Conformational flexibility correlates with glucose tolerance for point mutations in β-glucosidases - a computational...

Area of Science:

• Biotechnology and Bioengineering
• Enzyme Engineering
• Renewable Energy

Background:

• Beta-glucosidases are crucial for second-generation biofuel production, catalyzing the final step of lignocellulosic saccharification.
• Glucose inhibition of beta-glucosidases increases cellobiose concentration, hindering biofuel production efficiency.
• Engineering glucose-tolerant beta-glucosidases is essential for improving biofuel processes.

Purpose of the Study:

• To investigate the molecular mechanisms underlying glucose tolerance in beta-glucosidases.
• To analyze the structural and dynamic effects of mutations on enzyme activity and glucose tolerance.

Main Methods:

• High sampling accelerated molecular dynamics simulations.
• Analysis of wild-type and mutant GH1 beta-glucosidase (Bgl1A, Bgl1B) structures.
• Conformational cluster and free energy landscape profiling.

Main Results:

• Mutations conferring glucose tolerance enhanced the mobility of flexible loops (B and C loops, alpha-beta hairpin) near the active site.
• Increased loop mobility facilitated substrate channel closure, impeding glucose entry and promoting its exit.
• Structural analyses indicated that both stereochemical effects and altered loop dynamics contribute to glucose tolerance.

Conclusions:

• Rational design of glucose-tolerant beta-glucosidases can be achieved by modulating active site loop dynamics.
• These findings provide insights for developing industrially relevant enzymes for biofuel applications.
• Understanding enzyme-dynamics relationships is key for enzyme engineering.