Probing Carbohydrate Product Expulsion from a Processive Cellulase with Multiple Absolute Binding Free Energy Methods

Abstract

Understanding the enzymatic mechanism that cellulases employ to degrade cellulose is critical to efforts to efficiently utilize plant biomass as a sustainable energy resource. A key component of cellulase action on cellulose is product inhibition from monosaccharide and disaccharides in the product site of cellulase tunnel. The absolute binding free energy of cellobiose and glucose to the product site of the catalytic tunnel of the Family 7 cellobiohydrolase (Cel7A) of Trichoderma reesei (Hypocrea jecorina) was calculated using two different approaches: steered molecular dynamics (SMD) simulations and alchemical free energy perturbation molecular dynamics (FEP/MD) simulations. For the SMD approach, three methods based on Jarzynski's equality were used to construct the potential of mean force from multiple pulling trajectories. The calculated binding free energies, −14.4 kcal/mol using SMD and −11.2 kcal/mol using FEP/MD, are in good qualitative agreement. Analysis of the SMD pulling trajectories suggests that several protein residues (Arg-251, Asp-259, Asp-262, Trp-376, and Tyr-381) play key roles in cellobiose and glucose binding to the catalytic tunnel. Five mutations (R251A, D259A, D262A, W376A, and Y381A) were made computationally to measure the changes in free energy during the product expulsion process. The absolute binding free energies of cellobiose to the catalytic tunnel of these five mutants are −13.1, −6.0, −11.5, −7.5, and −8.8 kcal/mol, respectively. The results demonstrated that all of the mutants tested can lower the binding free energy of cellobiose, which provides potential applications in engineering the enzyme to accelerate the product expulsion process and improve the efficiency of biomass conversion.

Loop Motions Important to Product Expulsion in the Thermobifida fusca Glycoside Hydrolase Family 6 Cellobiohydrolase from Structural and Computational Studies

“…4, d (also referred to as the reaction coordinate), was increased with a speed of 1 Å/ns in 14 ns. We examined different pulling speeds, and 1 Å/ns was found to yield converged results similar to our previous work (15,59). The force constant is 5000 kcal/(mol ϫ Å 2 ).…”

“…Steered Molecular Dynamics (SMD) Simulations-The protocols for conducting SMD simulations on the TfuCel6B-cellodextrin complex and the TfuCel6B-cellulose complex follow our previous work on product inhibition (15,59). CHAMBER (61) was used to convert the protein structure file, coordinate file, and force field files in CHARMM format to Amber format.…”

Deciphering the Effect of the Different N-Glycosylation Sites on the Secretion, Activity, and Stability of Cellobiohydrolase I from Trichoderma reesei

“…Considering the relative positions of Asn45 and Asn384 along the catalytic tunnel, an assumption can be made that attachment of an Nglycan at Asn45 site would stabilize the substrate entrance region, including W40, which is key in recruiting individual substrate chains into the active channel to initiate processive hydrolysis (52). As for Asn384, glycosylation at this site seems to restrain the flexibility of the loop to which it is attached and may thus affect the binding mode of cellobiose relative to the exit tunnel (53). Upon temperature elevation, larger fluctuations of the loop may disturb the neighboring secondary structures and eventually their disassembly.…”

Genomics Review of Holocellulose Deconstruction by Aspergilli

“…2) suggest a tunnel-like conformation around the active site, fitting a single cellulosic chain at the reducing (GH7) or nonreducing (GH6) terminus (88)(89)(90). In contrast, endoglucanases (GH5 to -9, GH12, GH44, GH45, GH48, GH51, GH61, and GH124) are shaped by an open groove or cleft, into which a linear amorphous cellulose chain can fit randomly (Fig.…”