STRUCTURAL STUDIES OF GLYCOSYL HYDROLASE CLAN C ENZYMES
G. Sulzenbacher 3, C. Dupont2, V.S. Lamzin1 , K.S. Wilson3 & G.J. Davies3
1EMBL, Hamburg Outstation, DESY,
Notkestr. 85, 22603 Hamburg, Germany;
2Centre de Recherché en Microbiologie
Appliquée, Institut Armand-Frappier, Université du Québec,
Laval-des-Rapides, C.P. 100, Québec, Canada;
3Department of Chemistry, University of York,
Heslington, York YO1 5DD, England.
Glycosyl hydrolases, enzymes hydrolysing the glycosidic bond between two or more carbohydrates or between a carbohydrate and a non-carbohydrate moiety, have been classified into over 60 families (Henrissat, 1991). Recently a number of those families have been grouped into clans by protein sequence comparison methods, e.g. Hydrophobic Cluster Analysis, HCA, and the members of each clan are predicted to have related protein fold and catalytic machinery (Henrissat et al., 1995).
Glycosyl hydrolase Clan C groups glycosyl hydrolase families 11 and 12, which comprise the low molecular weight xylanases and cellulases, respectively. Family 12 cellulases act on the b-1,4-glycosidic bonds of cellulose, whereas family 11 xylanases perform hydrolysis of the b-1,4-glycosidic linkages in xylan. Glycosyl Hydrolase Clan C enzymes work in an endocatalytic fashion and lead to products with retained stereochemistry on the anomeric carbon. Structures of several family 11 xylanases have been solved in the last years (Havukainen et al., 1996; Rouvinen et al., 1990; Wakarchuk et al., 1994), whereas no 3D structure was available for the family 12 cellulases.
We have collected data on the crystal of the Bacillus pumilus xylanase at a pH where the enzyme has maximum activity to 1.15 A resolution on the EMBL X11 beam line and have refined the structure taking in account anisotropic displacement parameters. Recently we have also solved the X-ray structure of the family 12 endoglucanase CelB2 from Streptomyces lividans to 1.75 A resolution by MIR methods (Sulzenbacher et al., 1997). As predicted by HCA (Törrönen et al., 1993), this cellulase shares the "jelly-roll" topology observed for the family 11 xylanases, with two antiparallel b-sheets sitting on top of each other and enclosing a long open cleft on one side of the protein. The catalytic residues are located in this cleft at a distance of approximately 7 A, as it is expected from enzymes with endocatalytic activity. The similarity in fold and catalytic machinery between the two enzyme families is striking, with significant differences occurring only on the protein surfaces.
In order to elucidate the nature of substrate
binding to CelB2 we have determined the structure of the enzyme
in complex with dinitrophenyl-2F-cellotriose to 1.2 A resolution.
The ligand is present in the active site in 2 distinct
conformations, one of them corresponding to the cleaved substrate
and the other to the catalytic glycosyl-enzyme intermediate. This
structure sheds light on the differences in substrate specificity
between the family 11 xylanases and family 12 cellulases.