Kinetic and crystallographic analyses of the catalytic domain of chitinase from Pyrococcus furiosus - the role of conserved residues in the active site

The hyperthermostable chitinase from the hyperthermophilic archaeon Pyrococcus furiosus has a unique multidomain structure containing two chitin-binding domains and two catalytic domains, and exhibits strong crystalline chitin hydrolyzing activity at high temperature. In order to investigate the structure-function relationship of this chitinase, we analyzed one of the catalytic domains (AD2) using mutational and kinetic approaches, and determined the crystal structure of AD2 complexed with chito-oligosaccharide substrate. Kinetic studies showed that, among the acidic residues in the signature sequence of family 18 chitinases (DXDXE motif), the second Asp (D-2) and Glu (E) residues play critical roles in the catalysis of archaeal chitinase. Crystallographic analyses showed that the side-chain of the catalytic proton-donating E residue is restrained into the favorable conformer for proton donation by a hydrogen bond interaction with the adjacent D-2 residue. The comparison of active site conformations of family 18 chitinases provides a new criterion for the subclassification of family 18 chitinase based on the conformational change of the D-2 residue.