CRYSTAL-STRUCTURE OF THE CATALYTIC DOMAIN OF A THERMOPHILIC ENDOCELLULASE

One way to improve the economic feasibility of biomass conversion is t o enhance the catalytic efficiency of cellulases through protein engin eering. This requires that high-resolution structures of cellulases be available. Here we present the structure of E2cd, the catalytic domai n of the thermophilic endocellulase E2 from Thermomonospora fusca, as determined by X-ray crystallography. The structure was solved by multi ple isomorphous replacement at 2.6-angstrom resolution and has been re fined at 1.8-angstrom resolution to an R-value of 18.4% for all reflec tions between 10- and 1.8-angstrom resolution. The fold of E2cd is bas ed on an unusual parallel beta-barrel and is equivalent to the fold de termined for the catalytic domain of cellobiohydrolase II, an exocellu lase from Trichoderma reesei [Rouvinen et al. (1990) Science 249, 380- 385]. The active site cleft of the enzyme, approximately 11 angstrom d eep and running the entire length of the molecule, is seen to be compl etely free for ligand binding in the crystal. A 2.2-angstrom resolutio n analysis of crystals of E2cd complexed with cellobiose, an inhibitor , shows how cellobiose binds in the active site and interacts with sev eral residues which line the cleft. Catalytic roles are suggested for three aspartic acid residues at the active site. A comparison of the E 2cd and CBHII(cd) structures reveals a large difference in their activ e site accessibilities and supports the hypothesis that the main diffe rence between endo- and exocellulases is the degree to which their act ive sites are accessible to substrate.