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7 results on '"Cécile Giustini"'

1. 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

2. 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

3. Identification of Two Conserved Residues Involved in Copper Release from Chloroplast PIB-1-ATPases

Author

Cécile Giustini, Emeline Sautron, Thuy Van Dang, Daniel Salvi, Patrice Catty, Lucas Moyet, Serge Crouzy, Norbert Rolland, Daphné Seigneurin-Berny, 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), Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), 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 a l'Energie Atomique et aux Energies Alternatives, Centre National de la Recherche Scientifique, French National Institute for Agricultural Research, University of Grenoble Alpes, GRAL Labex [ANR-10-LABX-49-01], 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), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and 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)

Subjects
0301 basic medicine, Saccharomyces cerevisiae, Amino Acid Motifs, Arabidopsis, chemistry.chemical_element, Plant Biology, Biochemistry, Chloroplast membrane, Thylakoids, 03 medical and health sciences, chloroplast, ATPase, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, Histidine, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, Plastocyanin, Adenosine Triphosphatases, 030102 biochemistry & molecular biology, biology, Arabidopsis Proteins, food and beverages, Cell Biology, biology.organism_classification, Copper release site, Copper, Recombinant Proteins, Chloroplast, Lactococcus lactis, 030104 developmental biology, chemistry, PIB-1-ATPases, Thylakoid, plant biochemistry, copper transport, copper release site, Thylakoid Membrane Proteins, Cysteine
Abstract

International audience; Copper is an essential transition metal for living organisms. In the plant model Arabidopsis thaliana, half of the copper content is localized in the chloroplast and, as a cofactor of plastocyanin, copper is essential for photosynthesis. Within the chloroplast, copper delivery to plastocyanin involves two transporters of the PIB-1-ATPases subfamily, HMA6 at the chloroplast envelope and HMA8, in the thylakoid membranes. Both proteins are high affinity copper transporters but share distinct enzymatic properties. In the present work, the comparison of 140 sequences of PIB-1-ATPases revealed a conserved region unusually rich in histidine and cysteine residues in the TMA-L1 region of eukaryotic chloroplast copper ATPases. To evaluate the role of these residues, we mutated them in HMA6 and HMA8. Mutants of interest were selected from phenotypic tests in yeast and produced in Lactococcus lactis for further biochemical characterizations using phosphorylation assays from ATP and Pi. Combining functional and structural data, we highlight the importance of the cysteine and the first histidine of the Cx3Hx2H motif in the process of copper release from HMA6 and HMA8, and propose a copper pathway through the membrane domain of these transporters. Finally our work suggests a more general role of the histidine residue in the transport of copper by PIB-1-ATPases.

Published
2016

4. 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

5. 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

6. 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

7. 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

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