9IQB

Crystal structure of beta-glucosidase from Acetivibrio thermocellus

Summary for 9IQB

• Entry DOI: 10.2210/pdb9iqb/pdb
• Descriptor: Beta-glucosidase A, 2-AMINO-2-HYDROXYMETHYL-PROPANE-1,3-DIOL (3 entities in total)
• Functional Keywords: beta-glucosidase, gh1 family, cellobiose, hydrolase
• Biological source: Acetivibrio thermocellus
• Total number of polymer chains: 12
• Total formula weight: 631663.53

Authors

Kamale, C.,Bhaumik, P. (deposition date: 2024-07-12, release date: 2025-01-15, Last modification date: 2025-03-19)

Primary citation

Kamale, C.,Rauniyar, A.,Bhaumik, P.
Rational design facilitates the improvement of glucose tolerance and catalytic properties of a beta-glucosidase from Acetivibrio thermocellus.
Febs J., 292:1174-1196, 2025

PubMed Abstract: Cellulases are an ensemble of enzymes that hydrolyze cellulose chains into fermentable glucose and hence are widely used in bioethanol production. The last enzyme of the cellulose degradation pathway, β-glucosidase, is inhibited by its product, glucose. The product inhibition by glucose hinders cellulose hydrolysis limiting the saccharification during bioethanol production. Thus, engineered β-glucosidases with enhanced glucose tolerance and catalytic efficiency are essential. This study focuses on the rational engineering of β-glucosidase from Acetivibrio thermocellus (WT-AtGH1). Recombinant WT-AtGH1 exhibited activity on cellobiose and p-nitrophenyl-β-d-glucoside as substrates and retained around 80% of its activity over 48 h at 55 °C, pH 5.5. However, WT-AtGH1 showed low glucose tolerance of 380 mm as compared to the required IC value of > 800 mm for industrial use. Thus, a rational design approach was utilized to enhance the glucose tolerance of this enzyme. We determined the 3 Å resolution crystal structure of WT-AtGH1. The structure-based engineered G168W-AtGH1 and S242W-AtGH1 mutants exhibited improved glucose tolerance of 840 and 612 mm, respectively. Surprisingly, S242L-AtGH1 mutant showed ~ 2.5-fold increase in the catalytic efficiency as compared to WT-AtGH1. A combinatorial effect of improved glucose tolerance, as well as enhanced catalytic efficiency, was observed for the G168W-S242L-AtGH1 mutant. All the mutants with enhanced properties showed considerable stability at industrial operating conditions of 55 °C and pH 5.5. Thus, we present mutants of WT-AtGH1 with improved glucose tolerance and kinetic properties that have the potential to increase the efficiency of saccharification during biofuel production.

PubMed: 39764622
DOI: 10.1111/febs.17394

Experimental method

X-RAY DIFFRACTION (3 Å)