane lipids, Lipoxygenase, reactive electrophile species, Arabidopsis, gamma-ketols, Cyclopentanes, Plant Science, Horticulture, Biology, Quinone oxidoreductase, oxylipin, Biochemistry, Chloroplast membrane, Membrane Lipids, 03 medical and health sciences, Quinone Reductases, chloroplast, Inner membrane, alkenone reductase, Oxylipins, Jasmonate, Molecular Biology, ComputingMilieux_MISCELLANEOUS, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Arabidopsis Proteins, Quinones, Membrane Proteins, General Medicine, jasmonate, Chloroplast, Metabolic pathway, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], 030104 developmental biology, Fatty Acids, Unsaturated, Oxidation-Reduction
Abstract: International audience; Under oxidative stress conditions the lipid constituents of cells can undergo oxidation whose frequent consequence is the production of highly reactive alpha,beta-unsaturated carbonyls. These molecules are toxic because they can add to biomolecules (such as proteins and nucleic acids) and several enzyme activities cooperate to eliminate these reactive electrophile species. CeQORH (chloroplast envelope Quinone Oxidoreductase Homolog, At4g13010) is associated with the inner membrane of the chloroplast envelope and imported into the organelle by an alternative import pathway. In the present study, we show that the recombinant ceQORH exhibits the activity of a NADPH-dependent alpha,beta-unsaturated oxoene reductase reducing the double bond of medium-chain (C >= 9) to long-chain (18 carbon atoms) reactive electrophile species deriving from poly-unsaturated fatty acid peroxides. The best substrates of ceQORH are 13-lipoxygenase-derived gamma-ketols. gamma-Ketols are spontaneously produced in the chloroplast from the unstable allene oxide formed in the biochemical pathway leading to 12-oxo-phytodienoic acid, a precursor of the defense hormone jasmonate. In chloroplasts, ceQORH could detoxify 13-lipoxygenase-derived gamma-ketols at their production sites in the membranes. This finding opens new routes toward the understanding of gamma-ketols role and detoxification.
Published: 2016

17. The Water to Water Cycles in Microalgae

Author: Michel Matringe, Dimitris Petroutsos, Marcel Kuntz, Giorgio Forti, Leonardo Magneschi, Valeria Villanova, Giovanni Finazzi, Gilles Curien, Serena Flori, Cécile Giustini, Physiologie cellulaire et végétale (LPCV), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Centre National de la Recherche Scientifique (CNRS), Université Grenoble Alpes (UGA), Fermentalg SA, Istituto di Biofisica, Consiglio Nazionale delle Ricerche, University of Milan, Institut de Biosciences et de Biotechnologies de Grenoble (ex-IRTSV) (BIG), Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes (UGA), Institut National de la Recherche Agronomique (INRA), Agence Nationale de la Recherche [ANR-12-BIME-0005], Region Rhone-Alpes [Cible project], Marie Curie Initial Training Network Accliphot [FP7-PEPOPLE-2012-ITN, 316427], CNRS Defi [ENRS 2013], CEA Bioenergies program, Human Frontiers Science Program [HFSP0052], Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Curien G., Flori S., Villanova V., Magneschi L., Giustini C., Forti G., Matringe M., Petroutsos D., Kuntz M., Finazzi G., Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Università degli Studi di Milano = University of Milan (UNIMI), and Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])
Subjects: 0106 biological sciences, 0301 basic medicine, Light, Physiology, [SDV]Life Sciences [q-bio], Cell Respiration, Mehler reaction, Plastoquinone, Plant Science, Water to water cycles, Photosynthesis, 01 natural sciences, 03 medical and health sciences, chemistry.chemical_compound, Water Cycle, Microalgae, Electrochemical gradient, Photosystem, Organelles, biology, Chemistry, Electron transport, RuBisCO, food and beverages, Cell Biology, General Medicine, Electron transport chain, 030104 developmental biology, biology.protein, Biophysics, Photorespiration, Oxidoreductases, 010606 plant biology & botany
Abstract: In oxygenic photosynthesis, light produces ATP plus NADPH via linear electron transfer, i.e. the in-series activity of the two photosystems: PSI and PSII. This process, however, is thought not to be sufficient to provide enough ATP per NADPH for carbon assimilation in the Calvin-Benson-Bassham cycle. Thus, it is assumed that additional ATP can be generated by alternative electron pathways. These circuits produce an electrochemical proton gradient without NADPH synthesis, and, although they often represent a small proportion of the linear electron flow, they could have a huge importance in optimizing CO2 assimilation. In Viridiplantae, there is a consensus that alternative electron flow comprises cyclic electron flow around PSI and the water to water cycles. The latter processes include photosynthetic O-2 reduction via the Mehler reaction at PSI, the plastoquinone terminal oxidase downstream of PSII, photorespiration (the oxygenase activity of Rubisco) and the export of reducing equivalents towards the mitochondrial oxidases, through the malate shuttle. In this review, we summarize current knowledge about the role of the water to water cycles in photosynthesis, with a special focus on their occurrence and physiological roles in microalgae.
Published: 2016

18. No plastidial calmodulin-like proteins detected by two targeted mass-spectrometry approaches and GFP fusion proteins

Author: Daniel Salvi, Mathieu Baudet, Martine Neveu, Elisa Dell’Aglio, Myriam Ferro, Alexandra Kraut, David Macherel, Norbert Rolland, Gilles Curien, Martin-Laffon, Jacqueline, BLANC - Régulation de la dynamique du protéome chloroplastique - - Chloro-Pro2010 - ANR-10-BLAN-1610 - BLANC - VALID, Mediterranean Center for Environment and Biodiversity - - CeMEB2010 - ANR-10-LABX-0004 - LABX - VALID, Infrastructure Française de Protéomique - - ProFI2010 - ANR-10-INBS-0008 - INBS - VALID, Physiologie cellulaire et végétale (LPCV), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Etude de la dynamique des protéomes (EDyP ), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Institut de Recherche en Horticulture et Semences (IRHS), Université d'Angers (UA)-Institut National de la Recherche Agronomique (INRA)-AGROCAMPUS OUEST, ANR [ANR-2010-BLAN-1610-01 Chloro-Pro], Labex GRAL (Alliance Grenobloise pour la Biologie Structurale et Cellulaire Integrées : [ANR-10-LABEX-04], [ANR10-INSB-08 ProFI, Proteomics French Infrastructure], CEA DSV (IRTELIS International Program)., ANR-10-BLAN-1610,Chloro-Pro,Régulation de la dynamique du protéome chloroplastique(2010), ANR-10-LABX-0004,CeMEB,Mediterranean Center for Environment and Biodiversity(2010), ANR-10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Etude de la dynamique des protéomes (EDyP), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA), AGROCAMPUS OUEST-Institut National de la Recherche Agronomique (INRA)-Université d'Angers (UA), ANR–10–LABEX–04 ,GRAL,Labex, ANR10-INSB-08,ProFI,Proteomics French Infrastructure, Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), and Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)
Subjects: 0301 basic medicine, Proteomics, Calmodulin, Arabidopsis thaliana, Plastid, Plant Science, Vacuole, Mitochondrion, plastide, Biochemistry, Chloroplast, CaM-like protein, Purification protocol, 03 medical and health sciences, Cytosol, GFP fusion, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, calmoduline, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, protéomique, Molecular Biology, biology, chloroplaste, Mass spectrometry, Subcellular localization, Protein, Plant, Peroxisome, Cell biology, 030104 developmental biology, Targeted mass spectrometry, biology.protein, Calcium regulation, CaM-like proteins
Abstract: Supplementary data: http://dx.doi.org/10.1016/j.neps.2016.08.001; International audience; Background: CaM-like proteins (CMLs) are localized in the cytosol and others in organelles such as the mitochondria, the peroxisomes and the vacuole. To date, although several plastidial proteins were identified as CaM/CML interactors, no CMLs were assigned to the chloroplast. Absence of clues about the genetic identity of plastidial CMLs prevents investigating their regulatory role. Results: To improve our understanding of plastidial Ca2+ regulation, we attempted to identify plastidial CMLs with two large scale, CaM-specific proteomic approaches, and GFP-fusions. Conclusions: Despite the use of several different approaches no plastidial CML could be identified. GFP fusion of CML 35 CML36 and CML41 indicate a cytosolic localization.
Published: 2016

