Structure of the Bacillus agaradherans Family 5 Endoglucanase at 1.6 Å and Its Cellobiose Complex at 2.0 Å Resolution

The enzymatic degradation of cellulose, by cellulases, is not only industrially important in the food, paper, and textile industries but also a potentially useful method for the environmentally friendly recycling of municipal waste. An understanding of the structural and mechanistic requirements for the hydrolysis of the β-1,4 glycosidic bonds of cellulose is an essential prerequisite for beneficial engineering of cellulases for these processes. Cellulases have been classified into 13 of the 62 glycoside hydrolase families [Henrissat, B., and Bairoch, A. (1996) Biochem J. 316, 695−696]. The structure of the catalytic core of the family 5 endoglucanase, Cel5A, from the alkalophilic Bacillus agaradherans has been solved by multiple isomorphous replacement at 1.6 Å resolution. Cel5A has the (α/β)8 barrel structure and signature structural features typical of the grouping of glycoside hydrolase families known as clan GH-A, with the catalytic acid/base Glu 139 and nucleophile Glu 228 on barrel strands β4 and β7 as expected. In addition to the native enzyme, the 2.0 Å resolution structure of the cellobiose-bound form of the enzyme has also been determined. Cellobiose binds preferentially in the −2 and −3 subsites of the enzyme. Kinetic studies on the isolated catalytic core domain of Cel5A, using a series of reduced cellodextrins as substrates, suggest approximately five to six binding sites, consistent with the shape and size of the cleft observed by crystallography.