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J. Biol. Chem., Vol. 276, Issue 38, 35629-35635, September 21, 2001
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From the Department of Synthetic Chemistry and Biological
Chemistry, Graduate School of Engineering, Kyoto University,
Yoshida-honmachi, Sakyo-ku, Kyoto 606-8501, Japan, and Core
Research for Evolutional Science and Technology Program of Japan
Science and Technology Corporation (CREST-JST),
Kawaguchi, Saitama 332-0012, Japan
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.
Different Cleavage Specificities of the Dual Catalytic Domains in
Chitinase from the Hyperthermophilic Archaeon Thermococcus
kodakaraensis KOD1*
*
This work was supported by the Japan Science and Technology
Corporation for Core Research for Evolutional Science and Technology (T. I.) and by a grant-in-aid for the Japan Society for the Promotion of Science Fellows (to T. T.) from the Ministry of Education, Culture,
Sports, Science and Technology.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Synthetic Chemistry and Biological Chemistry, Graduate School of
Engineering, Kyoto University, Yoshida-honmachi, Sakyo-ku, Kyoto
606-8501, Japan. Tel.: 81-75-753-5568; Fax: 81-75-753-4703; E-mail:
imanaka@sbchem.kyoto-u.ac.jp.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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