Journal of Biological Chemistry
Volume 286, Issue 47, 25 November 2011, Pages 41028-41035
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Computational Biology
Multiple Functions of Aromatic-Carbohydrate Interactions in a Processive Cellulase Examined with Molecular Simulation*

https://doi.org/10.1074/jbc.M111.297713Get rights and content
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Proteins employ aromatic residues for carbohydrate binding in a wide range of biological functions. Glycoside hydrolases, which are ubiquitous in nature, typically exhibit tunnels, clefts, or pockets lined with aromatic residues for processing carbohydrates. Mutation of these aromatic residues often results in significant activity differences on insoluble and soluble substrates. However, the thermodynamic basis and molecular level role of these aromatic residues remain unknown. Here, we calculate the relative ligand binding free energy by mutating tryptophans in the Trichoderma reesei family 6 cellulase (Cel6A) to alanine. Removal of aromatic residues near the catalytic site has little impact on the ligand binding free energy, suggesting that aromatic residues immediately upstream of the active site are not directly involved in binding, but play a role in the glucopyranose ring distortion necessary for catalysis. Removal of aromatic residues at the entrance and exit of the Cel6A tunnel, however, dramatically impacts the binding affinity, suggesting that these residues play a role in chain acquisition and product stabilization, respectively. The roles suggested from differences in binding affinity are confirmed by molecular dynamics and normal mode analysis. Surprisingly, our results illustrate that aromatic-carbohydrate interactions vary dramatically depending on the position in the enzyme tunnel. As aromatic-carbohydrate interactions are present in all carbohydrate-active enzymes, these results have implications for understanding protein structure-function relationships in carbohydrate metabolism and recognition, carbon turnover in nature, and protein engineering strategies for biomass utilization. Generally, these results suggest that nature employs aromatic-carbohydrate interactions with a wide range of binding affinities for diverse functions.

Carbohydrate-binding Protein
Carbohydrate Processing
Enzyme Mutation
Molecular Modeling
Thermodynamics
Biomass Conversion
Carbohydrate-π Interactions
Free Energy Simulations
Ligand Binding
Thermodynamic Integration

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*

This work was supported by the Department of Energy Office of the Biomass Program.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Figs. S1–S13.