Thermal Sensitivity of the Enzymatic Activity of ß-Glucosidase: Simulations Lend Mechanistic Insights into Experimental Observations.

A crucial prerequisite for industrial applications of enzymes is the maintenance of specific activity across wide thermal ranges. ß-Glucosidase (EC 3.2.1.21) is an essential enzyme for converting cellulose in biomass to glucose. While the reaction mechanisms of ß-glucosidases from various thermal ranges (hyperthermophilic, thermophilic, and mesophilic) are similar, the factors underlying their thermal sensitivity remain obscure. The work presented here aims to unravel the molecular mechanisms underlying the thermal sensitivity of the enzymatic activity of the ß-glucosidase BglB from the bacterium Paenibacillus polymyxa. Experiments reveal a maximum enzymatic activity at 315 K, with a marked decrease in the activity below and above this temperature. Employing in silico simulations, we identified the crucial role of the active site tunnel residues in the thermal sensitivity. Specific tunnel residues were identified via energetic decomposition and protein-substrate hydrogen bond analyses. The experimentally observed trends in specific activity with temperature coincide with variations in overall binding free energy changes, showcasing a predominantly electrostatic effect that is consistent with enhanced catalytic pocket-substrate hydrogen bonding (HB) at Topt. The entropic advantage owing to the HB substate reorganization was found to facilitate better substrate binding at 315 K. This study elicits molecular-level insights into the associative mechanisms between thermally enabled fluctuations and enzymatic activity. Crucial differences emerge between molecular mechanisms involving the actual substrate (cellobiose) and a commonly employed chemical analogue. We posit that leveraging the role of fluctuations may reveal unexpected insights into enzyme behavior and offer novel paradigms for enzyme engineering.

Probing the dynamics between the substrate and the product towards glucose tolerance of Halothermothrix orenii ß-glucosidase.

Most ß-Glucosidase (B8CYA8) are prone to inhibition by glucose. Experimentally observed specific activity of B8CYA8 on 20 mM, 50 mM, and 100 mM p-nitrophenyl-ß-D-glucopyranoside (pNPGlc) substrate concentrations show surprise dependence on the presence of 0-3 M glucose at 335 K. We found that at high substrate concentration, the enzyme shows stimulation in specific activity with glucose and the glucose inhibition curve shifts toward the right with the increase in the substrate concentration. We employed atomistic molecular dynamics simulations of ß-Glucosidase from Halothermothrix orenii at different glucose and pNPGlc concentrations to provide microscopic explanations to the experimentally observed non-monotonic glucose concentration dependence of the enzyme activity. Our results show that accumulation of substrate (pNPGlc) near the B8CYA8 catalytic site residues E166 and E354 and in the active site tunnel increases up to 0.5 M glucose when the specific activity is the highest. The number of pNPGlc in the tunnel decreases drastically when glucose concentration is more than 0.5 M, and hence the specific activity decreases. Potential of mean force (PMF) calculations showed that the most favorable interaction between pNPGlc and ß-Glucosidase exists at 0.5 M glucose while at deficient and high glucose concentrations, the binding energy between the substrate and ß-Glucosidase is very low. These studies provide the molecular basis towards understanding inhibition and stimulation of ß-Glucosidase activity in the presence of glucose and may enable the optimum use of enzymes for the efficient conversion of high biomass loading saccharification reactions.Communicated by Ramaswamy H. Sarma.

Elucidating the regulation of glucose tolerance in a ß-glucosidase from Halothermothrix orenii by active site pocket engineering and computational analysis.

ß-Glucosidase catalyzes the hydrolysis of ß-1,4 linkage between two glucose molecules in cello-oligosaccharides and is prone to inhibition by the reaction product glucose. Relieving the glucose inhibition of ß-glucosidase is a significant challenge. Towards the goal of understanding how glucose interacts with ß-glucosidase, we expressed in Escherichia coli, the Hore_15280 gene encoding a ß-glucosidase in Halothermothrix orenii. Our results show that the enzyme is glucose tolerant, and its activity on p-nitrophenyl D-glucopyranoside stimulated in the presence of up to 0.5 M glucose. NMR analyses show the unexpected interactions between glucose and the ß-glucosidase at lower concentrations of glucose that, however, does not lead to enzyme inhibition. We identified non-conserved residues at the aglycone-binding and the gatekeeper site and show that increased hydrophobicity at the pocket entrance and a reduction in steric hindrances are critical towards enhanced substrate accessibility and significant improvement in activity. Analysis of structures and in combination with molecular dynamics simulations show that glucose increases the accessibility of the substrate by enhancing the structural flexibility of the active site pocket and may explain the stimulation in specific activity up to 0.5 M glucose. Such novel regulation of ß-glucosidase activity by its reaction product may offer novel ways of engineering glucose tolerance.

Understanding the glucose tolerance of an archaeon ß-glucosidase from Thermococcus sp.

