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Functional and Structural Insights Into Glycoside Hydrolase Family 130 Enzymes: Implications in Carbohydrate Foraging by Human Gut Bacteria

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Functional and Structural Insights Into Glycoside Hydrolase Family 130 Enzymes: Implications in Carbohydrate Foraging by Human Gut Bacteria TTHHÈÈSSEE En vue de l'obtention du DOCTORAT DE L’UNIVERSITÉ DE TOULOUSE Délivré par Discipline ou spécialité : Ingénieries Moléculaires et Enzymatiques Présentée et soutenue par Le Titre : JURY Ecole doctorale : Sciences Ecologiques, Vétérinaires, Agronomiques et Bioingénieries (SEVAB) Unité de recherche : LISBP, UMR CNRS 5504, UMR INRA 792, INSA Toulouse Directeur(s) de Thèse : Gabrielle POTOCKI-VÉRONÈSE, Directrice de Recherche INRA Elisabeth LAVILLE, Chargée de Recherche INRA THÈSE En vue de l’obtention du DOCTORAT de L’INSTITUT NATIONAL DES SCIENCES APPLIQUÉES DE TOULOUSE Présentée et soutenue publiquement le mardi 28 avril 2015 Spécialité : Sciences Ecologiques, Vétérinaires, Agronomiques et Bioingénieries Filière : Ingénieries Microbienne et Enzymatique Par Simon LADEVÈZE Functional and Structural insights into glycoside hydrolase family 130 enzymes: implications in carbohydrate foraging by human gut bacteria Directrices de thèse: Mme Gabrielle POTOCKI-VÉRONÈSE, Directrice de Recherche INRA Mme Elisabeth LAVILLE, Chargée de Recherche INRA 1 2 NOM: LADEVÈZE PRÉNOM: Simon TITRE: Apports Fonctionnels et Structuraux à la famille des glycoside hydrolase 130: implications dans la dégradation des glycanes par les bactéries de l'intestin humain. SPÉCIALITÉ: Sciences Ecologiques, Vétérinaires, Agronomiques et Bioingénieries FILIÈRE: Ingénieries Moléculaires et Enzymatiques ANNÉE: 2015 LIEU: INSA, TOULOUSE DIRECTRICES DE THÈSE: Mmes Gabrielle POTOCKI-VÉRONÈSE et Elisabeth LAVILLE RÉSUMÉ: Les relations entre bactéries intestinales, aliments et hôte jouent un rôle crucial dans le maintien de la santé humaine. La caractérisation fonctionnelle d’Uhgb_MP, une enzyme de la famille 130 des glycoside hydrolases découverte par métagénomique fonctionnelle, a révélé une nouvelle fonction de dégradation par phosphorolyse des polysaccharides de la paroi végétale et des glycanes de l'hôte tapissant l'épithélium intestinal. Les déterminants moléculaires de la spécificité d’Uhgb_MP vis-à-vis des mannosides ont été identifiés grâce à la résolution de sa structure cristallographique, sous forme apo et en complexe avec ses ligands. Un nouveau procédé de synthèse par phosphorolyse inverse d'oligosaccharides mannosylés à haute valeur ajoutée, a aussi été développé. Enfin, la caractérisation fonctionnelle de la protéine BACOVA_03624 issue de Bacteroides ovatus ATCC 8483, une bactérie intestinale hautement prévalente, a révélé que la famille GH130 comprend à la fois des glycoside-hydrolases et des glycoside-phosphorylases capables de dégrader les mannosides et les galactosides, et de les synthétiser par phosphorolyse inverse et/ou transglycosylation. L’ensemble de ces résultats, ainsi que l’identification d’inhibiteurs des enzymes de la famille GH130, ouvrent de nouvelles perspectives pour l'étude et le contrôle des interactions microbiote-hôte. MOTS-CLES: Microbiote intestinal, Mannanes, N-Glycans, Glycoside-phosphorylases, GH130. ECOLE DOCTORALE: Sciences Ecologiques, Vétérinaires, Agronomiques, et Bioingénieries (SEVAB) LABORATOIRE: Laboratoire d’Ingénierie des Systèmes Biologiques et Procédés (LISBP). UMR CNRS 5504, UMR INRA 792, INSA Toulouse. 3 SURNAME: LADEVÈZE FIRST NAME: Simon TITLE: Functional and structural insights into glycoside hydrolase family 130 enzymes: implications in carbohydrate foraging by human gut bacteria. SPECIALTY: Ecological, Veterinary, Agronomic Sciences and Bioingeneering FIELD: Enzymatic and Microbial Engeneering YEAR: 2015 PLACE: INSA, TOULOUSE THESIS DIRECTORS: Mrs Gabrielle POTOCKI-VÉRONÈSE and Elisabeth LAVILLE SUMMARY: The interplay between gut bacteria, food and host play a key role in human health. The functional characterization of Uhgb_MP, an enzyme belonging to the family 130 of glycoside hydrolases, discovered by functional metagenomics, revealed novel functions of plant cell wall polysaccharide and host glycan degradation by phosphorolysis. The molecular determinants of Uhgb_MP specificity towards mannosides were identified by solving its crystal structure, in apo form and in complex with its ligands. A new process of high added value mannosylated oligosaccharide synthesis by reverse-phosphorolysis was also developed. Finally, the functional characterization of the BACOVA_03624 protein from Bacteroides ovatus ATCC 8483, a highly prevalent gut bacterium, revealed that GH130 family both contains glycoside phosphorylases and glycoside hydrolases, which are able to degrade mannosides and galactosides, and to synthesize them by reverse-phosphorolysis and/or transglycosylation. All these results, together with the identification of GH130 enzyme inhibitors, open new perspectives for studying, and potentially also for controlling, interactions between host and gut microbes. KEYWORDS: Gut microbiota, Mannans, N-Glycans, Glycoside-phosphorylases, GH130. DOCTORATE SCHOOL: Ecological, Veterinary, Agronomic Sciences and Bioingeneering (SEVAB) LABORATORY: Laboratory of Biosystems and Chemical Engeneering (LISBP). UMR CNRS 5504, UMR INRA 792, INSA Toulouse. 