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1. Evolution of the Auxin Response Factors from charophyte ancestors.

Author

Raquel Martin-Arevalillo, Emmanuel Thévenon, Fanny Jégu, Thomas Vinos-Poyo, Teva Vernoux, François Parcy, and Renaud Dumas

Subjects
Genetics, QH426-470
Abstract

Auxin is a major developmental regulator in plants and the acquisition of a transcriptional response to auxin likely contributed to developmental innovations at the time of water-to-land transition. Auxin Response Factors (ARFs) Transcription Factors (TFs) that mediate auxin-dependent transcriptional changes are divided into A, B and C evolutive classes in land plants. The origin and nature of the first ARF proteins in algae is still debated. Here, we identify the most 'ancient' ARF homologue to date in the early divergent charophyte algae Chlorokybus atmophyticus, CaARF. Structural modelling combined with biochemical studies showed that CaARF already shares many features with modern ARFs: it is capable of oligomerization, interacts with the TOPLESS co-repressor and specifically binds Auxin Response Elements as dimer. In addition, CaARF possesses a DNA-binding specificity that differs from class A and B ARFs and that was maintained in class C ARF along plants evolution. Phylogenetic evidence together with CaARF biochemical properties indicate that the different classes of ARFs likely arose from an ancestral proto-ARF protein with class C-like features. The foundation of auxin signalling would have thus happened from a pre-existing hormone-independent transcriptional regulation together with the emergence of a functional hormone perception complex.

Published
2019
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2. A SAM oligomerization domain shapes the genomic binding landscape of the LEAFY transcription factor

Author

Camille Sayou, Max H. Nanao, Marc Jamin, David Posé, Emmanuel Thévenon, Laura Grégoire, Gabrielle Tichtinsky, Grégoire Denay, Felix Ott, Marta Peirats Llobet, Markus Schmid, Renaud Dumas, and François Parcy

Subjects
Science
Abstract

The LEAFY transcription factor is a master regulator of flower development in plants. Here the authors describe the structure of a LEAFY oligomerization domain and show that mutations that disrupt oligomerization alter its capacity to bind low affinity and poorly accessible target sites.

Published
2016
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3. Understanding the regulation of aspartate metabolism using a model based on measured kinetic parameters

Author

Gilles Curien, Olivier Bastien, Mylène Robert‐Genthon, Athel Cornish‐Bowden, María Luz Cárdenas, and Renaud Dumas

Subjects
allosteric regulation, Arabidopsis, aspartate metabolism, mathematical model, simulation, Biology (General), QH301-705.5, Medicine (General), R5-920
Abstract

Abstract The aspartate‐derived amino‐acid pathway from plants is well suited for analysing the function of the allosteric network of interactions in branched pathways. For this purpose, a detailed kinetic model of the system in the plant model Arabidopsis was constructed on the basis of in vitro kinetic measurements. The data, assembled into a mathematical model, reproduce in vivo measurements and also provide non‐intuitive predictions. A crucial result is the identification of allosteric interactions whose function is not to couple demand and supply but to maintain a high independence between fluxes in competing pathways. In addition, the model shows that enzyme isoforms are not functionally redundant, because they contribute unequally to the flux and its regulation. Another result is the identification of the threonine concentration as the most sensitive variable in the system, suggesting a regulatory role for threonine at a higher level of integration.

Published
2009
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4. Plant-TFClass: a structural classification for plant transcription factors

Author

Francois Parcy, Laura Turchi, Romain Blanc-Mathieu, Renaud Dumas, and Jérémy LUCAS

Abstract

Transcription factors (TFs) bind DNA at specific sequences to regulate gene expression. This universal process is achieved thanks to the DNA-binding domain (DBD) present in each TF. DBDs show a vast diversity of protein folds within and across organisms, ranging from simple long basic alpha helices to complex structural combinations of alpha, beta and loop folds. In mammals, the structural conformation of the DBDs and the way it establishes contact with DNA has been used to organize TFs in a hierarchical classification named TFClass. However, such classification is missing from plants that possess many DBD types absent from mammals. Here, we reviewed the numerous TF DBD 3D-structures and models available for plants to organize all plant TFs types following the TFClass hierarchy (Superclass/Class/Family/Subfamily). We classified most of the 55 recognized plant TF types within the existing TFClass framework. This extended classification led us to add six new classes and 34 new families corresponding to TF DBD structures absent in mammals. Plant-TFClass provides a unique resource for TF and TF binding sites comparison across TF families and across organisms.

Published
2022
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5. Auxin response factors are keys to the many auxin doors

Author

Coralie Cancé, Raquel Martin‐Arevalillo, Kenza Boubekeur, Renaud Dumas, Reproduction et développement des plantes (RDP), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Régulateurs du développement de la fleur (Flo_RE ), Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), ANR-18-CE12-0014,ChromAuxi,Décodage de la réponse auxine à l'interface ARFs-chromatine(2018), and ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010)

Subjects
Indoleacetic Acids, Physiology, Arabidopsis Proteins, fungi, middle region, Arabidopsis, food and beverages, Plant Science, Plants, interactions, DNA binding domain, auxin response factors (ARFs), PB1, Gene Expression Regulation, Plant, nuclear auxin pathway, heterocyclic compounds, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Transcription Factors
Abstract

International audience; In plants, most developmental programs depend on the action of auxin. The best described model of the auxin signaling pathway, which explains most, but not all, of the auxin transcriptional responses, relies on a de-repression mechanism. The auxin/indole-3-acetic acid repressors (Aux/IAAs) interact with the auxin response factors (ARFs), the transcription factors of the auxin signaling pathway, leading to repression of the ARF-controlled genes. Auxin induces Aux/IAA degradation, releases ARFs and activates transcription. However, this elegant model is not suitable for all ARFs. Indeed, in Arabidopsis, which has 22 ARFs, only five of them fit into the model since they are the ones able to interact with Aux/IAAs. The remaining 17 have a limited capacity to interact with the repressors, and their mechanisms of action are still unclear. The differential interactions between ARF and Aux/IAA proteins constitute one of many examples of the biochemical and structural diversification of ARFs that affect their action and therefore affect auxin transcriptional responses. A deeper understanding of the structural properties of ARFs is fundamental to obtaining a better explanation of the action of auxin in plants.

Published
2022
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6. LEAFY protein crystals with a honeycomb structure as a platform for selective preparation of outstanding stable bio-hybrid materials

Author

Sylvie Kieffer-Jaquinod, Philippe Carpentier, Stéphane Ravanel, Renaud Dumas, Julien Pérard, Olivier Hamelin, Stéphane Ménage, Alice Gogny, David Cobessi, Lucile Chiari, Catalyse Bioinorganique et Environnement (BioCE ), 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), 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)-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 (UGA), Biocatalyse (BIOCAT), European Synchrotron Radiation Facility (ESRF), Etude de la dynamique des protéomes (EDyP), BioSanté (UMR BioSanté), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), BioEnergie et Environnement (BEE), Plantes, Stress & Métaux (MetalStress), Physiologie cellulaire et végétale (LPCV), 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)-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), Institut de biologie structurale (IBS - UMR 5075), Régulateurs du développement de la fleur (Flo_RE ), 80|PRIME Program (CNRS), DRF Impulsion program (CEA), ANR-11-LABX-0003,ARCANE,Grenoble, une chimie bio-motivée(2011), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), ANR-10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), GON, Nathalie, Grenoble, une chimie bio-motivée - - ARCANE2011 - ANR-11-LABX-0003 - LABX - VALID, Grenoble Alliance for Integrated Structural Cell Biology - - GRAL2010 - ANR-10-LABX-0049 - LABX - VALID, and Infrastructure Française de Protéomique - - ProFI2010 - ANR-10-INBS-0008 - INBS - VALID

Subjects
Materials science, chemistry.chemical_element, Nanotechnology, 010402 general chemistry, 01 natural sciences, Mass Spectrometry, symbols.namesake, Honeycomb, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, General Materials Science, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Leafy, Enzymatic digestion, 010405 organic chemistry, Ginkgo biloba, Proteins, [PHYS.COND.CM-MS] Physics [physics]/Condensed Matter [cond-mat]/Materials Science [cond-mat.mtrl-sci], 0104 chemical sciences, Ruthenium, Plant Leaves, Honeycomb structure, chemistry, symbols, [PHYS.COND.CM-MS]Physics [physics]/Condensed Matter [cond-mat]/Materials Science [cond-mat.mtrl-sci], Raman spectroscopy, Hybrid material, Protein crystallization, Chromatography, Liquid
Abstract

International audience; Well-organized protein assemblies offer many properties that justify their use for the design of innovative bionanomaterials. Herein, crystals of the oligomerization domain of the LEAFY protein from Ginkgo biloba, organized in a honeycomb architecture, were used as a modular platform for the selective grafting of a ruthenium-based complex. The resulting bio-hybrid crystalline material was fully characterized by UV-visible and Raman spectroscopy and by mass spectrometry and LC-MS analysis after selective enzymatic digestion. Interestingly, insertion of complexes within the tubular structure affords an impressive increase in stability of the crystals, eluding the use of stabilizing cross-linking strategies.

Published
2021
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7. The LEAFY floral regulator displays pioneer transcription factor properties

Author

Chloe Zubieta, Gilles Vachon, Jeanne Loue-Manifel, Loïc GrandVuillemin, Ying Huang, Moussa Benhamed, Renaud Dumas, François Parcy, Jérémy Lucas, Xuelei Lai, Romain Blanc-Mathieu, Arnaud Stigliani, Hussein Daher, Emmanuel Thévenon, David Latrasse, and Eugenia Brun-Hernandez

Subjects
Histone, biology, Agamous, Arabidopsis, biology.protein, Nucleosome, biology.organism_classification, Leafy, Gene, Transcription factor, Chromatin, Cell biology
Abstract

Pioneer transcription factors (TFs) are a special category of TFs with the capacity to bind to closed chromatin regions in which DNA is wrapped around histones and often highly methylated. Subsequently, they are able to modify the chromatin state to initiate gene expression. In plants, LEAFY (LFY) is a master floral regulator and has been suggested to act as a pioneer TF in Arabidopsis. Here, we demonstrate that LFY is able to bind both methylated and non-methylated DNA using a combination of in vitro genome-wide binding experiments and structural modeling. Comparisons between regions bound by LFY in vivo and chromatin accessibility data suggest that LFY binds a subset of regions occupied by nucleosomes. We confirm that LFY is able to bind nucleosomal DNA in vitro using reconstituted nucleosomes. Finally, we show that constitutive LFY expression in seedling tissues is sufficient to induce chromatin accessibility in the LFY direct target genes, APETALA1 and AGAMOUS. Taken together, our study suggests that LFY possesses key pioneer TF features that contribute to launch the floral gene expression program.

Published
2021
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8. The LEAFY floral regulator displays pioneer transcription factor properties

Author

François Parcy, Chloe Zubieta, David Latrasse, Arnaud Stigliani, Ying Huang, Renaud Dumas, Moussa Benhamed, Xuelei Lai, Emmanuel Thévenon, Laura Turchi, Jeanne Loue-Manifel, Gilles Vachon, Eugenia Brun-Hernandez, Loïc GrandVuillemin, Hussein Daher, Romain Blanc-Mathieu, Jérémy Lucas, Régulateurs du développement de la fleur (Flo_RE ), Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), Institut des Sciences des Plantes de Paris-Saclay (IPS2 (UMR_9213 / UMR_1403)), Université d'Évry-Val-d'Essonne (UEVE)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), University of Copenhagen = Københavns Universitet (UCPH), Reproduction et développement des plantes (RDP), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut de biologie structurale (IBS - UMR 5075), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), ANR-16-CE92-0023,FLOPINET,Floral Pioneer Factors(2016), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), Université d'Évry-Val-d'Essonne (UEVE)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), University of Copenhagen = Københavns Universitet (KU), and École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL)

Subjects
0106 biological sciences, 0301 basic medicine, Arabidopsis, Flowers, Plant Science, Biology, 01 natural sciences, Chromatin remodeling, chromatin remodeling, Histones, 03 medical and health sciences, Gene Expression Regulation, Plant, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], flower development, Journal Article, Nucleosome, pioneer transcription factor, Molecular Biology, Leafy, Transcription factor, Regulation of gene expression, Arabidopsis Proteins, Agamous, [SDV.BV.BOT]Life Sciences [q-bio]/Vegetal Biology/Botanics, Plants, Genetically Modified, Chromatin, Nucleosomes, Cell biology, 030104 developmental biology, Histone, Seedlings, biology.protein, gene regulation, Transcription Factors, 010606 plant biology & botany
Abstract

International audience; Pioneer transcription factors (TFs) are a special category of TFs with the capacity to bind to closed chromatin regions in which DNA is wrapped around histones and may be highly methylated. Subsequently, pioneer TFs are able to modify the chromatin state to initiate gene expression. In plants, LEAFY (LFY) is a master floral regulator and has been suggested to act as a pioneer TF in Arabidopsis. Here, we demonstrate that LFY is able to bind both methylated and non-methylated DNA using a combination of in vitro genome-wide binding experiments and structural modeling. Comparisons between regions bound by LFY in vivo and chromatin accessibility data suggest that a subset of LFY bound regions is occupied by nucleosomes. We confirm that LFY is able to bind nucleosomal DNA in vitro using reconstituted nucleosomes. Finally, we show that constitutive LFY expression in seedling tissues is sufficient to induce chromatin accessibility in the LFY direct target genes, APETALA1 and AGAMOUS. Taken together, our study suggests that LFY possesses key pioneer TF features that contribute to launch the floral gene expression program.

