Substitution of distal and active site residues reduces product inhibition of E1 from Acidothermus Cellulolyticus

Summers, Samantha R.; Alamdari, Sarah; Kraft, Casey J.; Brunecky, Roman; Pfaendtner, Jim; Kaar, Joel L.

doi:10.1093/protein/gzab031

Title: Substitution of distal and active site residues reduces product inhibition of E1 from Acidothermus Cellulolyticus

Journal Article · 21 December 2021 · Protein Engineering, Design and Selection

DOI: https://doi.org/10.1093/protein/gzab031 · OSTI ID:1837094

Summers, Samantha R.; Alamdari, Sarah; Kraft, Casey J.; Brunecky, Roman; Pfaendtner, Jim;

Abstract 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.

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Research Organization:: National Renewable Energy Laboratory (NREL), Golden, CO (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Office of Workforce Development for Teachers & Scientists (WDTS); USDOE Office of Science (SC), Workforce Development for Teachers and Scientists (WDTS)

Grant/Contract Number:: AC36-08GO28308; SC0014664

OSTI ID:: 1837094

Report Number(s):: NREL/JA--2700-81872; gzab031

Journal Information:: Protein Engineering, Design and Selection, Journal Name: Protein Engineering, Design and Selection Vol. 34; ISSN 1741-0126

Publisher:: Oxford University PressCopyright Statement

Country of Publication:: United Kingdom

Language:: English

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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
cellobiose inhibition
cellulosic biofuels
endoglucanase
enzyme engineering
heteronuclear single quantum coherence NMR
molecular dynamics

Title: Substitution of distal and active site residues reduces product inhibition of E1 from Acidothermus Cellulolyticus

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