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Structural basis for glucose tolerance in GH1 β-glucosidases
Abstract and Figures
Product inhibition of β-glucosidases (BGs) by glucose is considered to be a limiting step in enzymatic technologies for plant-biomass saccharification. Remarkably, some β-glucosidases belonging to the GH1 family exhibit unusual properties, being tolerant to, or even stimulated by, high glucose concentrations. However, the structural basis for the glucose tolerance and stimulation of BGs is still elusive. To address this issue, the first crystal structure of a fungal β-glucosidase stimulated by glucose was solved in native and glucose-complexed forms, revealing that the shape and electrostatic properties of the entrance to the active site, including the +2 subsite, determine glucose tolerance. The aromatic Trp168 and the aliphatic Leu173 are conserved in glucose-tolerant GH1 enzymes and contribute to relieving enzyme inhibition by imposing constraints at the +2 subsite that limit the access of glucose to the -1 subsite. The GH1 family β-glucosidases are tenfold to 1000-fold more glucose tolerant than GH3 BGs, and comparative structural analysis shows a clear correlation between active-site accessibility and glucose tolerance. The active site of GH1 BGs is located in a deep and narrow cavity, which is in contrast to the shallow pocket in the GH3 family BGs. These findings shed light on the molecular basis for glucose tolerance and indicate that GH1 BGs are more suitable than GH3 BGs for biotechnological applications involving plant cell-wall saccharification.
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... Although the mechanism of glucose stimulation/inhibition remains elusive, structural analyses and literature data on glucose-tolerant GH1s allowed us to identify the residues likely involved in M-GH1 glucose tolerance [30]. In particular, we focused on the comparison of M-GH1 with the glucosestimulated β-glucosidase from H. insolens (HiBG, PDB: 4MDP [31]), which share 39.9% of sequence identity and reasonable structural homology (RMSD ~1.7 Å over 419 aligned residues, Fig. 10A). In HiBG, glucose tolerance has been ascribed to the presence of large hydrophobic residues at the subsite +2 ( Fig. 10 A, green sticks) which act by narrowing the tunnel that leads to the catalytic acidic residues and, consequently, limit product inhibition due to hindering of glucose entry to subsite -1. ...
... Although the molecular mechanisms of glucose tolerance in GH1 enzymes are still elusive, it is plausible that this feature depends on the shape and the electrostatic properties of active site entrance [29]. In glucose-stimulated HiBG, two hydrophobic residues (Trp168, Leu173) act as gatekeeping residues for the active site and reduce the size of subsite +2, thus limiting glucose entry to subsite -1 [31]. Sequence analysis indicates that the main difference between M-GH1 and HiBG is the conservative substitution of the gatekeeping Trp with a Phe (Phe173), while the Leu residue (Leu178) is conserved in M-GH1. ...
... and glucose-tolerant Humicola insolens βglucosidase (HiBG, PDB: 4MDP [31]) were also aligned, both shown in blue font. The sequence alignment was manually corrected based on 3D structure comparison. ...
Cold‐active enzymes support life at low temperatures due to their ability to maintain high activity in the cold and can be useful in several biotechnological applications. Although information on the mechanisms of enzyme cold adaptation is still too limited to devise general rules, it appears that very diverse structural and functional changes are exploited in different protein families and within the same family. In this context, we studied the cold adaptation mechanism and the functional properties of a member of the glycoside hydrolase family 1 (GH1) from the Antarctic bacterium Marinomonas sp. ef1. This enzyme exhibits all typical functional hallmarks of cold adaptation, including high catalytic activity at 5 °C, broad substrate specificity, low thermal stability, and higher lability of the active site compared to the overall structure. Analysis of the here‐reported crystal structure (1.8 Å resolution) and molecular dynamics simulations suggest that cold activity and thermolability may be due to a flexible region around the active site (residues 298–331), whereas the dynamic behavior of loops flanking the active site (residues 47–61 and 407–413) may favor enzyme‐substrate interactions at the optimal temperature of catalysis ( T opt ) by tethering together protein regions lining the active site. Stapling of the N‐terminus onto the surface of the β‐barrel is suggested to partly counterbalance protein flexibility, thus providing a stabilizing effect. The tolerance of the enzyme to glucose and galactose is accounted for by the presence of a “gatekeeping” hydrophobic residue (Leu178), located at the entrance of the active site.
