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Jean Duhamel, 4 april 2019, 11am, I... Internal CBMN seminar 7 March 2019... Sandrine VIllette, 7 february 2019,... Internal CBMN Seminar : newcomers ... Internal CBMN Seminar: A. Simon, 6 ... Marisela Velez, 28 november 2018, 2... Marc Bramkamp, 16 november 2018, 2p... Kazushi Kinbara, 12 november 2018, ... Internal CBMN Seminar: J. Lang, 8 N... Internal CBMN Seminars: A. Baudin, ... Plateforme production de protéine... Patrick Trouillas, 21 June 2018, 2p... Lucie Khemtemourian, 21 June 2018, ... Jonathan Faherty, 7th June, 11 AM, ... Yann Fichou, 3 May 2018, 2pm, IECB Internal CBMN seminars: A. Ciaccafa... Alain Roussel, 29/03/2018, 4 pm, EN... Simon Poly, 22 march 2018, 2pm, ENS... J.Crassous, M. Sallé & A. Martinez... O.Reinaud & V. Artero, 7/12/2017, 1... R. Dos santos Morais, 30/11/2017, 4... Christian Griesinger, November 24th... Takahiro Muraoka, 16/11/2017, 11am,... Vladimir Torbeev, 9/11/2017, 2pm, E... M. D. Smith & T.Constantieux, 3/11/... James Nowick, 15 september 2017, 11... Emmanuelle Thinon, 28 June 2017, 11... Caroline Tokarski, 15 June 2017, 4p... Christian Salesse, 8 June 2017, 4pm... Patrice Rey, 01 June 2017, 2h30 pm,... Christina Sizun, 18 May 2017, 4 pm,... Marisela Velez, 5 May 2017, 11am, I... Iqbal Choudhary, 27 April 2017, 4 p... Vincent Aucagne, 21 April 2017, 11 ... Sophie Zinn-Justin, 14 April 2017, ... Cécile Feuillie, 16 February 2017,... Félix M. Goñi, 8 december 2016, 4... Félix M. Goñi, 17 november 2016, ... Carl Creutz, 20 october 2016, 4 pm,... Diego Romero, 30 september 2016, 11... A. Ciaccafava, 8 september 2016, 14... A. Ramamoorthy, 16 June 2016, 16h,... Alexandre de Brevern, 2 june 2016, ... Isabelle Landrieu, 26 May, 16h, ENS... Frances Sepavoric, 12 May, 16h, ENS... Thomas Pradeu, 7 April 2016, 9h, EN... Françoise Argoul, 3 march 2016, 1... Corinne Loutelier-Bourhis, 16/12/20... Aristotelis XENAKIS, 3 december 201... Fabian Kiessling, 26 november 2015,... Michaël Molinari, 20 november 2015... Dipankar Das Sarma, 28 October 2015... Olivier Donard, 22 october 2015, 16... E. Morvan & A.Grelard, 17/12/2015, ... Christophe Cullin, 10 september 201... Brigitte Lindet, 18 June 2015, 16h,... Marion Decossas, 4 June 2015, 16h, ... Pascale Schellenberger, 21 Mai 2015... F. Leal-Calderon, 7 May 2015, 16h, ... Ibrahim Abdulhalim, 9 April 2015, 1... Manon Carré, 26 March 2015, 14h, ... C. Bure & JM Schmitter, 19 March 20... T. Ogata & H. Ihara, 17 March 2015,... Banafshe Larijani, 12 March 2015, 1...
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Jiarong Zhang thesis defense, Friday december 15th, 2 pm, ENSTBB amphitheater



The thesis defense will take place, Friday december 15th, at 2 pm, in the ENSTBB amphitheater., 146 rue Léo Saignat,  Bordeaux.

 

Etude de l'anthocyanidine synthase de Vitis Vinifera: substrats polyphénoliques et mécanismes réactionnels.

Abstract

 

Recombinant anthocyanidin synthase from Vitis vinifera (VvANS) has been expressed in E. coli, and purified by nickel affinity chromatography.

 

The production and purification of the iron-loaded enzyme has been developed in order to avoid uncontrolled non-enzymatic oxidation reactions in the presence of Fe(II) salt. A stable VvANS-Fe(II) complex is formed in the presence of 2-oxoglutarate and ascorbate, and this complex is catalytically active at ambient PO2 in the absence of Fe(II) salt.