Published
2021
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9. Characterization of the Bubblegum acyl-CoA synthetase of Microchloropsis gaditana

Author

Fabrice Rébeillé, Mariette Bedhomme, Juliette Jouhet, Laurent Poulet, Sébastien Leterme, Serge Crouzy, Séverine Collin, Renaud Dumas, Frédéric Laueffer, Eric Maréchal, Alberto Amato, Laurent Fourage, Morgane Michaud, Elodie Billey, Leonardo Magneschi, Amélie Andres-Robin, Giovanni Finazzi, LIPID, Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), Total Raffinage-Chimie, Light Photosynthesis & Metabolism (Photosynthesis), Régulateurs du développement de la fleur (Flo_RE ), Chimie computationnelle, modélisation et rayons X (COMX), 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 (UGA)-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 (UGA), CEA-Total partnership, Conseil Regional Auvergne-Rhône-Alpes, the European Commission, and Institut Carnot 3BCAR (LIPANG platform), ANR-11-BTBR-0008,OCEANOMICS,Biotechnologies et bioressources pour la valorisation des écosystèmes marins planctoniques(2011), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), and ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017)

Subjects
0106 biological sciences, eicosapentaenoic acid, Regular Issue, very-long-chain polyunsaturated fatty acid, Physiology, lipidosin, Plant Science, Cyanobacteria, 01 natural sciences, 03 medical and health sciences, chemistry.chemical_compound, Biosynthesis, Phosphatidylcholine, Coenzyme A Ligases, Genetics, Bubblegum Acyl-CoA synthase, Nannochloropsis, Photosynthesis, Plastid, 030304 developmental biology, 0303 health sciences, omega pathway, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Point mutation, Fatty Acids, [SDV.BV.BOT]Life Sciences [q-bio]/Vegetal Biology/Botanics, biology.organism_classification, Chloroplast, Cytosol, Metabolic pathway, chemistry, Biochemistry, lipids (amino acids, peptides, and proteins), Microchloropsis, Metabolic Networks and Pathways, 010606 plant biology & botany
Abstract

The metabolic pathways of glycerolipids are well described in cells containing chloroplasts limited by a two-membrane envelope but not in cells containing plastids limited by four membranes, including heterokonts. Fatty acids (FAs) produced in the plastid, palmitic and palmitoleic acids (16:0 and 16:1), are used in the cytosol for the synthesis of glycerolipids via various routes, requiring multiple acyl-Coenzyme A (CoA) synthetases (ACS). Here, we characterized an ACS of the Bubblegum subfamily in the photosynthetic eukaryote Microchloropsis gaditana, an oleaginous heterokont used for the production of lipids for multiple applications. Genome engineering with TALE-N allowed the generation of MgACSBG point mutations, but no knockout was obtained. Point mutations triggered an overall decrease of 16:1 in lipids, a specific increase of unsaturated 18-carbon acyls in phosphatidylcholine and decrease of 20-carbon acyls in the betaine lipid diacylglyceryl–trimethyl–homoserine. The profile of acyl-CoAs highlighted a decrease in 16:1-CoA and 18:3-CoA. Structural modeling supported that mutations affect accessibility of FA to the MgACSBG reaction site. Expression in yeast defective in acyl-CoA biosynthesis further confirmed that point mutations affect ACSBG activity. Altogether, this study supports a critical role of heterokont MgACSBG in the production of 16:1-CoA and 18:3-CoA. In M. gaditana mutants, the excess saturated and monounsaturated FAs were diverted to triacylglycerol, thus suggesting strategies to improve the oil content in this microalga.

Published
2021
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10. Self-Assembly of a Ginkgo Oligomerization Domain Creates a Sub-10-nm Honeycomb Architecture on Carbon and Silicon Surfaces with Customizable Pores: Implications for Nanoelectronics, Biosensing, and Biocatalysis

Author

Gregory Si Larbi, Denis Mariolle, Emmanuel Thévenon, François Parcy, Pierre-Henri Elchinger, Renaud Dumas, Pierre-Henri Jouneau, Denis Falconet, Raluca Tiron, Elise Jacquier, Régulateurs du développement de la fleur (Flo_RE ), Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé (SYMMES), 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 (UGA), Modélisation et Exploration des Matériaux (MEM), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), LIPID, Commissariat à l'énergie atomique et aux énergies alternatives - Laboratoire d'Electronique et de Technologie de l'Information (CEA-LETI), Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), CEA program Nanoprot 3D, DRF impulsion grant, ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), ANR-11-LABX-0003,ARCANE,Grenoble, une chimie bio-motivée(2011), and ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017)

Subjects
Materials science, Silicon, chemistry.chemical_element, Honeycomb (geometry), Nanotechnology, 02 engineering and technology, 010402 general chemistry, 021001 nanoscience & nanotechnology, 01 natural sciences, 0104 chemical sciences, Domain (software engineering), Nanoelectronics, chemistry, Biocatalysis, General Materials Science, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Self-assembly, 0210 nano-technology, Biosensor, Carbon, ComputingMilieux_MISCELLANEOUS
Abstract

International audience

Published
2021
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11. Crystal structure of the transcriptional repressor DdrO: insight into the metalloprotease/repressor-controlled radiation response in Deinococcus

Author

Philippe Roche, Fabrice Confalonieri, Pascale Servant, Romaric Magerand, David Lemaire, David Pignol, François Parcy, Pascal Arnoux, Géraldine Brandelet, Raquel Martin-Arevalillo, Laurence Blanchard, Jade Doloy, Nicolas Eugénie, Arjan de Groot, Marina I. Siponen, Renaud Dumas, Microbiologie Environnementale et Moléculaire (MEM), 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), Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Radiorésistance des bactéries et des archées (RBA), Département Biologie des Génomes (DBG), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Régulateurs du développement de la fleur (Flo_RE), 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), Interactions Protéine Métal (IPM), Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC), ANR-19-CE12-0010,NOVOREP,Réparation des dommages ou mort cellulaire chez les bactéries exposées aux stress, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), 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), ANR-19-CE12-0010,NOVOREP,Réparation des dommages ou mort cellulaire chez les bactéries exposées aux stress(2019), 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])-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), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay, Université Paris-Saclay, 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 Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-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 National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche en Cancérologie de Marseille (CRCM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Aix Marseille Université (AMU), MARAVAL, Alexandra, and Réparation des dommages ou mort cellulaire chez les bactéries exposées aux stress - - NOVOREP2019 - ANR-19-CE12-0010 - AAPG2019 - VALID

Subjects
Models, Molecular, DNA damage, [SDV]Life Sciences [q-bio], Repressor, [SDV.CAN]Life Sciences [q-bio]/Cancer, Cleavage (embryo), Crystallography, X-Ray, DNA-binding protein, Protein Structure, Secondary, 03 medical and health sciences, Protein structure, Structural Biology, Stress, Physiological, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Genetics, Deinococcus, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, Peptide sequence, 030304 developmental biology, 0303 health sciences, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, biology, 030306 microbiology, Gene Expression Regulation, Bacterial, biology.organism_classification, Protein Structure, Tertiary, [SDV] Life Sciences [q-bio], Repressor Proteins, Biophysics, Metalloproteases, Alpha helix, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, DNA Damage, Transcription Factors
Abstract

Exposure to harmful conditions such as radiation and desiccation induce oxidative stress and DNA damage. In radiation-resistant Deinococcus bacteria, the radiation/desiccation response is controlled by two proteins: the XRE family transcriptional repressor DdrO and the COG2856 metalloprotease IrrE. The latter cleaves and inactivates DdrO. Here, we report the biochemical characterization and crystal structure of DdrO, which is the first structure of a XRE protein targeted by a COG2856 protein. DdrO is composed of two domains that fold independently and are separated by a flexible linker. The N-terminal domain corresponds to the DNA-binding domain. The C-terminal domain, containing three alpha helices arranged in a novel fold, is required for DdrO dimerization. Cleavage by IrrE occurs in the loop between the last two helices of DdrO and abolishes dimerization and DNA binding. The cleavage site is hidden in the DdrO dimer structure, indicating that IrrE cleaves DdrO monomers or that the interaction with IrrE induces a structural change rendering accessible the cleavage site. Predicted COG2856/XRE regulatory protein pairs are found in many bacteria, and available data suggest two different molecular mechanisms for stress-induced gene expression: COG2856 protein-mediated cleavage or inhibition of oligomerization without cleavage of the XRE repressor.

Published
2019
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12. Evolution of the auxin response factors from charophyte ancestors

Author

François Parcy, Thomas Vinos-Poyo, Raquel Martin-Arevalillo, Fanny Jégu, Teva Vernoux, Renaud Dumas, Emmanuel Thévenon, Reproduction et développement des plantes (RDP), École normale supérieure - Lyon (ENS Lyon)-Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Régulateurs du développement de la fleur (Flo_RE), 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), ANR-12-BSV6-0005,AuxiFlo,Couplage entre le réseau transcriptionnel de l'auxine et l'identité florale(2012), ANR-18-CE12-0014,CHROMAUXI,DECODING AUXIN RESPONSE AT THE ARFS-CHROMATIN INTERFACE(2018), ANR-10- LABX-49-01,Labex GRAL,Labex GRAL, École normale supérieure de Lyon (ENS de Lyon)-Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), 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), French National Research Agency (ANR), ANR-12-BSV6-0005 Auxiflo ANR-18-CE12-0014 ChromAuxi, University Grenoble Alpes, Grenoble Alliance for Cell and Structural Biology ANR-10-LABX-49-01, ANR-18-CE12-0014,ChromAuxi,Décodage de la réponse auxine à l'interface ARFs-chromatine(2018), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Recherche Agronomique (INRA)-École normale supérieure - Lyon (ENS Lyon), 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), and 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)

Subjects
Cancer Research, Plant Evolution, Plant Science, QH426-470, Biochemistry, Database and Informatics Methods, 0302 clinical medicine, Plant Growth Regulators, Gene Expression Regulation, Plant, Transcriptional regulation, Arabidopsis thaliana, Auxin, Plant Hormones, [SDV.BDD]Life Sciences [q-bio]/Development Biology, Phylogeny, Genetics (clinical), Plant Proteins, Regulation of gene expression, Plant evolution, chemistry.chemical_classification, 0303 health sciences, biology, Plant Biochemistry, Chlorokybus, Eukaryota, food and beverages, Plants, Experimental Organism Systems, Multigene Family, Sequence Analysis, Research Article, Algae, Bioinformatics, Evolution, Arabidopsis Thaliana, Charophyceae, Auxin Response Factor, Sequence Databases, Receptors, Cell Surface, Brassica, Research and Analysis Methods, Response Elements, Evolution, Molecular, 03 medical and health sciences, Model Organisms, Sequence Motif Analysis, Plant and Algal Models, Phylogenetics, DNA-binding proteins, Genetics, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, Transcription factor, Ecology, Evolution, Behavior and Systematics, 030304 developmental biology, Evolutionary Biology, Indoleacetic Acids, auxine, fungi, Organisms, Biology and Life Sciences, Proteins, 15. Life on land, biology.organism_classification, Hormones, Organismal Evolution, Biological Databases, chemistry, Evolutionary biology, Animal Studies, Auxins, Sequence Alignment, 030217 neurology & neurosurgery, Transcription Factors
Abstract

Auxin is a major developmental regulator in plants and the acquisition of a transcriptional response to auxin likely contributed to developmental innovations at the time of water-to-land transition. Auxin Response Factors (ARFs) Transcription Factors (TFs) that mediate auxin-dependent transcriptional changes are divided into A, B and C evolutive classes in land plants. The origin and nature of the first ARF proteins in algae is still debated. Here, we identify the most ‘ancient’ ARF homologue to date in the early divergent charophyte algae Chlorokybus atmophyticus, CaARF. Structural modelling combined with biochemical studies showed that CaARF already shares many features with modern ARFs: it is capable of oligomerization, interacts with the TOPLESS co-repressor and specifically binds Auxin Response Elements as dimer. In addition, CaARF possesses a DNA-binding specificity that differs from class A and B ARFs and that was maintained in class C ARF along plants evolution. Phylogenetic evidence together with CaARF biochemical properties indicate that the different classes of ARFs likely arose from an ancestral proto-ARF protein with class C-like features. The foundation of auxin signalling would have thus happened from a pre-existing hormone-independent transcriptional regulation together with the emergence of a functional hormone perception complex., Author summary Plants transition from water to land was determining for the history of our planet, since it led to atmospheric and soil condition changes that promoted the appearance of other life forms. This transition initiated around 1 billion years ago from a Charophyte algae lineage that acquired features allowing it to adapt to the very different terrestrial conditions. Land plants coordinate their development with external stimuli through signalling mechanisms triggered by plant hormones. Therefore, evolution of these molecules and their signalling pathways likely played an important role in the aquatic to terrestrial move. In this manuscript we study the origin of auxin signalling, a plant hormone implicated in all plant developmental steps. Our studies suggest that out of the three families of proteins originally proposed to trigger auxin signalling in land plants, only one existed in Charophyte ancestors as a likely transcriptional repressor independent of auxin. We show that despite millions of years of evolution, this family of proteins has conserved its biochemical and structural properties that are found today in land plants. The results presented here provide an insight on how hormone signalling pathways could have evolved by co-opting a pre-existing hormone-independent transcriptional regulatory mechanism.

