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

Sinha, Sushant K.; Das, Shaon Kumar; Konar, Sukanya; Ghorai, Pradip Kr.; Das, Rahul; Datta, Supratim

doi:10.1016/j.ijbiomac.2020.04.036

Cited by 19 publications

(9 citation statements)

References 55 publications

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“…2). A comparison of the surface structures (Fig 3) showed that the active sites of Pf Bgl3A and Pf Bgl3B are open and shallow, whereas that of Pf Bgl1A is deep, narrow and tunnel-like consistent with reports on active site configuration for GH1 β-glucosidases (26, 28–31). This active site conformation is thought to improve the glucose tolerance of GH1 proteins by preventing the direct access of glucose to the active site (32).…”

Section: Resultssupporting

confidence: 85%

Profiling of the β-glucosidases identified in the genome ofPenicillium funiculosum: Insights from genomics, transcriptomics, proteomics and homology modelling studies

Okereke¹,

Gupta²,

Ogunyewo³

et al. 2022

Preprint

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Enzymatic lignocellulosic biomass conversion to bioethanol is dependent on efficient enzyme systems with β-glucosidase as a key component. In this study, we performed in-depth profiling of the various β-glucosidases present in the genome of the hypercellulolytic fungus;Penicillium funiculosumusing genomics, transcriptomics, proteomics and molecular dynamics simulation approaches. Of the eight β-glucosidase genes identified in theP. funiculosumgenome, three were found to be extracellular, as evidenced by presence of signal peptides and mass spectrometry. Among the three secreted β-glucosidase, two belonged to the GH3 and one belonged to GH1 families. Modelled structures of these proteins predicted a deep and narrow active site for the GH3 β-glucosidases (PfBgl3A andPfBgl3B) and a shallow open active site for the GH1 β-glucosidase (PfBgl1A). The enzymatic assays indicated thatP. funiculosumsecretome showed high β-glucosidase activities with prominent bands on 4-methylumbelliferyl β-D-glucopyranoside (MUG) zymogram. To understand the contributory effect of each of the three secreted β-glucosidases (PfBgls), the corresponding gene was deleted separately and the effect of the deletion on β-glucosidase activity of the secretome was examined. Although not the most abundant β-glucosidase,PfBgl3A was found to be the most significant one as evidenced by a 42 % reduction in β-glucosidase activity in the ΔPfBgl3A strain. To improve the thermostability, two mutants ofPfBgl3A were designed with the help of molecular dynamics (MD) simulation and were expressed in Pichia pastoris for evaluation. ThePfBgl3A mutant (Mutant A) gave 1.4 fold increase in the half-life (T1/2) of the enzyme at 50 °C.

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Section: Resultssupporting

confidence: 85%

Profiling of the β-glucosidases identified in the genome ofPenicillium funiculosum: Insights from genomics, transcriptomics, proteomics and homology modelling studies

Okereke¹,

Gupta²,

Ogunyewo³

et al. 2022

Preprint

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“…Experimentally, this hypothesis could be investigated by controlling the enzyme orientation on the surface, ensuring that the entrance to the catalytic site remains accessible after the enzyme immobilization, and monitoring the catalytic activity. One should also note that in the case of βGA, the broad size of the catalytic site, , which is located at the entrance of the central β-barrel, and its structural stability ensure that it remains open at all times. However, many proteins comprise a buried active site that is connected to the surface through internal tunnels and cavities (like globins or hydrogenases).…”

Section: Discussionmentioning

confidence: 99%

Between Two Walls: Modeling the Adsorption Behavior of β-Glucosidase A on Bare and SAM-Functionalized Gold Surfaces

et al. 2022

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The efficient immobilization of enzymes on surfaces remains a complex but central issue in the biomaterials field, which requires us to understand this process at the atomic level. Using a multi-scale approach combining all-atom molecular dynamics and coarsegrain Brownian dynamics simulations, we investigated the adsorption behavior of βglucosidase A (βGA) on bare and SAM-functionalized gold surfaces. We monitored the enzyme position and orientation during the MD trajectories, and measured the contacts it forms with both surfaces. While the adsorption process has little impact on the protein conformation, it can nonetheless perturb its mechanical properties and catalytic activity.Our results show that compared to the SAM-functionalized surface, the adsorption of βGA on bare gold is more stable, but also less specific, and more likely to disrupt the enzyme's function. This observation emphasizes the fact that the structural organization of proteins at the solid interface is a keypoint when designing devices based on enzyme immobilization, as one must find an acceptable stability-activity trade-off.

