The multi-ligand binding first family 35 Carbohydrate Binding Module (CBM35) of Clostridium thermocellum targets rhamnogalacturonan I.
Кључне речи
Апстрактан
Carbohydrate Binding Modules (CBMs) targeting cellulose, xylan and mannan have been reported, however, a CBM targeting rhamnogalacturonan I (RG I) has never been identified. We had studied earlier a rhamnogalacturonan lyase (CtRGL) from Clostridium thermocellum that was associated with a family 35 CBM, Rgl-CBM35. In this study we show that Rgl-CBM35 displays binding with β-d-glucuronic acid (β-D-GlcpA), Δ4,5-anhydro-d-galactopyranosyluronic acid (Δ4,5-GalpA), rhamnogalacturonan I, arabinan, galactan, glucuronoxylans and arabinoxylans. Rgl-CBM35 contains a conserved ligand binding site in the loops known for binding β-D-GlcpA and Δ4,5-GalpA moiety of unsaturated RG I and pectic-oligosaccharides. Mutagenesis revealed that Asn118 plays an important role in binding β-D-GlcpA, Δ4,5-GalpA, sugarbeet arabinan and potato galactan at its conserved ligand binding site present in surface exposed loops. EDTA-treated Rgl-CBM35 showed no affinity towards β-D-GlcpA and Δ4,5-GalpA underscoring Ca2+ mediated ligand recognition. Contrastingly, the EDTA-treated Rgl-CBM35 and its mutant N118A displayed affinity for sugarbeet arabinan and potato galactan. The curtailed affinity of Y37A/N118A and R69A/N118A double mutants towards sugarbeet arabinan emphasized the presence of a second ligand binding site. Rgl-CBM35 is the first CBM reported to primarily target RG I and also is the first member of family 35 CBM possessing at least two ligand binding sites.