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Research article - Peer-reviewed, 2014

The Mechanism of Cellulose Hydrolysis by a Two-Step, Retaining Cellobiohydrolase Elucidated by Structural and Transition Path Sampling Studies

Knott, Brandon C.; Haddad Momeni, Majid; Crowley, Michael F.; Mackenzie, Lloyd F.; Goetz, Andreas W.; Sandgren, Mats; Withers, Stephen G.; Ståhlberg, Jerry; Beckham, Gregg T.


Glycoside hydrolases (GHs) cleave glycosidic linkages in carbohydrates, typically via inverting or retaining mechanisms, the latter of which proceeds via a two-step mechanism that includes formation of a glycosyl-enzyme intermediate. We present two new structures of the catalytic domain of Hypocrea jecorina GH Family 7 cellobiohydrolase Cel7A, namely a Michaelis complex with a full cellononaose ligand and a glycosyl-enzyme intermediate, that reveal details of the 'static' reaction coordinate. We also employ transition path sampling to determine the 'dynamic' reaction coordinate for the catalytic cycle. The glycosylation reaction coordinate contains components of forming and breaking bonds and a conformational change in the nucleophile. Deglycosylation proceeds via a product-assisted mechanism wherein the glycosylation product, cellobiose, positions a water molecule for nucleophilic attack on the anomeric carbon of the glycosyl-enzyme intermediate. In concert with previous structures, the present results reveal the complete hydrolytic reaction coordinate for this naturally and industrially important enzyme family.

Published in

Journal of the American Chemical Society
2014, volume: 136, number: 1, pages: 321-329

Authors' information

Knott, Brandon C.
National Renewable Energy Laboratory
Haddad Momeni, Majid
Swedish University of Agricultural Sciences, Department of Molecular Biology
Crowley, Michael F.
National Renewable Energy Laboratory
Mackenzie, Lloyd F.
University of British Columbia
Goetz, Andreas W.
University of California San Diego
Swedish University of Agricultural Sciences, Department of Molecular Biology
Withers, Stephen G.
University of British Columbia
Swedish University of Agricultural Sciences, Department of Molecular Biology
Beckham, Gregg T.
National Renewable Energy Laboratory

UKÄ Subject classification

Biochemistry and Molecular Biology
Structural Biology
Renewable Bioenergy Research

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