Published
2019
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13. Capturing Auxin Response Factors Syntax Using DNA Binding Models

Author

Teva Vernoux, François Parcy, Raquel Martin-Arevalillo, Renaud Dumas, Victoria V. Mironova, Jérémy Lucas, Thomas Vinos-Poyo, Arnaud Stigliani, Addrien Bessy, Régulateurs du développement de la fleur (Flo_RE), 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), UMR 1417 PCV Laboratoire de Physiologie Cellulaire Végétale. Centre de recherche Auvergne-Rhône-Alpes, Institut National de la Recherche Agronomique (INRA), Institute of Cytology and Genetics, Russian Academy of Sciences [Moscow] (RAS), Reproduction et développement des plantes (RDP), École normale supérieure - Lyon (ENS Lyon)-Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Russian State Budget [0324-2018-0019], Russian Foundation for Basic Research [18-04-01130], ANR-12-BSV6-0005,AuxiFlo,Couplage entre le réseau transcriptionnel de l'auxine et l'identité florale(2012), ANR-10- LABX-49-01,Labex GRAL,Labex GRAL, Physiologie cellulaire et végétale [2016-2019] (LPCV [2016-2019]), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Novosibirsk State University (NSU), French National Research Agency (ANR) ANR-12-BSV6-0005, University Grenoble Alpes, Grenoble Alliance for Cell and Structural Biology ANR-10-LABX-49-01, Russian State Budget 0324-2019-0040, Russian Foundation for Basic Research (RFBR) 18-04-01130, 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), Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Recherche Agronomique (INRA)-École normale supérieure - Lyon (ENS Lyon), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), 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), and École normale supérieure de Lyon (ENS de Lyon)-Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL)

Subjects
0301 basic medicine, 0106 biological sciences, [SDV]Life Sciences [q-bio], Arabidopsis, Auxin Response Factor, Genomics, Plant Science, Computational biology, Biology, 01 natural sciences, Genome, DNA sequencing, 03 medical and health sciences, chemistry.chemical_compound, DNA binding model, Gene Expression Regulation, Plant, Consensus sequence, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, Auxin, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Binding site, Molecular Biology, Transcription factor, Gene, 030304 developmental biology, Regulation of gene expression, 0303 health sciences, Indoleacetic Acids, Arabidopsis Proteins, DNA, DNA-Binding Proteins, 030104 developmental biology, chemistry, Regulatory sequence, DAP-seq, 010606 plant biology & botany, Transcription Factors
Abstract

International audience; Auxin is a key hormone performing a wealth of functions throughout the life cycle of plants. It acts largely by regulating genes at the transcriptional level through a family of transcription factors called auxin response factors (ARFs). Even though all ARF monomers analyzed so far bind a similar DNA sequence, there is evidence that ARFs differ in their target genomic regions and regulated genes. Here, we report the use of position weight matrices (PWMs) to model ARF DNA binding specificity based on published DNA affinity purification sequencing (DAP-seq) data. We found that the genome binding of two ARFs (ARF2 and ARF5/Monopteros [MP]) differ largely because these two factors have different preferred ARF binding site (ARFbs) arrangements (orientation and spacing). We illustrated why PWMs are more versatile to reliably identify ARFbs than the widely used consensus sequences and demonstrated their power with biochemical experiments in the identification of the regulatory regions of IAA19, an well-characterized auxin-responsive gene. Finally, we combined gene regulation by auxin with ARF-bound regions and identified specific ARFbs configurations that are over-represented in auxin-upregulated genes, thus deciphering the ARFbs syntax functional for regulation. Our study provides a general method to exploit the potential of genome-wide DNA binding assays and to decode gene regulation.

Published
2019
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14. Structure of the

Author

Raquel, Martin-Arevalillo, Max H, Nanao, Antoine, Larrieu, Thomas, Vinos-Poyo, David, Mast, Carlos, Galvan-Ampudia, Géraldine, Brunoud, Teva, Vernoux, Renaud, Dumas, and François, Parcy

Subjects
Evolution, Molecular, Arabidopsis Proteins, Gene Expression Regulation, Plant, Amino Acid Motifs, Arabidopsis, food and beverages, Amino Acid Sequence, Protein Multimerization, Biological Sciences, Co-Repressor Proteins, Hydrophobic and Hydrophilic Interactions
Abstract

In most biological processes, genes have to be activated and/or repressed. In plants, the TOPLESS protein is essential for gene repression through its action as a corepressor bridging transcription factor with chromatin remodeling complexes. Here we combine biochemical and structural studies to describe the structure of TOPLESS, how it tetramerizes, and how it interacts with its protein partners. We show that both the tetramerization interface and the binding site for protein partners have been conserved since algae, highlighting the ancestrality of TOPLESS function. Comparison of this plant protein with one of its animal counterparts also shows how corepressors can use a common domain differently to achieve similar properties, illustrating the tinkering of evolution in transcriptional repression.

Published
2017

15. Plant SAM-Domain Proteins Start to Reveal Their Roles

Author

Renaud Dumas, Gilles Vachon, François Parcy, Chloe Zubieta, Grégoire Denay, 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), Institute for Developmental Genetics, ANR-10- LABX-49-01,Labex GRAL,Labex GRAL, ANR-11-BSV2-0005,CHARMFUL,Caractérisation de la fonction méristématique de LEAFY(2011), ANR-16-CE92-0023,FLOPINET,Floral Pioneer Factors, 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), ANR Gral [ANR-10-LABX-49-01], ANR projects Charmful and Flopinet, ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), ANR-16-CE92-0023,FLOPINET,Floral Pioneer Factors(2016), 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, Models, Molecular, Arabidopsis thaliana, [SDV]Life Sciences [q-bio], Regulator, Arabidopsis, Protein localisation, Plant Science, Computational biology, 01 natural sciences, Homology (biology), 03 medical and health sciences, Protein protein interaction domain, oligomerisation, Botany, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Protein Interaction Domains and Motifs, Amino Acid Sequence, Leafy, Phylogeny, Floral regulator, tRNA import component 1 and 2, biology, Arabidopsis Proteins, Oligomerisation domains, food and beverages, Plant, biology.organism_classification, LEAFY, SAM domain, Models, Structural, Sterile Alpha Motif, 030104 developmental biology, Transfer RNA, Protein structure, TRIC1/2, Sterile alpha motif, Sequence Alignment, 010606 plant biology & botany
Abstract

International audience; Proteins often act in complexes assembled via protein-protein interaction domains. The sterile alpha motif (SAM) domain is one of the most prominent interaction domains in animals and is present in proteins of diverse functions. This domain allows head-to-tail closed oligomerisation or polymer formation resulting in homo- and/or heterocomplexes that have been shown to be important for proper protein localisation and function. In plants this domain is also present but has been poorly studied except for recent studies on the LEAFY floral regulator and the tRNA import component (TRIC)1/2 proteins. Here we catalogue SAM domain-containing proteins from arabidopsis (Arabidopsis thaliana), compare plant and other eukaryotic SAM domains, and perform homology modelling to probe plant SAM domain interaction capabilities.

Published
2017
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16. A link between LEAFY and B-gene homologues in Welwitschia mirabilis sheds light on ancestral mechanisms prefiguring floral development

Author

Marie Monniaux, Charles P. Scutt, Edwige Moyroud, Renaud Dumas, Emmanuel Thévenon, Michael W. Frohlich, François Parcy, 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), Reproduction et développement des plantes (RDP), École normale supérieure de Lyon (ENS de Lyon)-Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Jodrell Laboratory, Royal Botanic Gardens [Kew], National Science Foundation (NSF) Plant Genome Research Program project DBI-0115684, National Science Foundation (NSF) DEB-9974374, ANR-07-BLAN-0211,Plant-TFcode,Cracking the code of transcriptional regulation: the key to the past and future evolution of plants(2007), 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), École normale supérieure - Lyon (ENS Lyon)-Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), Royal Botanic Garden , Kew, Moyroud, Edwige [0000-0001-7908-3205], Apollo - University of Cambridge Repository, 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), ANR-07-BLAN-0211,BLANC,Cracking the code of transcriptional regulation: the key to the past and future evolution of plants(2007), ANR-10- LABX-49-01,Labex GRAL,Labex GRAL, 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)-Institut National de la Recherche Agronomique (INRA), Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Institut National de la Recherche Agronomique (INRA)-École normale supérieure - Lyon (ENS Lyon)

Subjects
0301 basic medicine, flower origin, Physiology, Arabidopsis, Plant Science, PISTILLATA, LFY, Gene Expression Regulation, Plant, flower development, bisexual structure, MADS-box, Phylogeny, Plant Proteins, Regulation of gene expression, biology, Protein binding sites, food and beverages, NDLY, angiosperms, gymnosperms, Evolution, Flowers, Genes, Plant, 03 medical and health sciences, Gymnosperm, Welwitschia mirabilis, Phylogenetics, transcription factors, Botany, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Gene, Leafy, AP3, Binding Sites, Base Sequence, Sequence Homology, Amino Acid, Welwitschia, fungi, APETALA3, Plant, 15. Life on land, biology.organism_classification, Gene regulation, LEAFY, Plant Leaves, Kinetics, 030104 developmental biology, Evolutionary biology, Gene expression, Streptophyta
Abstract

International audience; Flowering plants evolved from an unidentified gymnosperm ancestor. Comparison of the mechanisms controlling development in angiosperm flowers and gymnosperm cones may help to elucidate the mysterious origin of the flower. We combined gene expression studies with protein behaviour characterization in Welwitschia mirabilis to test whether the known regulatory links between LEAFY and its MADS-box gene targets, central to flower development, might also contribute to gymnosperm reproductive development. We found that WelLFY, one of two LEAFY-like genes in Welwitschia, could be an upstream regulator of the MADS-box genes APETALA3/PISTILLATA-like (B-genes). We demonstrated that, even though their DNA-binding domains are extremely similar, WelLFY and its paralogue WelNDLY exhibit distinct DNA-binding specificities, and that, unlike WelNDLY, WelLFY shares with its angiosperm orthologue the capacity to bind promoters of Welwitschia B-genes. Finally, we identified several cis-elements mediating these interactions in Welwitschia and obtained evidence that the link between LFY homologues and B-genes is also conserved in two other gymnosperms, Pinus and Picea. Although functional approaches to investigate cone development in gymnosperms are limited, our state-of-the-art biophysical techniques, coupled with expression studies, provide evidence that crucial links, central to the control of floral development, may already have existed before the appearance of flowers.

Published
2017
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17. Biochemical and Structural Characterization of the Arabidopsis Bifunctional Enzyme Dethiobiotin Synthetase–Diaminopelargonic Acid Aminotransferase: Evidence for Substrate Channeling in Biotin Synthesis

Author

Virginie Pautre, David Cobessi, Claude Alban, Jean-Luc Ferrer, Renaud Dumas, Céline Meinguet, 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), 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), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS), 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), and Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG)

Subjects
Models, Molecular, 0106 biological sciences, Arabidopsis thaliana, DAPA, Arabidopsis, plant, Plant Science, vitamin B8, Ligands, 01 natural sciences, Protein Structure, Secondary, Substrate Specificity, chemistry.chemical_compound, Biotin, Catalytic Domain, Carbon-Nitrogen Ligases, Amino Acids, Research Articles, chemistry.chemical_classification, 0303 health sciences, biology, Recombinant Proteins, bifunctional enzyme, Biochemistry, Protein Binding, Subcellular Fractions, crystal structure, Molecular Sequence Data, Substrate channeling, physicochemical properties, Chimeric gene, 03 medical and health sciences, DTBS, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Phosphofructokinase 2, Amino Acid Sequence, Transaminases, 030304 developmental biology, Arabidopsis Proteins, substrate channeling, Active site, metabolic pathway, Cell Biology, biology.organism_classification, Fusion protein, Biosynthetic Pathways, Protein Structure, Tertiary, Enzyme, chemistry, kinetics, Mutation, Biocatalysis, biotin synthesis, biology.protein, Protein Multimerization, Holoenzymes, 010606 plant biology & botany
Abstract

International audience; Diaminopelargonic acid aminotransferase (DAPA-AT) and dethiobiotin synthetase (DTBS) catalyze the antepenultimate and the penultimate steps, respectively, of biotin synthesis. Whereas DAPA-AT and DTBS are encoded by distinct genes in bacteria, in biotin-synthesizing eukaryotes (plants and most fungi), both activities are carried out by a single enzyme encoded by a bifunctional gene originating from the fusion of prokaryotic monofunctional ancestor genes. In few angiosperms, including Arabidopsis thaliana, this chimeric gene (named BIO3-BIO1) also produces a bicistronic transcript potentially encoding separate monofunctional proteins that can be produced following an alternative splicing mechanism. The functional significance of the occurrence of a bifunctional enzyme in biotin synthesis pathway in eukaryotes and the relative implication of each of the potential enzyme forms (bifunctional versus monofunctional) in the plant biotin pathway are unknown. In this study, we demonstrate that the BIO3-BIO1 fusion protein is the sole protein form produced by the BIO3-BIO1 locus in Arabidopsis. The enzyme catalyzes both DAPA-AT and DTBS reactions in vitro and is targeted to mitochondria in vivo. Our biochemical and kinetic characterizations of the pure recombinant enzyme show that in the course of the reaction, the DAPA intermediate is directly transferred from the DAPA-AT active site to the DTBS active site. Analysis of several structures of the enzyme crystallized in complex with and without its ligands reveals key structural elements involved for acquisition of bifunctionality and brings, together with mutagenesis experiments, additional evidences for substrate channeling.

Published
2012
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18. 4-Hydroxyphenylpyruvate Dioxygenase Catalysis

Author

Adeline Y. Robin, Matthieu Graindorge, Michel Matringe, Serge Crouzy, Bertrand Lefèbvre, Corinne Raspail, Yohann Moreau, and Renaud Dumas

Subjects
chemistry.chemical_classification, 0303 health sciences, Stereochemistry, Cell Biology, 010402 general chemistry, 01 natural sciences, Biochemistry, Enzyme structure, 0104 chemical sciences, Enzyme catalysis, Hydroxylation, 03 medical and health sciences, chemistry.chemical_compound, Enzyme, chemistry, Dioxygenase, Shikimate pathway, Molecular Biology, 4-Hydroxyphenylpyruvate dioxygenase, 030304 developmental biology, Homogentisate 1,2-dioxygenase
Abstract

4-Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxyphenylpyruvate (HPP) into homogentisate. HPPD is the molecular target of very effective synthetic herbicides. HPPD inhibitors may also be useful in treating life-threatening tyrosinemia type I and are currently in trials for treatment of Parkinson disease. The reaction mechanism of this key enzyme in both plants and animals has not yet been fully elucidated. In this study, using site-directed mutagenesis supported by quantum mechanical/molecular mechanical theoretical calculations, we investigated the role of catalytic residues potentially interacting with the substrate/intermediates. These results highlight the following: (i) the central role of Gln-272, Gln-286, and Gln-358 in HPP binding and the first nucleophilic attack; (ii) the important movement of the aromatic ring of HPP during the reaction, and (iii) the key role played by Asn-261 and Ser-246 in C1 hydroxylation and the final ortho-rearrangement steps (numbering according to the Arabidopsis HPPD crystal structure 1SQD). Furthermore, this study reveals that the last step of the catalytic reaction, the 1,2 shift of the acetate side chain, which was believed to be unique to the HPPD activity, is also catalyzed by a structurally unrelated enzyme.

