title: Kinetic and crystallographic analyses of the catalytic domain of chitinase from Pyrococcus furiosus- the role of conserved residues in the active site.
abstract: 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.
authors.affiliation: Laboratory of Protein Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Tsuji
authors.forename: Shigenori
authors.initials: S
authors.lastname: Nishimura
authors.forename: Takashi
authors.initials: T
authors.lastname: Inui
authors.forename: Yuji
authors.initials: Y
authors.lastname: Kado
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Ishikawa
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Nakamura
authors.forename: Koichi
authors.initials: K
authors.lastname: Uegaki
mesh_terms: D020134:Catalytic Domain; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008958:Models, Molecular; D020027:Pyrococcus furiosus
chemical_list: D002688:Chitinases