title: Crystal structure of a "loopless" GH19 chitinase in complex with chitin tetrasaccharide spanning the catalytic center.
abstract: The structure of a GH19 chitinase from the moss Bryum coronatum (BcChi-A) in complex with the substrate was examined by X-ray crystallography and NMR spectroscopy in solution. The X-ray crystal structure of the inactive mutant of BcChi-A (BcChi-A-E61A) liganded with chitin tetramer (GlcNAc)4 revealed a clear electron density of the tetramer bound to subsites -2, -1, +1, and +2. Individual sugar residues were recognized by several amino acids at these subsites through a number of hydrogen bonds. This is the first crystal structure of GH19 chitinase liganded with oligosaccharide spanning the catalytic center. NMR titration experiments of chitin oligosaccharides into the BcChi-A-E61A solution showed that the binding mode observed in the crystal structure is similar to that in solution. The C-1 carbon of -1 GlcNAc, the Oε1 atom of the catalytic base (Glu70), and the Oγ atom of Ser102 form a "triangle" surrounding the catalytic water, and the arrangement structurally validated the proposed catalytic mechanism of GH19 chitinases. The glycosidic linkage between -1 and +1 sugars was found to be twisted and under strain. This situation may contribute to the reduction of activation energy for hydrolysis. The complex structure revealed a more refined mechanism of the chitinase catalysis.
authors.affiliation: Department of Advanced Bioscience, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Ohnuma
authors.affiliation: Department of Advanced Bioscience, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan.
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Umemoto
authors.affiliation: Department of Advanced Bioscience, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Nagata
authors.affiliation: Department of Advanced Bioscience, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan.
authors.forename: Shoko
authors.initials: S
authors.lastname: Shinya
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566, Japan. Electronic address: t-numata@aist.go.jp.
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Numata
authors.affiliation: Department of Bioscience and Biotechnology, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan.
authors.forename: Toki
authors.initials: T
authors.lastname: Taira
authors.affiliation: Department of Advanced Bioscience, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan. Electronic address: fukamizo@nara.kindai.ac.jp.
authors.forename: Tamo
authors.initials: T
authors.lastname: Fukamizo
mesh_terms: D019068:Bryopsida / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D020134:Catalytic Domain; D002686:Chitin / Q000737:chemistry* / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006860:Hydrogen Bonding; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009154:Mutation; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; D002686:Chitin; D002688:Chitinases
keywords: Catalytic mechanism; Chitin oligosaccharide; Crystal structure; GH19 chitinase; NMR spectroscopy