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Title: The Dissociation Mechanism of Processive Cellulases

Abstract

Cellulase enzymes deconstruct recalcitrant cellulose into soluble sugars, making them a biocatalyst of biotechnological interest for use in the nascent lignocellulosic bioeconomy. Cellobiohydrolases (CBHs) are cellulases capable of liberating many sugar molecules in a processive manner without dissociating from the substrate. Within the complete processive cycle of CBHs, dissociation from the cellulose substrate is rate limiting, but the molecular mechanism of this step is unknown. Here, we present a direct comparison of potential molecular mechanisms for dissociation via Hamiltonian replica exchange molecular dynamics of the model fungal CBH, Trichoderma reesei Cel7A. Computational rate estimates indicate that stepwise cellulose dethreading from the binding tunnel is 4 orders of magnitude faster than a clamshell mechanism, in which the substrate-enclosing loops open and release the substrate without reversing. We also present the crystal structure of a disulfide variant that covalently links substrate-enclosing loops on either side of the substrate-binding tunnel, which constitutes a CBH that can only dissociate via stepwise dethreading. Biochemical measurements indicate that this variant has a dissociation rate constant essentially equivalent to the wild type, implying that dethreading is likely the predominant mechanism for dissociation.

Authors:
ORCiD logo [1];  [2]; ORCiD logo [1];  [1];  [3];  [3];  [3];  [2];  [1]
  1. National Renewable Energy Laboratory (NREL), Golden, CO (United States)
  2. University of Tartu
  3. Swedish University of Agricultural Sciences
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE Office of Energy Efficiency and Renewable Energy (EERE), Bioenergy Technologies Office (EE-3B)
OSTI Identifier:
1574196
Report Number(s):
NREL/JA-2700-74624
DOE Contract Number:  
AC36-08GO28308
Resource Type:
Journal Article
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Name: Proceedings of the National Academy of Sciences of the United States of America
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; biofuels; cellulases; molecular mechanism; molecular simulation; crystal structure; BCPL

Citation Formats

Vermaas, Joshua, Kont, Riin, Beckham, Gregg T, Crowley, Michael F, Gudmundsson, Mikael, Sandgren, Mats, Stahlberg, Jerry, Valjamae, Priit, and Knott, Brandon C. The Dissociation Mechanism of Processive Cellulases. United States: N. p., 2019. Web. doi:10.1073/pnas.1913398116.
Vermaas, Joshua, Kont, Riin, Beckham, Gregg T, Crowley, Michael F, Gudmundsson, Mikael, Sandgren, Mats, Stahlberg, Jerry, Valjamae, Priit, & Knott, Brandon C. The Dissociation Mechanism of Processive Cellulases. United States. doi:10.1073/pnas.1913398116.
Vermaas, Joshua, Kont, Riin, Beckham, Gregg T, Crowley, Michael F, Gudmundsson, Mikael, Sandgren, Mats, Stahlberg, Jerry, Valjamae, Priit, and Knott, Brandon C. Wed . "The Dissociation Mechanism of Processive Cellulases". United States. doi:10.1073/pnas.1913398116.
@article{osti_1574196,
title = {The Dissociation Mechanism of Processive Cellulases},
author = {Vermaas, Joshua and Kont, Riin and Beckham, Gregg T and Crowley, Michael F and Gudmundsson, Mikael and Sandgren, Mats and Stahlberg, Jerry and Valjamae, Priit and Knott, Brandon C},
abstractNote = {Cellulase enzymes deconstruct recalcitrant cellulose into soluble sugars, making them a biocatalyst of biotechnological interest for use in the nascent lignocellulosic bioeconomy. Cellobiohydrolases (CBHs) are cellulases capable of liberating many sugar molecules in a processive manner without dissociating from the substrate. Within the complete processive cycle of CBHs, dissociation from the cellulose substrate is rate limiting, but the molecular mechanism of this step is unknown. Here, we present a direct comparison of potential molecular mechanisms for dissociation via Hamiltonian replica exchange molecular dynamics of the model fungal CBH, Trichoderma reesei Cel7A. Computational rate estimates indicate that stepwise cellulose dethreading from the binding tunnel is 4 orders of magnitude faster than a clamshell mechanism, in which the substrate-enclosing loops open and release the substrate without reversing. We also present the crystal structure of a disulfide variant that covalently links substrate-enclosing loops on either side of the substrate-binding tunnel, which constitutes a CBH that can only dissociate via stepwise dethreading. Biochemical measurements indicate that this variant has a dissociation rate constant essentially equivalent to the wild type, implying that dethreading is likely the predominant mechanism for dissociation.},
doi = {10.1073/pnas.1913398116},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = ,
volume = ,
place = {United States},
year = {2019},
month = {10}
}

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