4 Remerciements Ce manuscript vient conclure trois années et demie de thèse. Trois années et demie au cours desquelles beaucoup de travail a été fait. Pour autant, même si la bonne fortune à contribué à ce résultat, rien n’aurait été possible sans le concours de nombreuses personnes. Car une thèse, ce sont aussi des gens, des rencontres, une riche expérience de la vie. Que tous soient ici remerciés pour leur participation, matérielle, morale, de circonstance, ou plus régulière. Je tiens en premier lieu à remercier les membres de mon jury, Jean-Guy Berrin, Gideon Davies, Bernard Henrissat, Marion Leclerc, Magali Remaud-Siméon et Samuel Tranier pour me faire l’honneur de juger mon travail. Gabrielle, je souhaite te dire un immense merci. Immense, tout d’abord, pour avoir fait confiance, il y à près de quatre ans, à ce petit jeune, ce débarqué de la fac, cet inconnu au bataillon, à l’enthousiasme débordant et qui ne jurait à l’époque que par des cristaux en d’obscurs termes cristallographiques. Pour ton encadrement, ensuite, lors de ces années merveilleuses passées à travailler ensemble sur ces coquines enzymes, pour ta grande patience, mais aussi et surtout pour ta présence et ta grande honnêteté, aux antipodes de la langue de bois, qualités essentielles qui n’auraient pu me faire rêver de meilleures conditions pour l’accomplissement de cette thèse. Ton savoir, ta pertinence et ta rigueur ont été pour moi tout au long de ces années à la fois source d’admiration et d’inspiration. Pour la qualité de notre relation enfin, pour la joie partagée lors des bons moments, ainsi que pour le soutien lors des moins bons. Et finalement pour ton endurance, pour ce record inégalé d’avoir supporté deux thésards Gersois, venant du même petit village qui plus est… Elisabeth, merci également pour ta présence tout au long de ces années, pour tes conseils, et pour ton aide dans les analyses génomiques et métagénomiques en particulier. Ta gentillesse et ton humour m’ont toujours ravi, tout comme tes expressions cultes qui sont venues enrichir mon répertoire. Magali, merci de m’avoir si bien accueilli au sein de cette équipe, vivante, dynamique et chaleureuse. Ta pertinence, ta rigueur, ainsi que l’honnêteté et l’humanisme dont tu fais preuve afin de la faire vivre ont toujours ont toujours suscité en moi le respect. Je tiens ensuite à remercier l’ensemble de l’équipe Catalyse et Ingénierie Moléculaires et Enzymatiques, pour le dynamisme qui y règne, pour cette ambiance, décontractée et libre qui fut un excellent facteur d’intégration dès mon arrivée. Beaucoup de monde est présent dans cette équipe, ce qui participe à sa vivacité ; néanmoins il est fort dommage que la situation actuelle du monde nous interdise de partager davantage de moments ensembles. Tout d’abord merci à mes collègues de bureau, à ma voisine Marion qui a supporté mes bavardages, même s’il est vrai que mon silence peut s’acheter avec du 5 chocolat. Les grands délires que nous avons eus avec Yannick, Marlène, Etienne et Florent vont beaucoup me manquer, tout comme les « craquages » systématiques du vendredi après-midi, ou un peu plus tôt. Yannick, éternel fêtard et mélomane, ton soutien lors des soirées de manip fut une aide précieuse, occasion de riches découvertes musicales et d’intenses discussions philosophico-politiques. Thomas, heureusement que tu étais là afin de parfois arriver à trouver la motivation pour aller transpirer à Movida. Merci beaucoup à Morgane pour tes éclats de rire, et merci Marina, pour tous ces moments que nous avons partagés. Muchas gracias a Alvaro, a los Pablos y a Agustina, con quién pasé muchos buenos momentos, fue para mí una única oportunidad de practicar el idioma de Cervantes. Sin embargo, todavía no he decidido si prefiero el Real o el Barça, sobre todo cuando estamos probando vinos franceses. No olvidaré tampoco este concierto de massive attack en el castillo de Carcassona o la música trance del bikini. Alvaro, mi amigo, es así que entiendo la Unión Europea. Enfin merci à Marc pour ta gentillesse et ton humour, à Florence pour ta bonté, ainsi qu’à la douce Emilie qui cuisine divinement bien. Merci Laurence et merci Angeline, qui m’avez assisté à la paillasse et démultiplié mon travail, sans vous un tel projet n’aurait pu aboutir à une si belle conclusion et n’aurait jamais amassé des résultats dans de telles proportions. Merci à Nelly et Laurence pour leur aide sur le plateau HPLC, vous m’avez tiré de pas mal de galères et m’avez tiré d’un grand nombre de prises de tête avec chroméléon.
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