title: The family 6 Carbohydrate Binding Module (CtCBM6) of glucuronoxylanase (CtXynGH30) of Clostridium thermocellum binds decorated and undecorated xylans through cleft A.
abstract: 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 Ca(2+) 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.
authors: Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India.
authors: Anil Kumar
authors: AK
authors: Verma
authors: CIISA-Faculdade de Medicina Veterinária, Avenida da Universidade Técnica, 1300-477 Lisbon, Portugal.
authors: Pedro
authors: P
authors: Bule
authors: CIISA-Faculdade de Medicina Veterinária, Avenida da Universidade Técnica, 1300-477 Lisbon, Portugal.
authors: Teresa
authors: T
authors: Ribeiro
authors: CIISA-Faculdade de Medicina Veterinária, Avenida da Universidade Técnica, 1300-477 Lisbon, Portugal.
authors: Joana L A
authors: JL
authors: Brás
authors: Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, India.
authors: Joyeeta
authors: J
authors: Mukherjee
authors: Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, India.
authors: Munishwar N
authors: MN
authors: Gupta
authors: CIISA-Faculdade de Medicina Veterinária, Avenida da Universidade Técnica, 1300-477 Lisbon, Portugal.
authors: Carlos M G A
authors: CM
authors: Fontes
authors: Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India. Electronic address: arungoyl@iitg.ernet.in.
authors: Arun
authors: A
authors: Goyal
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002151:Calorimetry; D002942:Circular Dichroism; D048013:Acetivibrio thermocellus / Q000201:enzymology*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D056004:Molecular Dynamics Simulation; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D014990:Xylans / Q000378:metabolism*; D014995:Xylosidases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D014990:Xylans; D014995:Xylosidases; C068045:glucuronoxylan xylanohydrolase
keywords: Binding cleft; Clostridium thermocellum; Ligand binding affinity and isothermal titration calorimetry