Multiple Functions Of The Key Residues And Processive Mode In Cellobiohydrolase A From Clostridium Thermocellum Obtained From Molecular Dynamics Simulation

With the depletion of oil resources and human demand for energy growth,the search for cheap,clean renewable energy has become a common goal of researchers worldwide.Meanwhile,cellulose is a highly stable polymeric component of the plant cell wall and is the most abundant renewable resource of carbon and energy on earth.Therefore,the utilization and transformation of cellulose biomass is a highly significant subject in solving environmental pollution and energy crisis.Cellulose is a polysaccharide formed by the polymerization of a few hundred to over several thousand ?-(1,4)-D-glucose units,and it can be degraded into small cellodextrin or glucose by glycoside hydrolase(GH).GHs are currently categorized into 135 families,which are catalogued on the Carbohydrate–Active Enzymes database(http://www.cazy.org).Cellulase is a type of GH,which has high diversity.It is effective in hydrolyzing cellulose biomass into carbohydrates,which will be further transformed into bio-based fuels,chemicals,and materials.More than 170 members are included in GHs of Family 9.the enzymes in this family have two feathers,including a(?/?)6-barrel fold in the structure and an inverting mechanism in catalytic hydrolysis.In the inverting enzyme,a carboxylic acidic residue donates a proton to the glycosidic oxygen,while a general base activates the nucleophilic water molecule to attack the anomeric carbon.For all cellulases in Family 9,the determined acidic residues are glutamic acid,while the determined base residues are aspartic acid.As a member of Family9,our identified sugar-binding subsites of-2 to +2 from the nonreduced end to the reduced end were observed in the active cleft,while the cleaving position is between-1 and +1 subsites.This result indicates that the energy of the Cbh9A-substrate complex will be lower when the sugar units are located at certain binding sites.Among the residues around the-1 and +1 subsites,Asp383,Asp386,and Glu795 are conserved in all enzymes of Family 9 and are considered as the acid(Glu795)and base(Asp383 or Asp386)in the inverting mechanism.Residues Tyr555 and Trp678 considerably affect catalytic activity,but their mechanisms are still unknown.To investigate the mechanism of Tyr555 and Trp678,conventional molecular dynamics(MD),steered molecular dynamics(SMD),and free energy calculation were performed to simulate the processive process of wild type(WT)-Cbh9 A,Y555S mutant and W678 G mutant,which gain insight into whether Tyr555 and Trp678 affects the processivity of the Cbh9 A.Analysis of simulation results suggest that the active cleft is widened in Y555 S and W678 G mutants.And the binding free energies between the substrate and WT-Cbh9 A are lower than those of Y555 S and W678 G mutants.Residue decomposition analysis illustrated that the interactions between the substrate and the residues of Asp383,Gly548,Trp616 and Asp789 even nearly disappearing from the two mutants.Furthermore,the pull forces and energy barrier in Y555 S and W678 G mutants were reduced significantly during the SMD simulation compared with the WT-Cbh9 A.This condition indicated the substrate has weaker interaction with the protein in Y555 S and W678 G mutants.Consequently,The Tyr555 and Trp678 may be effect the processivity through control the width of the active cleft.Meanwhile,In the process mode of Cbh9 A,Asp383,Asp386,Glu795,Tyr555,Trp678,Trp791,Asp789 and Trp616 are key residues are also play the crucial roles.Moreover,we believe the selective hydrolysis mechanism of Cbh9 A may be due to the different orientation of Glc(-1)and the interaction between Trp678 and Glc(-2).This work provides theoretical information for cellulase modification and mutation.