title: Critical roles of Asp270 and Trp273 in the α-repeat of the carbohydrate-binding module of endo-1,3-β-glucanase for laminarin-binding avidity.
abstract: A carbohydrate-binding module from family 13 (CBM13), appended to the catalytic domain of endo-1,3-β-glucanase from Cellulosimicrobium cellulans, was overexpressed in E. coli, and its interactions with β-glucans, laminarin and laminarioligosaccharides, were analyzed using surface plasmon resonance biosensor and isothermal titration calorimetry. The association constants for laminarin and laminarioligosaccharides were determined to be approximately 10(6) M(-1) and 10(4) M(-1), respectively, indicating that 2 or 3 binding sites in the α-, β-, and γ-repeats of CBM13 are involved in laminarin binding in a cooperative manner. The binding avidity is approximately 2-orders higher than the monovalent binding affinity. Mutational analysis of the conserved Asp residues in the respective repeats showed that the α-repeat primarily contributes to β-glucan binding. A Trp residue is predicted to be exposed to the solvent only in the α-repeat and would contribute to β-glucan binding. The α-repeat bound β-glucan with an affinity of approximately 10(4) M(-1), and the other repeats additionally bound laminarin, resulting in the increased binding avidity. This binding is unique compared to the recognition mode of another CBM13 from Streptomyces lividans xylanase.
authors.affiliation: Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Hangi-cho, Shimogamo, Sakyo-ku, Japan.
authors.forename: Tomonari
authors.initials: T
authors.lastname: Tamashiro
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Tanabe
authors.forename: Teikichi
authors.initials: T
authors.lastname: Ikura
authors.forename: Nobutoshi
authors.initials: N
authors.lastname: Ito
authors.forename: Masayuki
authors.initials: M
authors.lastname: Oda
mesh_terms: D000192:Actinomycetales / Q000201:enzymology*; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry*; D001665:Binding Sites; D002151:Calorimetry; D020134:Catalytic Domain / Q000502:physiology; D002942:Circular Dichroism; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000737:chemistry*; D005936:Glucans; D037102:Lectins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020449:Repetitive Sequences, Amino Acid; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology; D020349:Surface Plasmon Resonance; D014364:Tryptophan / Q000737:chemistry*; D047071:beta-Glucans / Q000378:metabolism
chemical_list: D005936:Glucans; D037102:Lectins; D011134:Polysaccharides; D047071:beta-Glucans; D001224:Aspartic Acid; D014364:Tryptophan; C008247:laminaran; D004693:Glucan Endo-1,3-beta-D-Glucosidase