ß-glucosidase hydrolyzes the ß-1,4 linkage of cellobiose, a product generated from the action of endoglucanase and cellobiohydrolase on cellulose, and generates glucose. Accumulated glucose during saccharification leads to product inhibition of ß-glucosidase, which in turn cause an accumulation of cellobiose and inhibition of other cellulolytic enzymes. Thus, glucose tolerant and active ß-glucosidase is required for the efficient saccharification of biomass. O08324 is a glucose tolerant ß-glucosidase isolated from archaeon Thermococcus sp. which shows no loss in enzyme specific activity in the presence of up to 4 M glucose and is active at 78 °C. Since O08324 has such high glucose tolerance, knowing the rationale for glucose tolerance will be helpful in engineering glucose tolerant ß-glucosidase. In the present study, we designed mutations at eleven sites across the gatekeeper, aglycone, and glycone region. Based on the kinetic studies of O08324 mutants, the gatekeeper residues at positions 160, 166, 167, 168, and the aglycone binding residue 156 were identified to play a role in glucose inhibition. However, only residues at the tunnel entrance, and not all gatekeeper residues contribute to glucose tolerance. This study sheds some light on the unusual glucose tolerance of O08321 archaeal GH1 ß-glucosidase.

Probing the Effect of Glucose on the Activity and Stability of ß-Glucosidase: An All-Atom Molecular Dynamics Simulation Investigation.

ß-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 ß-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 ß-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 ß-glucosidase.

Recyclable Thermoresponsive Polymer-ß-Glucosidase Conjugate with Intact Hydrolysis Activity.

ß-Glucosidase (BG) catalyzes the hydrolysis of cellobiose to glucose and is a rate-limiting enzyme in the conversion of lignocellulosic biomass to sugars toward biofuels. Since the cost of enzyme is a major contributor to biofuel economics, we report the bioconjugation of a temperature-responsive polymer with the highly active thermophilic ß-glucosidase (B8CYA8) from Halothermothrix orenii toward improving enzyme recyclability. The bioconjugate, with a lower critical solution temperature (LCST) of 33 °C withstands high temperatures up to 70 °C. Though the secondary structure of the enzyme in the conjugate is slightly distorted with a higher percentage of ß-sheet like structure, the stability and specific activity of B8CYA8 in the conjugate remains unaltered up to 30 °C and retains more than 70% specific activity of the unmodified enzyme at 70 °C. The conjugate can be reused for ß-glucosidic bond cleavage of cellobiose for at least four cycles without any significant loss in specific activity.

Exploiting non-conserved residues to improve activity and stability of Halothermothrix orenii ß-glucosidase.

ß-glucosidase (EC 3.2.1.21; BG) cleaves ß-glucosidic linkages in disaccharide or glucose-substituted molecules. In an effort towards designing better BGs, we focused on the role of non-conserved residues across an otherwise homologous BG active site tunnel and designed mutants across the aglycone-binding site (V169C) and the gatekeeper residues (I246A) of the active site tunnel. We expressed in Escherichia coli, the Hore_15280 gene encoding a ß-glucosidase (BG) in Halothermothrix orenii. The overexpressed and purified wild-type (B8CYA8) has a high specific activity of 345 µmol/min/mg on pNPGlc and a half-life of 1.13 h when assayed with pNPGlc at pH 7.1 and 70 °C. The specific activities of V169C and I246A were 1.7 and 1.2 times higher than that of wild-type (WT) enzyme with the model substrate pNPGlc, while the activity on the natural substrate cellobiose was slightly higher to the WT. The two mutants were kinetically stable with 4.4- to 11-fold longer half-life compared to the WT enzyme. When the two mutations were combined to generate the V169C/I246A mutant, the specific activity increased to nearly twofold higher than WT on both substrates and the half-life increased fivefold. The two single mutants also show enhanced saccharification of insoluble natural biomass on supplementation of Trichoderma viride cellulase cocktail. These enhanced properties suggest the need for a closer look at the active site tunnel of these enzymes, especially across residues that are not conserved towards improving catalytic efficiencies.

ß-Glucosidase from the hyperthermophilic archaeon Thermococcus sp. is a salt-tolerant enzyme that is stabilized by its reaction product glucose.

ß-Glucosidase (BG) is widely applied in the biofuel's industry, as part of a cellulase cocktail to catalyze the hydrolysis of the ß-1,4 linkages that join two glucose molecules in a cellulose polymer. The hydrolysis step is generally recognized as the major limiting step in the development of efficient enzyme-based technologies for the conversion of lignocellulosic biomass to sugars and the production of biofuels due to the accumulation of the reaction product, glucose. Relieving this glucose inhibition of BG is therefore a major challenge. In this study, O08324, a putative BG gene encoded in the hyperthermophilic archaeon Thermococcus sp., was cloned and overexpressed in Escherichia coli. O08324 showed maximum activity between pH 5-6.8 and at 78 °C and was thermostable with a half-life of 860 min at 78 °C in the presence of 1.5 M glucose. O08324 was not inhibited by glucose up to the highest assayable concentration of 4 M and also shows no decrease in activity in the presence of up to 4 M of sodium chloride or potassium chloride. O08324 supplementation of Trichoderma viride cellulase enhanced glucose production by more than 50 % compared to a commercially available BG, when Avicel (10 %, w/v) was used as a substrate at 37 °C. Multiple sequence alignments across previously reported glucose-tolerant BGs shows that many conserved residues previously implicated in glucose tolerance are not conserved in this BG suggesting a need for a relook at understanding the molecular basis of glucose tolerance.