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1. 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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2. 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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3. Impaired photoprotection in Phaeodactylum tricornutum KEA3 mutants reveals the proton regulatory circuit of diatoms light acclimation

Author

Cécile Giustini, Xue Zhao, Yufang Pan, Anna Matuszyńska, Benjamin Bailleul, Alexander V. Ruban, Florence Courtois, Hanhua Hu, Mattia Storti, Erika Guglielmino, Jhoanell Angulo, Claire Seydoux, Vasco Giovagnetti, Giovanni Finazzi, and Guillaume Allorent

Subjects
chemistry.chemical_compound, Light intensity, Diatom, biology, Chemistry, Non-photochemical quenching, Photoprotection, Antiporter, Diadinoxanthin, Biophysics, Phaeodactylum tricornutum, biology.organism_classification, Photosynthesis
Abstract

Diatoms are amongst the most successful clades of oceanic phytoplankton, significantly contributing to photosynthesis on Earth. Their ecological success likely stems from their ability to acclimate to changing environmental conditions, including e.g. variable light intensity. Diatoms are outstanding at dissipating light energy exceeding the maximum photosynthetic electron transfer (PET) capacity of via Non Photochemical Quenching (NPQ). While the molecular effectors of this process, as well as the role of the Proton Motive Force (PMF) in its regulation are known, the putative regulators of the PET/PMF relationship in diatoms remain unidentified. Here, we demonstrate that the H+/K+ antiporter KEA3 is the main regulator of the coupling between PMF and PET in the model diatom Phaeodactylum tricornutum. By controlling the PMF, it modulates NPQ responses at the onset of illumination, during transients and in steady state conditions. Under intermittent light KEA3 absence results in reduced fitness. Using a parsimonious model including only two components, KEA3 and the diadinoxanthin de-epoxidase, we can describe most of the feedback loops observed between PET and NPQ. This two-components regulatory system allows for efficient responses to fast (minutes) or slow (e.g. diel) changes in light environment, thanks to the presence of a regulatory Ca2+-binding domain in KEA3 that controls its activity. This circuit is likely finely tuned by the NPQ effector proteins LHCX, providing diatoms with the required flexibility to thrive in different ocean provinces.One sentence summaryThe author(s) responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plcell/pages/General-Instructions) is Giovanni Finazzi.

Published
2021

4. 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

5. 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

6. 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

7. 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

8. Generation of Mutants of Nuclear-Encoded Plastid Proteins Using CRISPR/Cas9 in the Diatom Phaeodactylum tricornutum

Author

Guillaume, Allorent, Erika, Guglielmino, Cécile, Giustini, and Florence, Courtois

Subjects
Cell Nucleus, Diatoms, Gene Editing, Chloroplast Proteins, Gene Knockout Techniques, Mutation, Biolistics, CRISPR-Cas Systems
Abstract

Genome modifications in microalgae are becoming a widespread and mandatory tool for research in both fundamental and applied biology. Among genome editing methods in these photosynthetic organisms, CRISPR/Cas9 offers a specific, powerful and efficient tool for genome engineering by inducing mutations in targeted regions of the genome. Here we described a protocol that allows the generation of knockout mutants by CRISPR/Cas9 in the diatom Phaeodactylum tricornutum using biolistic transformation.

Published
2018

9. Generation of Mutants of Nuclear-Encoded Plastid Proteins Using CRISPR/Cas9 in the Diatom Phaeodactylum tricornutum

Author

Cécile Giustini, Florence Courtois, Erika Guglielmino, Guillaume Allorent, 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), Marechal Eric, 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), 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
0106 biological sciences, 0301 basic medicine, Biolistic transformation, biology, Cas9, Mutant, Computational biology, biology.organism_classification, 01 natural sciences, Genome, Phaeodactylum tricornutum, Genome engineering, 03 medical and health sciences, Transformation (genetics), 030104 developmental biology, Genome editing, CRISPR, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, CRISPR/Cas9, 010606 plant biology & botany
Abstract

International audience; Genome modifications in microalgae are becoming a widespread and mandatory tool for research in both fundamental and applied biology. Among genome editing methods in these photosynthetic organisms, CRISPR/Cas9 offers a specific, powerful and efficient tool for genome engineering by inducing mutations in targeted regions of the genome. Here we described a protocol that allows the generation of knockout mutants by CRISPR/Cas9 in the diatom Phaeodactylum tricornutum using biolistic transformation.