19. Nitrite–nitric oxide control of mitochondrial respiration at the frontier of anoxia

Author: Ljudmilla Borisjuk, David Macherel, Marie-Hélène Avelange-Macherel, Abdelilah Benamar, H. Ahmed Mostefai, Gilles Curien, Ramaroson Andriantsitohaina, Hardy Rolletschek, Physiologie Moléculaire des Semences (PMS), Université d'Angers (UA)-Institut National de la Recherche Agronomique (INRA)-Institut National d'Horticulture, Leibniz Institute of Plant Genetics and Crop Plant Research [Gatersleben] (IPK-Gatersleben), Laboratoire de physiologie cellulaire végétale (LPCV), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), Biologie Neurovasculaire Intégrée (BNVI), Université d'Angers (UA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), Institut für Pflanzengenetik und Kulturpflanzenforschung, Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut National d'Horticulture-Institut National de la Recherche Agronomique (INRA)-Université d'Angers (UA), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
Subjects: 0106 biological sciences, Bioenergetics, Nitrite, Cell Respiration, Biophysics, plant, Mitochondrion, bioenergetics, 01 natural sciences, Biochemistry, mathematical analysis, Nitric oxide, 03 medical and health sciences, chemistry.chemical_compound, Oxygen homeostasis, Respiration, Animals, Cytochrome c oxidase, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Nitrites, Pisum sativum, 030304 developmental biology, 0303 health sciences, biology, hypoxia, Electron Spin Resonance Spectroscopy, Peas, anoxia, Cell Biology, Plants, Cell biology, Oxygen, mitochondria, chemistry, germination, Coenzyme Q – cytochrome c reductase, Seeds, Mammalia, biology.protein, Oxidation-Reduction, seed, respiration, 010606 plant biology & botany
Abstract: International audience; Actively respiring animal and plant tissues experience hypoxia because of mitochondrial O(2) consumption. Controlling oxygen balance is a critical issue that involves in mammals hypoxia-inducible factor (HIF) mediated transcriptional regulation, cytochrome oxidase (COX) subunit adjustment and nitric oxide (NO) as a mediator in vasodilatation and oxygen homeostasis. In plants, NO, mainly derived from nitrite, is also an important signalling molecule. We describe here a mechanism by which mitochondrial respiration is adjusted to prevent a tissue to reach anoxia. During pea seed germination, the internal atmosphere was strongly hypoxic due to very active mitochondrial respiration. There was no sign of fermentation, suggesting a down-regulation of O(2) consumption near anoxia. Mitochondria were found to finely regulate their surrounding O(2) level through a nitrite-dependent NO production, which was ascertained using electron paramagnetic resonance (EPR) spin trapping of NO within membranes. At low O(2), nitrite is reduced into NO, likely at complex III, and in turn reversibly inhibits COX, provoking a rise to a higher steady state level of oxygen. Since NO can be re-oxidized into nitrite chemically or by COX, a nitrite-NO pool is maintained, preventing mitochondrial anoxia. Such an evolutionarily conserved mechanism should have an important role for oxygen homeostasis in tissues undergoing hypoxia.
Published: 2008
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20. Regulation of One-Carbon Metabolism in Arabidopsis: The N-Terminal Regulatory Domain of Cystathionine γ-Synthase Is Cleaved in Response to Folate Starvation

Author: Guo Fang Zhang, Fabrice Rébeillé, Dominique Van Der Straeten, Bernadette Gambonnet, Samuel Jabrin, Karen Loizeau, Stéphane Ravanel, Willy E. Lambert, and Gilles Curien
Subjects: 0106 biological sciences, Physiology, Carbon-Oxygen Lyases, Molecular Sequence Data, Arabidopsis, Plant Science, 01 natural sciences, 03 medical and health sciences, chemistry.chemical_compound, Folic Acid, Methionine, Gene Expression Regulation, Plant, Genetics, Post-translational regulation, Amino Acid Sequence, Cells, Cultured, 030304 developmental biology, 2. Zero hunger, chemistry.chemical_classification, 0303 health sciences, biology, Metabolism, biology.organism_classification, Cystathionine beta synthase, Carbon, Amino acid, Carbon-Sulfur Lyases, Metabolic pathway, Enzyme, chemistry, Biochemistry, biology.protein, Folic Acid Antagonists, Research Article, 010606 plant biology & botany
Abstract: In all organisms, control of folate homeostasis is of vital importance to sustain the demand for one-carbon (C1) units that are essential in major metabolic pathways. In this study we induced folate deficiency in Arabidopsis (Arabidopsis thaliana) cells by using two antifolate inhibitors. This treatment triggered a rapid and important decrease in the pool of folates with significant modification in the distribution of C1-substituted folate coenzymes, suggesting an adaptive response to favor a preferential shuttling of the flux of C1 units to the synthesis of nucleotides over the synthesis of methionine (Met). Metabolic profiling of folate-deficient cells indicated important perturbation of the activated methyl cycle because of the impairment of Met synthases that are deprived of their substrate 5-methyl-tetrahydrofolate. Intriguingly, S-adenosyl-Met and Met pools declined during the initial period of folate starvation but were further restored to typical levels. Reestablishment of Met and S-adenosyl-Met homeostasis was concomitant with a previously unknown posttranslational modification that consists in the removal of 92 amino acids at the N terminus of cystathionine γ-synthase (CGS), the first specific enzyme for Met synthesis. Rescue experiments and analysis of different stresses indicated that CGS processing is specifically associated with perturbation of the folates pool. Also, CGS processing involves chloroplastic serine-type proteases that are expressed in various plant species subjected to folate starvation. We suggest that a metabolic effector, to date unidentified, can modulate CGS activity in vivo through an interaction with the N-terminal domain of the enzyme and that removal of this domain can suppress this regulation.
Published: 2007

21. Analytical ultracentrifugation and preliminary X-ray studies of the chloroplast envelope quinone oxidoreductase homologue from Arabidopsis thaliana

Author: Cécile Giustini, Norbert Rolland, Jean Luc Ferrer, David Cobessi, Gilles Curien, Sarah Mas y mas, Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Beamline FIP-BM30A ESRF, ANR-10-INBS-0005-02,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-10- LABX-49-01,Labex GRAL,Labex GRAL, Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), FRISBI project (ANR-10-INSB-05-0, GRAL project (ANR-10- LABX-49-01), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Arabidopis thaliana, Chloroplasts, Quinone oxidoreductase homologue, Substrate specificity, Molecular Sequence Data, Biophysics, Arabidopsis, Quinone oxidoreductase, Biochemistry, Chloroplast membrane, Research Communications, Structural Biology, Oxidoreductase, Transit Peptide, Genetics, NAD(P)H Dehydrogenase (Quinone), [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, Peptide sequence, Purification, Transit peptide, chemistry.chemical_classification, biology, Arabidopsis Proteins, Protein, Membrane, food and beverages, Plant, Crystallisation, Condensed Matter Physics, biology.organism_classification, Chloroplast, chemistry, Chloroplast envelope, Enzyme, Analytical ultracentrifugation, Crystallization, Ultracentrifugation, CeQORH, Cloning
Abstract: Quinone oxidoreductases reduce a broad range of quinones and are widely distributed among living organisms. The chloroplast envelope quinone oxidoreductase homologue (ceQORH) fromArabidopsis thalianabinds NADPH, lacks a classical N-terminal and cleavable chloroplast transit peptide, and is transported through the chloroplast envelope membrane by an unknown alternative pathway without cleavage of its internal chloroplast targeting sequence. To unravel the fold of this targeting sequence and its substrate specificity, ceQORH fromA. thalianawas overexpressed inEscherichia coli, purified and crystallized. Crystals of apo ceQORH were obtained and a complete data set was collected at 2.34 Å resolution. The crystals belonged to space groupC2221, with two molecules in the asymmetric unit.
Published: 2015

22. Allosteric monofunctional aspartate kinases fromArabidopsis

Author: Gilles Curien, Renaud Dumas, Mathieu Laurencin, and Mylène Robert-Genthon
Subjects: 0106 biological sciences, endocrine system diseases, Allosteric regulation, Lysine, complex mixtures, 01 natural sciences, Biochemistry, 03 medical and health sciences, Enzyme activator, Aspartate kinase, Molecular Biology, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, biology, Kinase, nutritional and metabolic diseases, Cell Biology, Molecular biology, Enzyme assay, Aspartate carbamoyltransferase, Enzyme, chemistry, biology.protein, bacteria, hormones, hormone substitutes, and hormone antagonists, 010606 plant biology & botany
Abstract: Plant monofunctional aspartate kinase is unique among all aspartate kinases, showing synergistic inhibition by lysine and S-adenosyl-l-methionine (SAM). The Arabidopsis genome contains three genes for monofunctional aspartate kinases. We show that aspartate kinase 2 and aspartate kinase 3 are inhibited only by lysine, and that aspartate kinase 1 is inhibited in a synergistic manner by lysine and SAM. In the absence of SAM, aspartate kinase 1 displayed low apparent affinity for lysine compared to aspartate kinase 2 and aspartate kinase 3. In the presence of SAM, the apparent affinity of aspartate kinase 1 for lysine increased considerably, with K(0.5) values for lysine inhibition similar to those of aspartate kinase 2 and aspartate kinase 3. For all three enzymes, the inhibition resulted from an increase in the apparent K(m) values for the substrates ATP and aspartate. The mechanism of aspartate kinase 1 synergistic inhibition was characterized. Inhibition by lysine alone was fast, whereas synergistic inhibition by lysine plus SAM was very slow. SAM by itself had no effect on the enzyme activity, in accordance with equilibrium binding analyses indicating that SAM binding to aspartate kinase 1 requires prior binding of lysine. The three-dimensional structure of the aspartate kinase 1-Lys-SAM complex has been solved [Mas-Droux C, Curien G, Robert-Genthon M, Laurencin M, Ferrer JL & Dumas R (2006) Plant Cell18, 1681-1692]. Taken together, the data suggest that, upon binding to the inactive aspartate kinase 1-Lys complex, SAM promotes a slow conformational transition leading to formation of a stable aspartate kinase 1-Lys-SAM complex. The increase in aspartate kinase 1 apparent affinity for lysine in the presence of SAM thus results from the displacement of the unfavorable equilibrium between aspartate kinase 1 and aspartate kinase 1-Lys towards the inactive form.
Published: 2006

