ABSTRACT
Cellulases from GH9 family show endo-, exo- or processive endocellulase activity, but the reason behind the variation is unclear. A GH9 recombinant endoglucanase, AtGH9C-CBM3A-CBM3B from Acetivibrio thermocellus was structurally characterized for conformation, binding and dynamics assessment. Modeled AtGH9C-CBM3A-CBM3B depicted (α/α)6-barrel structure with Asp98, Asp101 and Glu489 acting as catalytic triad. CD results revealed 25.2 % α-helix, 18.4 % ß-sheet and rest 56.4 % of random coils, corroborating with predictions from PSIPRED and SOPMA. MD simulation of AtGH9C-CBM3A-CBM3B bound cellotetraose showed structural stability and global compactness with lowered RMSD values (1.5 nm) as compared with only AtGH9C-CBM3A-CBM3B (1.8 nm) for 200 ns. Higher fluctuation in RMSF values in far-positioned CBM3B pointed to its redundancy in substrate binding. Docking studies showed maximum binding with cellotetraose (ΔG = -5.05 kcal/mol), with reduced affinity towards ligands with degree of polymerization (DP) lower (DP < 4) or higher than 4 (DP > 4). Processivity index displayed the enzyme to be processive with loop 3 (342-379 aa) possibly blocking the non-reducing end of cellulose chain, resulting in cellotetraose release. SAXS analysis of AtGH9C-CBM3A-CBM3B at 5 mg/mL displayed monodispersed state with fist-and-elbow shape in solution. Negative zeta potential of -24 mV at 5 mg/mL indicated stability and free from aggregation.