Poster Presentation 1B-16

 

Catalytically Enhanced Endocellulase Cel5A

from Acidothermus cellulolyticus

 

 

  John O. Baker*, James R. McCarley1, Rebecca Lovett1,2, Ching-Hsing Yu1, William S. Adney, David Templeton, Todd B. Vinzant, Stephen R. Decker, Joshua Sakon1

and Michael E. Himmel

 

 

National Bioenergy Center, National Renewable Energy Laboratory

1617 Cole Boulevard, Golden, CO 80401

Phone:  (303)384-7770
Fax:  (303)384-7752
john_baker@nrel.gov

 

1Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701

 

2NSF-summer undergraduate research participant, current address Department of Chemistry, Southern Oregon University, Ashland, OR 97520, USA

 

 

 

When Tyr245 in endocellulase Cel5A from Acidothermus cellulolyticus was changed to Gly (Y245G) by designed mutation, the value of Ki for inhibition of the enzyme by the product cellobiose was increased more than 1,480%.  This reduction in product inhibition enabled the mutant enzyme (used in conjunction with Trichoderma reesei cellobiohydrolase-I) to release soluble sugars from biomass cellulose at a rate as much as 40% greater than that achieved by the wild-type enzyme.  The mutant was designed on the basis of the crystal structure [Sakon et al. (1996) Biochemistry 35 10648-10660] of the wild type enzyme/substrate complex (at a resolution of 2.4 Å) which provided insights into the enzyme mechanism at the atomic level and identified Tyr245 as a key residue interacting with a leaving group.  To determine the origin of the change in activity, the crystal structure of Y245G was solved at 2.4 Å resolution to an R-factor of 0.19 (R-free = 0.25).  To obtain additional information on the enzyme-product interactions, density functional calculations were performed on representative fragments of the wild type Cel5A and Y245G.  The combined results indicate that the loss of the platform (Y245G) and of a hydrogen bond (from a conformational change in Gln247) reduces the binding energy between product and enzyme by several kcal/mol.  Both kinetic and structural analysis thus relates the increased enzymatic activity to reduced product inhibition.  The biochemical and kinetic properties of this modified enzyme and its utility in biomass conversion, as a member of new ternary cellulase systems, will be discussed.