Bioethanol, a sustainable alternative to fossil fuels, is produced from cellulose via enzymatic saccharification. β-Glucosidase, a key enzyme in this process, hydrolyses disaccharides into glucose but is limited by feedback inhibition. This study investigated GH1 β-glucosidase from a soil metagenome (UnBGl1) to understand and overcome this limitation. We solved the near-atomic resolution crystal structure of UnBGl1 at 1.15 Å as an apo enzyme and report the first high-resolution crystal structures of the enzyme in its pre-hydrolytic state as a cellobiose complex and covalent intermediate-bound state, capturing key stages of its catalytic mechanism. Structural analysis revealed three crucial glucose-binding subsites in UnBGl1's crater. Glucose binding at the -1 subsite induced a 1.4 Å shift in E370, increasing the distance between catalytic glutamates and reducing enzymatic activity. The C188V variant, generated by rational engineering, significantly improved glucose tolerance to 2.5 M. Additionally, the H261W mutation at the +2 subsite enhanced kinetic properties by improving cellobiose affinity ( K m = 22.87 ± 1.1 mM) and shifted the optimal pH to 5.5 from 6.0. Comparative structural analysis with other glucose-tolerant GH1 β-glucosidases revealed conserved residues at the -1 subsite crucial for substrate stabilization and +1 subsite residues interacting with glucose, offering targets for further optimization. Engineered UnBGl1 variants retained high stability and activity on sugarcane bagasse, demonstrating their potential for industrial cellulase cocktails. These findings provide a robust framework for engineering β-glucosidases with enhanced glucose tolerance and catalytic efficiency, paving the way for improved bioethanol production and contributing to sustainable energy solutions.

Rewiring Catalytic Craters: A Path for Engineering β-Glucosidases to Improve Glucose Tolerance