CRYSTAL-STRUCTURE OF THERMOSTABLE FAMILY-5 ENDOCELLULASE E1 FROM ACIDOTHERMUS-CELLULOLYTICUS IN COMPLEX WITH CELLOTETRAOSE

The crystal structure of the catalytic domain of the thermostable endo cellulase E1 from Acidothermus cellulolyticus in complex with cellotet raose has been solved by multiple isomorphous replacement and refined at 2.4 Angstrom resolution to an R-factor of 0.18 (R(free) = 0.24). E1 cd is a member of the 4/7 superfamily of hydrolases, and as expected, its structure is an (alpha/beta)(8) barrel, which constitutes a protot ype for family 5-subfamily 1 cellulases. The cellotetraose molecule bi nds in a manner consistent with the expected Michaelis complex for the glycosylation half-reaction and reveals that all eight residues conse rved in family 5 enzymes are involved in recognition of the glycosyl g roup attacked during cleavage. Whereas only three residues are conserv ed in the whole 4/7 superfamily (the Asn/Glu duo and the Glu from whic h the name is derived), structural comparisons show that all eight res idues conserved in family 5 have functional equivalents in the other 4 /7 superfamily members, strengthening the case that mechanistic detail s are conserved throughout the superfamily. On the basis of the struct ure, a detailed sequence of physical steps of the cleavage mechanism i s proposed. 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. Finally, purely str uctural based comparisons are used to show that significant difference s exist in structural similarity scores resulting from different metho ds and suggest that caution should be exercised in interpreting such r esults in terms of implied evolutional relationships.