Regulation of plant polygalacturonases activity through a disulfide redox switch mechanism
Mihaela Sakic (France)1; Adrien Lemaire (France)1; Vanessa Ung (New Zealand)2; François Jobert (France)1; Solène Bassard (France)1; Paulo Marcelo (France)1; Sebastien Rigaud (France)1; Jean-Xavier Fontaine (France)1; Roland Molinié (France)1; Valérie Lefebvre (France)1; Taku Demura (Japan)3; Davide Mercadante (New Zealand)2; Jérôme Pelloux (France)1; Josip Safran (France)1;
1 - Université Picardie Jules Verne; 2 - University of Auckland; 3 - Nara Institute of Science and Technology;
Keywords: polygalacturonases; oxidoreductases; reduction;
Abstract Topics: Theme 1: Pectins: Structure, Remodeling, and Function
Type of Presentation: Oral Communication

Abstract text: Polygalacturonases (PGs) are key cell wall–remodelling enzymes that catalyse pectin depolymerization, shaping plant’s primary cell wall architecture. The in vivo regulation of plant PGs remains poorly understood. Here, we identified a novel redox-based mechanism controlling the activity of Arabidopsis thaliana ADPG2. Structural analysis revealed a high number of cysteine residues forming disulfide bridges, particularly in the loops surrounding the active site. We propose that these structural elements act as redox-sensitive switches. We showed that chemical reduction decreases enzymatic activity in vitro, consistent with molecular dynamics (MD) simulations showing changes in loop dynamics between reduced and oxidized states. Site-directed mutagenesis of loop cysteines strongly impaired enzyme activity and processivity without altering global protein structure, demonstrating their regulatory role. We further identified Arabidopsis GILT1 (gamma-inducible thiol reductase 1) as a candidate oxidoreductase mediating this regulation. Microscale thermophoresis confirmed nanomolar binding affinities between ADPG2 and GILT1, supporting a direct interaction while redox proteomic showed reduction of loop cysteines. This mechanism introduces a redox-based layer of PG regulation at the cell wall, providing new insights into the dynamic control of pectin degradation during plant development.

Direct interaction between ADPG2 and GILT1 confirmed with AlphaFold3 and Microscale thermophoresis (MST). A. AlphaFold3 simulation highlights the implication of disulfide bridges in the interaction between ADPG2 (orange) and GILT1 (turquoise). B. GILT1 active site situated in the proximity of the ADPG2 loop disulfide bridge. C. Molecular scale thermophoresis confirms in vitro binding between the two enzymes.