Insights Into The Process Of Product Expulsion In Cellobiohydrolase Ce16A From Trichoderma Reesei By Molecular Dynamics Simulation

Cellulose,a homopolymer of?-1,4-linked glucosyl units,is the predominant component of the plant cell wall.Native cellulose is an insoluble material.However,enzyme cocktails from fungi and bacteria can synergistically cooperate to completely degrade cellulose into glucose.Cellulase can be divided into three classes:endoglucanases?EGs?,cellobiohydrolases?CBHs?,and?-glucosidases.EGs randomly cleave glycosidic linkages primarily in the amorphous regions of cellulose to produce various cellooligosaccharides.CBHs preferentially attach to the ends of carbohydrate chains and hydrolyze cellooligosaccharides into cellobiose,which is subsequently decomposed into glucose by?-glucosidases.Glycoside hydrolase cellulase family 6 from Trichoderma reesei?TrCe16A?is an important cellobiohydrolase to hydrolyze cellooligosaccharide into cellobiose.The knowledge of enzymatic mechanisms is critical for improving the conversion efficiency of cellulose into ethanol or other chemicals.However,the process of product expulsion,a key component of enzymatic depolymerization,from TrCe16A has not yet been described in detail.Here,conventional molecular dynamics and steered molecular dynamics were applied to study product expulsion from TrCe16A.The tunnel of TrCe16A contains four binding subsites in-2 to+2 for glucosyl units.Moreover,experiment has shown that+3 and+4 subsites exist at the entrance of the active site.In the investigation of homologous Humicola insolens Ce16A?HiCe16A?,which shares 64%sequence identity with TrCe16A,Varrot et al.obtained a crystal structure of wild-type CBH HiCe16A in complex with a fluorescent substrate?PDB1OCB?.They found that two new binding sites at the-3 and-4 subsites are present at the tunnel exit.Key feature of this complex is that the C?–C?bond of Tyr103 rotates by approximately 125°to open the-3 subsite,whereas the-3 subsite is blocked by Tyr103 in the majority of the crystal structure.Tyr103 may be a crucial residue in product expulsion given that it exhibits two different posthydrolytic conformations.In one conformation,Tyr103 rotates to open the-3 subsite.However,Tyr103 does not rotate in the other conformation.Three different routes for product expulsion were proposed on the basis of the two different conformations.The total energy barriers of the three routes were calculated through SMD simulations.The total energy barrier of product expulsion through Route 1,in which Tyr103 does not rotate,was 22.2kcal·mol^-1.The total energy barriers of product expulsion through Routes 2 and 3,in which Tyr103 rotates to open the-3 subsite,were 10.3 and 14.4 kcal·mol^-1,respectively.Therefore,Routes 2 and 3 have lower energy barriers than Route 1,and Route 2 is the thermodynamically optimal route for product expulsion.Consequently,the rotation of Tyr103 may be crucial for product release from TrCe16A.Results of this work have potential applications in cellulose.