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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

2013; Elsevier BV; Volume: 288; Issue: 26 Linguagem: Inglês

10.1074/jbc.m113.462135

1083-351X

Takao Arimori, Noriko Kawamoto, Shoko Shinya, Nobuo Okazaki, Masami Nakazawa, Kazutaka Miyatake, Tamo Fukamizo, Mitsuhiro Ueda, Taro Tamada,

Biofuel production and bioconversion

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. 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.