Structural changes of the active site tunnel of Humicola insolens cellobiohydrolase, Cel6A, upon oligosaccharide binding

The mechanisms of crystalline cellulose degradation by cellulases are of pa ramount importance for the exploitation of these enzymes in applied process es, such as biomass conversion. Cellulases have traditionally been classifi ed into cellobiohydrolases, which are effective in the degradation of cryst alline materials, and endoglucanases, which appear to act on "soluble" regi ons of the substrate. Humicola insolens Cel6A (CBH II) is a cellobiohydrola se from glycoside hydrolase family 6 whose native structure has been determ ined at 1.9 Angstrom resolution [Varrot, A., Hastrup, S., Schulein, M., and Davies, G. J. (1999) Biochem. J. 337, 297-304]. Here we present the struct ure of the catalytic core domain of Humicola insolens cellobiohydrolase II Cel6A in complex with glucose/cellotetraose at 1.7 Angstrom resolution. Cry stals of Cel6A, grown in the presence of cellobiose, reveal six binding sub sites, with a single glucose moiety bound in the -2 subsite and cellotetrao se in the +1 to +4 subsites. The complex structure is strongly supportive o f the assignment of Asp 226 as the catalytic acid and consistent with propo sals that Asp 405 acts as the catalytic base. The structure undergoes sever al conformational changes upon substrate binding,. the most significant of which is a closing of the two active site loops (residues 174-196 and 397-4 35) with main-chain movements of up to 4.5 Angstrom observed. This complex not only defines the polysaccharide-enzyme interactions but also provides t he first three-dimensional demonstration of conformational change in this c lass of enzymes.