STRUCTURAL CHARACTERIZATION OF AN ENDO β-1,3-GLUCANASE OF FAMILY 81 GLYCOSIDE HYDROLASE (CtLam81A) FROM CLOSTRIDIUM THERMOCELLUM

The gene sequence of a family 81 glycoside hydrolase from Clostridium thermocellum Cthe_0660, (GenBank accession number: ABN51485.1) was retrieved from CAZy (Carbohydrate-Active Enzyme) database. The gene (2151 bp) encoding the catalytic module of Cthe_0660, an endo β-1,3-glucanase (CtLam81A) cloned, expressed earlier was used in this study. The secondary structure analysis of CtLam81A by PsiPred showed the presence of 23% α-helices and 23% β-strands whereas, CD analysis showed 24% of α-helices and 24% of β-strands. The secondary structure analysis by Circular Dichroism corroborated the PsiPred results. The 3D modeled structure of CtLam81 generated by Swiss-Model server and energy minimized by Swiss-PDB viewer displayed an N-terminal â-sandwich domain, a (α/α)6 domain and a short β-sandwich domain at C-terminal. The quality assessment of modelled structure by Ramachandran plot displayed 94% residues in favoured region, 4.6% in allowed region and 1.4% in outlier region. The superposition of CtLam81A modeled structure with its closest homologue Bh0236 (PDB ID: 5t49) from Bacillus halodurans indicated that Glu515 acts as a catalytic base, while Asp438 acts as a catalytic acid. Multiple sequence alignment showed that these catalytic residues are conserved within the family 81 glycoside hydrolase. CtLam81A showed lowest binding energy against laminaripentaose and the amino acid residues, Tyr363, Trp364, Tyr430, Trp588, Trp589 and Trp637create the binding pocket at active site to hold the ligand. Keywords : Family 81 glycoside hydrolase; β-1,3-glucanase; Laminari-oligosaccharides; Circular Dichroism; Homology modeling; Docking; Clostridium thermocellum  The gene sequence of a family 81 glycoside hydrolase from Clostridium thermocellum Cthe_0660, (GenBank accession number: ABN51485.1) was retrieved from CAZy (Carbohydrate-Active Enzyme) database. The gene (2151 bp) encoding the catalytic module of Cthe_0660, an endo �-1,3-glucanase (CtLam81A) cloned, expressed earlier was used in this study. The secondary structure analysis of CtLam81A by PsiPred showed the presence of 23% �-helices and 23% �-strands whereas, CD analysis showed 24% of �-helices and 24% of �-strands. The secondary structure analysis by Circular Dichroism corroborated the PsiPred results. The 3D modeled structure of CtLam81 generated by Swiss-Model server and energy minimized by Swiss-PDB viewer displayed an N-terminal â-sandwich domain, a (�/�)6 domain and a short �-sandwich domain at C-terminal. The quality assessment of modelled structure by Ramachandran plot displayed 94% residues in favoured region, 4.6% in allowed region and 1.4% in outlier region. The superposition of CtLam81A modeled structure with its closest homologue Bh0236 (PDB ID: 5t49) from Bacillus halodurans indicated that Glu515 acts as a catalytic base, while Asp438 acts as a catalytic acid. Multiple sequence alignment showed that these catalytic residues are conserved within the family 81 glycoside hydrolase. CtLam81A showed lowest binding energy against laminaripentaose and the amino acid residues, Tyr363, Trp364, Tyr430, Trp588, Trp589 and Trp637create the binding pocket at active site to hold the ligand. Key words: Family 81 glycoside hydrolase; �-1,3-glucanase; Laminari-oligosaccharides; Circular Dichroism; Homology modeling; Docking; Clostridium thermocellum