The mechanism of cellulose hydrolysis by a two-step, retaining cellobiohydrolase elucidated by structural and transition path sampling studies

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

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

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. 
Original languageEnglish
JournalJournal of the American Chemical Society
Volume136
Issue number1
Pages (from-to)321-329
Number of pages9
ISSN0002-7863
DOIs
Publication statusPublished - 2014
Externally publishedYes

Keywords

  • Chemistry (all)
  • Catalysis
  • Biochemistry
  • Colloid and Surface Chemistry
  • Cellobiohydrolase Cel7A
  • Cellulose hydrolysis
  • Conformational change
  • Glycoside hydrolases
  • Glycosyl-enzyme intermediate
  • Glycosylation reactions
  • Hydrolytic reactions
  • Transition path sampling
  • Esterification
  • Glycosylation
  • Enzymes
  • Cellulose
  • Cellulose 1,4 beta cellobiosidase
  • Conformational transition
  • Deglycosylation
  • Enzyme active site
  • Enzyme structure
  • Hydrolysis
  • Hypocrea jecorina
  • Sampling

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