Control of Substrate Conformation by Hydrogen Bonding in a Retaining β-Endoglycosidase

Bacterial β-glycosidases are hydrolytic enzymes that depolymerize polysaccharides such as β-cellulose, β-glucans and β-xylans from different sources, offering diverse biomedical and industrial uses. It has been shown that a conformational change of the substrate, from a relaxed ⁴ C₁ conformation to a distorted ¹ S₃ /^1,4 B conformation of the reactive sugar, is necessary for catalysis. However, the molecular determinants that stabilize the substrate's distortion are poorly understood. Here we use quantum mechanics/molecular mechanics (QM/MM)-based molecular dynamics methods to assess the impact of the interaction between the reactive sugar, i. e. the one at subsite -1, and the catalytic nucleophile (a glutamate) on substrate conformation. We show that the hydrogen bond involving the C2 exocyclic group and the nucleophile controls substrate conformation: its presence preserves sugar distortion, whereas its absence (e.g. in an enzyme mutant) knocks it out. We also show that 2-deoxy-2-fluoro derivatives, widely used to trap the reaction intermediates by X-ray crystallography, reproduce the conformation of the hydrolysable substrate at the experimental conditions. These results highlight the importance of the 2-OH⋅⋅⋅nucleophile interaction in substrate recognition and catalysis in endo-glycosidases and can inform mutational campaigns aimed to search for more efficient enzymes.