title: Structure, dynamics, and specificity of endoglucanase D from Clostridium cellulovorans.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA; Great Lakes Bioenergy Research Center, University of Wisconsin, Madison, WI 53706, USA.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Bianchetti
authors.forename: Phillip
authors.initials: P
authors.lastname: Brumm
authors.forename: Robert W
authors.initials: RW
authors.lastname: Smith
authors.forename: Kevin
authors.initials: K
authors.lastname: Dyer
authors.forename: Greg L
authors.initials: GL
authors.lastname: Hura
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Rutkoski
authors.forename: George N
authors.initials: GN
authors.lastname: Phillips
mesh_terms: D002241:Carbohydrates / Q000737:chemistry; D020134:Catalytic Domain; D002480:Cellulase / Q000737:chemistry* / Q000378:metabolism*; D002482:Cellulose / Q000737:chemistry / Q000378:metabolism; D046974:Clostridium cellulovorans / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D009844:Oligosaccharides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D013379:Substrate Specificity
chemical_list: D002241:Carbohydrates; D009844:Oligosaccharides; D002482:Cellulose; D002480:Cellulase
keywords: 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