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“…Our group recently reported the stimulation of a β-glucosidase in the presence of glucose but without transglycosylation. The β-glucosidase from Halothermothrix orenii is stimulated up to 0.5 M glucose due to the increased access of the substrate with the increase in active site pocket structural flexibility . The activation of β-glucosidases in the presence of glucose could be due to the simple competition between the glucose and nonproductive binding of substrate molecules at additional sites during enzymatic reaction .…”

Section: Results and Discussionmentioning

confidence: 99%

“…However, most β-glucosidases are prone to glucose inhibition, and the consequent accumulation further inhibits cellobiohydrolase and endoglucanase to result in inefficient biomass hydrolysis. Therefore, the identification of efficient β-glucosidases has been an ongoing effort. − Recently, there were reports of glucose-tolerant GH1 β-glucosidase that can tolerate very high glucose concentrations. ,,− Glucose tolerance has been linked to stimulatory effects by low concentration of glucose, transglycosylation, allosteric effects, , and nonproductive substrate binding . The higher glucose-tolerant enzymes thus far are all reported from the GH1 family.…”

Section: Introductionmentioning

confidence: 99%

Role of Conformational Change and Glucose Binding Sites in the Enhanced Glucose Tolerance of Agrobacterium tumefaciens 5A GH1 β-Glucosidase Mutants

et al. 2021

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β-Glucosidases are often inhibited by their reaction product glucose and a barrier to the efficient lignocellulosic biomass hydrolysis to glucose. We had previously reported the mutants, C174V, and H229S, with a nearly 2-fold increased glucose tolerance over the wild type (WT), H0HC94, encoded in Agrobacterium tumefaciens 5A (apparent K i , Glc = 686 mM). We report our steady−state and time-resolved intrinsic fluorescence spectroscopy, circular dichroism, and isothermal titration calorimetry (ITC) studies to further understand increased glucose tolerance. Changes in the mutants' emission intensity and the differential change in quenching rate in the absence and presence of glucose reflect changes in protein conformation by glucose. Time-resolved lifetime and anisotropy measurements further indicated the microenvironment differences across solvent-exposed tryptophan residues and a higher hydrodynamic radius due to glucose binding, respectively. ITC measurements confirmed the increase of glucose binding sites in the mutants. The experiment results were supported by molecular dynamics simulations, which revealed significant variations in the glucose−protein hydrogen-bonding profiles. Protein structure network analysis of the simulated structures further indicates the mutants' conformation change than the WT. Computational studies also indicated additional glucose binding sites in mutants. Our results indicate the role of glucose binding in modulating the enzyme response to glucose.

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

Abstract: Elucidating the regulation of glucose tolerance through the interaction between the reaction product and 2 active site pocket residues of a β-glucosidase from Halothermothrix orenii

Cited by 19 publications

References 55 publications

Profiling of the β-glucosidases identified in the genome ofPenicillium funiculosum: Insights from genomics, transcriptomics, proteomics and homology modelling studies

Profiling of the β-glucosidases identified in the genome ofPenicillium funiculosum: Insights from genomics, transcriptomics, proteomics and homology modelling studies

Between Two Walls: Modeling the Adsorption Behavior of β-Glucosidase A on Bare and SAM-Functionalized Gold Surfaces

Role of Conformational Change and Glucose Binding Sites in the Enhanced Glucose Tolerance of Agrobacterium tumefaciens 5A GH1 β-Glucosidase Mutants

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