Insights into ligand-induced conformational change in Cel5A from Bacillus agaradhaerens revealed by a catalytically active crystal form

Glycoside hydrolases are ubiquitous enzymes involved in a diverse array of biological processes, from the breakdown of biomass, through to viral invas ion and cellular signalling. Endoglucanase Cel5A from Bacillus agaradhaeren s, classified into glycoside hydrolase family 5, has been studied in a cata lytically inactive crystal form at low pH conditions, in which native and c omplex structures revealed the importance of ring distortion during catalys is. Here, we present the structure of Cel5A in a new crystal form obtained at higher pH values in which the enzyme is active "in-crystal". Native, cel lotriosyl-enzyme intermediate and beta-D-cellobiose structures were solved at 1.95, 1.75 and 2.1 Angstrom resolution, respectively. These structures r eveal two classes of conformational change: those caused by crystal-packing and pH, with others- induced upon substrate binding. At pH 7 a histidine r esidue, His206, implicated in substrate-binding and catalysis, but previous ly far removed from the substrate-binding cleft, moves over 10 Angstrom int o the active site cleft in order to interact with the substrate in the +2 s ubsite. Occupation of the -1 subsite by substrate induces a -loop closure t o optimise protein-ligand interactions. Cel5A, along with the unrelated fam ily 45 and family 6 cellulases, provides further evidence of substantial co nformational change in response to ligand binding for this class of hydroly tic enzyme. (C) 2000 Academic Press.