Substitution of Distal and Active Site Residues Reduces Product Inhibition of E1 from Acidothermus Cellulolyticus

Samantha Summers, Sarah Alamdari, Casey Kraft, Roman Brunecky, Jim Pfaendtner, Joel Kaar

Research output: Contribution to journalArticlepeer-review

2 Scopus Citations


Cellulases are largely afflicted by inhibition from their reaction products, especially at high-substrate loading, which represents a major challenge for biomass processing. This challenge was overcome for endoglucanase 1 (E1) from Acidothermus cellulolyticus by identifying a large conformational change involving distal residues upon binding cellobiose. Having introduced alanine substitutions at each of these residues, we identified several mutations that reduced cellobiose inhibition of E1, including W212A, W213A, Q247A, W249A and F250A. One of the mutations (W212A) resulted in a 47-fold decrease in binding affinity of cellobiose as well as a 5-fold increase in the kcat. The mutation further increased E1 activity on Avicel and dilute-acid treated corn stover and enhanced its productivity at high-substrate loadings. These findings were corroborated by funnel metadynamics, which showed that the W212A substitution led to reduced affinity for cellobiose in the +1 and +2 binding sites due to rearrangement of key cellobiose-binding residues.

Original languageAmerican English
Article numberArticle No. gzab031
JournalProtein Engineering, Design and Selection
StatePublished - 2021

Bibliographical note

Publisher Copyright:
© 2021 The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail:

NREL Publication Number

  • NREL/JA-2700-81872


  • cellobiose inhibition
  • cellulosic biofuels
  • endoglucanase
  • enzyme engineering
  • heteronuclear single quantum coherence NMR
  • molecular dynamics


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