title: Interaction of a goose-type lysozyme with chitin oligosaccharides as determined by NMR spectroscopy.
abstract: The interaction between a goose-type lysozyme from ostrich egg white (OEL) and chitin oligosaccharides [(GlcNAc)(n) (n = 2, 4 and 6)] was studied by nuclear magnetic resonance (NMR) spectroscopy. A stable isotope-labelled OEL was produced in Pichia pastoris, and backbone resonance assignments for the wild-type and an inactive mutant (E73A OEL) were achieved using modern multi-dimensional NMR techniques. NMR titration was performed with (GlcNAc)(n) for mapping the interaction sites of the individual oligosaccharides based on the shifts in the two-dimensional heteronuclear single quantum correlation (HSQC) resonances. In wild-type OEL, the interaction sites for (GlcNAc)(n) were basically similar to those determined by X-ray crystallography. In E73A OEL, however, the interaction sites were spread more widely over the substrate-binding cleft than expected, due to the multiple modes of binding. The association constant for E73A OEL and (GlcNAc)(6) calculated from the shifts in the Asp97 resonance (7.2 × 10(3) M(-1)) was comparable with that obtained by isothermal titration calorimetry (5.3 × 10(3) M(-1)). The interaction was enthalpy-driven as judged from the thermodynamic parameters (ΔH = -6.1 kcal/mol and TΔS = -1.0 kcal/mol). This study provided novel insights into the oligosaccharide binding mechanism and the catalytic residues of the enzymes belonging to family GH-23.
authors.affiliation: Department of Advanced Bioscience, Kinki University, Nakamachi, Nara, Japan.
authors.forename: Shoko
authors.initials: S
authors.lastname: Shinya
authors.forename: Takayuki
authors.initials: T
authors.lastname: Ohnuma
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Kawamura
authors.forename: Takao
authors.initials: T
authors.lastname: Torikata
authors.forename: Shigenori
authors.initials: S
authors.lastname: Nishimura
authors.forename: Etsuko
authors.initials: E
authors.lastname: Katoh
authors.forename: Tamo
authors.initials: T
authors.lastname: Fukamizo
mesh_terms: D000818:Animals; D002686:Chitin / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009113:Muramidase / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism*; D011485:Protein Binding; D020310:Struthioniformes
chemical_list: D009844:Oligosaccharides; D002686:Chitin; D009113:Muramidase