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J. Biol. Chem., Vol. 277, Issue 17, 14695-14702, April 26, 2002

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Biochemical and Genetic Properties of Paenibacillus Glycosyl Hydrolase Having Chitosanase Activity and Discoidin Domain^*

Hisashi Kimoto^§, Hideo Kusaoke^¶, Ikkyu Yamamoto, Yutaka Fujii, Takashi Onodera^¶, and Akira Taketo^¶

From the Departments of  Biochemistry I and  Chemistry, Faculty of Medicine, Fukui Medical University, 23-3 Shimoaizuki, Matsuoka, Fukui 910-1193, Japan and the ^¶ Department of Applied Physics and Chemistry, Fukui University of Technology, 3-6-1 Gakuen, Fukui 910, Japan

Cells of "Paenibacillus fukuinensis" D2 produced chitosanase into surrounding medium, in the presence of colloidal chitosan or glucosamine. The gene of this enzyme was cloned, sequenced, and subjected to site-directed mutation and deletion analyses. The nucleotide sequence indicated that the chitosanase was composed of 797 amino acids and its molecular weight was 85,610. Unlike conventional family 46 chitosanases, the enzyme has family 8 glycosyl hydrolase catalytic domain, at the amino-terminal side, and discoidin domain at the carboxyl-terminal region. Expression of the cloned gene in Escherichia coli revealed -1,4-glucanase function, besides chitosanase activity. Analyses by zymography and immunoblotting suggested that the active enzyme was, after removal of signal peptide, produced from inactive 81-kDa form by proteolysis at the carboxyl-terminal region. Replacements of Glu¹¹⁵ and Asp¹⁷⁶, highly conserved residues in the family 8 glycosylase region, with Gln and Asn caused simultaneous loss of chitosanase and glucanase activities, suggesting that these residues formed part of the catalytic site. Truncation experiments demonstrated indispensability of an amino-terminal region spanning 425 residues adjacent to the signal peptide.

The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession number(s) AB006819.

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