THE ROLES OF KEY RESIDUES IN THE 4/7 SUPERFAMILY OF GLYCOSYL HYDROLASES REVEALED BY CELLULASE:SUBSTRATE COMPLEX. Joshua Sakon^l, Steven Thomas², Michael Himmel² and P. Andrew Karplus¹, Section of Biochemistry, Molecular and Cell Biology, Cornell University,Ithaca, NY 14853¹, National Renewable Energy Labs, Golden, CO 80401²

Cellulase E1 from Acidothermus cellulolyticus is a member of a large superfamily of [[beta]]-glycosyl hydrolases characterized by a retaining mechanism and ([[alpha]]/[[beta]])₈-barrel fold with three invariant active site residues: an adjacent Asn-Glu pair at the end of [[beta]]- strand 4, in which the Glu serves as the acid/base in catalysis, and a nucleophilic Glu residue at the end of [[beta]]-strand 7. The superfamily, encompasses families 1 ([[beta]]-glucosidase, lactase phlorizin hydrolase, 6-phospho [[beta]]-glucosidase, 6-phospho [[beta]] galactosidase, [[beta]]-galactosidase, cyanogenic [[beta]]-glucosidase), 2 ([[beta]]- galactosidase, [[beta]]-glucuronidase), 5 (cellulase, [[beta]]-mannase), 10 (xylanase), 17 ([[beta]]-1,3-1,4-glucanase), 30 (glucocerebrosidase), 35 ([[beta]]-galactosidase), 39 ([[alpha]]-L-Iduronidase) and 42 ([[beta]]-galactosidase). The crystal structure of the catalytic domain of E 1 complexed to cellotetraose solved by multiple isomorphous replacement method and refined to 2.4 Å resolution (R=17.9 % and R_free=23.8%), reveals the functional interactions made by the three known conserved residues: the nucleophilic glutamate is poised to attack the anomeric carbon; the acid/base glutamate hydrogen-bonds to the glycosidic oxygen; and the conserved asparagine hydrogen-bonds to the C2-hydroxyl near the cleavage site. A close approach of two key glutamate residues provides an elegant mechanism for the shift in the pK_a of the acid/base for the glycosylation and deglycosylation half-reactions. The structure also identifies and defines the roles of five further residues which are well-conserved within the superfamily. The structure is entirely consistent with a large body of kinetic data observed for wild-type and mutated forms of superfamily members, and allows us to extend the known chemical mechanism with a detailed sequence of physical steps that we propose are involved in catalysis by the enzymes. This superfamily includes a large number of cellulases, so the insights will aid protein engineering efforts to improve cellulase activities for use in biomass conversion.