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 106 M−1 and 104 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 104 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.
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Abbreviations
- CBM:
-
Carbohydrate-binding module
- SPR:
-
Surface plasmon resonance
- ITC:
-
Isothermal titration calorimetry
- His-tag:
-
Polyhistidine-tag
- PCR:
-
Polymerase chain reaction
- Ni-NTA:
-
Ni-nitrilotriacetic acid
- SDS-PAGE:
-
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- CD:
-
Circular dichroism
- DLS:
-
Dynamic light scattering
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Acknowledgments
The authors thank Mr. Takahiro Maruno of Osaka University and Prof. Yuji Kobayashi of Osaka University of Pharmaceutical Sciences for technical support and helpful discussion. This work was partly performed under the Joint Usage/Research Program of Medical Research Institute, Tokyo Medical and Dental University.
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Tamashiro, T., Tanabe, Y., Ikura, T. et al. 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. Glycoconj J 29, 77–85 (2012). https://doi.org/10.1007/s10719-011-9366-x
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DOI: https://doi.org/10.1007/s10719-011-9366-x