CRYSTAL-STRUCTURE OF THE FAMILY-7 ENDOGLUCANASE-I (CEL7B) FROM HUMICOLA INSOLENS AT 2.2 ANGSTROM RESOLUTION AND IDENTIFICATION OF THE CATALYTIC NUCLEOPHILE BY TRAPPING OF THE COVALENT GLYCOSYL-ENZYME INTERMEDIATE

Cellulose is the major polysaccharide component of the plant cell wall and the most abundant naturally produced macromolecule on Earth. The enzymic degradation of cellulose, by cellulases, is therefore of great environmental and commercial significance. Cellulases are found in 12 of the glycoside hydrolase families classified according to their ami no acid sequence similarities. Endoglucanase I (Cel7B), from the soft- rot fungus Humicola insolens, is a family 7 enzyme. The structure of t he native form of Cel7B from H. insolens at 2.2 Angstrom resolution ha s been solved by molecular replacement using the known Trichoderma ree sei cellobiohydrolase I [Divne, Stahlberg, Reinikainen, Ruohonen, Pett ersson, Knowles, Teeri and Jones (1994) Science 265, 524-528] structur e as the search model. Cel7B catalyses hydrolysis of the beta-1,4 glyc osidic linkages in cellulose with net retention of anomeric configurat ion. The catalytic nucleophile at the active site of Cel7B has been id entified as Glu-197 by trapping of a 2-deoxy-2-fluorocellotriosyl enzy me intermediate and identification of the labelled peptide in peptic d igests by tandem MS. Site-directed mutagenesis of both Glu-197 and the prospective catalytic acid, Glu-202, results in inactive enzyme, conf irming the critical role of these groups for catalysis.