Published
2011
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19. A Glimpse beyond Structures in Auxin-Dependent Transcription

Author

François Parcy, Renaud Dumas, Teva Vernoux, 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), Reproduction et développement des plantes (RDP), Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Recherche Agronomique (INRA)-École normale supérieure - Lyon (ENS Lyon), European Community 283570, Gral Labex, ANR-12-BSV6-0005, ANR-12-BSV6-0005,AuxiFlo,Couplage entre le réseau transcriptionnel de l'auxine et l'identité florale(2012), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), European Project: 283570,EC:FP7:INFRA,FP7-INFRASTRUCTURES-2011-1,BIOSTRUCT-X(2011), 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), École normale supérieure - Lyon (ENS Lyon)-Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), ANR-10- LABX-49-01,Labex GRAL,Labex GRAL, 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), and École normale supérieure de Lyon (ENS de Lyon)-Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL)

Subjects
0301 basic medicine, Transcription, Genetic, Domain structures, Repressor, Plant Science, Biology, 03 medical and health sciences, chemistry.chemical_compound, Transcriptional regulation, Gene Expression Regulation, Plant, Auxin, Transcription (biology), Phytohormone, heterocyclic compounds, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Signalling pathway, DNA binding, Transcription factor, ComputingMilieux_MISCELLANEOUS, Plant Proteins, chemistry.chemical_classification, Indoleacetic Acids, fungi, food and beverages, Hedgehog signaling pathway, Cell biology, 030104 developmental biology, chemistry, Biochemistry, Signal transduction, DNA, Signal Transduction, Transcription Factors
Abstract

International audience; Auxin response factors (ARFs), transcription factors (TFs), and their Aux/IAA (IAA) repressors are central components of the auxin signalling pathway. They interact as homo- and heteromultimers. The structure of their interacting domains revealed a PB1 fold mediating electrostatic interactions through positive and negative faces. Detailed structural analysis revealed additional hydrophobic and polar determinants and started unveiling an ARF/IAA interaction code. Structural progress also shed new light on the DNA binding mode of ARFs showing how they dimerize to bind repeated DNA elements. Here, we discuss the in vitro and in vivo significance of these structural properties for the ARF family of TFs and identify some critical missing information on how specificity might be achieved in the auxin signalling pathway.

Published
2016
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20. 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
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21. The Myb-domain protein ULTRAPETALA1 INTERACTING FACTOR 1 controls floral meristem activities in Arabidopsis

Author

Irene López-Vidriero, José Manuel Franco-Zorrilla, François Parcy, Robert Blanvillain, Renaud Dumas, Emmanuel Thévenon, Patrice Morel, Cristel C. Carles, Christophe Trehin, Fanny Moreau, Carles, Christel, 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), Department of Plant Molecular Genetics, Centro Nacional de Biotecnologia, Consejo Superior de Investigaciones Científicas [Spain] (CSIC), Reproduction et développement des plantes (RDP), École normale supérieure - Lyon (ENS Lyon)-Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), ANR-10-JCJC-1206,ChromFlow,Dynamique de l'activation de la chromatine au cours de la morphogenèse florale(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), Consejo Superior de Investigaciones Científicas [Madrid] (CSIC), Rhone-Alpes county (Allocation Doctorale de Recherche, Cluster 7) [12-01293101], Centre National de la Recherche Scientifique (CNRS-Higher Education Chair) [0428-64], University of Grenoble-Alpes (Chaire Initiative Universitaire Alpes, UGA-UJF), French National Agency Young Researcher Grant [ChromFlow ANR JCJC] [SVSE2-1206 01], Centro Nacional de Biotecnología [Madrid] (CNB-CSIC), and École normale supérieure de Lyon (ENS de Lyon)-Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL)

Subjects
0301 basic medicine, Transcription, Genetic, Arabidopsis thaliana, Protein domain, Meristem, Molecular Sequence Data, Morphogenesis, Arabidopsis, Repressor, Electrophoretic Mobility Shift Assay, Flowers, 03 medical and health sciences, Gene Expression Regulation, Plant, WUSCHEL, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, MYB, Protein Interaction Domains and Motifs, Amino Acid Sequence, Molecular Biology, Gene, Transcription factor, transcription factor, Genetics, Homeodomain Proteins, Stem cell, biology, Plant Stems, Arabidopsis Proteins, Stem Cells, fungi, food and beverages, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Plant, biology.organism_classification, Protein Structure, Tertiary, Plant Leaves, 030104 developmental biology, Flower, Myb transcription factor, Sequence Alignment, Developmental Biology, Transcription Factors
Abstract

International audience; Higher plants continuously and iteratively produce new above-ground organs in the form of leaves, stems and flowers. These organs arise from shoot apical meristems whose homeostasis depends on coordination between self-renewal of stem cells and their differentiation into organ founder cells. This coordination is stringently controlled by the central transcription factor WUSCHEL (WUS), which is both necessary and sufficient for stem cell specification inArabidopsis thaliana ULTRAPETALA1 (ULT1) was previously identified as a plant-specific, negative regulator ofWUSexpression. However, molecular mechanisms underlying this regulation remain unknown. ULT1 protein contains a SAND putative DNA-binding domain and a B-box, previously proposed as a protein interaction domain in eukaryotes. Here, we characterise a novel partner of ULT1, named ULT1 INTERACTING FACTOR 1 (UIF1), which contains a Myb domain and an EAR motif. UIF1 and ULT1 function in the same pathway for regulation of organ number in the flower. Moreover, UIF1 displays DNA-binding activity and specifically binds toWUSregulatory elements. We thus provide genetic and molecular evidence that UIF1 and ULT1 work together in floral meristem homeostasis, probably by direct repression ofWUSexpression.

Published
2016
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22. Allosteric monofunctional aspartate kinases fromArabidopsis

Author

Gilles Curien, Renaud Dumas, Mathieu Laurencin, and Mylène Robert-Genthon

Subjects
0106 biological sciences, endocrine system diseases, Allosteric regulation, Lysine, complex mixtures, 01 natural sciences, Biochemistry, 03 medical and health sciences, Enzyme activator, Aspartate kinase, Molecular Biology, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, biology, Kinase, nutritional and metabolic diseases, Cell Biology, Molecular biology, Enzyme assay, Aspartate carbamoyltransferase, Enzyme, chemistry, biology.protein, bacteria, hormones, hormone substitutes, and hormone antagonists, 010606 plant biology & botany
Abstract

Plant monofunctional aspartate kinase is unique among all aspartate kinases, showing synergistic inhibition by lysine and S-adenosyl-l-methionine (SAM). The Arabidopsis genome contains three genes for monofunctional aspartate kinases. We show that aspartate kinase 2 and aspartate kinase 3 are inhibited only by lysine, and that aspartate kinase 1 is inhibited in a synergistic manner by lysine and SAM. In the absence of SAM, aspartate kinase 1 displayed low apparent affinity for lysine compared to aspartate kinase 2 and aspartate kinase 3. In the presence of SAM, the apparent affinity of aspartate kinase 1 for lysine increased considerably, with K(0.5) values for lysine inhibition similar to those of aspartate kinase 2 and aspartate kinase 3. For all three enzymes, the inhibition resulted from an increase in the apparent K(m) values for the substrates ATP and aspartate. The mechanism of aspartate kinase 1 synergistic inhibition was characterized. Inhibition by lysine alone was fast, whereas synergistic inhibition by lysine plus SAM was very slow. SAM by itself had no effect on the enzyme activity, in accordance with equilibrium binding analyses indicating that SAM binding to aspartate kinase 1 requires prior binding of lysine. The three-dimensional structure of the aspartate kinase 1-Lys-SAM complex has been solved [Mas-Droux C, Curien G, Robert-Genthon M, Laurencin M, Ferrer JL & Dumas R (2006) Plant Cell18, 1681-1692]. Taken together, the data suggest that, upon binding to the inactive aspartate kinase 1-Lys complex, SAM promotes a slow conformational transition leading to formation of a stable aspartate kinase 1-Lys-SAM complex. The increase in aspartate kinase 1 apparent affinity for lysine in the presence of SAM thus results from the displacement of the unfavorable equilibrium between aspartate kinase 1 and aspartate kinase 1-Lys towards the inactive form.

Published
2006
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23. Allosteric Threonine Synthase

Author

Valérie Biou, Renaud Dumas, and Corine Mas-Droux

Subjects
0303 health sciences, biology, Stereochemistry, 030302 biochemistry & molecular biology, Allosteric regulation, Active site, Cell Biology, Lyase, Biochemistry, Cystathionine beta synthase, 03 medical and health sciences, chemistry.chemical_compound, Threonine synthase, chemistry, biology.protein, Threonine, Pyridoxal phosphate, Binding site, Molecular Biology, 030304 developmental biology
Abstract

Threonine synthase (TS) is a fold-type II pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the ultimate step of threonine synthesis in plants and microorganisms. Unlike the enzyme from microorganisms, plant TS is activated by S-adenosylmethionine (AdoMet). The mechanism of activation has remained unknown up to now. We report here the crystallographic structures of Arabidopsis thaliana TS in complex with PLP (aTS) and with PLP and AdoMet (aTS-AdoMet), which show with atomic detail how AdoMet activates TS. The aTS structure reveals a PLP orientation never previously observed for a type II PLP-dependent enzyme and explains the low activity of plant TS in the absence of its allosteric activator. The aTS-AdoMet structure shows that activation of the enzyme upon AdoMet binding triggers a large reorganization of active site loops in one monomer of the structural dimer and allows the displacement of PLP to its active conformation. Comparison with other TS structures shows that activation of the second monomer may be triggered by substrate binding. This structure also discloses a novel fold for two AdoMet binding sites located at the dimer interface, each site containing two AdoMet effectors bound in tandem. Moreover, aTS-AdoMet is the first structure of an enzyme that uses AdoMet as an allosteric effector.

Published
2006
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24. Identification of Six Novel Allosteric Effectors of Arabidopsis thaliana Aspartate Kinase-Homoserine Dehydrogenase Isoforms

Author

Stéphane Ravanel, Gilles Curien, Renaud Dumas, and Mylène Robert

Subjects
0106 biological sciences, chemistry.chemical_classification, Alanine, Homoserine dehydrogenase, 0303 health sciences, Cell Biology, Biology, 01 natural sciences, Biochemistry, 03 medical and health sciences, chemistry.chemical_compound, Aspartate carbamoyltransferase, chemistry, Biosynthesis, Aspartate kinase, Threonine, Molecular Biology, Essential amino acid, Amino acid synthesis, 030304 developmental biology, 010606 plant biology & botany
Abstract

The Arabidopsis genome contains two genes predicted to code for bifunctional aspartate kinase-homoserine dehydrogenase enzymes (isoforms I and II). These two activities catalyze the first and the third steps toward the synthesis of the essential amino acids threonine, isoleucine, and methionine. We first characterized the kinetic and regulatory properties of the recombinant enzymes, showing that they mainly differ with respect to the inhibition of the homoserine dehydrogenase activity by threonine. A systematic search for other allosteric effectors allowed us to identify an additional inhibitor (leucine) and 5 activators (alanine, cysteine, isoleucine, serine, and valine) equally efficient on aspartate kinase I activity (4-fold activation). The six effectors of aspartate kinase I were all activators of aspartate kinase II activity (13-fold activation) and displayed a similar specificity for the enzyme. No synergy between different effectors could be observed. The activation, which resulted from a decrease in the Km values for the substrates, was detected using low substrates concentrations. Amino acid quantification revealed that alanine and threonine were much more abundant than the other effectors in Arabidopsis leaf chloroplasts. In vitro kinetics in the presence of physiological concentrations of the seven allosteric effectors confirmed that aspartate kinase I and II activities were highly sensitive to changes in alanine and threonine concentrations. Thus, physiological context rather than enzyme structure sets the specificity of the allosteric control. Stimulation by alanine may play the role of a feed forward activation of the aspartate-derived amino acid pathway in plant.

Published
2005
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25. Crystal Structures of Cobalamin-independent Methionine Synthase Complexed with Zinc, Homocysteine, and Methyltetrahydrofolate

Author

Jean-Luc Ferrer, Renaud Dumas, Mylène Robert, Stéphane Ravanel, Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)

Subjects
Models, Molecular, Homocysteine, Protein Conformation, Arabidopsis, Crystallography, X-Ray, biosynthèse, 5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase, 01 natural sciences, Biochemistry, chemistry.chemical_compound, Methionine, Protein structure, site actif, Methionine synthase, Pterin, Tetrahydrofolates, 0303 health sciences, Fourier Analysis, biology, zinc, méthionine, homocystéine, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Vitamin B 12, plante expérimentale, Plasmids, Stereochemistry, chemistry.chemical_element, Zinc, folate, 010402 general chemistry, site de liaison, 03 medical and health sciences, Folic Acid, Cations, Escherichia coli, Histidine, Cysteine, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Binding site, Molecular Biology, 030304 developmental biology, Binding Sites, arabidopsis thaliana, Active site, Methyltransferases, Cell Biology, Protein Structure, Tertiary, 0104 chemical sciences, chemistry, structure tridimensionnelle, biology.protein, Sulfur
Abstract

International audience; Cobalamin-independent methionine synthase (MetE) catalyzes the synthesis of methionine by a direct transfer of the methyl group of N5-methyltetrahydrofolate (CH3-H4PteGlun) to the sulfur atom of homocysteine (Hcy). We report here the first crystal structure of this metalloenzyme under different forms, free or complexed with the Hcy and folate substrates. The Arabidopsis thaliana MetE (AtMetE) crystals reveal a monomeric structure built by two (βα)8 barrels making a deep groove at their interface. The active site is located at the surface of the C-terminal domain, facing the large interdomain cleft. Inside the active site, His647, Cys649, and Cys733 are involved in zinc coordination, whereas Asp605, Ile437, and Ser439 interact with Hcy. Opposite the zinc/Hcy binding site, a cationic loop (residues 507–529) belonging to the C-terminal domain anchors the first glutamyl residue of CH3-H4PteGlu5. The pterin moiety of CH3-H4PteGlu5 is stacked with Trp567, enabling the N5-methyl group to protrude in the direction of the zinc atom. These data suggest a structural role of the N-terminal domain of AtMetE in the stabilization of loop 507–529 and in the interaction with the poly-glutamate chain of CH3-H4PteGlun. Comparison of AtMetE structures reveals that the addition of Hcy does not lead to a direct coordination of the sulfur atom with zinc but to a reorganization of the zinc binding site with a stronger coordination to Cys649, Cys733, and a water molecule.

