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

•CtCBM6 has two potential binding clefts, cleft A and cleft B.•Cleft A key residues are Tyr28, Phe84 and Asn112.•Cleft B key residues Tyr34 and Tyr104 but inaccessible due to hindrance by Pro68.•CtCBM6 displays higher affinity towards wheat- and rye-arabinoxylans.•Site-directed mutagenesis revealed Tyr 28 and Phe84 involved in the ligand binding.

CtCBM6 of glucuronoxylan-xylanohydrolase (CtXynGH30) 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 CtCBM6 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 CtCBM6 and that Ca2+ ions did not affect its structure stability and binding affinity significantly. The CtCBM6 structure was modeled and refined and CD spectrum displayed 44% β-strands supporting the predicted structure. CtCBM6 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 CtCBM6 interacts with carbohydrates through cleft A, which recognizes equally well both decorated and un-decorated xylans.

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