23. Identification of Six Novel Allosteric Effectors of Arabidopsis thaliana Aspartate Kinase-Homoserine Dehydrogenase Isoforms

Author: Stéphane Ravanel, Gilles Curien, Renaud Dumas, and Mylène Robert
Subjects: 0106 biological sciences, chemistry.chemical_classification, Alanine, Homoserine dehydrogenase, 0303 health sciences, Cell Biology, Biology, 01 natural sciences, Biochemistry, 03 medical and health sciences, chemistry.chemical_compound, Aspartate carbamoyltransferase, chemistry, Biosynthesis, Aspartate kinase, Threonine, Molecular Biology, Essential amino acid, Amino acid synthesis, 030304 developmental biology, 010606 plant biology & botany
Abstract: The Arabidopsis genome contains two genes predicted to code for bifunctional aspartate kinase-homoserine dehydrogenase enzymes (isoforms I and II). These two activities catalyze the first and the third steps toward the synthesis of the essential amino acids threonine, isoleucine, and methionine. We first characterized the kinetic and regulatory properties of the recombinant enzymes, showing that they mainly differ with respect to the inhibition of the homoserine dehydrogenase activity by threonine. A systematic search for other allosteric effectors allowed us to identify an additional inhibitor (leucine) and 5 activators (alanine, cysteine, isoleucine, serine, and valine) equally efficient on aspartate kinase I activity (4-fold activation). The six effectors of aspartate kinase I were all activators of aspartate kinase II activity (13-fold activation) and displayed a similar specificity for the enzyme. No synergy between different effectors could be observed. The activation, which resulted from a decrease in the Km values for the substrates, was detected using low substrates concentrations. Amino acid quantification revealed that alanine and threonine were much more abundant than the other effectors in Arabidopsis leaf chloroplasts. In vitro kinetics in the presence of physiological concentrations of the seven allosteric effectors confirmed that aspartate kinase I and II activities were highly sensitive to changes in alanine and threonine concentrations. Thus, physiological context rather than enzyme structure sets the specificity of the allosteric control. Stimulation by alanine may play the role of a feed forward activation of the aspartate-derived amino acid pathway in plant.
Published: 2005

24. Methionine Metabolism in Plants

Author: Fabrice Rébeillé, Samuel Jabrin, Roland Douce, Maryse A. Block, Gilles Curien, Pascal Rippert, and Stéphane Ravanel
Subjects: 0106 biological sciences, chemistry.chemical_classification, 0303 health sciences, Methyltransferase, biology, food and beverages, Cell Biology, Methylation, Metabolism, 01 natural sciences, Biochemistry, Cystathionine beta synthase, Chloroplast, 03 medical and health sciences, Cytosol, Enzyme, chemistry, biology.protein, Methionine synthase, Molecular Biology, 030304 developmental biology, 010606 plant biology & botany
Abstract: The subcellular distribution of Met and S-adenosylmethionine (AdoMet) metabolism in plant cells discloses a complex partition between the cytosol and the organelles. In the present work we show that Arabidopsis contains three functional isoforms of vitamin B12-independent methionine synthase (MS), the enzyme that catalyzes the methylation of homocysteine to Met with 5-methyltetrahydrofolate as methyl group donor. One MS isoform is present in chloroplasts and is most likely required to methylate homocysteine that is synthesized de novo in this compartment. Thus, chloroplasts are autonomous and are the unique site for de novo Met synthesis in plant cells. The additional MS isoforms are present in the cytosol and are most probably involved in the regeneration of Met from homocysteine produced in the course of the activated methyl cycle. Although Met synthesis can occur in chloroplasts, there is no evidence that AdoMet is synthesized anywhere but the cytosol. In accordance with this proposal, we show that AdoMet is transported into chloroplasts by a carrier-mediated facilitated diffusion process. This carrier is able to catalyze the uniport uptake of AdoMet into chloroplasts as well as the exchange between cytosolic AdoMet and chloroplastic AdoMet or S-adenosylhomocysteine. The obvious function for the carrier is to sustain methylation reactions and other AdoMet-dependent functions in chloroplasts and probably to remove S-adenosylhomocysteine generated in the stroma by methyltransferase activities. Therefore, the chloroplastic AdoMet carrier serves as a link between cytosolic and chloroplastic one-carbon metabolism.
Published: 2004

25. A kinetic model of the branch-point between the methionine and threonine biosynthesis pathways in Arabidopsis thaliana

Author: Stéphane Ravanel, Renaud Dumas, and Gilles Curien
Subjects: chemistry.chemical_classification, Methionine, biology, Context (language use), Biochemistry, Cystathionine beta synthase, chemistry.chemical_compound, Enzyme, Threonine synthase, Allosteric enzyme, chemistry, biology.protein, Threonine, Cysteine
Abstract: This work proposes a model of the metabolic branch-point between the methionine and threonine biosynthesis pathways in Arabidopsis thaliana which involves kinetic competition for phosphohomoserine between the allosteric enzyme threonine synthase and the two-substrate enzyme cystathionine γ-synthase. Threonine synthase is activated by S-adenosylmethionine and inhibited by AMP. Cystathionine γ-synthase condenses phosphohomoserine to cysteine via a ping-pong mechanism. Reactions are irreversible and inhibited by inorganic phosphate. The modelling procedure included an examination of the kinetic links, the determination of the operating conditions in chloroplasts and the establishment of a computer model using the enzyme rate equations. To test the model, the branch-point was reconstituted with purified enzymes. The computer model showed a partial agreement with the in vitro results. The model was subsequently improved and was then found consistent with flux partition in vitro and in vivo. Under near physiological conditions, S-adenosylmethionine, but not AMP, modulates the partition of a steady-state flux of phosphohomoserine. The computer model indicates a high sensitivity of cystathionine flux to enzyme and S-adenosylmethionine concentrations. Cystathionine flux is sensitive to modulation of threonine flux whereas the reverse is not true. The cystathionine γ-synthase kinetic mechanism favours a low sensitivity of the fluxes to cysteine. Though sensitivity to inorganic phosphate is low, its concentration conditions the dynamics of the system. Threonine synthase and cystathionine γ-synthase display similar kinetic efficiencies in the metabolic context considered and are first-order for the phosphohomoserine substrate. Under these conditions outflows are coordinated.
Published: 2003

26. Three different classes of aminotransferases evolved prephenate aminotransferase functionality in arogenate-competent microorganisms

Author: Michel Matringe, Gilles Curien, Alexandra Kraut, Lucas Moyet, Matthieu Graindorge, Cécile Giustini, Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'étude de la dynamique des protéomes (LEDyP), Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), French National Institute for Agricultural Research (INRA), CNRS, Commissariat a l'Energie Atomique et aux Energies Alternatives (CEA), University of Grenoble Alpes, GRAL Labex (Grenoble Alliance for Integrated Structural Cell Biology) [ANR-10-LABEX-04], Curien, Gilles, Matringe, Michel, Martin-Laffon, Jacqueline, Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
Subjects: Transamination, phenylalanine, enzymatic activity, Phenylalanine, MESH: Amino Acids, Dicarboxylic, Biochemistry, MESH: Cyclohexenes, MESH: Tyrosine, chemistry.chemical_compound, Aromatic amino acids, Pyridoxal phosphate, Tyrosine, Rhodobacter, prephenate aminotransferase, microorganisms, bacteria, MESH: Bacterial Proteins, MESH: Evolution, Molecular, 2. Zero hunger, chemistry.chemical_classification, Synechococcus, aminotransferases, Streptomyces, 3. Good health, Amino acid, Amino Acids, Dicarboxylic, Pyridoxal Phosphate, arogenate, aminoacide, MESH: Pyridoxal Phosphate, Rhizobium, Biology, Microbiology, amination, bacterial metabolism, Mycobacterium, Evolution, Molecular, Bacterial Proteins, MESH: Transaminases, Cyclohexenes, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Nitrosomonas, Molecular Biology, Transaminases, MESH: Humans, Cell Biology, metabolic pathway, aromatic aminoacid biosynthesis, Metabolic pathway, enzyme, MESH: Bacteria, Enzyme, Metabolism, chemistry, tyrosine
Abstract: Background: The genes encoding prephenate aminotransferase in arogenate-competent microorganisms for tyrosine synthesis are still unknown. Results: Three different classes of aminotransferase use prephenate as an amino acceptor. Conclusion: Prephenate aminotransferase functionality has arisen more than once during evolution. Significance: This first identification of prephenate aminotransferases in arogenate competent microorganisms affords a better understanding of the origin and fixation of the arogenate route during evolution. The aromatic amino acids phenylalanine and tyrosine represent essential sources of high value natural aromatic compounds for human health and industry. Depending on the organism, alternative routes exist for their synthesis. Phenylalanine and tyrosine are synthesized either via phenylpyruvate/4-hydroxyphenylpyruvate or via arogenate. In arogenate-competent microorganisms, an aminotransferase is required for the transamination of prephenate into arogenate, but the identity of the genes is still unknown. We present here the first identification of prephenate aminotransferases (PATs) in seven arogenate-competent microorganisms and the discovery that PAT activity is provided by three different classes of aminotransferase, which belong to two different fold types of pyridoxal phosphate enzymes: an aspartate aminotransferase subgroup 1 in tested - and -proteobacteria, a branched-chain aminotransferase in tested cyanobacteria, and an N-succinyldiaminopimelate aminotransferase in tested actinobacteria and in the -proteobacterium Nitrosomonas europaea. Recombinant PAT enzymes exhibit high activity toward prephenate, indicating that the corresponding genes encode bona fide PAT. PAT functionality was acquired without other modification of substrate specificity and is not a general catalytic property of the three classes of aminotransferases.
Published: 2014

