Effects of Tyr555 and Trp678 on the processivity of cellobiohydrolase A from Ruminiclostridium thermocellum: A simulation study.

Cellobiohydrolase A from Ruminiclostridium thermocellum (Cbh9A) is a processive exoglucanase from family 9 and is an important cellobiohydrolase that hydrolyzes cello-oligosaccharide into cellobiose. Residues Tyr555 and Trp678 considerably affect catalytic activity, but their mechanisms are still unknown. To investigate how the Tyr555 and Trp678 affect the processivity of Cbh9A, conventional molecular dynamics, steered molecular dynamics, and free energy calculation were performed to simulate the processive process of wild type (WT)-Cbh9A, Y555S mutant, and W678G mutant. Analysis of simulation results suggests that the binding free energies between the substrate and WT-Cbh9A are lower than those of Y555S and W678G mutants. The pull forces and energy barrier in Y555S and W678G mutants also reduced significantly during the steered molecular dynamics (SMD) simulation compared with that of the WT-Cbh9A. And the potential mean force calculations showed that the pulling energy barrier of Y555S and W678G mutants is much lower than that of WT-Cbh9A.