Deciphering the structure of a distinctive trimodular cellulosomal licheninase (RfGH16_21), a family 16 glycoside hydrolase from Ruminococcus flavefaciens by computational and experimental methods

Abstract

In order to know the insights of a unique naturally existing trimodular licheninase from GH16 family, sub-family 21 (RfGH16_21) from Ruminococcus flavefaciens, its structure was modeled to understand its functional relations to reveal information regarding modifying the enzyme for improved properties with enhanced catalytic efficiency. Homology modeling revealed three tandem repeats of β-jelly roll like folds linked by natural linkers. Catalytic pockets and the catalytically important amino acids in each tandem repeat of RfGH16_21 determined by multiple sequence alignment and structure superposition with its homologues indicated that two Glu residues are involved in a retaining-type of catalytic mechanism. Sequential molecular docking revealed maximum binding energy with mixed linked cellotriose showing that cellotriose is the lowest oligomeric hydrolysed product formed by the catalytic action of endo-β-1,3–1,4-glucanase. Molecular dynamic (MD) simulation of RfGH16_21-cellotriose complex confirmed the structural specificity of catalytic residues and increased stability of enzyme in presence of ligand as compared to simulated RfGH16_21 alone. The binding affinity of cellotriose towards the three tandem repeats of RfGH16_21 was also confirmed by calculating total binding Gibbs free energy, i.e. −100.8 ± 2.6 KJ/mol, by using g_mmpbsa tool. The stability of the protein was determined by protein melting analysis that showed Ca2+ and Mg2+ ions imparted structural stability to RfGH16_21. Dynamic light scattering analysis of RfGH16_21 showed monodispersity and hydrodynamic radius of 4.0 nm at 2.0 mg/mL protein concentration, which was comparable with the radius of gyration of 3.2 nm determined by MD simulation showing the protein to be in monomeric form. Communicated by Ramaswamy H. Sarma