27. Crystal structure of threonine synthase from Arabidopsis thaliana

Author: Karine Thomazeau, Valérie Biou, Gilles Curien, and Renaud Dumas
Subjects: S-Adenosylmethionine, Methyltransferase, Stereochemistry, Carbon-Oxygen Lyases, Molecular Sequence Data, Allosteric regulation, Arabidopsis, Biochemistry, Article, Protein Structure, Secondary, Enzyme activator, chemistry.chemical_compound, Amino Acid Sequence, Methionine synthase, S-Adenosyl methionine, Threonine, Binding site, Protein Structure, Quaternary, Molecular Biology, Conserved Sequence, Binding Sites, Crystallography, biology, Protein Structure, Tertiary, Enzyme Activation, Threonine synthase, chemistry, Pyridoxal Phosphate, biology.protein, Oxidation-Reduction
Abstract: Threonine synthase (TS) is a PLP-dependent enzyme that catalyzes the last reaction in the synthesis of threonine from aspartate. In plants, the methionine pathway shares the same substrate, O-phospho-L-homoserine (OPH), and TS is activated by S-adenosyl-methionine (SAM), a downstream product of methionine synthesis. This positive allosteric effect triggered by the product of another pathway is specific to plants. The crystal structure of Arabidopsis thaliana apo threonine synthase was solved at 2.25 A resolution from triclinic crystals using MAD data from the selenomethionated protein. The structure reveals a four-domain dimer with a two-stranded beta-sheet arm protruding from one monomer onto the other. This domain swap could form a lever through which the allosteric effect is transmitted. The N-terminal domain (domain 1) has a unique fold and is partially disordered, whereas the structural core (domains 2 and 3) shares the functional domain of PLP enzymes of the same family. It also has similarities with SAM-dependent methyltransferases. Structure comparisons allowed us to propose potential sites for pyridoxal-phosphate and SAM binding on TS; they are close to regions that are disordered in the absence of these molecules.
Published: 2001

28. Transport, Compartmentation, and Metabolism of Homoserine in Higher Plant Cells

Author: Serge Aubert, Richard Bligny, Roland Douce, Gilles Curien, and Elisabeth Gout
Subjects: biology, Physiology, Homoserine kinase, Homoserine, Plant Science, Metabolism, Acer pseudoplatanus, Plant cell, biology.organism_classification, chemistry.chemical_compound, Threonine synthase, chemistry, Biochemistry, Proton transport, Genetics, biology.protein, Threonine
Abstract: The transport, compartmentation, and metabolism of homoserine was characterized in two strains of meristematic higher plant cells, the dicotyledonous sycamore (Acer pseudoplatanus) and the monocotyledonous weedEchinochloa colonum. Homoserine is an intermediate in the synthesis of the aspartate-derived amino acids methionine, threonine (Thr), and isoleucine. Using 13C-nuclear magnetic resonance, we showed that homoserine actively entered the cells via a high-affinity proton-symport carrier (Kmapproximately 50–60 μm) at the maximum rate of 8 ± 0.5 μmol h−1 g−1 cell wet weight, and in competition with serine or Thr. We could visualize the compartmentation of homoserine, and observed that it accumulated at a concentration 4 to 5 times higher in the cytoplasm than in the large vacuolar compartment.31P-nuclear magnetic resonance permitted us to analyze the phosphorylation of homoserine. When sycamore cells were incubated with 100 μm homoserine, phosphohomoserine steadily accumulated in the cytoplasmic compartment over 24 h at the constant rate of 0.7 μmol h−1 g−1 cell wet weight, indicating that homoserine kinase was not inhibited in vivo by its product, phosphohomoserine. The rate of metabolism of phosphohomoserine was much lower (0.06 μmol h−1 g−1 cell wet weight) and essentially sustained Thr accumulation. Similarly, homoserine was actively incorporated by E. colonum cells. However, in contrast to what was seen in sycamore cells, large accumulations of Thr were observed, whereas the intracellular concentration of homoserine remained low, and phosphohomoserine did not accumulate. These differences with sycamore cells were attributed to the presence of a higher Thr synthase activity in this strain of monocot cells.
Published: 1998

29. Analytical Kinetic Modeling: A Practical Procedure

Author: María Luz Cárdenas, Gilles Curien, and Athel Cornish-Bowden
Subjects: chemistry.chemical_classification, 0303 health sciences, Computer science, 030302 biochemistry & molecular biology, 03 medical and health sciences, Metabolic pathway, Range (mathematics), Enzyme, chemistry, Simple (abstract algebra), Metabolic control analysis, Applied mathematics, Enzyme kinetics, 030304 developmental biology
Abstract: This chapter describes a practical procedure to dissect metabolic systems, simplify them, and use or derive enzyme rate equations in order to build a mathematical model of a metabolic system and run simulations. We first deal with a simple example, modeling a single enzyme that follows Michaelis-Menten kinetics and operates in the middle of an unbranched metabolic pathway. Next we describe the rules that can be followed to isolate sub-systems from their environment to simulate their behavior. Finally we use examples to show how to derive suitable rate equations, simpler than those needed for mechanistic studies, though adequate to describe the behavior over the physiological range of conditions.Many of the general characteristics of kinetic models will be obvious to readers familiar with the theory of metabolic control analysis (Cornish-Bowden, Fundamentals of Enzyme Kinetics, Wiley-Blackwell, Weinheim, 327-380, 2012), but here we shall not assume such knowledge, as the chapter is directed toward practical application rather than theory.
Published: 2013

30. Complementary biochemical approaches applied to the identification of plastidial calmodulin-binding proteins

Author: Sabine Brugière, Daniel Salvi, Michel Matringe, Faustine Delpierre, Gilles Curien, Daphné Seigneurin-Berny, Cécile Giustini, Elisa Dell’Aglio, Lucas Moyet, Norbert Rolland, Mathieu Baudet, Myriam Ferro, Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA), Institut de Biosciences et de Biotechnologies de Grenoble (ex-IRTSV) (BIG), Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes (UGA), Labex GRAL (Alliance Grenobloise pour la Biologie Structurale et Cellulaire Intégrées), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG)
Subjects: 0106 biological sciences, calmodulin, CaM-binding protein, Chloroplasts, animal structures, Calmodulin, Arabidopsis thaliana, proteome, Arabidopsis, plant, 01 natural sciences, 03 medical and health sciences, affinity chromatography, chloroplast, Spinacia oleracea, Organelle, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, Plant Proteins, 030304 developmental biology, mass spectrometry, 0303 health sciences, calcium, biology, Arabidopsis Proteins, Gene Expression Profiling, biology.organism_classification, Calmodulin-binding proteins, 3. Good health, Plant Leaves, Chloroplast, Phosphotransferases (Alcohol Group Acceptor), Biochemistry, Proteome, biology.protein, Calmodulin-Binding Proteins, NAD+ kinase, Signal transduction, metabolism, Protein Binding, Signal Transduction, 010606 plant biology & botany, Biotechnology
Abstract: International audience; Ca(2+)/Calmodulin (CaM)-dependent signaling pathways play a major role in the modulation of cell responses in eukaryotes. In the chloroplast, few proteins such as the NAD(+) kinase 2 have been previously shown to interact with CaM, but a general picture of the role of Ca(2+)/CaM signaling in this organelle is still lacking. Using CaM-affinity chromatography and mass spectrometry, we identified 210 candidate CaM-binding proteins from different Arabidopsis and spinach chloroplast sub-fractions. A subset of these proteins was validated by an optimized in vitro CaM-binding assay. In addition, we designed two fluorescence anisotropy assays to quantitatively characterize the binding parameters and applied those assays to NAD(+) kinase 2 and selected candidate proteins. On the basis of our results, there might be many more plastidial CaM-binding proteins than previously estimated. In addition, we showed that an array of complementary biochemical techniques is necessary in order to characterize the mode of interaction of candidate proteins with CaM.
Published: 2013

31. Characterization of anArabidopsis thalianacDNA encoding an S-adenosylmethionine-sensitive threonine synthase Threonine synthase from higher plants