Published
2018

10. 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

11. 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

12. 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

13. Crystal structure of norcoclaurine-6- O -methyltransferase, a key rate-limiting step in the synthesis of benzylisoquinoline alkaloids

Author

Renaud Dumas, Matthieu Graindorge, Adeline Robin, Michel Matringe, Cécile Giustini, 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), INRA, European Project: 283570,EC:FP7:INFRA,FP7-INFRASTRUCTURES-2011-1,BIOSTRUCT-X(2011), and 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)

Subjects
0106 biological sciences, 0301 basic medicine, Berberine, Protein Conformation, [SDV]Life Sciences [q-bio], Norcoclaurine - 6-0-methyltransferase, Plant Science, Crystallography, X-Ray, 01 natural sciences, chemistry.chemical_compound, Thalictrum, Benzylisoquinoline, Enzyme Inhibitors, ComputingMilieux_MISCELLANEOUS, Plant Proteins, chemistry.chemical_classification, Alkaloid biosynthesis, biology, Alkaloid, Biochemistry, Thalictrum flavum, Antimicrobial agent, Benzylisoquinolines, 03 medical and health sciences, Escherichia coli, Genetics, Sanguinarine, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Secondary metabolism, Benzophenanthridines, Reticuline, Binding Sites, Crystal structure, Active site, Methyltransferases, Cell Biology, Plant, Isoquinolines, 030104 developmental biology, Enzyme, chemistry, biology.protein, Protein Multimerization, Ranunculaceae, 010606 plant biology & botany
Abstract

International audience; Growing pharmaceutical interest in benzylisoquinoline alkaloids (BIA) coupled with their chemical complexity make metabolic engineering of microbes to create alternative platforms of production an increasingly attractive proposition. However, precise knowledge of rate-limiting enzymes and negative feedback inhibition by end-products of BIA metabolism is of paramount importance for this emerging field of synthetic biology. In this work we report the structural characterization of (S)-norcoclaurine-6-O-methyltransferase (6OMT), a key rate-limiting step enzyme involved in the synthesis of reticuline, the final intermediate to be shared between the different end-products of BIA metabolism, such as morphine, papaverine, berberine and sanguinarine. Four different crystal structures of the enzyme from Thalictrum flavum (Tf 6OMT) were solved: the apoenzyme, the complex with S-adenosyl-l-homocysteine (SAH), the complexe with SAH and the substrate and the complex with SAH and a feedback inhibitor, sanguinarine. The Tf 6OMT structural study provides a molecular understanding of its substrate specificity, active site structure and reaction mechanism. This study also clarifies the inhibition of Tf 6OMT by previously suggested feedback inhibitors. It reveals its high and time-dependent sensitivity toward sanguinarine. Significance Statement Many plants produce plant-derived alkaloid secondary metabolites that find uses as drugs including benzylisoquinoline alkaloids. A molecular understanding of the reaction mechanism of (S)-norcoclaurine-6-O-methyltransferase is provided, which also clarifies the biochemical inhibition of this key rate-limiting enzyme by feedback inhibitors berberine and sanguinarine.

Published
2016

15. Dynamics Characterization of Fully Hydrated Bacterial Cell Walls by Solid-State NMR: Evidence for Cooperative Binding of Metal Ions

Author

Thomas Kern, Bernard Joris, Nhat Khai Bui, Jean-Pierre Simorre, Sabine Hediger, Waldemar Vollmer, Mathilde Giffard, Catherine M. Bougault, Ana Maria Amoroso, and Cécile Giustini

Subjects
Staphylococcus aureus, Peptidoglycan, Biochemistry, Catalysis, Bacterial cell structure, Divalent, Cell wall, 03 medical and health sciences, chemistry.chemical_compound, Colloid and Surface Chemistry, Cell Wall, Escherichia coli, Magnesium, Nuclear Magnetic Resonance, Biomolecular, 030304 developmental biology, Ions, chemistry.chemical_classification, Manganese, 0303 health sciences, Teichoic acid, Binding Sites, Molecular Structure, 030306 microbiology, Water, Cooperative binding, General Chemistry, Nuclear magnetic resonance spectroscopy, Teichoic Acids, Solid-state nuclear magnetic resonance, chemistry, Biophysics, Thermodynamics, Bacillus subtilis
Abstract

The bacterial cell wall maintains a cell's integrity while allowing growth and division. It is made up of peptidoglycan (PG), a biopolymer forming a multigigadalton bag-like structure, and, additionally in gram-positive bacteria, of covalently linked anionic polymers collectively called teichoic acids. These anionic polymers are thought to play important roles in host-cell adhesion, inflammation, and immune activation. In this Article, we compare the flexibility and the organization of peptidoglycans from gram-negative bacteria (E. coli) with its counterpart from different gram-positive bacteria using solid-state nuclear magnetic resonance spectroscopy (NMR) under magic-angle sample spinning (MAS). The NMR fingerprints suggest an identical local conformation of the PG in all of these bacterial species. Dynamics in the peptidoglycan network decreases from E. coli to B. subtilis and from B. subtilis to S. aureus and correlates mainly with the degree of peptide cross-linkage. For intact bacterial cells and isolated cell walls, we show that (31)P solid-state NMR is particularly well adapted to characterize and differentiate wall teichoic acids of different species. We have further observed complexation with divalent ions, highlighting an important structural aspect of gram-positive cell wall architecture. We propose a new model for the interaction of divalent cations with both wall teichoic acids and carbonyl groups of peptidoglycan.