Published
2004
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26. A kinetic model of the branch-point between the methionine and threonine biosynthesis pathways in Arabidopsis thaliana

Author

Stéphane Ravanel, Renaud Dumas, and Gilles Curien

Subjects
chemistry.chemical_classification, Methionine, biology, Context (language use), Biochemistry, Cystathionine beta synthase, chemistry.chemical_compound, Enzyme, Threonine synthase, Allosteric enzyme, chemistry, biology.protein, Threonine, Cysteine
Abstract

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

Published
2003
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27. Evolution. Response to Comment on 'A promiscuous intermediate underlies the evolution of LEAFY DNA binding specificity'

Author

Renaud Dumas, Detlef Weigel, Norman Warthmann, François Parcy, Yong Zhang, Gane Ka-Shu Wong, Michael Melkonian, Samuel F. Brockington, Emmanuel Thévenon, Camille Sayou, Marie Monniaux, Edwige Moyroud, Max H. Nanao, Hicham Chahtane, Department of Plant Sciences, University of Cambridge [UK] (CAM), 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), Unit for Virus Host-Cell Interactions, Université Grenoble Alpes (UGA), European Molecular Biology Laboratory, Research School of Biology (Australian National University), Botanisches Institut, Universität Köln, Beijing Genomics Institute, Department of Biological Sciences [Edmonton], University of Alberta, Max Planck Institute for Developmental Biology, Max-Planck-Gesellschaft, Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Research School of Biology [Canberra, Australia], Australian National University (ANU), Universität zu Köln = University of Cologne, Beijing Genomics Institute [Shenzhen] (BGI), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS), and Universität zu Köln

Subjects
0106 biological sciences, DNA, Plant, Evolution, [SDV]Life Sciences [q-bio], Computational biology, DNA binding specificity, Biology, 010603 evolutionary biology, 01 natural sciences, LEAFY gene, Evolution, Molecular, 03 medical and health sciences, chemistry.chemical_compound, Phylogenetics, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Leafy, Transcription factor, Plant Proteins, 030304 developmental biology, Genetics, Technical response to a comment, 0303 health sciences, Multidisciplinary, Dna binding specificity, Plant, Evolutionary transitions, DNA-Binding Proteins, Flower development, chemistry, Identification (biology), DNA, Transcription Factors
Abstract

Brunkard et al . propose that the identification of novel LEAFY sequences contradicts our model of evolution through promiscuous intermediates. Based on the debate surrounding land plant phylogeny and on our analysis of these interesting novel sequences, we explain why there is no solid evidence to disprove our model.

Published
2015
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28. Overproduction, Purification, and Characterization of Recombinant Bifunctional Threonine-Sensitive Aspartate Kinase-Homoserine Dehydrogenase from Arabidopsis thaliana

Author

Peter M. Wessel, Renaud Dumas, and Stephane Paris

Subjects
Threonine, Homoserine dehydrogenase, chemistry.chemical_classification, biology, Arabidopsis, Homoserine, Gene Expression, Dehydrogenase, Recombinant Proteins, Cofactor, Kinetics, chemistry.chemical_compound, Enzyme, Biochemistry, chemistry, Aspartokinase Homoserine Dehydrogenase, Escherichia coli, biology.protein, Aspartate kinase, Phosphofructokinase 2, Cloning, Molecular, Biotechnology
Abstract

In plant, the first and the third steps of the synthesis of methionine and threonine are catalyzed by a bifunctional enzyme, aspartate kinase-homoserine dehydrogenase (AK-HSDH). In this study, we report the first purification and characterization of a highly active threonine-sensitive AK-HSDH from plants ( Arabidopsis thaliana ). The specific activities corresponding to the forward reaction of AK and reverse reaction of HSDH of AK-HSDH were 5.4 μmol of aspartyl phosphate produced min −1 mg −1 of protein and 18.8 μmol of NADPH formed min −1 mg −1 of protein, respectively. These values are 200-fold higher than those reported previously for partially purified plant enzymes. AK-HSDH exhibited hyperbolic kinetics for aspartate, ATP, homoserine, and NADP with K M values of 11.6 mM, 5.5 mM, 5.2 mM, and 166 μM, respectively. Threonine was found to inhibit both AK and HSDH activities by decreasing the affinity of the enzyme for its substrates and cofactors. In the absence of threonine, AK-HSDH behaved as an oligomer of 470 kDa. Addition of the effector converted the enzyme into a tetrameric form of 320 kDa.

Published
2002
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29. Overproduction, Purification, and Characterization of Recombinant Aspartate Semialdehyde Dehydrogenase from Arabidopsis thaliana

Author

Renaud Dumas, Stephane Paris, and Peter M. Wessel

Subjects
DNA, Complementary, Aspartate-semialdehyde dehydrogenase, Molecular Sequence Data, Lysine, Arabidopsis, Gene Expression, Biology, chemistry.chemical_compound, Sequence Analysis, Protein, Escherichia coli, Arabidopsis thaliana, Amino Acid Sequence, Cloning, Molecular, Threonine, Overproduction, chemistry.chemical_classification, Methionine, Sequence Homology, Amino Acid, Aspartate-Semialdehyde Dehydrogenase, food and beverages, biology.organism_classification, Molecular biology, Recombinant Proteins, Amino acid, Solutions, Kinetics, Enzyme, chemistry, Biochemistry, Biotechnology
Abstract

In plant and microorganisms, aspartate semialdehyde dehydrogenase (ASDH) produces the branch point intermediate between the lysine and threonine/methionine pathways. In this study, we report the first cDNA cloning, purification, and characterization of a plant ASDH. The Arabidopsis thaliana ASDH is an homodimeric enzyme composed of subunits of 36 kDa. The plant enzyme exhibited a specific activity of 26 micromol NADPH oxidized min(-1) mg(-1) of protein with a K(M) value for NADPH of 92 microM. ASDH showed cooperative behavior for aspartyl phosphate with a K(0.5) value of 37 microM.

Published
2002
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30. Biochemical and biophysical characterization of the selenium-binding and reducing site in Arabidopsis thaliana homologue to mammals selenium-binding protein 1

Author

Renaud Dumas, Géraldine Sarret, Denis Testemale, Florie Schild, Véronique Hugouvieux, Andrés Palencia, Agnès Jourdain, Jacques Bourguignon, Vincent Forge, David Cobessi, Chloe Zubieta, Sylvie Kieffer-Jaquinod, Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), European Molecular Biology Laboratory [Heidelberg] (EMBL), Unit for Virus Host-Cell Interactions [Grenoble] (UVHCI), Université Joseph Fourier - Grenoble 1 (UJF)-European Molecular Biology Laboratory [Grenoble] (EMBL)-Centre National de la Recherche Scientifique (CNRS), 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), Institut des Sciences de la Terre (ISTerre), Université Joseph Fourier - Grenoble 1 (UJF)-Institut Français des Sciences et Technologies des Transports, de l'Aménagement et des Réseaux (IFSTTAR)-Institut national des sciences de l'Univers (INSU - CNRS)-Institut de recherche pour le développement [IRD] : UR219-PRES Université de Grenoble-Université Savoie Mont Blanc (USMB [Université de Savoie] [Université de Chambéry])-Centre National de la Recherche Scientifique (CNRS), 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), Matériaux, Rayonnements, Structure (NEEL - MRS), Institut Néel (NEEL), Université Joseph Fourier - Grenoble 1 (UJF)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS), Rhone-Alpes region, Biologie et Amelioration des Plantes Department of Institut National de la Recherche Agronomique, CEA Toxicology program, Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Unit of Virus Host Cell Interactions (UVHCI), Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS), 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), Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Université Savoie Mont Blanc (USMB [Université de Savoie] [Université de Chambéry])-PRES Université de Grenoble-Institut de recherche pour le développement [IRD] : UR219-Institut national des sciences de l'Univers (INSU - CNRS)-Institut Français des Sciences et Technologies des Transports, de l'Aménagement et des Réseaux (IFSTTAR)-Université Joseph Fourier - Grenoble 1 (UJF), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), MRS - Matériaux, Rayonnements, Structure, Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS), 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]), Centre National de la Recherche Scientifique (CNRS)-European Molecular Biology Laboratory [Grenoble] (EMBL)-Université Joseph Fourier - Grenoble 1 (UJF), 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), Matériaux, Rayonnements, Structure (MRS), and GON, Nathalie

Subjects
0106 biological sciences, Proteomics, Arabidopsis, Molecular Conformation, Plant Biology, Plasma protein binding, Selenium-Binding Proteins, Ligands, 01 natural sciences, Biochemistry, Gene Expression Regulation, Plant, [SDV.BV] Life Sciences [q-bio]/Vegetal Biology, Selenium binding, caractérisation biochimique, 0303 health sciences, Vegetal Biology, Plant Biochemistry, plant biochemistry, x-ray absorption spectroscopy, protein chemical modification, proteomics, selenium, food and beverages, Recombinant Proteins, [SDV.TOX] Life Sciences [q-bio]/Toxicology, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, [SDV.TOX]Life Sciences [q-bio]/Toxicology, Thermodynamics, circulatory and respiratory physiology, Protein Binding, inorganic chemicals, Molecular Sequence Data, [SDV.BBM.BP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, chemistry.chemical_element, Protein Chemical Modification, Biology, 03 medical and health sciences, Selenium, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Animals, Humans, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, cardiovascular diseases, Amino Acid Sequence, Cysteine, Binding site, Molecular Biology, 030304 developmental biology, Binding Sites, Sequence Homology, Amino Acid, Ligand, Arabidopsis Proteins, arabidopsis thaliana, Isothermal titration calorimetry, Cell Biology, biology.organism_classification, X-ray Absorption Spectroscopy, chemistry, 13. Climate action, Mutagenesis, Site-Directed, Carrier Proteins, Biologie végétale, 010606 plant biology & botany
Abstract

Background: The selenium-binding site in selenium-binding protein (SBP) homologues was not identified. Results: The Arabidopsis thaliana SBP1 selenium-binding site was characterized as a R-S-Se(II)-S-R-type complex involving Cys(21) and Cys(22). Conclusion: This is the first identification of the selenium-binding site in any SBP. Significance: It is an important step toward a better understanding of the link between selenium binding and function of SBP. The function of selenium-binding protein 1 (SBP1), present in almost all organisms, has not yet been established. In mammals, SBP1 is known to bind the essential element selenium but the binding site has not been identified. In addition, the SBP family has numerous potential metal-binding sites that may play a role in detoxification pathways in plants. In Arabidopsis thaliana, AtSBP1 over-expression increases tolerance to two toxic compounds for plants, selenium and cadmium, often found as soil pollutants. For a better understanding of AtSBP1 function in detoxification mechanisms, we investigated the chelating properties of the protein toward different ligands with a focus on selenium using biochemical and biophysical techniques. Thermal shift assays together with inductively coupled plasma mass spectrometry revealed that AtSBP1 binds selenium after incubation with selenite (SeO32-) with a ligand to protein molar ratio of 1:1. Isothermal titration calorimetry confirmed the 1:1 stoichiometry and revealed an unexpectedly large value of binding enthalpy suggesting a covalent bond between selenium and AtSBP1. Titration of reduced Cys residues and comparative mass spectrometry on AtSBP1 and the purified selenium-AtSBP1 complex identified Cys(21) and Cys(22) as being responsible for the binding of one selenium. These results were validated by site-directed mutagenesis. Selenium K-edge x-ray absorption near edge spectroscopy performed on the selenium-AtSBP1 complex demonstrated that AtSBP1 reduced SeO32- to form a R-S-Se(II)-S-R-type complex. The capacity of AtSBP1 to bind different metals and selenium is discussed with respect to the potential function of AtSBP1 in detoxification mechanisms and selenium metabolism.

Published
2014
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31. Molecular basis for AUXIN RESPONSE FACTOR protein interaction and the control of auxin response repression

Author

Max H. Nanao, Lucia C. Strader, Joseph M. Jez, Renaud Dumas, David A. Korasick, Tom J. Guilfoyle, Gretchen Hagen, Corey S. Westfall, Soon Goo Lee, Department of Biology, Washington University, University of Washington [Seattle], Unit of Virus Host- Cell Interactions, Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), European Molecular Biology Laboratory [Grenoble] (EMBL), Laboratoire de physiologie cellulaire végétale (LPCV), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS), Department of Biochemistry, University of Missouri, University of Missouri [Columbia] (Mizzou), University of Missouri System-University of Missouri System, National Science Foundation (NSF) Graduate Research Fellowship Program (2011101911), National Institutes of Health (R00 GM089987-03), US Department of Agriculture-National Institute of Food and Agriculture Fellowship Program (MOW-2010-05240), Université Grenoble Alpes (UGA), 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), University of Missouri [Columbia], 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
crystal structure, Arabidopsis thaliana, Molecular Sequence Data, Arabidopsis, plant, Plasma protein binding, phytohormone, medicine.disease_cause, Auxin, Gene expression, medicine, heterocyclic compounds, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, Transcription factor, Psychological repression, transcription factor, Plant Proteins, chemistry.chemical_classification, Mutation, Multidisciplinary, Indoleacetic Acids, Sequence Homology, Amino Acid, biology, fungi, food and beverages, Biological Sciences, biology.organism_classification, Cell biology, chemistry, Biochemistry, gene expression, auxin
Abstract

International audience; In plants, the AUXIN RESPONSE FACTOR (ARF) transcription factor family regulates gene expression in response to auxin. In the absence of auxin, ARF transcription factors are repressed by interaction with AUXIN/INDOLE 3-ACETIC ACID (Aux/IAA) proteins. Although the C termini of ARF and Aux/IAA proteins facilitate their homo- and heterooligomerization, the molecular basis for this interaction remained undefined. The crystal structure of the C-terminal interaction domain of Arabidopsis ARF7 reveals a Phox and Bem1p (PB1) domain that provides both positive and negative electrostatic interfaces for directional protein interaction. Mutation of interface residues in the ARF7 PB1 domain yields monomeric protein and abolishes interaction with both itself and IAA17. Expression of a stabilized Aux/IAA protein (i.e., IAA16) bearing PB1 mutations in Arabidopsis suggests a multimerization requirement for ARF protein repression, leading to a refined auxin-signaling model.

