Constrained Catalytic Itinerary of a Retaining 3,6-Anhydro-D-Galactosidase, a Key Enzyme in Red Algal Cell Wall Degradation - Archive ouverte HAL
Journal Articles Angewandte Chemie International Edition Year : 2024

Constrained Catalytic Itinerary of a Retaining 3,6-Anhydro-D-Galactosidase, a Key Enzyme in Red Algal Cell Wall Degradation

Abstract

The marine Bacteroidota Zobellia galactanivorans has a polysaccharide utilization locus dedicated to the catabolism of the red algal cell wall galactan carrageenan and its unique and industrially important α‐3,6‐anhydro‐D‐galactose (ADG) monosaccharide. Here we present the first analysis of the specific molecular interactions that the exo‐(α‐1,3)‐3,6‐anhydro‐D‐galactosidase Zg GH129 uses to cope with the strict steric restrictions imposed by its bicyclic ADG substrate — which is ring flipped relative to D‐galactose. Crystallographic snapshots of key catalytic states obtained with the natural substrate and novel chemical tools designed to mimic species along the reaction coordinate, together with quantum mechanics/molecular mechanics (QM/MM) metadynamics methods and kinetic studies, demonstrate a retaining mechanism where the second step is rate limiting. The conformational landscape of the constrained 3,6‐anhydro‐D‐galactopyranose ring proceeds through enzyme glycosylation B$_{1,4}$ → [E$_4$]$^≠$ → E$_4$/$^1$C$_4$ and deglycosylation E$_4/^1$C$_4$ → [E$_4$]$^≠$ → B$_{1,4}$ itineraries limited to the Southern Hemisphere of the Cremer–Pople sphere. These results demonstrate the conformational changes throughout catalysis in a non‐standard, sterically restrained, bicyclic monosaccharide, and provide a molecular framework for mechanism‐based inhibitor design for anhydro‐type carbohydrate‐processing enzymes and for future applications involving carrageenan degradation. In addition, our study provides a rare example of distinct niche‐based conformational itineraries within the same carbohydrate‐active enzyme family.
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Dates and versions

hal-04700900 , version 1 (18-09-2024)

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Michael Wallace, Irene Cuxart, Thomas Roret, Laura Guée, Aleksandra Debowski, et al.. Constrained Catalytic Itinerary of a Retaining 3,6-Anhydro-D-Galactosidase, a Key Enzyme in Red Algal Cell Wall Degradation. Angewandte Chemie International Edition, inPress, Online ahead of print. ⟨10.1002/anie.202411171⟩. ⟨hal-04700900⟩
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