Published
2010

16. 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

17. 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

18. 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

19. 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

20. Central domain of DivIB caps the C-terminal regions of the FtsL/DivIC coiled-coil rod

Author

Cécile Giustini, Frank Gabel, Soizic Masson, André Zapun, Thomas Kern, Jean-Pierre Simorre, Thierry Vernet, Audrey Le Gouellec, Philip Callow, 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), 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), 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), Le Gouellec, Audrey, 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, Magnetic Resonance Spectroscopy, MESH: Protein Structure, Quaternary, [SDV]Life Sciences [q-bio], MESH: Protein Structure, Secondary, Cell Cycle Proteins, MESH: Amino Acid Sequence, Biochemistry, Protein Structure, Secondary, MESH: Protein Structure, Tertiary, Protein structure, X-Ray Diffraction, MESH: Peptide Fragments, Peptide sequence, MESH: Bacterial Proteins, Coiled coil, 0303 health sciences, MESH: Protein Multimerization, MESH: X-Ray Diffraction, Transmembrane protein, Cell biology, MESH: Surface Plasmon Resonance, MESH: Neutron Diffraction, [SDV] Life Sciences [q-bio], Neutron Diffraction, Streptococcus pneumoniae, Protein Structure and Folding, MESH: Membrane Proteins, MESH: Streptococcus pneumoniae, MESH: Models, Molecular, Molecular Sequence Data, MESH: Sequence Alignment, Sequence alignment, Bacterial genome size, Biology, 03 medical and health sciences, MESH: Cell Cycle Proteins, Bacterial Proteins, Scattering, Small Angle, Amino Acid Sequence, Protein Structure, Quaternary, Cell Cycle Protein, Molecular Biology, MESH: Scattering, Small Angle, 030304 developmental biology, MESH: Molecular Sequence Data, 030306 microbiology, MESH: Magnetic Resonance Spectroscopy, Membrane Proteins, Cell Biology, Surface Plasmon Resonance, Peptide Fragments, Protein Structure, Tertiary, MESH: Solubility, MESH: Extracellular Space, Solubility, Membrane protein, Protein Multimerization, Extracellular Space, Sequence Alignment
Abstract

International audience; DivIB(FtsQ), FtsL, and DivIC(FtsB) are enigmatic membrane proteins that are central to the process of bacterial cell division. DivIB(FtsQ) is dispensable in specific conditions in some species, and appears to be absent in other bacterial species. The presence of FtsL and DivIC(FtsB) appears to be conserved despite very low sequence conservation. The three proteins form a complex at the division site, FtsL and DivIC(FtsB) being associated through their extracellular coiled-coil region. We report here structural investigations by NMR, small-angle neutron and x-ray scattering, and interaction studies by surface plasmon resonance, of the complex of DivIB, FtsL, and DivIC from Streptococcus pneumoniae, using soluble truncated forms of the proteins. We found that one side of the "bean"-shaped central beta-domain of DivIB interacts with the C-terminal regions of the dimer of FtsL and DivIC. This finding is corroborated by sequence comparisons across bacterial genomes. Indeed, DivIB is absent from species with shorter FtsL and DivIC proteins that have an extracellular domain consisting only of the coiled-coil segment without C-terminal conserved regions (Campylobacterales). We propose that the main role of the interaction of DivIB with FtsL and DivIC is to help the formation, or to stabilize, the coiled-coil of the latter proteins. The coiled-coil of FtsL and DivIC, itself or with transmembrane regions, could be free to interact with other partners.

Published
2009

21. Toward the characterization of peptidoglycan structure and protein-peptidoglycan interactions by solid-state NMR spectroscopy

Author

Bernard Joris, Sabine Hediger, Jean-Pierre Simorre, Patrick Müller, Cécile Giustini, Catherine M. Bougault, Thomas Kern, and Waldemar Vollmer

Subjects
Peptidoglycan metabolism, Carbon Isotopes, Magnetic Resonance Spectroscopy, Nitrogen Isotopes, Stereochemistry, Proteins, General Chemistry, Nuclear magnetic resonance spectroscopy, Peptidoglycan, medicine.disease_cause, Biochemistry, Catalysis, Characterization (materials science), carbohydrates (lipids), NMR spectra database, chemistry.chemical_compound, Colloid and Surface Chemistry, Solid-state nuclear magnetic resonance, chemistry, medicine, Escherichia coli, Spectroscopy
Abstract

Solid-state NMR spectroscopy is applied to intact peptidoglycan sacculi of the Gram-negative bacterium Escherichia coli. High-quality solid-state NMR spectra allow atom-resolved investigation of the peptidoglycan structure and dynamics as well as the study of protein-peptidoglycan interactions.

Published
2008

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