Published
2014
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32. Enzymology, Structure, and Dynamics of Acetohydroxy Acid Isomeroreductase

Author

Renaud Dumas, Frédéric Halgand, Roland Douce, Ronald G. Duggleby, and Valérie Biou

Subjects
chemistry.chemical_classification, Sequence Homology, Amino Acid, Chemistry, Stereochemistry, Ketol-Acid Reductoisomerase, Molecular Sequence Data, Nucleic acid sequence, General Medicine, General Chemistry, Catalysis, Amino acid, Alcohol Oxidoreductases, Structure-Activity Relationship, Enzyme, Biochemistry, Valine, Amino Acid Sequence, Leucine, Isoleucine, Site-directed mutagenesis, Pyruvate decarboxylase, Subcellular Fractions
Abstract

Acetohydroxy acid isomeroreductase is a key enzyme involved in the biosynthetic pathway of the amino acids isoleucine, valine, and leucine. This enzyme is of great interest in agrochemical research because it is present only in plants and microorganisms, making it a potential target for specific herbicides and fungicides. Moreover, it catalyzes an unusual two-step reaction that is of great fundamental interest. With a view to characterizing both the mechanism of inhibition by potential herbicides and the complex reaction mechanism, various techniques of enzymology, molecular biology, mass spectrometry, X-ray crystallography, and theoretical simulation have been used. The results and conclusions of these studies are described briefly in this paper.

Published
2001
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33. Crystal structure of threonine synthase from Arabidopsis thaliana

Author

Karine Thomazeau, Valérie Biou, Gilles Curien, and Renaud Dumas

Subjects
S-Adenosylmethionine, Methyltransferase, Stereochemistry, Carbon-Oxygen Lyases, Molecular Sequence Data, Allosteric regulation, Arabidopsis, Biochemistry, Article, Protein Structure, Secondary, Enzyme activator, chemistry.chemical_compound, Amino Acid Sequence, Methionine synthase, S-Adenosyl methionine, Threonine, Binding site, Protein Structure, Quaternary, Molecular Biology, Conserved Sequence, Binding Sites, Crystallography, biology, Protein Structure, Tertiary, Enzyme Activation, Threonine synthase, chemistry, Pyridoxal Phosphate, biology.protein, Oxidation-Reduction
Abstract

Threonine synthase (TS) is a PLP-dependent enzyme that catalyzes the last reaction in the synthesis of threonine from aspartate. In plants, the methionine pathway shares the same substrate, O-phospho-L-homoserine (OPH), and TS is activated by S-adenosyl-methionine (SAM), a downstream product of methionine synthesis. This positive allosteric effect triggered by the product of another pathway is specific to plants. The crystal structure of Arabidopsis thaliana apo threonine synthase was solved at 2.25 A resolution from triclinic crystals using MAD data from the selenomethionated protein. The structure reveals a four-domain dimer with a two-stranded beta-sheet arm protruding from one monomer onto the other. This domain swap could form a lever through which the allosteric effect is transmitted. The N-terminal domain (domain 1) has a unique fold and is partially disordered, whereas the structural core (domains 2 and 3) shares the functional domain of PLP enzymes of the same family. It also has similarities with SAM-dependent methyltransferases. Structure comparisons allowed us to propose potential sites for pyridoxal-phosphate and SAM binding on TS; they are close to regions that are disordered in the absence of these molecules.

Published
2001
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34. Evidence for Two Distinct Effector-Binding Sites in Threonine Deaminase by Site-Directed Mutagenesis, Kinetic, and Binding Experiments

Author

Peter M. Wessel, Renaud Dumas, Emmanuelle Graciet, and Roland Douce

Subjects
Arabidopsis, Biology, medicine.disease_cause, Biochemistry, Gene Expression Regulation, Enzymologic, Threonine Dehydratase, Valine, medicine, Isoleucine, Threonine, Binding site, Site-directed mutagenesis, Escherichia coli, chemistry.chemical_classification, Binding Sites, Mutagenesis, Models, Theoretical, Recombinant Proteins, Protein Structure, Tertiary, Kinetics, Enzyme, chemistry, Mutation, Mutagenesis, Site-Directed
Abstract

A three-dimensional structure comparison between the dimeric regulatory serine-binding domain of Escherichia coli D-3-phosphoglycerate dehydrogenase [Schuller, D. J., Grant, G. A., and Banaszak, L. J. (1995) Nat. Struct. Biol. 2, 69-76] and the regulatory domain of E. coli threonine deaminase [Gallagher, D. T., Gilliland, G. L., Xiao, G., Zondlo, J., Fisher, K. E., Chinchilla, D. , and Eisenstein, E. (1998) Structure 6, 465-475] led us to make the hypothesis that threonine deaminase could have two binding sites per monomer. To test this hypothesis about the corresponding plant enzyme, site-directed mutagenesis was carried out on the recombinant Arabidopsis thaliana threonine deaminase. Kinetic and binding experiments demonstrated for the first time that each regulatory domain of the monomers of A. thaliana threonine deaminase possesses two different effector-binding sites constituted in part by Y449 and Y543. Our results demonstrate that Y449 belongs to a high-affinity binding site whose interaction with a first isoleucine induces conformational modifications yielding a conformer displaying a higher activity and with enhanced ability to bind a second isoleucine on a lower-affinity binding site containing Y543. Isoleucine interaction with this latter binding site is responsible for conformational modifications leading to final inhibition of the enzyme. Y449 interacts with both regulators, isoleucine and valine. However, interaction of valine with the high-affinity binding site induces different conformational modifications leading to reversal of isoleucine binding and reversal of inhibition.

Published
2000
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35. A Hybrid-Potential Free-Energy Study of the Isomerization Step of the Acetohydroxy Acid Isomeroreductase Reaction

Author

Flavien Proust-De Martin, Martin J. Field, and and Renaud Dumas

Subjects
chemistry.chemical_classification, Colloid and Surface Chemistry, Enzyme, chemistry, General Chemistry, Acetohydroxy acid isomeroreductase, Photochemistry, Biochemistry, Combinatorial chemistry, Isomerization, Catalysis, Amino acid
Abstract

The enzyme acetohydroxy acid isomeroreductase is a promising target for the design of herbicides because it is an essential enzyme for the synthesis of branched-chain amino acids in plants but is a...

Published
2000
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36. Structure of spinach acetohydroxyacid isomeroreductase complexed with its reaction product dihydroxymethylvalerate, manganese and (phospho)-ADP-ribose

Author

Renaud Dumas, Frédéric Halgand, Valérie Biou, Karine Thomazeau, Eric Forest, and Roland Douce

Subjects
Models, Molecular, Protein Conformation, Stereochemistry, Ketol-Acid Reductoisomerase, Molecular Conformation, chemistry.chemical_element, Manganese, Reaction intermediate, Crystallography, X-Ray, Divalent, chemistry.chemical_compound, Spinacia oleracea, Structural Biology, Valerates, Alkyl, chemistry.chemical_classification, Adenosine Diphosphate Ribose, Nicotinamide, biology, Magnesium, Active site, General Medicine, Recombinant Proteins, Alcohol Oxidoreductases, Enzyme, chemistry, biology.protein, Dimerization
Abstract

Acetohydroxyacid isomeroreductase catalyses a two-step reaction composed of an alkyl migration followed by an NADPH-dependent reduction. Both steps require a divalent cation and the first step has a strong preference for magnesium. Manganese ions are highly unfavourable to the reaction: only 3% residual activity is observed in the presence of this cation. Acetohydroxyacid isomeroreductase has been crystallized with its substrate, 2-aceto-2-hydroxybutyrate (AHB), Mn(2+) and NADPH. The 1.6 A resolution electron-density map showed the reaction product (2,3-dihydroxy-3-methylvalerate, DHMV) and a density corresponding to (phospho)-ADP-ribose instead of the whole NADP(+). This is one of the few structures of an enzyme complexed with its reaction product. The structure of this complex was refined to an R factor of 19.3% and an R(free) of 22.5%. The overall structure of the enzyme is very similar to that of the complex with the reaction-intermediate analogue IpOHA [N-hydroxy-N-isopropyloxamate; Biou et al. (1997), EMBO J. 16, 3405-3415]. However, the active site shows some differences: the nicotinamide is cleaved and the surrounding amino acids have rearranged accordingly. Comparison between the structures corresponding to the reaction intermediate and to the end of the reaction allowed the proposal of a reaction scheme. Taking this result into account, the enzyme was crystallized with Ni(2+) and Zn(2+), for which only 0.02% residual activity were measured; however, the crystals of AHB/Zn/NADPH and of AHB/Ni/NADPH also contain the reaction product. Moreover, mass-spectrometry measurements confirmed the -cleavage of nicotinamide.

Published
2000
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37. Stepwise building of a 115-kDa macromolecular edifice monitored by electrospray mass spectrometry

Author

Renaud Dumas, Laurent Serani, Frédéric Halgand, Eric Forest, Olivier Laprévote, and Bhupesh C. Das

Subjects
Electrospray, Chromatography, Protein mass spectrometry, Chemistry, Ligand (biochemistry), Biochemistry, Magnesium ion, Sample preparation in mass spectrometry, Ion source, Stoichiometry, Macromolecule
Abstract

The macromolecular complexes formed by the enzyme acetohydroxy acid isomeroreductase with NADPH, magnesium ions and the competitive inhibitor N-hydroxy-N-isopropyloxamate (IpOHA) were analysed by electrospray mass spectrometry. Each ligand was added successively to a protein solution, allowing the stoichiometry of the whole macromolecular edifice (115 583 Da) to be unambiguously determined. The combination of an electrospray ion source with the high mass range magnetic instrument used in the present studies proved to be a very powerful tool for characterizing, in a specific manner, the quaternary structures of proteins by single mass measurements.

Published
1999
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38. A Loop Deletion in the Plant Acetohydroxy Acid Isomeroreductase Homodimer Generates an Active Monomer with Reduced Stability and Altered Magnesium Affinity

Author

Roland Douce, Valérie Biou, Renaud Dumas, and Peter M. Wessel

Subjects
Models, Molecular, Stereochemistry, Phenylalanine, Dimer, Ketol-Acid Reductoisomerase, chemistry.chemical_element, Cooperativity, Biochemistry, Cofactor, Serine, chemistry.chemical_compound, Spinacia oleracea, Enzyme Stability, Magnesium, chemistry.chemical_classification, Binding Sites, biology, Recombinant Proteins, Enzyme Activation, Alcohol Oxidoreductases, Kinetics, Monomer, Enzyme, chemistry, Mutagenesis, Site-Directed, biology.protein, Thermodynamics, Protein quaternary structure, Dimerization
Abstract

Plant acetohydroxy acid isomeroreductase is a stable homodimer which catalyzes in the presence of magnesium an alkyl migration followed by a NADPH-dependent reduction. Since the enzyme exhibits no kinetic cooperativity either for its cofactor (NADPH and magnesium) or for its substrates, the reason for dimerization of this enzyme was not obvious. Recently, crystallographic studies [Biou, V., et al. (1997) EMBO J. 16, 3405-3415] revealed that the loop of residues 422-431 plays a major part in the dimer interface. To understand the role of the quaternary structure of the enzyme, we have deleted residues 423-430 and substituted Phe 431 for serine. This mutant was further overproduced in Escherichia coli, purified to homogeneity, and characterized. Gel filtration and thermodynamic experiments disclosed that this mutant behaves as an active monomer with reduced thermal stability. Furthermore, kinetic and fluorescence experiments showed that the behavior of the monomer with respect to magnesium was greatly altered. These results demonstrate the function of the quaternary structure of plant acetohydroxy acid isomeroreductase in the stabilization of the tertiary structure but also in the stabilization of a high-affinity magnesium binding site.

Published
1998
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39. Kinetic and Mass Spectrometric Analyses of the Interactions between Plant Acetohydroxy Acid Isomeroreductase and Thiadiazole Derivatives

Author

Roland Douce, Renaud Dumas, Dominique Job, Françoise Vives, Frédéric Halgand, Eric Forest, Richard Cantegril, Jean-Pierre Andrieu, Valérie Biou, Jean Gagnon, and Jens Andersen

Subjects
chemistry.chemical_classification, Stereochemistry, Chemistry, Ketol-Acid Reductoisomerase, Molecular Sequence Data, Branched-chain amino acid, food and beverages, Plants, Acetohydroxy acid isomeroreductase, Biochemistry, Mass spectrometric, Mass Spectrometry, Adduct, Alcohol Oxidoreductases, Kinetics, chemistry.chemical_compound, Enzyme, Non-competitive inhibition, Covalent bond, Thiadiazoles, Amino Acid Sequence, Enzyme Inhibitors, Binding site
Abstract

Plant acetohydroxy acid isomeroreductase (EC 1.1.1.86), the second enzyme of the branched chain amino acid biosynthetic pathway, has been submitted to high-throughput screening for herbicide discovery. We report here the discovery of a new class of compounds belonging to the thiadiazole family, which exhibit a strong inhibitory effect on this plant enzyme. Kinetic analyses revealed that these compounds act as either reversible or irreversible noncompetitive inhibitors of the plant enzyme. Reversibility or irreversibility of these compounds can be attributed to the nature of the additional groups of the thiadiazole ring favoring or not favoring the formation of a covalent adduct. Mass spectrometric experiments on the complex between an irreversible compound belonging to the thiadiazole family and the plant enzyme identified Cys498 as the binding site of the inhibitor.

