Avis de Soutenance
Monsieur Thibaud LAFFARGUE
Ingénieries microbienne et enzymatique
Soutiendra publiquement ses travaux de thèse intitulés
Voies enzymatiques pour la phosphorylation d’alpha-glucanes
dirigés par Madame Magali REMAUD-SIMEON et Madame Claire MOULIS
Soutenance prévue le mercredi 31 mai 2023 à 14h00
Lieu : Bâtiment 12, 135 avenue de Rangueil 31077 Toulouse CEDEX 4
Salle : Amphithéâtre Sophie Germain
Composition du jury
- Mme Magali REMAUD-SIMEON – Institut National des Sciences Appliquées de Toulouse Directrice de thèse
- M. Pedro COUTINHO – Université Aix-Marseille Rapporteur
- Mme Claire MOULIS – Institut National des Sciences Appliquées de Toulouse Co-directrice de thèse
- Mme Leila LO LEGGIO – University of Copenhagen Rapporteure
- M. Gianluca CIOCI – Institut National des Sciences Appliquées de Toulouse Examinateur
- M. Christophe COLLEONI – Université de Lille Examinateur
Summary: Polysaccharides are of growing interest in our modern society, as they are biobased and biodegradable. However, in their natural form, they usually present less attractive properties than petro-sourced polymers. The introduction of new functions on their backbones makes it possible to diversify their structures and physico-chemical properties, giving them a competitive advantage over synthetic polymers, and also opening new types of applications. Phosphorylation is a particular example of functionalization process that leads to an interesting diversity of molecules. To date, like most of functionalization processes, it is carried out through chemical means, which involves the use of toxic solvents and high energy costs, and also often leads to polysaccharide chain degradation. One way to overcome these drawbacks is to use enzymatic catalysts to perform the reaction, as enzymes work mostly in water and under mild conditions. In this work, we focused on the phosphorylation of a particular class of bacterial α-glucans, produced by GH13 and GH70 α-transglucosylases. These enzymes polymerize the glucosyl units of sucrose in high molar mass glucans with various types of osidic linkages, depending on the enzyme specificity. To explore enzymatic routes for the phosphorylation of these glucans, we first screened a set of different hexokinases synthesizing glucose-6-phosphate, as well as the dikinase from Solanum tuberosum known to phosphorylate starch and to be involved in its metabolism. After this initial screening, the dikinase StGWD1 was selected as a working tool. This enzyme catalyses the transfer of the β-phosphate of ATP on the free C6-OH of starch. Its recombinant form as well as truncated versions, designed from models obtained using AlphaFold2, were subjected to X-ray experiments and examined by SAXS. Our study allowed us to propose a new organization of the protein in 5 domains, comprising two CBM45, a central domain, a domain containing the catalytic His and an ATP-binding domain. We also solved the first 3D-structure for the central domain of the enzyme, at 3.0 Å resolution. This domain adopts a unique fold and has no equivalent in the PDB. Our data also support the existence of a swivelling movement of the catalytic histidine domain between the central domain and the ATP-binding domain, allowing the autophosphorylation of the enzyme and subsequent transfer to the glucan. In addition, the two CBM45 would not play the same role, one of the CBM45 being far from the enzyme core. We also identified contact surfaces between the domains, and amino acids potentially critical for those domain interactions and for catalysis. In parallel, we characterized the activity of StGWD1 on crystalline maltodextrins and established methods to analyse the different reaction products. We used conditions that enabled us to obtain in vitro a substitution degree close to that obtained in planta. The enzyme was also tested to phosphorylate the set of bacterial α-glucans produced from sucrose. Glucans predominantly composed of α-1,6, α-1,2 or α-1,3 linkages were not substrates for StGWD1, suggesting that the specificity of the CBMs for the glucan to be phosphorylated is important. In contrast, various amylose-like glucans varying in size and crystallinity, which are produced by GH13 amylosucrase were successfully phosphorylated to levels comparable to that of potato tuber starch. Amylopectin and glycogen elongated with amylosucrase were also good substrates. This work has improved our understanding of StGWD1 and provided further insights into the structure-function relationships of this enzyme. It also offers interesting perspectives for guiding enzyme engineering and making StGWD1 more efficient for the phosphorylation of amylosucrase-derived polysaccharides as well as α-glucans of different structure.
