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J. Biol. Chem., Vol. 276, Issue 44, 41343-41349, November 2, 2001
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From the Four exposed aromatic residues, two in the
N-terminal domain (Trp-69 and Trp-33) and two in the catalytic domain
(Trp-245 and Phe-232) of Serratia marcescens chitinase A,
are linearly aligned with the deep catalytic cleft. To investigate the
importance of these residues in the binding activity and hydrolyzing
activity against insoluble chitin, site-directed mutagenesis to alanine was carried out. The substitution of Trp-69, Trp-33, or Trp-245 significantly reduced the binding activity to both highly crystalline
Roles of the Exposed Aromatic Residues in Crystalline
Chitin Hydrolysis by Chitinase A from Serratia marcescens
2170*
,,,
Department of Applied Biological Chemistry,
Faculty of Agriculture, Niigata University, 8050 Ikarashi-2, Niigata
950-2181, Japan, the § Department of BioEngineering, Nagaoka
University of Technology, Nagaoka, Niigata 940-2188, Japan, and
¶ Wood Research Institute, Kyoto University, Uji, Kyoto, 611-0011, Japan
-chitin and colloidal chitin. The substitution of Phe-232, which is
located closest to the catalytic cleft, did not affect the binding
activity. On the other hand, the hydrolyzing activity against
-chitin microfibrils was significantly reduced by the substitution
of any one of the four aromatic residues including Phe-232. None of the
mutations reduced the hydrolyzing activity against soluble substrates.
These results clearly demonstrate that the four exposed aromatic
residues are essential determinants for crystalline chitin hydrolysis.
Three of them, two in the N-terminal domain and one in the catalytic
domain, play vital roles in the chitin binding. Phe-232 appeared to be
important for guiding the chitin chain into the catalytic cleft. Based
on these observations, a model for processive hydrolysis of crystalline
chitin by chitinase A is proposed.
*
This work was supported in part by Grant-in-aid for
Scientific Research 12660070 from the Ministry of Education, Science, and Culture of Japan.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. Tel.:
81-25-262-6647; Fax: 81-25-262-6854; E-mail:
wata@agr.niigata-u.ac.jp.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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