Author: Renaud Dumas, Stéphane Ravanel, Gilles Curien, and Roland Douce
Subjects: S-Adenosylmethionine, DNA, Complementary, Arabidopsis thaliana, Carbon-Oxygen Lyases, Molecular Sequence Data, Mutant, Arabidopsis, Biophysics, Lyases, medicine.disease_cause, Chloroplast, Polymerase Chain Reaction, Biochemistry, Structural Biology, Complementary DNA, Threonine synthase activity, Escherichia coli, Genetics, medicine, Amino Acid Sequence, Cloning, Molecular, Pyridoxal 5′-phosphate, Molecular Biology, Conserved Sequence, DNA Primers, chemistry.chemical_classification, Base Sequence, Sequence Homology, Amino Acid, biology, Threonine synthase, Lysine, food and beverages, Cell Biology, Chromatography, Ion Exchange, biology.organism_classification, Molecular biology, Recombinant Proteins, o-Phospho-l-homoserine, Complementation, Kinetics, Enzyme, chemistry, Chromatography, Gel, biology.protein
Abstract: An Arabidopsis thaliana cDNA encoding an Sadenosylmethionine-sensitive threonine synthase (EC 4.2.99.2) has been isolated by functional complementation of an Escherichia coli mutant devoid of threonine synthase activity. Threonine synthase from A. thaliana was shown to be synthesized with a transit peptide. The recombinant protein is activated by Sadenosylmethionine in the same range as the plant threonine synthase and evidence is presented for an involvement of the N-terminal part of the mature enzyme in the sensitivity to Sadenosylmethionine.
Published: 1996

32. The biosynthetic capacities of the plastids and integration between cytoplasmic and chloroplast processes

Author: Giovanni Finazzi, Marcel Kuntz, Gilles Curien, Daphné Seigneurin-Berny, Norbert Rolland, Eric Maréchal, Michel Matringe, Stéphane Ravanel, Institut de Biosciences et de Biotechnologies de Grenoble (ex-IRTSV) (BIG), Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes (UGA), Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
Subjects: 0106 biological sciences, MESH: Oxidation-Reduction, Cytoplasm, Chloroplasts, envelope transporters, plant, MESH: Carbon Dioxide, 01 natural sciences, Chloroplast Proteins, MESH: Cell Compartmentation, Cell Compartmentation, MESH: Chloroplast Proteins, Plastids, Photosynthesis, plastid, MESH: Evolution, Molecular, MESH: Photosynthesis, 0303 health sciences, MESH: Symbiosis, Endosymbiosis, MESH: Proteomics, food and beverages, MESH: Plastids, MESH: Cyanobacteria, Cell biology, Chloroplast, Protein Transport, Oxidation-Reduction, MESH: Protein Transport, Nuclear gene, chloroplast/cytosol exchange, review, Biology, Cyanobacteria, Evolution, Molecular, 03 medical and health sciences, proteomics, Plant Cells, Genetics, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Plastid, Symbiosis, 030304 developmental biology, MESH: Chloroplasts, MESH: Cytoplasm, fungi, Carbon Dioxide, MESH: Plant Cells, Cytosol, MESH: Organelle Size, Organelle Size, cell compartment, metabolism, 010606 plant biology & botany
Abstract: Document Type : Book Chapter; International audience; Plastids are semiautonomous organelles derived from cyanobacterial ancestors. Following endosymbiosis, plastids have evolved to optimize their functions, thereby limiting metabolic redundancy with other cell compartments. Contemporary plastids have also recruited proteins produced by the nuclear genome of the host cell. In addition, many genes acquired from the cyanobacterial ancestor evolved to code for proteins that are targeted to cell compartments other than the plastid. Consequently, metabolic pathways are now a patchwork of enzymes of diverse origins, located in various cell compartments. Because of this, a wide range of metabolites and ions traffic between the plastids and other cell compartments. In this review, we provide a comprehensive analysis of the well-known, and of the as yet uncharacterized, chloroplast/cytosol exchange processes, which can be deduced from what is currently known about compartmentation of plant-cell metabolism.
Published: 2012

33. The many faces of aspartate kinases

Author: Renaud Dumas, Adeline Y. Robin, David Cobessi, Gilles Curien, Jean-Luc Ferrer, Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG)
Subjects: Allosteric regulation, Biophysics, review, Biochemistry, MESH: Allosteric Regulation, 03 medical and health sciences, MESH: Protein Structure, Tertiary, ACT domain, Allosteric Regulation, regulatory domain, MESH: Aspartate Kinase, Aspartate kinase, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, structure, Aspartate Kinase, Binding site, effector binding site, Molecular Biology, 030304 developmental biology, 0303 health sciences, allostery, Binding Sites, biology, MESH: Kinetics, Kinase, Effector, 030302 biochemistry & molecular biology, biosynthetic pathway, Cell biology, Protein Structure, Tertiary, enzyme, Kinetics, Allosteric enzyme, MESH: Binding Sites, biology.protein, metabolism, Function (biology)
Abstract: International audience; Based on recent X-ray structures and biochemical characterizations of aspartate kinases from different species, we show in this review how various organizations of a regulatory domain have contributed to the different mechanisms of control observed in aspartate kinases allowing simple to complex allosteric controls in branched pathways. The aim of this review is to show the relationships between domain organization, effector binding sites, mechanism of inhibition and regulatory function of an allosteric enzyme in a biosynthetic pathway.
Published: 2011

34. Branched-chain-amino-acid biosynthesis in plants: molecular cloning and characterization of the gene encoding acetohydroxy acid isomeroreductase (ketol-acid reductoisomerase) from Arabidopsis thaliana (thale cress)

Author: Roland Douce, Renaud Dumas, Gilles Curien, and R T DeRose
Subjects: RNA Splicing, Ketol-Acid Reductoisomerase, Molecular Sequence Data, Restriction Mapping, Branched-chain amino acid, Arabidopsis, Molecular cloning, Biology, Genes, Plant, Biochemistry, chemistry.chemical_compound, Complementary DNA, Coding region, Arabidopsis thaliana, Amino Acid Sequence, Cloning, Molecular, Molecular Biology, Gene, Genetics, Base Sequence, Nucleic acid sequence, Hydrogen Bonding, Cell Biology, biology.organism_classification, Introns, Alcohol Oxidoreductases, genomic DNA, Oligodeoxyribonucleotides, chemistry, Nucleic Acid Conformation, Sequence Alignment, Research Article
Abstract: Towards the goal of gaining a better understanding of the molecular mechanisms controlling branched-chain-amino-acid biosynthesis in plants, we have isolated, sequenced and characterized a gene encoding acetohydroxy acid isomero-reductase (ketol-acid reductoisomerase) from Arabidopsis thaliana (thale cress). Comparison between the acetohydroxy acid isomeroreductase cDNA and the genomic sequence has allowed us to determine the exon structure of the coding region. The isolated acetohydroxy acid isomeroreductase gene is distributed over approx. 4.5 kbp and contains nine introns (79-347 bp). The transcriptional start site was found to be 52 bp upstream of the translational initiation site. Southern-blot analysis of A. thaliana genomic DNA shows that the acetohydroxy acid isomeroreductase is encoded by a single-copy gene.
Published: 1993

35. Nucleotide sequence and characterization of a cDNA encoding the acetohydroxy acid isomeroreductase from Arabidopsis thaliana

Author: Renaud Dumas, Gilles Curien, and Roland Douce
Subjects: Molecular Sequence Data, Arabidopsis, Sequence alignment, Plant Science, Biology, Genes, Plant, Conserved sequence, Transit Peptide, Complementary DNA, Consensus Sequence, Genetics, Amino Acid Sequence, Peptide sequence, Plant Proteins, chemistry.chemical_classification, Base Sequence, 2-Acetolactate Mutase, Protein primary structure, Nucleic acid sequence, food and beverages, General Medicine, Amino acid, chemistry, Biochemistry, Sequence Alignment, Agronomy and Crop Science
Abstract: The primary structure of acetohydroxy acid isomeroreductase from Arabidopsis thaliana was deduced from two overlapping cDNA. The full-length cDNA sequence predicts an amino acid sequence for the protein precursor of 591 residues including a putative transit peptide of 67 amino acids. Comparison of the A. thaliana and spinach acetohydroxy acid isomeroreductases reveals that the sequences are conserved in the mature protein regions, but divergent in the transit peptides and around their putative processing site.
Published: 1993

36. Identification of a plant gene encoding glutamate/aspartate-prephenate aminotransferase: the last homeless enzyme of aromatic amino acids biosynthesis

