Abstract
β-Glucosidase (EC 3.2.1.21) plays an essential role in the removal of glycosyl residues from disaccharide cellobiose to produce glucose during the hydrolysis of lignocellulosic biomass. Although there exist a few β-glucosidase that are tolerant to large concentrations of glucose, these enzymes are typically prone to glucose inhibition. Understanding the basis of this inhibition is important for the production of cheaper biofuels from lignocellulose. In this study, all-atom molecular dynamics simulation at different temperatures and glucose concentrations was used to understand the molecular basis of glucose inhibition of GH1 β-glucosidase (B8CYA8) from Halothermothrix orenii. Our results show that glucose induces a broadening of the active site tunnel through residues lining the tunnel and facilitates the accumulation of glucose. In particular, we observed that glucose accumulates at the tunnel entrance and near the catalytic sites to block substrate accessibility and inhibit enzyme activity. The reduction of enzyme activity was also confirmed experimentally through specific activity measurements in the presence of 0–2.5 M glucose. We also show that the increase in glucose concentrations leads to a decrease in the number of water molecules inside the tunnel to affect substrate hydrolysis. Overall, the results help in understanding the role of residues along the active site tunnel for the engineering of glucose-tolerant β-glucosidase.
Highlights
The production of biofuels from lignocellulosic biomass could be broadly divided into three steps pretreatment of the biomass to reduce cellulose recalcitrance, enzymatic conversion of the polysaccharides in pretreated biomass to sugars, and the conversion of the sugars to fuels by the same or other microbes through fermentative processes
Endoglucanase (EC 3.2.1.4) randomly cleave the β-1,4 glycosidic linkages of cellulose, cellobiohydrolase (EC 3.2.1.91) attack the cellulose chain ends to produce a dimer of glucose called cellobiose, which is linked by a β-1,4 glycosidic bond. βGlucosidase (EC 3.2.1.21) hydrolyze the β-1,4 linkage in cellobiose to produce glucose.[3−6] The final hydrolysis step is generally recognized as a limiting step in the conversion of lignocellulosic biomass to sugars as β-glucosidase are inhibited by its reaction product glucose.[4,6,7]
Molecular dynamics (MD) simulations at different temperatures and glucose concentrations assist us in understanding the effect of glucose on B8CYA8 activity
Summary
The production of biofuels from lignocellulosic biomass could be broadly divided into three steps pretreatment of the biomass to reduce cellulose recalcitrance, enzymatic conversion of the polysaccharides in pretreated biomass to sugars, and the conversion of the sugars to fuels by the same or other microbes through fermentative processes. ΒGlucosidase (EC 3.2.1.21) hydrolyze the β-1,4 linkage in cellobiose to produce glucose.[3−6] The final hydrolysis step is generally recognized as a limiting step in the conversion of lignocellulosic biomass to sugars as β-glucosidase are inhibited by its reaction product glucose.[4,6,7] The efficient and economic hydrolysis by cellulase requires enzymes that are active in high glucose concentrations. ACS Omega help gain deeper insights into the structure−function relationship.[13−16] Computational modeling and MD simulation have been reported toward understanding catalytic activity and thermostability of cellulases.[15,17−23] there are no reports of theoretical studies for a molecular-level understanding of β-glucosidase inhibition or the effect of glucose on the structural stability of such enzymes. The time step was of 1.0 fs and all the properties were computed from the trajectories stored at an interval of 4.0 ps during the production run
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