Probing carbohydrate product expulsion from a processive cellulase with multiple absolute binding free energy...

Area of Science:

• Biochemistry
• Enzymology
• Biomass Conversion

Background:

• Cellulases are crucial enzymes for degrading cellulose into sugars.
• Product inhibition by sugars (monosaccharides and disaccharides) limits cellulase efficiency.
• Understanding cellulase-product interactions is vital for improving biomass utilization for sustainable energy.

Purpose of the Study:

• To calculate the binding free energy of cellobiose and glucose to the catalytic tunnel of Trichoderma reesei Family 7 cellobiohydrolase (Cel7A).
• To identify key protein residues involved in product binding.
• To computationally engineer Cel7A to enhance product expulsion and improve biomass conversion efficiency.

Main Methods:

• Utilized steered molecular dynamics (SMD) simulations and alchemical free energy perturbation molecular dynamics (FEP/MD) simulations.
• Applied Jarzynski's equality to derive potential of mean force from SMD trajectories.
• Performed computational mutagenesis of key residues (Arg-251, Asp-259, Asp-262, Trp-376, Tyr-381) to assess binding free energy changes.

Main Results:

• Calculated absolute binding free energies of -14.4 kcal/mol (SMD) and -11.2 kcal/mol (FEP/MD) for cellobiose and glucose.
• Identified Arg-251, Asp-259, Asp-262, Trp-376, and Tyr-381 as critical for product binding.
• Mutations significantly reduced cellobiose binding free energy, with values ranging from -6.0 to -13.1 kcal/mol.

Conclusions:

• The study provides quantitative insights into product inhibition mechanisms in Cel7A.
• Computational mutagenesis demonstrates potential for engineering cellulases to accelerate product release.
• These findings offer a pathway to enhance enzyme efficiency for improved biomass conversion and sustainable energy production.