Crystal Structures of the Catalytic Domain of a Novel Glycohydrolase Family 23 Chitinase from Ralstonia sp. A-471 Reveals a Unique Arrangement of the Catalytic Residues for Inverting Chitin Hydrolysis

• Published in: The Journal of biological chemistry Vol. 288; no. 26; pp. 18696 - 18706
• Main Authors: Arimori, Takao, Kawamoto, Noriko, Shinya, Shoko, Okazaki, Nobuo, Nakazawa, Masami, Miyatake, Kazutaka, Fukamizo, Tamo, Ueda, Mitsuhiro, Tamada, Taro
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 28.06.2013; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Animals; Bacterial Proteins - chemistry; Catalytic Domain; Chitin - chemistry; Chitinase; Chitinases - chemistry; Crystal Structure; Crystallography, X-Ray; Gadus morhua; Glycoside Hydrolases; Glycoside Hydrolases - chemistry; Hydrolysis; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Mutagenesis Site Specific; Mutagenesis, Site-Directed; Mutation; Oligosaccharide; Plant Proteins; Protein Structure and Folding; Ralstonia - enzymology; Sequence Homology, Amino Acid; Substrate Specificity; Chitinase; Crystal Structure; Glycoside Hydrolases; Oligosaccharide; Mutagenesis Site Specific
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.462135

Chitinase C from Ralstonia sp. A-471 (Ra-ChiC) has a catalytic domain sequence similar to goose-type (G-type) lysozymes and, unlike other chitinases, belongs to glycohydrolase (GH) family 23. Using NMR spectroscopy, however, Ra-ChiC was found to interact only with the chitin dimer but not with the peptidoglycan fragment. Here we report the crystal structures of wild-type, E141Q, and E162Q of the catalytic domain of Ra-ChiC with or without chitin oligosaccharides. Ra-ChiC has a substrate-binding site including a tunnel-shaped cavity, which determines the substrate specificity. Mutation analyses based on this structural information indicated that a highly conserved Glu-141 acts as a catalytic acid, and that Asp-226 located at the roof of the tunnel activates a water molecule as a catalytic base. The unique arrangement of the catalytic residues makes a clear contrast to the other GH23 members and also to inverting GH19 chitinases. Background: Chitinase C from Ralstonia sp. A-471 (Ra-ChiC) is a chitinase that was first found in glycohydrolase family 23. Results: The crystal structure of Ra-ChiC exhibited a tunnel-shaped conformation in its active site. Conclusion: The tunnel-shaped conformation is essential for a unique arrangement of the catalytic residues and substrate specificity. Significance: This is the first report on the tunnel-shaped binding site of an inverting chitinase.