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J Mol Biol. 2013 Nov 15;425(22):4267-85. doi: 10.1016/j.jmb.2013.05.030. Epub 2013 Jun 8.

Structure, dynamics, and specificity of endoglucanase D from Clostridium cellulovorans.

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Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA; Great Lakes Bioenergy Research Center, University of Wisconsin, Madison, WI 53706, USA.


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 β-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-Å-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 Trp162 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 Trp162 plays a direct role in substrate binding.


CBM; EngD; GH; PDB; PT; Protein Data Bank; SAXS; X-ray crystallography; carbohydrate-binding module; cellulase; cellulose degradation; endoglucanase; endoglucanase D; glycosyl hydrolase; proline/threonine-rich; small-angle X-ray scattering

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