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

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

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.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Carbohydrates / chemistry
  • Catalytic Domain
  • Cellulase / chemistry*
  • Cellulase / metabolism*
  • Cellulose / chemistry
  • Cellulose / metabolism
  • Clostridium cellulovorans / metabolism*
  • Hydrolysis
  • Kinetics
  • Molecular Dynamics Simulation
  • Oligosaccharides / chemistry
  • Protein Binding
  • Protein Conformation
  • Protein Interaction Domains and Motifs
  • Substrate Specificity

Substances

  • Carbohydrates
  • Oligosaccharides
  • Cellulose
  • Cellulase

Associated data

  • PDB/3NDY
  • PDB/3NDZ