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Title: Multiple Functions of Aromatic-Carbohydrate Interactions in a Processive Cellulase Examined with Molecular Simulation

Abstract

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 residuesimmediately 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 rolessuggested 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 presentmore » in all carbohydrate-active enzymes, these results have implications for understanding proteinstructure-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.« less

Authors:
; ; ; ; ; ;
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE Office of Energy Efficiency and Renewable Energy (EERE)
OSTI Identifier:
1573191
Report Number(s):
NREL/JA-5100-52948
DOE Contract Number:  
AC36-08GO28308
Resource Type:
Journal Article
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 286
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; carbohydrate-binding; carbohydrate processing; enzyme mutation; molecular modeling; thermodynamics biomass conversion; free energy simulations; ligand binding; thermodynamic integration

Citation Formats

Payne, Christina M., Bomble, Yannick J., Taylor, Courtney B., McCabe, Clare, Himmel, Michael E., Crowley, Michael F., and Beckham, Gregg T. Multiple Functions of Aromatic-Carbohydrate Interactions in a Processive Cellulase Examined with Molecular Simulation. United States: N. p., 2011. Web. doi:https://dx.doi.org/10.1074/jbc.M111.297713.
Payne, Christina M., Bomble, Yannick J., Taylor, Courtney B., McCabe, Clare, Himmel, Michael E., Crowley, Michael F., & Beckham, Gregg T. Multiple Functions of Aromatic-Carbohydrate Interactions in a Processive Cellulase Examined with Molecular Simulation. United States. doi:https://dx.doi.org/10.1074/jbc.M111.297713.
Payne, Christina M., Bomble, Yannick J., Taylor, Courtney B., McCabe, Clare, Himmel, Michael E., Crowley, Michael F., and Beckham, Gregg T. Fri . "Multiple Functions of Aromatic-Carbohydrate Interactions in a Processive Cellulase Examined with Molecular Simulation". United States. doi:https://dx.doi.org/10.1074/jbc.M111.297713.
@article{osti_1573191,
title = {Multiple Functions of Aromatic-Carbohydrate Interactions in a Processive Cellulase Examined with Molecular Simulation},
author = {Payne, Christina M. and Bomble, Yannick J. and Taylor, Courtney B. and McCabe, Clare and Himmel, Michael E. and Crowley, Michael F. and Beckham, Gregg T.},
abstractNote = {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 residuesimmediately 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 rolessuggested 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 proteinstructure-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.},
doi = {https://dx.doi.org/10.1074/jbc.M111.297713},
journal = {Journal of Biological Chemistry},
number = ,
volume = 286,
place = {United States},
year = {2011},
month = {11}
}

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