Author: Cécile Giustini, Alexandra Kraut, Michel Matringe, Matthieu Graindorge, Gilles Curien, Anne-Claire Jacomin, Laboratoire de physiologie cellulaire végétale (LPCV), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), Laboratoire d'étude de la dynamique des protéomes (LEDyP), Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
Subjects: 0106 biological sciences, Arabidopsis thaliana, Transamination, Arabidopsis, Phenylalanine, plant, 01 natural sciences, Biochemistry, Mass Spectrometry, MESH: Recombinant Proteins, chemistry.chemical_compound, Structural Biology, Aromatic amino acids, aromatic amino acids biosynthesis, MESH: Arabidopsis, Tyrosine, prephenate aminotransferase, Cells, Cultured, chemistry.chemical_classification, 0303 health sciences, biology, MESH: Kinetics, MESH: Glutamic Acid, Recombinant Proteins, Amino acid, MESH: Amino Acids, Aromatic, Electrophoresis, Polyacrylamide Gel, Aromatic amino acid, MESH: Cells, Cultured, purification, Biophysics, Glutamic Acid, MESH: Arabidopsis Proteins, Aspartate aminotransferase, 03 medical and health sciences, Amino Acids, Aromatic, MESH: Aspartate Aminotransferases, Biosynthesis, MESH: Transaminases, Genetics, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Aspartate Aminotransferases, Molecular Biology, Transaminases, 030304 developmental biology, MESH: Mass Spectrometry, Arabidopsis Proteins, Cell Biology, Molecular biology, Enzyme assay, enzyme, Enzyme, Metabolism, chemistry, kinetics, biochemical properties, biology.protein, Enzymology, 010606 plant biology & botany, MESH: Electrophoresis, Polyacrylamide Gel
Abstract: International audience; In all organisms synthesising phenylalanine and/or tyrosine via arogenate, a prephenate aminotransferase is required for the transamination of prephenate into arogenate. The identity of the gene encoding this enzyme in the organisms where this activity occurs is still unknown. Glutamate/aspartate-prephenate aminotransferase (PAT) is thus the last homeless enzyme in the aromatic amino acids pathway. We report on the purification, mass spectrometry identification and biochemical characterization of Arabidopsis thaliana prephenate aminotransferase. Our data revealed that this activity is housed by the prokaryotic-type plastidic aspartate aminotransferase (At2g22250). This represents the first identification of a gene encoding PAT.
Published: 2010

37. A new mode of dimerization of allosteric enzymes with ACT domains revealed by the crystal structure of the aspartate kinase from Cyanobacteria

Author: Adeline Y. Robin, Gilles Curien, Renaud Dumas, Jean-Luc Ferrer, Mylène Robert-Genthon, David Cobessi, Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Models, Molecular, Threonine, MESH: Protein Structure, Quaternary, MESH: Protein Structure, Secondary, MESH: Amino Acid Sequence, MESH: Synechocystis, Crystallography, X-Ray, cyanobacteria, Protein Structure, Secondary, Protein structure, ACT domain, Structural Biology, MESH: Threonine, 0303 health sciences, allostery, biology, 030302 biochemistry & molecular biology, Synechocystis, Prephenate dehydrogenase, Biochemistry, Chorismate mutase, Dimerization, MESH: Models, Molecular, Protein Binding, crystal structure, Stereochemistry, Molecular Sequence Data, 03 medical and health sciences, MESH: Aspartate Kinase, MESH: Protein Binding, Aspartate kinase, MESH: Lysine, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, structure, Amino Acid Sequence, Aspartate Kinase, Protein Structure, Quaternary, Molecular Biology, 030304 developmental biology, MESH: Molecular Sequence Data, Binding Sites, Lysine, metabolic pathway, biology.organism_classification, MESH: Crystallography, X-Ray, enzyme, Allosteric enzyme, MESH: Binding Sites, MESH: Dimerization, essential aminoacid, biology.protein
Abstract: International audience; Aspartate kinases (AKs) can be divided in two subhomology divisions, AKalpha and AKbeta, depending on the presence of an extra sequence of about 60 amino acids, which is found only in the N-terminus of all AKalpha's. To date, the structures of AKalpha failed to provide a role for this additional N-terminal sequence. In this study, the structure of the AKbeta from the Cyanobacteria Synechocystis reveals that this supplementary sequence is linked to the dimerization mode of AKs. Its absence in AKbeta leads to the dimerization by the catalytic domain instead of involving the ACT domains [Pfam 01842; small regulatory domains initially found in AK, chorismate mutase and TyrA (prephenate dehydrogenase)] as observed in AKalpha. Thus, the structural analysis of the Synechocystis AKbeta revealed a dimer with a novel architecture. The four ACT domains of each monomer interact together and do not make any contact with those of the second monomer. The enzyme is inhibited synergistically by threonine and lysine with the binding of threonine first. The interaction between ACT1 and ACT4 or between ACT2 and ACT3 generates a threonine binding site and a lysine binding site at each interface, making a total of eight regulatory sites per dimer and allowing a fine-tuning of the AK activity by the end products, threonine and lysine.
Published: 2010

38. Amino acid biosynthesis: New architectures in allosteric enzymes

Author: Valérie Biou, Gilles Curien, Mylène Robert-Genthon, Jean-Luc Ferrer, Renaud Dumas, Corine Mas-Droux, Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'enzymologie et biochimie structurales (LEBS), Centre National de la Recherche Scientifique (CNRS), Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Models, Molecular, Physiology, aminoacid biosynthesis, plant, Plant Science, yeast, Protein Structure, Secondary, chemistry.chemical_compound, ACT domain, structural biology, Amino Acids, bacteria, Amino acid synthesis, Plant Proteins, acetohydroxyacid synthase, chemistry.chemical_classification, 0303 health sciences, allostery, 030302 biochemistry & molecular biology, dihydrodipicolinate synthase, Enzymes, Biochemistry, threonine synthase, aspartate kinase, Carbon-Oxygen Lyases, Allosteric regulation, review, Biology, 03 medical and health sciences, Allosteric Regulation, Bacterial Proteins, Biosynthesis, Genetics, Aspartate kinase, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, crystallography, 030304 developmental biology, Homoserine dehydrogenase, S-adenosylmethionine, allosteric control, metabolic pathway, homoserine dehydrogenase, isopropylmalate synthase, Protein Structure, Tertiary, enzyme, Enzyme, chemistry, Allosteric enzyme, kinetics, biology.protein, metabolism
Abstract: This review focuses on the allosteric controls in the Aspartate-derived and the branched-chain amino acid biosynthetic pathways examined both from kinetic and structural points of view. The objective is to show the differences that exist among the plant and microbial worlds concerning the allosteric regulation of these pathways and to unveil the structural bases of this diversity. Indeed, crystallographic structures of enzymes from these pathways have been determined in bacteria, fungi and plants, providing a wonderful opportunity to obtain insight into the acquisition and modulation of allosteric controls in the course of evolution. This will be examined using two enzymes, threonine synthase and the ACT domain containing enzyme aspartate kinase. In a last part, as many enzymes in these pathways display regulatory domains containing the conserved ACT module, the organization of ACT domains in this kind of allosteric enzymes will be reviewed, providing explanations for the variety of allosteric effectors and type of controls observed.
Published: 2008

39. Simplified modelling of metabolic pathways for flux prediction and optimisation: lessons from an in vitro reconstruction of the upper part of glycolysis

Author: Christine Dillmann, Dominique de Vienne, Julie B. Fiévet, Gilles Curien, Génétique Végétale (GV), Institut National de la Recherche Agronomique (INRA)-Université Paris-Sud - Paris 11 (UP11)-Institut National Agronomique Paris-Grignon (INA P-G)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
Subjects: In Vitro Techniques, Context (language use), Biology, Models, Biological, Biochemistry, Set (abstract data type), Metabolic engineering, modelling, 03 medical and health sciences, metabolic network, Computer Simulation, Integrative biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, biochemical pathway, 030304 developmental biology, 0303 health sciences, Quantitative Biology::Molecular Networks, 030302 biochemistry & molecular biology, Cell Biology, Models, Theoretical, glycolysis, Metabolic pathway, enzyme, Metabolism, flux computation, kinetics, Biological system, metabolic engineering, Flux (metabolism), Algorithms, Research Article
Abstract: Explicit modelling of metabolic networks relies on well-known mathematical tools and specialized computer programs. However, identifying and estimating the values of the very numerous enzyme parameters inherent to the models remain a tedious and difficult task, and the rate equations of the reactions are usually not known in sufficient detail. A way to circumvent this problem is to use ‘non-mechanistic’ models, which may account for the behaviour of the systems with a limited number of parameters. Working on the first part of glycolysis reconstituted in vitro, we showed how to derive, from titration experiments, values of effective enzyme activity parameters that do not include explicitly any of the classical kinetic constants. With a maximum of only two parameters per enzyme, this approach produced very good estimates for the flux values, and enabled us to determine the optimization conditions of the system, i.e. to calculate the set of enzyme concentrations that maximizes the flux. This fast and easy method should be valuable in the context of integrative biology or for metabolic engineering, where the challenge is to deal with the dramatic increase in the number of parameters when the systems become complex.
Published: 2006

40. A novel organization of ACT domains in allosteric enzymes revealed by the crystal structure of Arabidopsis aspartate kinase

