title: Crystal structure and chitin oligosaccharide-binding mode of a 'loopful' family GH19 chitinase from rye, Secale cereale, seeds.
abstract: The substrate-binding mode of a 26-kDa GH19 chitinase from rye, Secale cereale, seeds (RSC-c) was investigated by crystallography, site-directed mutagenesis and NMR spectroscopy. The crystal structure of RSC-c in a complex with an N-acetylglucosamine tetramer, (GlcNAc)(4) , was successfully solved, and revealed the binding mode of the tetramer to be an aglycon-binding site, subsites +1, +2, +3, and +4. These are the first crystallographic data showing the oligosaccharide-binding mode of a family GH19 chitinase. From HPLC analysis of the enzymatic reaction products, mutation of Trp72 to alanine was found to affect the product distribution obtained from the substrate, p-nitrophenyl penta-N-acetyl-β-chitopentaoside. Mutational experiments confirmed the crystallographic finding that the Trp72 side chain interacts with the +4 moiety of the bound substrate. To further confirm the crystallographic data, binding experiments were also conducted in solution using NMR spectroscopy. Several signals in the (1) H-(15) N HSQC spectrum of the stable isotope-labeled RSC-c were affected upon addition of (GlcNAc)(4) . Signal assignments revealed that most signals responsive to the addition of (GlcNAc)(4) are derived from amino acids located at the surface of the aglycon-binding site. The binding mode deduced from NMR binding experiments in solution was consistent with that from the crystal structure.
authors:  Department of Advanced Bioscience, Kinki University, Nara, Japan Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.
authors: Takayuki
authors: T
authors: Ohnuma
authors:  Department of Advanced Bioscience, Kinki University, Nara, Japan Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.
authors: Tomoyuki
authors: T
authors: Numata
authors:  Department of Advanced Bioscience, Kinki University, Nara, Japan Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.
authors: Takuo
authors: T
authors: Osawa
authors:  Department of Advanced Bioscience, Kinki University, Nara, Japan Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.
authors: Hideko
authors: H
authors: Inanaga
authors:  Department of Advanced Bioscience, Kinki University, Nara, Japan Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.
authors: Yoko
authors: Y
authors: Okazaki
authors:  Department of Advanced Bioscience, Kinki University, Nara, Japan Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.
authors: Shoko
authors: S
authors: Shinya
authors:  Department of Advanced Bioscience, Kinki University, Nara, Japan Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.
authors: Kaori
authors: K
authors: Kondo
authors:  Department of Advanced Bioscience, Kinki University, Nara, Japan Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.
authors: Tatsuya
authors: T
authors: Fukuda
authors:  Department of Advanced Bioscience, Kinki University, Nara, Japan Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.
authors: Tamo
authors: T
authors: Fukamizo
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D018360:Crystallography, X-Ray; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012434:Secale / Q000201:enzymology*; D012639:Seeds / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D002686:Chitin; D002688:Chitinases