Published
1998
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40. A promiscuous intermediate underlies the evolution of LEAFY DNA binding specificity

Author

Camille Sayou, Norman Warthmann, Samuel F. Brockington, François Parcy, Michael Melkonian, Hicham Chahtane, Yong Zhang, Renaud Dumas, Edwige Moyroud, Max H. Nanao, Gane Ka-Shu Wong, Emmanuel Thévenon, Detlef Weigel, Marie Monniaux, 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), Unit of Virus Host- Cell Interactions, Université Grenoble Alpes (UGA), European Molecular Biology Laboratory [Grenoble] (EMBL), Department of Plant Sciences, University of Cambridge [UK] (CAM), Department of Molecular Biology [Tübingen], Max Planck Institute for Developmental Biology, Max-Planck-Gesellschaft-Max-Planck-Gesellschaft, Botanisches Institut, Universität Köln, Beijing Genomics Institute (BGI), Beijing Genomics Institute, Department of Biological Sciences [Edmonton], University of Alberta, funds from the Max Planck Society, Coopérations et Mobilités Internationales Rhône-Alpes, FP7 P-CUBE number 227764, Alberta Ministry of Enterprise and Advanced Education, Alberta Innovates Technology Futures, Innovates Centre of Research Excellence, Musea Ventures, and BGI-Shenzhen, Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Universität zu Köln = University of Cologne, Beijing Genomics Institute [Shenzhen] (BGI), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS), and Universität zu Köln

Subjects
Genetics, Multidisciplinary, Cell division, Mechanism (biology), fungi, Regulator, evolutionary transitions, plant, DNA binding specificity, Biology, LEAFY gene, transcription factors, Gene duplication, evolution, flower development, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Gene, Transcription factor, Leafy, Function (biology)
Abstract

LEAFY Evolution It is generally believed that redundancy across gene copies allows for the evolution of novel function in proteins. However, it is less clear how single-copy genes with crucial function may evolve. Sayou et al. (p. 645 , published online 16 January; see the Perspective by Kovach and Lamb ) examined the evolution of the essential plant transcription factor LEAFY, which is generally found as a single-copy gene. LEAFY homologs in taxa representing the major evolutionary branches of the land plants and algae exhibited three classes of LEAFY binding sites. Structural analysis identified amino acid changes in the proteins, that were responsible for contacts with specific DNA motifs and allowed the likely effects of specific amino acid changes over the evolution of land plants to be resolved.

Published
2014
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41. Structural Basis for the Oligomerization of the MADS Domain Transcription Factor SEPALLATA3 in Arabidopsis

Author

Romain Marcellin, Vanessa M. Conn, Simon J. Conn, Luca Costa, Rainer Melzer, Renaud Dumas, Darren J. Hart, François Parcy, Chloe Zubieta, S. Puranik, Catarina S. Silva, Samira Acajjaoui, Elizabeth Brown, Anthony Vial, Günter Theißen, Max H. Nanao, Struct Biol Grp, European Synchrotron Radiation Facility (ESRF), Centre pour la biologie du cancer, University of South Australia, University of South Australia [Adelaide], SA Pathology and the University of South Australia, Centre for Cancer Biology, Université de Montpellier (UM), Department of Genetics, Friedrich-Schiller-Universität Jena, Integrated Structural Biology Grenoble, Unit of Virus Host Cell Interactions, Université Grenoble Alpes (UGA), Departement Genetique, Laboratoire de Physiologie Cellulaire and Végétale, 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), European Molecular Biology Laboratory, Unit for Virus Host-Cell Interactions, Laboratoire de Physiologie Cellulaire and Végétale - UMR5168, Puranik, Sriharsha, Acajjaoui, Samira, Conn, Simon, Costa, Luca, Conn, Vanessa, Vial, Anthony, Marcellin, Romain, Melzer, Rainer, Brown, Elizabeth, Hart, Darren, Theiben, Günter, Silva, Catarina S, Parcy, François, Dumas, Renaud, Nanao, Max, Zubieta, Chloe, Friedrich-Schiller-Universität = Friedrich Schiller University Jena [Jena, Germany], Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, DNA, Plant, [SDV]Life Sciences [q-bio], Molecular Sequence Data, Arabidopsis, MADS Domain Proteins, Plant Science, homeotic genes, HOMEOTIC GENES, Crystallography, X-Ray, Microscopy, Atomic Force, SEED PLANTS, Protein–protein interaction, chemistry.chemical_compound, Structure-Activity Relationship, Transcription (biology), CRYSTAL-STRUCTURE, Amino Acid Sequence, Promoter Regions, Genetic, Transcription factor, FLOWER DEVELOPMENT, BOX GENES, MADS-box, Research Articles, Genetics, Homeodomain Proteins, biology, plants, Arabidopsis Proteins, ANTIRRHINUM-MAJUS, Cell Biology, IN-VITRO, biology.organism_classification, Protein Structure, Tertiary, PROTEIN INTERACTIONS, chemistry, TERNARY COMPLEX-FORMATION, Biophysics, Chromatography, Gel, FLORAL QUARTETS, Mutant Proteins, Protein Multimerization, Homeotic gene, seed, DNA, Alpha helix, Protein Binding, Transcription Factors
Abstract

In plants, MADS domain transcription factors act as central regulators of diverse developmental pathways. In Arabidopsis thaliana, one of the most central members of this family is SEPALLATA3 (SEP3), which is involved in many aspects of plant reproduction, including floral meristem and floral organ development. SEP3 has been shown to form homo and heterooligomeric complexes with other MADS domain transcription factors through its intervening (I) and keratin-like (K) domains. SEP3 function depends on its ability to form specific protein-protein complexes; however, the atomic level determinants of oligomerization are poorly understood. Here, we report the 2.5-Å crystal structure of a small portion of the intervening and the complete keratin-like domain of SEP3. The domains form two amphipathic alpha helices separated by a rigid kink, which prevents intramolecular association and presents separate dimerization and tetramerization interfaces comprising predominantly hydrophobic patches. Mutations to the tetramerization interface demonstrate the importance of highly conserved hydrophobic residues for tetramer stability. Atomic force microscopy was used to show SEP3-DNA interactions and the role of oligomerization in DNA binding and conformation. Based on these data, the oligomerization patterns of the larger family of MADS domain transcription factors can be predicted and manipulated based on the primary sequence.

Published
2014
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42. Structural basis for oligomerization of auxin transcriptional regulators

Author

Meryl Mazzoleni, Hanbing Li, Renaud Dumas, David Mast, Tom J. Guilfoyle, Teva Vernoux, Max H. Nanao, Emmanuel Thévenon, Shucai Wang, Thomas Vinos-Poyo, Géraldine Brunoud, François Parcy, Stéphanie Lainé, Gretchen Hagen, Unit of Virus Host- Cell Interactions, Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), European Molecular Biology Laboratory [Grenoble] (EMBL), 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), Reproduction et développement des plantes (RDP), Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Recherche Agronomique (INRA)-École normale supérieure - Lyon (ENS Lyon), Key Laboratory of Molecular Epigenetics of MOE, University of Changchun, Department of Biochemistry, University of Missouri, University of Missouri [Columbia] (Mizzou), University of Missouri System-University of Missouri System, Program for Introducing Talents to Universities (B07017), A startup fund from Northeast Normal University (www.nenu.edu.cn)', University of Missouri F21 Program, Parcy, François, Vernoux, TEVA, Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), École normale supérieure de Lyon (ENS de Lyon)-Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Université Grenoble Alpes (UGA), École normale supérieure - Lyon (ENS Lyon)-Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), and University of Missouri [Columbia]

Subjects
0106 biological sciences, Protein domain, Arabidopsis, General Physics and Astronomy, Repressor, plant, Biology, phytohormone, Crystallography, X-Ray, 01 natural sciences, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Plant Growth Regulators, Gene Expression Regulation, Plant, Transcription (biology), Auxin, Gene expression, Morphogenesis, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, Protein Interaction Domains and Motifs, heterocyclic compounds, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, régulateur de transcription, Transcription factor, transcription factor, 030304 developmental biology, Regulation of gene expression, chemistry.chemical_classification, 0303 health sciences, Multidisciplinary, Indoleacetic Acids, auxine, Arabidopsis Proteins, fungi, food and beverages, General Chemistry, biology.organism_classification, Protein Structure, Tertiary, Cell biology, DNA-Binding Proteins, chemistry, Biochemistry, Plant hormone, auxin, gene regulation, Signal Transduction, Transcription Factors, 010606 plant biology & botany
Abstract

Supplementary Information Supplementary Figures 1-3 and Supplementary Tables 1-2; International audience; The plant hormone auxin is a key morphogenetic regulator acting from embryogenesis onwards. Transcriptional events in response to auxin are mediated by the auxin response factor (ARF) transcription factors and the Aux/IAA (IAA) transcriptional repressors. At low auxin concentrations, IAA repressors associate with ARF proteins and recruit corepressors that prevent auxin-induced gene expression. At higher auxin concentrations, IAAs are degraded and ARFs become free to regulate auxin-responsive genes. The interaction between ARFs and IAAs is thus central to auxin signalling and occurs through the highly conserved domain III/IV present in both types of proteins. Here, we report the crystal structure of ARF5 domain III/IV and reveal the molecular determinants of ARF-IAA interactions. We further provide evidence that ARFs have the potential to oligomerize, a property that could be important for gene regulation in response to auxin.

Published
2014
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43. Characterization of anArabidopsis thalianacDNA encoding an S-adenosylmethionine-sensitive threonine synthase Threonine synthase from higher plants

Author

Renaud Dumas, Stéphane Ravanel, Gilles Curien, and Roland Douce

Subjects
S-Adenosylmethionine, DNA, Complementary, Arabidopsis thaliana, Carbon-Oxygen Lyases, Molecular Sequence Data, Mutant, Arabidopsis, Biophysics, Lyases, medicine.disease_cause, Chloroplast, Polymerase Chain Reaction, Biochemistry, Structural Biology, Complementary DNA, Threonine synthase activity, Escherichia coli, Genetics, medicine, Amino Acid Sequence, Cloning, Molecular, Pyridoxal 5′-phosphate, Molecular Biology, Conserved Sequence, DNA Primers, chemistry.chemical_classification, Base Sequence, Sequence Homology, Amino Acid, biology, Threonine synthase, Lysine, food and beverages, Cell Biology, Chromatography, Ion Exchange, biology.organism_classification, Molecular biology, Recombinant Proteins, o-Phospho-l-homoserine, Complementation, Kinetics, Enzyme, chemistry, Chromatography, Gel, biology.protein
Abstract

An Arabidopsis thaliana cDNA encoding an Sadenosylmethionine-sensitive threonine synthase (EC 4.2.99.2) has been isolated by functional complementation of an Escherichia coli mutant devoid of threonine synthase activity. Threonine synthase from A. thaliana was shown to be synthesized with a transit peptide. The recombinant protein is activated by Sadenosylmethionine in the same range as the plant threonine synthase and evidence is presented for an involvement of the N-terminal part of the mature enzyme in the sensitivity to Sadenosylmethionine.

Published
1996
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44. Cytokinin signalling inhibitory fields provide robustness to phyllotaxis

Author

Pierre Chambrier, Fabrice Besnard, Jonathan Legrand, Yann Guédon, Frédérique Rozier, Emmanuel Thévenon, Christophe Godin, Vincent Mirabet, François Parcy, Benjamin Marteaux, Géraldine Brunoud, Teva Vernoux, Yassin Refahi, Anthony Bishopp, Etienne Farcot, Stéphanie Lainé, Yrjö Helariutta, Jan Traas, Renaud Dumas, Valérie Morin, Pradeep Das, Arezki Boudaoud, Coralie Cellier, Reproduction et développement des plantes (RDP), École normale supérieure de Lyon (ENS de Lyon)-Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Modeling plant morphogenesis at different scales, from genes to phenotype (VIRTUAL PLANTS), Inria Sophia Antipolis - Méditerranée (CRISAM), Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de la Recherche Agronomique (INRA)-Amélioration génétique et adaptation des plantes méditerranéennes et tropicales (UMR AGAP), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro)-Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Laboratoire Joliot Curie, École normale supérieure de Lyon (ENS de Lyon)-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)-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), HiLIFE - Institute of Biotechnology [Helsinki] (BI), Helsinki Institute of Life Science (HiLIFE), Helsingin yliopisto = Helsingfors universitet = University of Helsinki-Helsingin yliopisto = Helsingfors universitet = University of Helsinki, Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Recherche Agronomique (INRA)-École normale supérieure - Lyon (ENS Lyon), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro), École normale supérieure - Lyon (ENS Lyon)-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), University of Helsinki-University of Helsinki, École normale supérieure - Lyon (ENS Lyon)-Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro)-Institut National de la Recherche Agronomique (INRA)-Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro)-Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0106 biological sciences, Cytokinins, F60 - Physiologie et biochimie végétale, [INFO.INFO-OH]Computer Science [cs]/Other [cs.OH], Meristem, Arabidopsis, F62 - Physiologie végétale - Croissance et développement, 01 natural sciences, 03 medical and health sciences, chemistry.chemical_compound, Plant Growth Regulators, Auxin, Botany, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, Multidisciplinary, biology, Indoleacetic Acids, Arabidopsis Proteins, fungi, Spatiotemporal pattern, Robustness (evolution), food and beverages, Biological Transport, Phyllotaxis, biology.organism_classification, [INFO.INFO-MO]Computer Science [cs]/Modeling and Simulation, Cell biology, chemistry, Cytokinin, Plant hormone, Plant Shoots, 010606 plant biology & botany, Signal Transduction
Abstract

How biological systems generate reproducible patterns with high precision is a central question in science1. The shoot apical meristem (SAM), a specialized tissue producing plant aerial organs, is a developmental system of choice to address this question. Organs are periodically initiated at the SAM at specific spatial positions and this spatiotemporal pattern defines phyllotaxis. Accumulation of the plant hormone auxin triggers organ initiation2, 3, 4, 5, whereas auxin depletion around organs generates inhibitory fields that are thought to be sufficient to maintain these patterns and their dynamics4, 6, 7, 8, 9, 10, 11, 12, 13. Here we show that another type of hormone-based inhibitory fields, generated directly downstream of auxin by intercellular movement of the cytokinin signalling inhibitor ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6 (AHP6)14, is involved in regulating phyllotactic patterns. We demonstrate that AHP6-based fields establish patterns of cytokinin signalling in the meristem that contribute to the robustness of phyllotaxis by imposing a temporal sequence on organ initiation. Our findings indicate that not one but two distinct hormone-based fields may be required for achieving temporal precision during formation of reiterative structures at the SAM, thus indicating an original mechanism for providing robustness to a dynamic developmental system.

