The family 6 Carbohydrate Binding Module (CtCBM6) of glucuronoxylanase (CtXynGH30) of Clostridium thermocellum binds decorated and undecorated xylans through cleft A.

Ct CBM6 of glucuronoxylan-xylanohydrolase ( Ct XynGH30) from Clostridium thermocellum was cloned, expressed and purified as a soluble ∼14 kDa protein. Quantitative binding analysis with soluble polysaccharides by affinity electrophoresis and ITC revealed that Ct CBM6 displays similar affinity towards decorated and undecorated xylans by binding wheat- and rye-arabinoxylans, beechwood-, birchwood- and oatspelt-xylan. Protein melting studies confirmed thermostable nature of Ct CBM6 and that Ca 2+ ions did not affect its structure stability and binding affinity significantly. The Ct CBM6 structure was modeled and refined and CD spectrum displayed 44% β-strands supporting the predicted structure. Ct CBM6 displays a jelly roll β-sandwich fold presenting two potential carbohydrate binding clefts, A and B. The cleft A, is located between two loops connecting β4–β5 and β8–β9 strands. Tyr28 and Phe84 present on these loops make a planar hydrophobic binding surface to accommodate sugar ring of ligand. The cleft B, is located on concave surface of β-sandwich fold. Tyr34 and Tyr104 make a planar hydrophobic platform, which may be inaccessible to ligand due to hindrance by Pro68. Site-directed mutagenesis revealed Tyr28 and Phe84 in cleft A, playing a major role in ligand binding. The results suggest that Ct CBM6 interacts with carbohydrates through cleft A, which recognizes equally well both decorated and un-decorated xylans. [ABSTRACT FROM AUTHOR]