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.

Tryptophan introduction can change β-glucan binding ability of the carbohydrate-binding module of endo-1,3-β-glucanase

“…Using mutational experiments, we showed that Asp270 primarily contributes to the binding of laminarioligosaccharides and laminarin, and Asp311 and Asp355 additionally contribute to the multivalent binding of laminarin. 9) We also showed that the unique α-repeat preference for β-glucan binding of CBM-DK is due to the Trp273 residue in α-repeat, which could be exposed to the solvent and interact with β-glucan. In the amino acid sequence of CBM-DK, Trp273 corresponding residues in βand γ-repeats are Asp314 and Gly358, respectively (Fig.…”

“…The protein with an N-terminal polyhistidine-tag was overexpressed in E. coli, and purified as described previously. 9) The protein concentration was calculated from absorbance of 280 nm using molar extinction coefficient, 3.5 × 10 4 M −1 cm −1 for the wild-type and its modified values for the mutants, depending on the number of Trp.…”

“…6,7) The CBMs in family 13 have a β-trefoil fold displaying the pseudo-3-fold symmetry arising from imperfect tandem α-, β-, and γ-repeats, each of which has a putative carbohydratebinding site. 8) We recently found that the α-repeat of CBM-DK primarily contributes to β-glucan binding, 9) a property different from other CBMs in family 13 from Streptomyces lividans xylanase 10A and Streptomyces olivaceoviridis E-86 xylanase. 10,11) The CBM13 lectinlike xylan binding domain from Streptomyces lividans xylanase 10A was reported to have different repeat preferences for the respective substrates.…”

“…In the SPR system, the multivalent binding of laminarin can involve several CBM molecules immobilized on the sensor chip, increasing the binding strength depending on the laminarin concentration, as reported previously. 9) In contrast, ITC can detect the interaction of laminarin with a single CBM-DK molecule. The SPR and ITC results clearly showed that the α-repeat preference of CBM-DK for β-glucan binding could be switched by Trp introduction in other repeats.…”

Characterization of a recombinant multifunctional glycoside hydrolase family 3 β-xylosidase/α-l-arabinofuranosidase/β-glucosidase from Cellulosimicrobium cellulans sp. 21

“…Previously, several bifunctional ␣-l-arabinosidases/␤-xylosidases and multifunctional ␤-glucosidases/␤-xylosidases/␣-arabinosidases belonging to GH family 3 have been characterized [11][12][13]. Cellulosimicrobium cellulans, previously designated as Oerskovia xanthineolytica, is an actinobacteria with yeast-lytic glucanases activity [14,15]. Previously, C. cellulans was reported to produce hemicellulose-degradation enzymes in culture medium when using corn cob as sole carbon source [16].…”