Published
2013
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45. Inhibition of p-Aminobenzoate and Folate Syntheses in Plants and Apicomplexan Parasites by Natural Product Rubreserine

Author

Jeroen Van Daele, Fabrice Rébeillé, Roland Douce, Caroline Barette, Cordelia Bisanz, Djeneb Camara, Willy E. Lambert, Esmare Human, Eric Maréchal, Marie-France Cesbron-Delauw, Renaud Dumas, Dominique Van Der Straeten, Bernice Barnard, Lyn-Marie Birkholtz, Christophe P. Stove, 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 Adaptation et pathogénie des micro-organismes [Grenoble] (LAPM), Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS), Groupe Plateforme et Moyens Scientifiques et techniques communs / Centre de Criblage pour Molécules Bio-Actives (GPMS / CMBA), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratory of Toxicology, State University of Ghent, Malaria Research Group, University of Pretoria [South Africa], Laboratory of Functional Plant Biology, Ghent University [Belgium] (UGENT), Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF), Universiteit Gent = Ghent University [Belgium] (UGENT), Region Rhone-Alpes, Ghent University [BOF09-GOA-004], Agence Nationale de la Recherche PlasmoExpress grant, Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Genetics and Chemogenomics (GenChem), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Universiteit Gent = Ghent University (UGENT)

Subjects
0106 biological sciences, Plasmodium, one carbon metabolism, Arabidopsis thaliana, Physostigmine, Arabidopsis, plant, 01 natural sciences, Biochemistry, chemistry.chemical_compound, high troughput screening, 0303 health sciences, biology, Antiparasitic Agents, antifolate drugs, Recombinant Proteins, 3. Good health, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Antifolate, parasite, CHORISMATE-UTILIZING ENZYMES, DE-NOVO SYNTHESIS, PLASMODIUM-FALCIPARUM, TOXOPLASMA-GONDII, AMINODEOXYCHORISMATE SYNTHASE, ANTHRANILATE SYNTHASE, 4-AMINO-4-DEOXYCHORISMATE SYNTHASE, ISOCHORISMATE SYNTHASE, GMP SYNTHETASE, GLUCOSE ESTER, Growth inhibition, 4-Aminobenzoic Acid, Toxoplasma, Aminodeoxychorismate synthase, Plasmodium falciparum, GAT-ADCS, folate, glutamine amidotransferase aminodeoxychorismate synthase, 03 medical and health sciences, Inhibitory Concentration 50, Folic Acid, pABA biosynthesis, P aminobenzoate, parasitic diseases, 4-Aminobenzoic acid, Escherichia coli, biosynthesis inhibitor, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, 030304 developmental biology, Glutamine amidotransferase, Dose-Response Relationship, Drug, Plant Extracts, rubreserine, cofactor, Cell Biology, Antiparasitic agent, Kinetics, chemistry, Models, Chemical, Isochorismate synthase, biology.protein, Apicomplexa, metabolism, 010606 plant biology & botany, Phytotherapy
Abstract

International audience; Glutamine amidotransferase/aminodeoxychorismate synthase (GAT-ADCS) is a bifunctional enzyme involved in the synthesis of p-aminobenzoate, a central component part of folate cofactors. GAT-ADCS is found in eukaryotic organisms autonomous for folate biosynthesis, such as plants or parasites of the phylum Apicomplexa. Based on an automated screening to search for new inhibitors of folate biosynthesis, we found that rubreserine was able to inhibit the glutamine amidotransferase activity of the plant GAT-ADCS with an apparent IC(50) of about 8 μm. The growth rates of Arabidopsis thaliana, Toxoplasma gondii, and Plasmodium falciparum were inhibited by rubreserine with respective IC(50) values of 65, 20, and 1 μm. The correlation between folate biosynthesis and growth inhibition was studied with Arabidopsis and Toxoplasma. In both organisms, the folate content was decreased by 40-50% in the presence of rubreserine. In both organisms, the addition of p-aminobenzoate or 5-formyltetrahydrofolate in the external medium restored the growth for inhibitor concentrations up to the IC(50) value, indicating that, within this range of concentrations, rubreserine was specific for folate biosynthesis. Rubreserine appeared to be more efficient than sulfonamides, antifolate drugs known to inhibit the invasion and proliferation of T. gondii in human fibroblasts. Altogether, these results validate the use of the bifunctional GAT-ADCS as an efficient drug target in eukaryotic cells and indicate that the chemical structure of rubreserine presents interesting anti-parasitic (toxoplasmosis, malaria) potential.

Published
2012
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46. Crystallization and Preliminary Crystallographic Data for Acetohydroxy Acid Isomeroreductase from Spinacia oleracea

Author

Roland Douce, Claudine Cohen-Addad, Eva Pebay-Peyroula, Renaud Dumas, and Dominique Job

Subjects
chemistry.chemical_classification, Spinacia, biology, Molecular mass, Ketol-Acid Reductoisomerase, Reaction intermediate, Crystallography, X-Ray, biology.organism_classification, law.invention, Alcohol Oxidoreductases, Acetic acid, chemistry.chemical_compound, Crystallography, Enzyme, chemistry, Spinacia oleracea, Structural Biology, law, Spinach, Ammonium, Crystallization, Molecular Biology
Abstract

Acetohydroxy acid isomeroreductase (EC 1.1.1.86) is one of the enzymes involved in branched-chain amino acid biosynthesis. The enzyme from spinach (Spinacia oleracea) leaves has been crystallized using the hanging drop vapour diffusion method. The free enzyme crystallized from polymethylene glycol solutions, but these crystals were unsuitable for X-ray diffraction analysis. In the presence of NADPH, Mg(2+) and a reaction intermediate analogue (2-dimethylphosphinoyl-2-hydroxy acetic acid (Hoe 704) or N-hydroxy-N-isopropyloxamate (IpOHA)), much better crystals were obtained. Crystals grown from ammonium sulphate belong to space group P2(1) with cell dimensions a + 193.78(7) A, b = 63.69(2) A, c = 112.84(1) A and beta = 121.22(1) degrees. The molecular mass of the protein, the volume of the unit cell, and crystal density measurements indicated that the asymmetric unit contains two dimers. X-ray diffraction patterns showed measurable reflections to beyond 2.5 A.

Published
1994
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47. The many faces of aspartate kinases

Author

Renaud Dumas, Adeline Y. Robin, David Cobessi, Gilles Curien, Jean-Luc Ferrer, Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG)

Subjects
Allosteric regulation, Biophysics, review, Biochemistry, MESH: Allosteric Regulation, 03 medical and health sciences, MESH: Protein Structure, Tertiary, ACT domain, Allosteric Regulation, regulatory domain, MESH: Aspartate Kinase, Aspartate kinase, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, structure, Aspartate Kinase, Binding site, effector binding site, Molecular Biology, 030304 developmental biology, 0303 health sciences, allostery, Binding Sites, biology, MESH: Kinetics, Kinase, Effector, 030302 biochemistry & molecular biology, biosynthetic pathway, Cell biology, Protein Structure, Tertiary, enzyme, Kinetics, Allosteric enzyme, MESH: Binding Sites, biology.protein, metabolism, Function (biology)
Abstract

International audience; Based on recent X-ray structures and biochemical characterizations of aspartate kinases from different species, we show in this review how various organizations of a regulatory domain have contributed to the different mechanisms of control observed in aspartate kinases allowing simple to complex allosteric controls in branched pathways. The aim of this review is to show the relationships between domain organization, effector binding sites, mechanism of inhibition and regulatory function of an allosteric enzyme in a biosynthetic pathway.

Published
2011
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48. The synthesis of pABA: Coupling between the glutamine amidotransferase and aminodeoxychorismate synthase domains of the bifunctional aminodeoxychorismate synthase from Arabidopsis thaliana

Author

Djeneb Camara, Fabrice Rébeillé, Céline Richefeu-Contesto, Bernadette Gambonnet, Renaud Dumas, 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), Région Rhône-Alpes (Cluster 9, 'Plantacter'), 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
Aminodeoxychorismate synthase, Arabidopsis thaliana, Stereochemistry, Biophysics, Arabidopsis, catalytic activity, plant, glutamine amidotransferase, 010402 general chemistry, folate, chorismate, 01 natural sciences, Biochemistry, 03 medical and health sciences, chemistry.chemical_compound, Folic Acid, Biosynthesis, ADC synthase, P aminobenzoate, 4-Aminobenzoic acid, aminodeoxychorismate synthase, Phosphofructokinase 2, Carbon-Nitrogen Ligases, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, Transaminases, 030304 developmental biology, Glutamine amidotransferase, 0303 health sciences, ATP synthase, biology, vitamin, Recombinant Proteins, p-aminobenzoate, 0104 chemical sciences, Protein Structure, Tertiary, Glutamine, body regions, enzyme, chemistry, kinetics, biology.protein, biosynthesis, 4-Aminobenzoic Acid, Sequence Analysis
Abstract

International audience; Aminodeoxychorismate (ADC) synthase in plants is a bifunctional enzyme containing glutamine amidotransferase (GAT) and ADC synthase (ADCS) domains. The GAT domain releases NH(3) from glutamine and the ADCS domain uses NH(3) to aminate chorismate. This enzyme is involved in folate (vitamin B9) biosynthesis. We produced a stable recombinant GAT-ADCS from Arabidopsis. Its kinetic properties were characterized, and activities and coupling of the two domains assessed. Both domains could operate independently, but not at their optimal capacities. When coupled, the activity of one domain modified the catalytic properties of the other. The GAT activity increased in the presence of chorismate, an activation process that probably involved conformational changes. The ADCS catalytic efficiency was 10(4) fold higher with glutamine than with NH(4)Cl, indicating that NH(3) released from glutamine and used for ADC synthesis did not equilibrate with the external medium. We observed that the GAT activity was always higher than that of ADCS, the excess of NH(3) being released in the external medium. In addition, we observed that ADC accumulation retro-inhibited ADCS activity. Altogether, these results indicate that channeling of NH(3) between the two domains and/or amination of chorismate are the limiting step of the whole process, and that ADC cannot accumulate.

Published
2011
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49. Branched-chain-amino-acid biosynthesis in plants: molecular cloning and characterization of the gene encoding acetohydroxy acid isomeroreductase (ketol-acid reductoisomerase) from Arabidopsis thaliana (thale cress)

Author

Roland Douce, Renaud Dumas, Gilles Curien, and R T DeRose

Subjects
RNA Splicing, Ketol-Acid Reductoisomerase, Molecular Sequence Data, Restriction Mapping, Branched-chain amino acid, Arabidopsis, Molecular cloning, Biology, Genes, Plant, Biochemistry, chemistry.chemical_compound, Complementary DNA, Coding region, Arabidopsis thaliana, Amino Acid Sequence, Cloning, Molecular, Molecular Biology, Gene, Genetics, Base Sequence, Nucleic acid sequence, Hydrogen Bonding, Cell Biology, biology.organism_classification, Introns, Alcohol Oxidoreductases, genomic DNA, Oligodeoxyribonucleotides, chemistry, Nucleic Acid Conformation, Sequence Alignment, Research Article
Abstract

Towards the goal of gaining a better understanding of the molecular mechanisms controlling branched-chain-amino-acid biosynthesis in plants, we have isolated, sequenced and characterized a gene encoding acetohydroxy acid isomero-reductase (ketol-acid reductoisomerase) from Arabidopsis thaliana (thale cress). Comparison between the acetohydroxy acid isomeroreductase cDNA and the genomic sequence has allowed us to determine the exon structure of the coding region. The isolated acetohydroxy acid isomeroreductase gene is distributed over approx. 4.5 kbp and contains nine introns (79-347 bp). The transcriptional start site was found to be 52 bp upstream of the translational initiation site. Southern-blot analysis of A. thaliana genomic DNA shows that the acetohydroxy acid isomeroreductase is encoded by a single-copy gene.

Published
1993
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50. Nucleotide sequence and characterization of a cDNA encoding the acetohydroxy acid isomeroreductase from Arabidopsis thaliana

Author

Renaud Dumas, Gilles Curien, and Roland Douce

Subjects
Molecular Sequence Data, Arabidopsis, Sequence alignment, Plant Science, Biology, Genes, Plant, Conserved sequence, Transit Peptide, Complementary DNA, Consensus Sequence, Genetics, Amino Acid Sequence, Peptide sequence, Plant Proteins, chemistry.chemical_classification, Base Sequence, 2-Acetolactate Mutase, Protein primary structure, Nucleic acid sequence, food and beverages, General Medicine, Amino acid, chemistry, Biochemistry, Sequence Alignment, Agronomy and Crop Science
Abstract

The primary structure of acetohydroxy acid isomeroreductase from Arabidopsis thaliana was deduced from two overlapping cDNA. The full-length cDNA sequence predicts an amino acid sequence for the protein precursor of 591 residues including a putative transit peptide of 67 amino acids. Comparison of the A. thaliana and spinach acetohydroxy acid isomeroreductases reveals that the sequences are conserved in the mature protein regions, but divergent in the transit peptides and around their putative processing site.

Published
1993
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