PhD defence will take place on March 31st at 2 PM in room 401 of TBI.
Thesis is entitled: Structure–Dynamics Relationships of Two Enzymes Involved in Polysaccharide Biosynthesis and Functionalization.
The presentation will be in English.
Jury composition:
- Ms. Mirjam CZJZEK, Reviewer, CNRS, Roscoff
- Mr. Marcelo GUERIN, Reviewer, CSIC, Barcelona
- Ms. Célia PLISSON CHASTANG, Examiner, CNRS, Toulouse
- Mr. Daniele DE SANCTIS, Examiner, ESRF, Grenoble
- Ms. Magali Remaud-Siméon, Invited member, INSA, Toulouse
- Ms. Claire MOULIS, Thesis Director, INSA, Toulouse
- Mr. Gianluca CIOCI, Thesis Co-Director, INRAE, Toulouse
Abstract:
This thesis aimed to elucidate the structure-function-dynamics relationships of two large, multidomain carbohydrate-active enzymes involved in glucan synthesis and functionalization: the plant glucan water dikinase StGWD1 and a glycoside phosphorylase from the GH161 family recently isolated from a human gut metagenome. To address the lack of structural data available for these enzyme families, an integrative structural biology strategy combining cryo-electron microscopy, X-ray crystallography, SAXS, molecular modelling, and biochemical and interaction studies was implemented. Structural and functional analyses of StGWD1 revealed its multimodular architecture, including the organization and distinct roles of its CBM45 modules, and provided the first high-resolution structural insights into its catalytic core, supporting a catalytic mechanism involving domain flexibility and histidine-domain swiveling during phosphate transfer. Complementary studies on CBM45 modules established the first experimental structures of CBM45 family members and clarified their contribution to substrate targeting and positioning. Cryo-EM investigations further enabled the reconstruction of the catalytic core of the full-length enzyme and refined current mechanistic models of glucan phosphorylation. In parallel, this work provided the first experimental structural characterization of a GH161 β-1,3-glucan phosphorylase, including high-resolution cryo-EM structures captured in multiple catalytic states. These results demonstrated the central role of loop dynamics, particularly gate loop motion, and highlighted the importance of inter-subunit communication in catalysis. Altogether, this work advances the molecular understanding of large glucan-active enzymes and demonstrates the power of cryo-EM to capture enzyme conformational landscapes. These findings open new perspectives for rational or semi-rational engineering of these CAZymes for the tailored enzymatic synthesis and functionalization of carbohydrate-based biomolecules.
