title: Insights into ascorbate regeneration in plants: investigating the redox and structural properties of dehydroascorbate reductases from Populus trichocarpa.
abstract: Dehydroascorbate reductases (DHARs), enzymes belonging to the GST superfamily, catalyse the GSH-dependent reduction of dehydroascorbate into ascorbate in plants. By maintaining a reduced ascorbate pool, they notably participate to H2O2 detoxification catalysed by ascorbate peroxidases (APXs). Despite this central role, the catalytic mechanism used by DHARs is still not well understood and there is no supportive 3D structure. In this context, we have performed a thorough biochemical and structural analysis of the three poplar DHARs and coupled this to the analysis of their transcript expression patterns and subcellular localizations. The transcripts for these genes are mainly detected in reproductive and green organs and the corresponding proteins are expressed in plastids, in the cytosol and in the nucleus, but not in mitochondria and peroxisomes where ascorbate regeneration is obviously necessary. Comparing the kinetic properties and the sensitivity to GSSG-mediated oxidation of DHAR2 and DHAR3A, exhibiting 1 or 3 cysteinyl residues respectively, we observed that the presence of additional cysteines in DHAR3A modifies the regeneration mechanism of the catalytic cysteine by forming different redox states. Finally, from the 3D structure of DHAR3A solved by NMR, we were able to map the residues important for the binding of both substrates (GSH and DHA), showing that DHAR active site is very selective for DHA recognition and providing further insights into the catalytic mechanism and the roles of the additional cysteines found in some DHARs.
authors.affiliation: Université de Lorraine, UMR 1136 Interactions Arbres/Microorganismes, Faculté des Sciences et Technologies, 54506 Vandœuvre-lès-Nancy, France INRA, UMR 1136 Interactions Arbres/Microorganismes, Centre INRA Nancy Lorraine, 54280 Champenoux, France.
authors.forename: Pierre-Alexandre
authors.initials: PA
authors.lastname: Lallement
authors.affiliation: Université de Lorraine, UMR 1136 Interactions Arbres/Microorganismes, Faculté des Sciences et Technologies, 54506 Vandœuvre-lès-Nancy, France INRA, UMR 1136 Interactions Arbres/Microorganismes, Centre INRA Nancy Lorraine, 54280 Champenoux, France.
authors.forename: Thomas
authors.initials: T
authors.lastname: Roret
authors.affiliation: Université de Lorraine, CRM2, UMR 7036, 54506 Vandœuvre-lès-Nancy, France CNRS, CRM2, UMR 7036, 54506 Vandœuvre-lès-Nancy, France.
authors.forename: Pascale
authors.initials: P
authors.lastname: Tsan
authors.affiliation: Institut de Biologie Moléculaire des Plantes, CNRS-UPR 2357, 67084 Strasbourg, France.
authors.forename: José M
authors.initials: JM
authors.lastname: Gualberto
authors.affiliation: Université de Lorraine, UMR 1136 Interactions Arbres/Microorganismes, Faculté des Sciences et Technologies, 54506 Vandœuvre-lès-Nancy, France INRA, UMR 1136 Interactions Arbres/Microorganismes, Centre INRA Nancy Lorraine, 54280 Champenoux, France.
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Girardet
authors.affiliation: Université de Lorraine, CRM2, UMR 7036, 54506 Vandœuvre-lès-Nancy, France CNRS, CRM2, UMR 7036, 54506 Vandœuvre-lès-Nancy, France.
authors.forename: Claude
authors.initials: C
authors.lastname: Didierjean
authors.affiliation: Université de Lorraine, UMR 1136 Interactions Arbres/Microorganismes, Faculté des Sciences et Technologies, 54506 Vandœuvre-lès-Nancy, France INRA, UMR 1136 Interactions Arbres/Microorganismes, Centre INRA Nancy Lorraine, 54280 Champenoux, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Rouhier
authors.affiliation: Université de Lorraine, UMR 1136 Interactions Arbres/Microorganismes, Faculté des Sciences et Technologies, 54506 Vandœuvre-lès-Nancy, France INRA, UMR 1136 Interactions Arbres/Microorganismes, Centre INRA Nancy Lorraine, 54280 Champenoux, France arnaud.hecker@univ-lorraine.fr.
authors.forename: Arnaud
authors.initials: A
authors.lastname: Hecker
mesh_terms: D001205:Ascorbic Acid / Q000378:metabolism*; D001665:Binding Sites; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D018506:Gene Expression Regulation, Plant / Q000502:physiology*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D032107:Populus / Q000378:metabolism*; D011487:Protein Conformation; D014026:Nicotiana
chemical_list: D010940:Plant Proteins; D010088:Oxidoreductases; C020666:glutathione dehydrogenase (ascorbate); D001205:Ascorbic Acid
keywords: Populus trichocarpa; ascorbate recycling; catalytic cysteine residue; dehydroascorbate reductases; glutathione; nuclear magnetic resonance