CRYSTAL-STRUCTURE OF THE CATALYTIC DOMAIN OF THE BETA-1,4-GLYCANASE CEX FROM CELLULOMONAS-FIMI

beta-1,4-Glycanases, principally cellulases and xylanases, are respons ible for the hydrolysis of plant biomass. The bifunctional beta-1,4-xy lanase/glucanase Cex from the bacterium Cellulomonas fimi, one of a la rge family of cellulases/xylanases, depolymerizes oligosaccharides and releases a disaccharide unit from the substrate nonreducing end. Hydr olysis occurs with net retention of the anomeric configuration of the sugar through a double-displacement mechanism involving a covalent gly cosyl-enzyme intermediate. The active site nucleophile, Glu233, has be en unambiguously identified by trapping of such an intermediate [Tull et al. (1991) J. Biol. Chem. 266, 15621-15625] and the acid/base catal yst, Glu127, by detailed kinetic analysis of mutants [MacLeod et al. ( 1994) Biochemistry 33, 6371-6376]. However, little is known about the enzyme's overall folding and its active site architecture. We report h ere the high-resolution crystal structure of the catalytic domain of C ex. The atomic structure refinement results in a model that includes 2 400 protein atoms and 45 water molecules, with an R-factor of 0.217 fo r data extending to 1.8-Angstrom resolution. The protein forms an eigh t-parallel-stranded alpha/beta-barrel, which is a novel folding patter n for a microbial beta-glycanase. The active site, inferred from the l ocation of Glu233, Glu127, and other conserved residues, is an open cl eft on the carboxy-terminal end of the alpha/beta-barrel. An extensive hydrogen-bonding network stabilizes the ionization states of the key residues; in particular, the Asp235-His205-Glu233 hydrogen-bonding net work may play a role in modulating the ionization state of Glu233 and in controlling local charge balance during the reaction.