title: Tryptophan introduction can change β-glucan binding ability of the carbohydrate-binding module of endo-1,3-β-glucanase.
abstract: Endo-1,3-β-glucanase from Cellulosimicrobium cellulans DK-1 has a carbohydrate-binding module (CBM-DK) at the C-terminal side of a catalytic domain. Out of the imperfect tandem α-, β-, and γ-repeats in CBM-DK, the α-repeat primarily contributes to β-glucan binding. This unique feature is derived from Trp273 in α-repeat, whose corresponding residues in β- and γ-repeats are Asp314 and Gly358, respectively. In this study, we generated Trp-switched mutants, W273A/D314W, D270A/W273A/D314W, W273A/G358W, and D270A/W273A/G358W, and analyzed their binding abilities toward laminarioligosaccharides and laminarin. While the binding affinities of D270A/W273A and W273A mutants were either lost or much lower than that of the wild-type, those of Trp-switched mutants recovered, indicating that a Trp introduction in β- or γ-repeat can substitute the α-repeat by primarily contributing to β-glucan binding. Thus, we have successfully engineered a CBM-DK that binds to laminarin by a mechanism different from that of the wild-type, but with similar affinity.
authors.affiliation: a Graduate School of Life and Environmental Sciences, Kyoto Prefectural University , Kyoto , Japan.
authors.forename: Ayako
authors.initials: A
authors.lastname: Miki
authors.affiliation: a Graduate School of Life and Environmental Sciences, Kyoto Prefectural University , Kyoto , Japan.
authors.forename: Satomi
authors.initials: S
authors.lastname: Inaba
authors.affiliation: b Graduate School of Engineering, Osaka University , Suita , Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Maruno
authors.affiliation: b Graduate School of Engineering, Osaka University , Suita , Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Kobayashi
authors.affiliation: a Graduate School of Life and Environmental Sciences, Kyoto Prefectural University , Kyoto , Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Oda
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007795:Laminaria / Q000201:enzymology; D009154:Mutation; D011485:Protein Binding; D012091:Repetitive Sequences, Nucleic Acid; D014364:Tryptophan; D000070660:Wolfiporia / Q000201:enzymology; D047071:beta-Glucans / Q000378:metabolism*
chemical_list: D047071:beta-Glucans; D014364:Tryptophan; D004693:Glucan Endo-1,3-beta-D-Glucosidase
keywords: isothermal titration calorimetry; protein engineering; repeat preference for β-glucan binding; surface plasmon resonance biosensor