Structure, Dynamics, and Specificity of Endoglucanase D from Clostridium cellulovorans

JOURNAL OF MOLECULAR BIOLOGY

卷:425

Article

Bianchetti, Christopher M.^1,2  Brumm, Phillip^2,3  Smith, Robert W.^1,2  Dyer, Kevin⁴  Hura, Greg L.⁴  Rutkoski, Thomas J.^1,2  Phillips, George N., Jr.^1,2,5 

[1] Univ Wisconsin, Dept Biochem, Madison, WI 53706 USA

[2] Univ Wisconsin, Great Lakes Bioenergy Res Ctr, Madison, WI 53706 USA

[3] Lucigen Corp & C5 6 Technol, Madison, WI 53562 USA

[4] Univ Calif Berkeley, Lawrence Berkeley Natl Lab, Phys Biosci Div, Berkeley, CA 94720 USA

[5] Rice Univ, Dept Biochem & Cell Biol, Houston, TX 77005 USA

关键词: cellulase;    endoglucanase;    cellulose degradation;    small-angle X-ray scattering;    X-ray crystallography;   

DOI  :  10.1016/j.jmb.2013.05.030

来源: Elsevier

【 摘 要 】

The enzymatic degradation of cellulose is a critical step in the biological conversion of plant biomass into an abundant renewable energy source. An understanding of the structural and dynamic features that cellulases utilize to bind a single strand of crystalline cellulose and hydrolyze the beta-1,4-glycosidic bonds of cellulose to produce fermentable sugars would greatly facilitate the engineering of improved cellulases for the large-scale conversion of plant biomass. Endoglucanase D (EngD) from Clostridium cellulovorans is a modular enzyme comprising an N-terminal catalytic domain and a C-terminal carbohydrate-binding module, which is attached via a flexible linker. Here, we present the 2.1-angstrom-resolution crystal structures of full-length EngD with and without cellotriose bound, solution small-angle X-ray scattering (SAXS) studies of the full-length enzyme, the characterization of the active cleft glucose binding subsites, and substrate specificity of EngD on soluble and insoluble polymeric carbohydrates. SAXS data support a model in which the linker is flexible, allowing EngD to adopt an extended conformation in solution. The cellotriose-bound EngD structure revealed an extended active-site cleft that contains seven glucose-binding subsites, but unlike the majority of structurally determined endocellulases, the active-site cleft of EngD is partially enclosed by Trpl 62 and Tyr232. EngD variants, which lack Trp162, showed a significant reduction in activity and an alteration in the distribution of cellohexaose degradation products, suggesting that Trpl 62 plays a direct role in substrate binding. (C) 2013 Elsevier Ltd. All rights reserved.

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