Author: Jean-Luc Ferrer, Mylène Robert-Genthon, Corine Mas-Droux, Renaud Dumas, Mathieu Laurencin, Gilles Curien, Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG)
Subjects: Models, Molecular, S-Adenosylmethionine, Arabidopsis thaliana, crystallization, Arabidopsis, plant, Plant Science, Crystallography, X-Ray, Adenosine Triphosphate, ACT domain, Catalytic Domain, Serine, Transferase, Research Articles, Aspartate kinase, 0303 health sciences, allostery, biology, Effector, 030302 biochemistry & molecular biology, X Ray structure, Isoenzymes, SAM, Biochemistry, allosteric effector, Dimerization, Protein subunit, Molecular Sequence Data, Allosteric regulation, essential aminoacids biosynthesis, 03 medical and health sciences, Allosteric Regulation, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Protein Structure, Quaternary, 030304 developmental biology, Aspartic Acid, Binding Sites, lysine, Arabidopsis Proteins, Cell Biology, metabolic pathway, biology.organism_classification, Protein Subunits, enzyme, Allosteric enzyme, biology.protein
Abstract: Asp kinase catalyzes the first step of the Asp-derived essential amino acid pathway in plants and microorganisms. Depending on the source organism, this enzyme contains up to four regulatory ACT domains and exhibits several isoforms under the control of a great variety of allosteric effectors. We report here the dimeric structure of a Lys and S-adenosylmethionine–sensitive Asp kinase isoform from Arabidopsis thaliana in complex with its two inhibitors. This work reveals the structure of an Asp kinase and an enzyme containing two ACT domains cocrystallized with its effectors. Only one ACT domain (ACT1) is implicated in effector binding. A loop involved in the binding of Lys and S-adenosylmethionine provides an explanation for the synergistic inhibition by these effectors. The presence of S-adenosylmethionine in the regulatory domain indicates that ACT domains are also able to bind nucleotides. The organization of ACT domains in the present structure is different from that observed in Thr deaminase and in the regulatory subunit of acetohydroxyacid synthase III.
Published: 2006

41. Identification of six novel allosteric effectors of Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase isoforms. Physiological context sets the specificity

Author: Gilles, Curien, Stéphane, Ravanel, Mylène, Robert, and Renaud, Dumas
Subjects: Threonine, Chloroplasts, DNA, Complementary, Arabidopsis, Models, Biological, Adenosine Triphosphate, Methionine, Gene Expression Regulation, Plant, Genes, Reporter, Escherichia coli, Homoserine Dehydrogenase, Protein Isoforms, Aspartate Kinase, Enzyme Inhibitors, Isoleucine, Aspartic Acid, Alanine, Dose-Response Relationship, Drug, Temperature, Recombinant Proteins, Enzyme Activation, Kinetics, Spectrophotometry, Allosteric Site, Genome, Plant, Plasmids
Abstract: The Arabidopsis genome contains two genes predicted to code for bifunctional aspartate kinase-homoserine dehydrogenase enzymes (isoforms I and II). These two activities catalyze the first and the third steps toward the synthesis of the essential amino acids threonine, isoleucine, and methionine. We first characterized the kinetic and regulatory properties of the recombinant enzymes, showing that they mainly differ with respect to the inhibition of the homoserine dehydrogenase activity by threonine. A systematic search for other allosteric effectors allowed us to identify an additional inhibitor (leucine) and 5 activators (alanine, cysteine, isoleucine, serine, and valine) equally efficient on aspartate kinase I activity (4-fold activation). The six effectors of aspartate kinase I were all activators of aspartate kinase II activity (13-fold activation) and displayed a similar specificity for the enzyme. No synergy between different effectors could be observed. The activation, which resulted from a decrease in the Km values for the substrates, was detected using low substrates concentrations. Amino acid quantification revealed that alanine and threonine were much more abundant than the other effectors in Arabidopsis leaf chloroplasts. In vitro kinetics in the presence of physiological concentrations of the seven allosteric effectors confirmed that aspartate kinase I and II activities were highly sensitive to changes in alanine and threonine concentrations. Thus, physiological context rather than enzyme structure sets the specificity of the allosteric control. Stimulation by alanine may play the role of a feed forward activation of the aspartate-derived amino acid pathway in plant.
Published: 2005

42. Methionine metabolism in plants: chloroplasts are autonomous for de novo methionine synthesis and can import S-adenosylmethionine from the cytosol

Author: Stéphane, Ravanel, Maryse A, Block, Pascal, Rippert, Samuel, Jabrin, Gilles, Curien, Fabrice, Rébeillé, and Roland, Douce
Subjects: S-Adenosylmethionine, Chloroplasts, DNA, Complementary, Time Factors, Dose-Response Relationship, Drug, Blotting, Western, Green Fluorescent Proteins, Immunoblotting, Molecular Sequence Data, Arabidopsis, Peas, Models, Biological, Mitochondria, Diffusion, Kinetics, Luminescent Proteins, Vitamin B 12, Cytosol, Methionine, Escherichia coli, Protein Isoforms, Plastids, Cloning, Molecular, Homocysteine, Phylogeny
Abstract: The subcellular distribution of Met and S-adenosylmethionine (AdoMet) metabolism in plant cells discloses a complex partition between the cytosol and the organelles. In the present work we show that Arabidopsis contains three functional isoforms of vitamin B(12)-independent methionine synthase (MS), the enzyme that catalyzes the methylation of homocysteine to Met with 5-methyltetrahydrofolate as methyl group donor. One MS isoform is present in chloroplasts and is most likely required to methylate homocysteine that is synthesized de novo in this compartment. Thus, chloroplasts are autonomous and are the unique site for de novo Met synthesis in plant cells. The additional MS isoforms are present in the cytosol and are most probably involved in the regeneration of Met from homocysteine produced in the course of the activated methyl cycle. Although Met synthesis can occur in chloroplasts, there is no evidence that AdoMet is synthesized anywhere but the cytosol. In accordance with this proposal, we show that AdoMet is transported into chloroplasts by a carrier-mediated facilitated diffusion process. This carrier is able to catalyze the uniport uptake of AdoMet into chloroplasts as well as the exchange between cytosolic AdoMet and chloroplastic AdoMet or S-adenosylhomocysteine. The obvious function for the carrier is to sustain methylation reactions and other AdoMet-dependent functions in chloroplasts and probably to remove S-adenosylhomocysteine generated in the stroma by methyltransferase activities. Therefore, the chloroplastic AdoMet carrier serves as a link between cytosolic and chloroplastic one-carbon metabolism.
Published: 2004

43. A kinetic model of the branch-point between the methionine and threonine biosynthesis pathways in Arabidopsis thaliana

Author: Gilles, Curien, Stéphane, Ravanel, and Renaud, Dumas
Subjects: Threonine, Kinetics, Methionine, Models, Chemical, Carbon-Oxygen Lyases, Molecular Sequence Data, Arabidopsis, Reproducibility of Results, Computer Simulation, Sensitivity and Specificity, Adenosine Monophosphate
Abstract: This work proposes a model of the metabolic branch-point between the methionine and threonine biosynthesis pathways in Arabidopsis thaliana which involves kinetic competition for phosphohomoserine between the allosteric enzyme threonine synthase and the two-substrate enzyme cystathionine gamma-synthase. Threonine synthase is activated by S-adenosylmethionine and inhibited by AMP. Cystathionine gamma-synthase condenses phosphohomoserine to cysteine via a ping-pong mechanism. Reactions are irreversible and inhibited by inorganic phosphate. The modelling procedure included an examination of the kinetic links, the determination of the operating conditions in chloroplasts and the establishment of a computer model using the enzyme rate equations. To test the model, the branch-point was reconstituted with purified enzymes. The computer model showed a partial agreement with the in vitro results. The model was subsequently improved and was then found consistent with flux partition in vitro and in vivo. Under near physiological conditions, S-adenosylmethionine, but not AMP, modulates the partition of a steady-state flux of phosphohomoserine. The computer model indicates a high sensitivity of cystathionine flux to enzyme and S-adenosylmethionine concentrations. Cystathionine flux is sensitive to modulation of threonine flux whereas the reverse is not true. The cystathionine gamma-synthase kinetic mechanism favours a low sensitivity of the fluxes to cysteine. Though sensitivity to inorganic phosphate is low, its concentration conditions the dynamics of the system. Threonine synthase and cystathionine gamma-synthase display similar kinetic efficiencies in the metabolic context considered and are first-order for the phosphohomoserine substrate. Under these conditions outflows are coordinated.
Published: 2003

44. Mechanism of control of Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase by threonine

Author: Renaud Dumas, Claire Viemon, Gilles Curien, Stéphane Paris, Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Bayer SAS, and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)
Subjects: Threonine, Molecular Sequence Data, Mutant, Arabidopsis, Dehydrogenase, Biology, Biochemistry, gène thra, Structure-Activity Relationship, 03 medical and health sciences, Aspartate kinase, Amino Acid Sequence, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], inhibition enzymatique, Molecular Biology, Plant Proteins, 030304 developmental biology, chemistry.chemical_classification, Homoserine dehydrogenase, 0303 health sciences, Aspartate kinase activity, BIOCHIMIE, ASPARTOKINASE, 030302 biochemistry & molecular biology, Cell Biology, séquence nucléotidique, désaminase, Amino acid, Enzyme Activation, Kinetics, thréonine, Enzyme, chemistry, protéine, Aspartokinase Homoserine Dehydrogenase, Mutation, structure enzymatique, escherichia coli
Abstract: International audience; The regulatory domain of the bifunctional threonine-sensitive aspartate kinase homoserine dehydrogenase contains two homologous subdomains defined by a common loop-αhelix-loop-β strand-loop-β strand motif. This motif is homologous with that found in the two subdomains of the biosynthetic threonine-deaminase regulatory domain. Comparisons of the primary and secondary structures of the two enzymes allowed us to predict the location and identity of the amino acid residues potentially involved in two threonine-binding sites ofArabidopsis thaliana aspartate kinase-homoserine dehydrogenase. These amino acids were then mutated and activity measurements were carried out to test this hypothesis. Steady-state kinetic experiments on the wild-type and mutant enzymes demonstrated that each regulatory domain of the monomers of aspartate kinase-homoserine dehydrogenase possesses two nonequivalent threonine-binding sites constituted in part by Gln443 and Gln524. Our results also demonstrated that threonine interaction with Gln443 leads to inhibition of aspartate kinase activity and facilitates the binding of a second threonine on Gln524. Interaction of this second threonine with Gln524 leads to inhibition of homoserine dehydrogenase activity.
Published: 2003

