La chitotriosidase (CHIT1) est une chitinase humaine appartenant à la famille glycosyl hydrolase 18 (GH18) qui hydrolyse la chitine. CHIT1 présente plusieurs caractéristiques enzymatiques conservées dans la famille GH18 qui ne sont pas complètement comprises. Pour renforcer nos connaissances sur le mécanisme catalytique de CHIT1 et de la famille GH18, j'ai amélioré la résolution des structures obtenues par diffraction de rayon-X du domaine catalytique de CHIT1. Ces structures correspondent à la forme apo de CHIT1, pseudo-apo ainsi qu’en complexe avec la chitobiose ont été obtenues à des résolutions comprises entre 0.95Å et 1.10Å. Mes résultats m’ont permis de proposer un nouveau mécanisme d’hydrolyse des chaines chito-oligosaccharidiques. En outre, grâce à une nouvelle stratégie de cristallisation, la première structure cristalline de CHIT1 complète a pu être obtenue à une résolution de 1.95Å. Mon étude donne de nouvelles perspectives sur le mode d'action de CHIT1 et les caractéristiques enzymatiques conservées dans la famille GH18. / Chitotriosidase (CHIT1) is a human chitinase belonging to the glycosyl hydrolase family 18 (GH18), a highly conserved enzyme family. GH18 enzymes hydrolyze chitin, a N-acetyl glucosamine polymer. CHIT1 is characterized by many enzymatic features that are conserved in GH18 and not completely understood. To increase our knowledge on the catalytic mechanism in CHIT1 and GH18 family, I improved the X-ray resolution crystal structure of CHIT1 catalytic domain in apo and pseudo apo forms as well as in complex with a synthetic substrate to a resolution range between 0.95Å and at 1.10Å. My results allow me to suggest a new mechanism for chito-oligosaccharide chains hydrolysis. Moreover, thanks to a new a crystallogenesis strategy, I obtained the first crystal structure of full length CHIT1 at 1.95Å resolution. My study presents many structural and mechanistic aspects of CHIT1 which gives new insights onto its mode of action and shed light into the conserved enzymatic features in GH18 chitinase family.

All cells and extracellular matrices of prokaryotes and eukaryotes are made up of glycans, the carbohydrate macromolecules that play a predominant role in cell-to-cell interaction, protection, stabilization, and barrier functions. Glycans are also central to human microbiome-host interactions where bacterial glycans are recognized by innate immune signaling pathways, and host mucins are a major nutrient source for various gut bacteria. Many microorganisms encode glycoside hydrolases (GHs) to utilize the available host cell surface glycans as a nutrient source and to modulate host protein function. The GHs are divided into families having conserved linkage specificity within each family and individual family members can be specific for dramatically divergent macromolecular substrates. In general, within a given GH family very few members have been biochemically characterized and the substrate specificity is poorly understood. GH genes are abundant in the human gut microbiome and culture-enriched metagenomics identified more than 10,000 distinct bacterial GH genes in an individual. The focus of this thesis is endo-β-N-acetylglucosaminidases (ENGases) encoded by GH18 and GH85 families. Bioinformatic analysis shows that the predicted proteins within each of these GH families fell into separate clusters in the Sequence Similarity Networks of each family. The hypothesis of this project is that human microbiome-encoded ENGases from the same GH family differ in their substrate specificities and within the SSN network of the same GH family, enzymes with similar substrate specificity may fall in the same cluster. In this work, I established conditions for overexpression of GH18 and GH85 proteins and investigated the activity of these enzymes on various substrates. / Thesis / Master of Science (MSc) / All the cell surfaces of animals, plants, and microbes are coated with sugars, also known as glycans. These sugars on the cell surface act as a barrier and protect them from the external environment. Glycans on the cells of both microbes and humans are essential for basic interactions between them. Many bacteria produce enzymes such as glycoside hydrolases to obtain nutrients from dietary sugars and alter the sugars on host proteins. There are various families of these enzymes, and they act on specific sugars and cleavage sites. The substrate specificities and characterization of these enzymes from most bacteria found in the human microbiome have not been studied in detail. My work focuses on developing standard enzyme assays for determining specific substrate specificities. This tool can be used to reshape glycans and understand their role in cell processes.