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J. Biochem, 2004, Vol. 136, No. 2 163-168
© 2004 The Japanese Biochemical Society

BIOCHEMISTRY

Importance of Exposed Aromatic Residues in Chitinase B from Serratia marcescens 2170 for Crystalline Chitin Hydrolysis

Fuminori Katouno1, Masashi Taguchi1, Kengo Sakurai1, Taku Uchiyama1, Naoki Nikaidou1, Takamasa Nonaka2, Junji Sugiyama3 and Takeshi Watanabe1,*

1 Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, 8050 Ikarashi-2, Niigata 950-2181; 2 Department of BioEngineering, Nagaoka University of Technology, Nagaoka 940-2188; and 3 Wood Research Institute, Kyoto University, Uji 611-0011

Chitinase B (ChiB) of S. marcescens has five exposed aromatic residues linearly aligned toward the catalytic cleft, Tyr481 and Trp479 in the C-terminal domain, and Trp252, Tyr240 and Phe190 in the catalytic domain. To determine the contribution of these residues to the hydrolysis of crystalline ß-chitin, site-directed mutagenesis, to replace them by alanine, was carried out. The Y481A, W479A, W252A, and Y240A mutations all decreased the binding activity and hydrolyzing activity toward ß-chitin microfibrils. Substitution of Trp residues affected the binding activity more severely than that of Tyr residues. The F190A mutation decreased neither the binding activity nor the hydrolyzing activity. None of the mutations decreased the hydrolyzing activity toward soluble substrates. These results suggest that ChiB hydrolyzes crystalline ß-chitin via a mechanism in which four exposed aromatic residues play important roles, similar to the mechanism of hydrolysis by ChiA of this bacterium, although the directions of hydrolysis of the two chitinases are opposite.

* To whom correspondence should be addressed. Tel: +81-25-262-6647, Fax: +81-25-262-6854, E-mail: wata{at}agr.niigata-u.ac.jp


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