CE43 - Bioéconomie, de la biomasse aux usages : chimie, matériaux, procédés et approches systémiques

Design of molecular assemblies to assess glycosylation role on hemicellulases activity – LABEL

Submission summary

Post-translational glycosylation (PTG) impacts the intrinsic features of proteins, including properties and biological functions. Despite numerous data on PTG, its role in glycoside hydrolases (GHs) is far from been fully elucidated, especially regarding enzyme-substrate interactions and hydrolytic efficiency. Focusing on lignocellulolytic enzymes that are widely used in biorefinery applications, the question that arises is whether carbohydrate moieties appended to these enzymes can guide their diffusion towards native substrates and if these glycosyl decorations can play a role in maintaining the spatial enzyme-substrate proximity during catalysis? To answer these questions, we should first develop a system that allows access to a well-defined set of glycoprotein models without the drawback of commonly used protein expression systems leading to either unglycosylated or glycoforms. Therefore, achieving tailored PTG remains challenging. Secondly, we need a set of convenient and reliable visualization methods to monitor the glycoproteins in contact with their substrates. LABEL aims to devise solutions to overcome these experimental challenges and tackle the unexplored question of substrate targeting by glycosylated enzymes. Two alternative approaches will be used to generate homogeneous precision-glycosylated GHs by genetic code expansion. Site(s)-specific incorporation of non-canonical amino acids and bioorthogonal ligation reactions onto a carbohydrate clickable platform molecules will provide fluorescent glycosylated hemicellulases as functional tools. These will be used to investigate how glycosylation affects substrate interactions and, more generally, lignocellulosic biomass deconstruction. To achieve this, combined approach implementing traditional biochemical and biophysical characterizations as well as advanced microscopy and spectroscopic techniques will be applied. Studies on labeled glycosylated hemicellulases will aim at elucidating molecular interactions and catalytic properties, including enzyme diffusion, affinity and activity, within model matrices of increasing complexity. Overall, LABEL will bring new perspectives for enzyme glycoengineering.

Project coordination

Régis FAURE (Institut National des Sciences Appliquées Toulouse)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

TBI Institut National des Sciences Appliquées Toulouse
CERMAV CERMAV
FARE Institut national de recherche pour l'agriculture, l'alimentation et l'environnement

Help of the ANR 400,276 euros
Beginning and duration of the scientific project: February 2023 - 36 Months

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