title: Different cleavage specificities of the dual catalytic domains in chitinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1.
abstract: The chitinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1, Tk-ChiA, has an interesting multidomain structure containing dual catalytic domains and triple chitin-binding domains. To determine the biochemical properties of each domain, we constructed deletion mutant genes corresponding to the individual catalytic domains and purified the recombinant proteins. A synergistic effect was observed when chitin was degraded in the presence of both catalytic domains, suggesting different cleavage specificity of these domains. Analyses of degradation products from N-acetyl-chitooligosaccharides and their chromogenic derivatives with thin layer chromatography indicated that the N-terminal catalytic domain mainly hydrolyzed the second glycosidic bond from the nonreducing end of the oligomers, whereas the C-terminal domain randomly hydrolyzed glycosidic bonds other than the first bond from the nonreducing end. Both catalytic domains formed diacetyl-chitobiose as a major end product and possessed transglycosylation activity. Further analysis of degradation products from colloidal chitin with high performance liquid chromatography showed that the N-terminal catalytic domain exclusively liberated diacetyl-chitobiose, whereas reactions with the C-terminal domain led to N-acetyl-chitooligosaccharides of various lengths. These results demonstrated that the N-terminal and C-terminal catalytic domains functioned as exo- and endochitinases, respectively. The biochemical results provide a physiological explanation for the presence of two catalytic domains with different specificity and suggest a cooperative function between the two on a single polypeptide in the degradation of chitin.
authors.affiliation: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida-honmachi, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Tanaka
authors.forename: T
authors.initials: T
authors.lastname: Fukui
authors.forename: T
authors.initials: T
authors.lastname: Imanaka
mesh_terms: D001483:Base Sequence; D020134:Catalytic Domain; D002688:Chitinases / Q000378:metabolism*; D017931:DNA Primers; D006868:Hydrolysis; D016296:Mutagenesis; D017384:Sequence Deletion; D013379:Substrate Specificity; D019712:Thermococcus / Q000201:enzymology*
chemical_list: D017931:DNA Primers; D002688:Chitinases