45. MAD on threonine synthase: the phasing power of oxidized selenomethionine

Author: Valérie Biou, Gilles Curien, Renaud Dumas, Andrew Thompson, and Karine Thomazeau
Subjects: Models, Molecular, biology, Chemistry, Protein Conformation, Carbon-Oxygen Lyases, Arabidopsis, chemistry.chemical_element, General Medicine, Phase problem, Crystallography, X-Ray, Signal, Wavelength, Crystallography, Threonine synthase, Protein structure, Spectrometry, Fluorescence, Structural Biology, Atom, biology.protein, Molecule, Crystallization, Selenomethionine, Oxidation-Reduction, Selenium
Abstract: The use of selenomethionine and anomalous dispersion has become the most widely used way of solving the phase problem for de novo protein structure determination. In this paper, MAD data collected from oxidized and reduced selenomethionine-containing protein are described, and it is shown that oxidized selenomethionine has a very strong phasing power and can be efficiently used if the oxidation is uniform. The comparison was performed on threonine synthase crystals. For example, the phasing power of the oxidized data is doubled for the dispersive signal and is 20% stronger for the anomalous signal at the peak wavelength. The strength of the anomalous signal can be used to improve the signal when a protein contains few methionines or for single anomalous dispersion. The oxidation of some selenomethionines shows in the electron-density map through the presence of water molecules within hydrogen-bonding distance of the putative O atom.
Published: 2001

46. Biochemical and molecular bases of the carbon flux regulation between threonine and methionine in plants

Author: V. Verne, Roland Douce, Gilles Curien, Dominique Job, Michel Droux, J. Yaxley, Bertrand Gakière, and Stéphane Ravanel
Subjects: chemistry.chemical_classification, Methionine, biology, Homoserine, biology.organism_classification, Amino acid, chemistry.chemical_compound, chemistry, Biochemistry, biology.protein, Methionine synthase, Threonine, Bacteria, Carbon flux
Abstract: Methionine and threonine are two essential amino acids belonging to the aspartate-derived family of amino acids. In plants, they both originate from the same precursor, O-phosphohomoserine (OPH) (Fig. 1). This contrasts with the situation seen in bacteria where branching between the two pathways occurs upstream of OPH, at the level of homoserine (Saint-Girons et al, 1998). Despite intensive studies (for reviews, see Giovanelli et al., 1980; Leustek, 1996; Azevedo et al., 1997; Hell, 1997; Ravanel, 1997; Ravanel et al., 1998b), the specific mechanisms that are responsible for homeostatic regulation of methionine and threonine in plants are not fully understood.
Published: 1999

47. Allosteric activation of Arabidopsis threonine synthase by S-adenosylmethionine

Author: Roland Douce, Renaud Dumas, Dominique Job, and Gilles Curien
Subjects: S-Adenosylmethionine, Stereochemistry, Allosteric regulation, Carbon-Oxygen Lyases, Arabidopsis, Biochemistry, Models, Biological, Homoserine, Enzyme kinetics, Threonine, Amino acid synthesis, Plant Proteins, chemistry.chemical_classification, Homoserine dehydrogenase, biology, Receptor protein serine/threonine kinase, Recombinant Proteins, Enzyme Activation, Metabolic pathway, Kinetics, Threonine synthase, Spectrometry, Fluorescence, chemistry, Models, Chemical, biology.protein, Allosteric Site, Protein Binding
Abstract: Plant threonine synthase, in contrast to its bacterial counterpart, is strongly stimulated by S-adenosylmethionine via a noncovalent interaction (Giovanelli et al. (1984) Plant. Physiol. 76, 285- 292). The mechanism of activation remained, however, largely unknown. To further characterize this unusual role for S-adenosylmethionine, the Arabidopsis thaliana threonine synthase was overexpressed in Escherichia coli, purified to homogeneity, and then used for kinetic and enzyme-bound pyridoxal 5'- phosphate fluorescence equilibrium-binding experiments. We observed that the activating effect of S-adenosylmethionine results from an 8-fold increase in the rate of catalysis and from a 25-fold decrease in the Km value for the O-phosphohomoserine substrate. The data can be well fitted by a kinetic model assuming binding of two S-adenosylmethionine molecules on the native enzyme. We suggest that the dramatic modifications of the enzyme kinetic properties originate most presumably from an allosteric and cooperative transition induced by S-adenosylmethionine. This transition occurs at a much faster rate in the presence of the substrate than in its absence. The aspartate derived amino acids pathway in plants has raised strong interests over the past few years when the inhibition of several enzymes in the pathway was proved to be herbicidal (1). More recently, the possibility of obtaining transgenic plants with increased levels of the essential amino acids methionine, lysine, and threonine (2) has contributed to stimulate researches on this metabolic pathway in plants. These agrochemical and nutritional applications require detailed studies of the enzyme kinetics and regulatory mechanisms. Our study of plant threonine synthase takes place in this context. Plant threonine synthase (EC 4.2.99.2), a pyridoxal 5'- phosphate dependent enzyme, catalyzes the last step of threonine formation (3), converting O-phosphohomoserine (OPH) 1 to threonine and phosphate.
Published: 1998

48. Biosynthesis of Branched-Chain Amino Acids in Plants: Structure and Function of Acetohydroxy Acid Isomeroreductase

Author: Dominique Job, Gilles Curien, Roland Douce, Eva Pebay-Peyroula, Valérie Biou, Renaud Dumas, Claudine Cohen-Addad, Marie-Christine Butikofer, and Françoise Vives
Subjects: chemistry.chemical_classification, chemistry.chemical_compound, Biochemistry, Biosynthesis, chemistry, Chain (algebraic topology), Acetohydroxy acid isomeroreductase, Structure and function, Amino acid
Published: 1995

49. Understanding the regulation of aspartate metabolism using a model based on measured kinetic parameters

Author: Olivier Bastien, Athel Cornish-Bowden, Renaud Dumas, Mylène Robert-Genthon, María Luz Cárdenas, Gilles Curien, Laboratoire de physiologie cellulaire végétale (LPCV), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), Bioénergétique et Ingénierie des Protéines (BIP ), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0106 biological sciences, Chloroplasts, Arabidopsis thaliana, Arabidopsis, Aspartate metabolism, plant, 01 natural sciences, enzyme kinetics, Threonine, Amino Acids, ComputingMilieux_MISCELLANEOUS, 0303 health sciences, aspartate, Mathematical model, Applied Mathematics, simulation, Recombinant Proteins, Computational Theory and Mathematics, Biochemistry, General Agricultural and Biological Sciences, Biological system, aminoacid, flux distribution, Information Systems, metabolism, metabolic pathway, mathematical model, allosteric regulation, threonine, Allosteric regulation, Biology, Kinetic energy, Models, Biological, General Biochemistry, Genetics and Molecular Biology, Article, 03 medical and health sciences, aspartate metabolism, SDV:BBM, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Computer Simulation, 030304 developmental biology, Aspartic Acid, General Immunology and Microbiology, Kinetic model, Reproducibility of Results, biology.organism_classification, Kinetics, Function (biology), 010606 plant biology & botany
Abstract: International audience; The aspartate-derived amino-acid pathway from plants is well suited for analysing the function of the allosteric network of interactions in branched pathways. For this purpose, a detailed kinetic model of the system in the plant model Arabidopsis was constructed on the basis of in vitro kinetic measurements. The data, assembled into a mathematical model, reproduce in vivo measurements and also provide non-intuitive predictions. A crucial result is the identification of allosteric interactions whose function is not to couple demand and supply but to maintain a high independence between fluxes in competing pathways. In addition, the model shows that enzyme isoforms are not functionally redundant, because they contribute unequally to the flux and its regulation. Another result is the identification of the threonine concentration as the most sensitive variable in the system, suggesting a regulatory role for threonine at a higher level of integration.
Published: 2009

50. Threonine synthase fromArabidopsis thaliana: 2.2 Å resolution structure and reaction proposal

Author: R. Dumas, Valérie Biou, Gilles Curien, and K. Thomazeau
Subjects: Threonine synthase, biology, Biochemistry, Structural Biology, Chemistry, Resolution (electron density), biology.protein, Arabidopsis thaliana, biology.organism_classification
Published: 2000

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