 

The transformation of (+)-catechin by VvANS has been studied with and without ascorbate, by using either the VvANS-Fe(II) complex or the holoenzyme co-incubated with ferrous sulfate, to investigate the role of ascorbate. No enzyme activity has been observed in the absence of ascorbate, which means that it is an essential enzyme cofactor. A covalent adduct ascorbate-cyanidin is produced in vitro, but only in the absence of glutathione (GSH), another major nucleophilic and reducing agent.

The two stereoisomers of leucocyanidin (3,4-cis et 3,4-trans flavan-diols) which were expected to behave as substrates of Vv-ANS,are not commercial and were synthesizedby reduction of dihydroquercetin by NaBH4, and characterized by proton NMR. The analysis of the two stereoisomers by means of tandem mass spectrometry (MS/MS) shows that their fragmentation pathways are distinct and may be used to distinguish them during their production. The two stereoisomers are stable in frozen aqueous medium at -20°C.

Twelve flavonoids of four distinct families (flavanones, dihydroflavonols, flavan-3-ols et flavan-3,4-diols) were tested as potential substrates of VvANS. All enzymatic products were purified by means of reverse-phase HPLC and characterized by MS/MS, with the following results:

1) Only dihydroflavonols of (2R,3R) configuration are accepted as substrates by VvANS, the activity decreasing with the number of hydroxyl groups of ring B.

2) Only flavan-3-ols or flavan-3,4-diols of (2R,3S) configuration having either a catechol or three vicinal phenolic OH on ring B are accepted as substrates.

3) Naringenin is not substrate of Vv-ANS, most likely because a C3 hydroxyl group is missing.

4) 3,4-cis-leucocyanidin (natural stereoisomer) is transformed into two major products, quercetin and flavan-3,3,4-triol (gem-diol at C3) instead of cyanidin. 3,4-trans-leucocyanidin is also a substrate of VvANS, but it is transformed into five products, (+)-DHQ, (-)-epiDHQ, cyanidin, quercetin and flavan-3,3,4-triol (gem-diol at C3, stereoisomer of the previous one). A flavan-3,3-gem-diol intermediate derived from (+)-catechin has also been observed by real-time monitoring of the transformation of (+)-catechin by MS, as well as a flav-2-en-3-ol intermediate. All these results are compatible with the hypothesis that the first catalytic step of ANS is a C3-hydroxylation that systematically produces a gem-diol.

Glutathione GSH is a powerful nucleophilic and reducing agent as well as a free radical scavenger, which is abundant in grape berries. We therefore studied its effect on VvANS activity with all identified substrates. GSH has no effect on the transformation of dihydroflavonols and flavan-3,4-diols, but it considerably modifies the transformation pattern of (+)-catechin and (+)-gallocatechin. In the presence of (+)-catechin and GSH, we observe two major products, cyanidin and a cyanidin-glutathione thioether, with production yields which are much higher than in the absence of GSH. Moreover, the ascorbate-cyanidin covalent adduct and the (+)-catechin dimer that had been obtained in the absence of GSH have disappeared. Our data suggest that the cyanidin-glutathione adduct is a C4-thioether which is in equilibrium between the two keto-enolic tautomeric forms at C3, and decomposes into cyanidin and GSH. In the presence of (+)-gallocatechin, a similar delphinidin-glutathione thioether adduct is also observed. In order to test the possible specificity of GSH as a cofactor, three other mercaptans (thiomalate, cysteine and cysteamine) were tested, and no similar product was observed, which suggests that GSH is a specific ligand, and might be a coenzyme of VvANS.

 

Our results suggest that anthocyanidins could be produced in vivo from a flavan-3ol substrate (catechin or gallocatechin) via a glutathione thioether intermediate, whereas the natural 3,4-cis stereoisomer of leucocyanidin is not transformed into cyanidin by VvANS.

 

Supervision: Jean Chaudière.

Team : Structure et Activité des Macromolécules Biologiques.

 

Jury:

 

Jalila Simmaan, Chargée de Recherche, CNRS, Université d'Aix-Marseille, Rapporteur

Véronique Cheynier, Directrice de Recherche, INRA, Université de Montpellier, Rapporteur

Sandrine Boschi, Professeur, Université de Lorraine, Examinateur

Olivier Dangles, Professeur, Université d'Avignon, Examinateur

Jean-Marie Schmitter, Professeur, Université de Bordeaux, Examinateur

Claudine Trossat-Magnin, Maître de Conférence, Université de Bordeaux, Invitée.

 

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