Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 110, Issue 36, Pages 14646-14651Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1309106110
Keywords
biofuels; cellulase; post-translational modification; carbohydrate recognition
Categories
Funding
- BioEnergy Technologies Office
- University of Colorado Boulder
- University College Ghent
- National Science Foundation through Extreme Science and Engineering Discovery Environment resources on the National Institute for Computational Science Kraken and Athena clusters [TG-MCB090159]
- National Renewable Energy Laboratory Computational Sciences Center
- US Department of Energy (DOE) Energy Efficiency and Renewable Energy [DE-AC36-08GO28308]
- ORNL Center for Structural Molecular Biology [ERKP291]
- US DOE Office of Science, Office of Biological and Environmental Research
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Plant cell-wall polysaccharides represent a vast source of food in nature. To depolymerize polysaccharides to soluble sugars, many organisms use multifunctional enzyme mixtures consisting of glycoside hydrolases, lytic polysaccharide mono-oxygenases, polysaccharide lyases, and carbohydrate esterases, as well as accessory, redox-active enzymes for lignin depolymerization. Many of these enzymes that degrade lignocellulose are multimodular with carbohydrate-binding modules (CBMs) and catalytic domains connected by flexible, glycosylated linkers. These linkers have long been thought to simply serve as a tether between structured domains or to act in an inchworm-like fashion during catalytic action. To examine linker function, we performed molecular dynamics (MD) simulations of the Trichoderma reesei Family 6 and Family 7 cellobiohydrolases (TrCel6A and TrCel7A, respectively) bound to cellulose. During these simulations, the glycosylated linkers bind directly to cellulose, suggesting a previously unknown role in enzyme action. The prediction from the MD simulations was examined experimentally by measuring the binding affinity of the Cel7A CBM and the natively glycosylated Cel7A CBM-linker. On crystalline cellulose, the glycosylated linker enhances the binding affinity over the CBM alone by an order of magnitude. The MD simulations before and after binding of the linker also suggest that the bound linker may affect enzyme action due to significant damping in the enzyme fluctuations. Together, these results suggest that glycosylated linkers in carbohydrate-active enzymes, which are intrinsically disordered proteins in solution, aid in dynamic binding during the enzymatic deconstruction of plant cell walls.
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