... The acidic catalytic residues are Glu353, which acts as a nucleophile, and Glu166, which serves as the acid/base catalyst. Other residues, including His121, Trp122, Asn165, Tyr296, Trp399, Glu406, Trp407, and Phe415, which are essential for substrate recognition and binding, are also conserved (de Giuseppe et al., 2014). However, due to limitations of homology modeling methods with low sequence identity (approximately 60%), the actual crystallographic structure of PgBgl1 is necessary for a comprehensive structural comparative analysis. ...
The novel β-glucosidase gene (pgbgl1) of glycoside hydrolase (GH) family 1 from the psychrotrophic bacterium Psychrobacillus glaciei sp. PB01 was successfully expressed in Escherichia coli BL21 (DE3). The deduced PgBgl1 contained 447 amino acid residues with a calculated molecular mass of 51.4 kDa. PgBgl1 showed its maximum activity at pH 7.0 and 40 °C, and still retained over 10% activity at 0 °C, suggesting that the recombinant PgBgl1 is a cold-adapted enzyme. The substrate specificity, Km, Vmax, and Kcat/Km for the p-Nitrophenyl-β-D-glucopyranoside (pNPG) as the substrate were 1063.89 U/mg, 0.36 mM, 1208.31 U/mg and 3871.92/s, respectively. Furthermore, PgBgl1 demonstrated remarkable stimulation of monosaccharides such as glucose, xylose, and galactose, as well as NaCl. PgBgl1 also demonstrated a high capacity to convert the primary soybean isoflavone glycosides (daidzin, genistin, and glycitin) into their respective aglycones. Overall, PgBgl1 exhibited high catalytic activity towards aryl glycosides, suggesting promising application prospects in the food, animal feed, and pharmaceutical industries.
... Nonetheless, the accumulation of glucose, a product arising from cellulose hydrolysis, hampers the efficacy of β-glucosidase-mediated degradation. As a result, the quest for high-glucose-tolerant β-glucosidases has gained prominence (de Giuseppe et al., 2014). Elevated temperature conditions offer manifold advantages, including augmented enzyme-substrate interaction rates, reduced contamination risks, heightened matrix solubility, and accelerated diffusion rates (Schröder et al., 2014;Ratuchne and Knob, 2021). ...
Introduction
β-Glucosidase serves as the pivotal rate-limiting enzyme in the cellulose degradation process, facilitating the hydrolysis of cellobiose and cellooligosaccharides into glucose. However, the widespread application of numerous β-glucosidases is hindered by their limited thermostability and low glucose tolerance, particularly in elevated-temperature and high-glucose environments.
Methods
This study presents an analysis of a β-glucosidase gene belonging to the GH1 family, denoted lqbg8, which was isolated from the metagenomic repository of Hehua hot spring located in Tengchong, China. Subsequently, the gene was cloned and heterologously expressed in Escherichia coli BL21(DE3). Post expression, the recombinant β-glucosidase (LQBG8) underwent purification through a Ni affinity chromatography column, thereby enabling the in-depth exploration of its enzymatic properties.
Results
LQBG8 had an optimal temperature of 70°C and an optimum pH of 5.6. LQBG8 retained 100 and 70% of its maximum activity after 2-h incubation periods at 65°C and 70°C, respectively. Moreover, even following exposure to pH ranges of 3.0–10.0 for 24 h, LQBG8 retained approximately 80% of its initial activity. Notably, the enzymatic prowess of LQBG8 remained substantial at glucose concentrations of up to 3 M, with a retention of over 60% relative activity. The kinetic parameters of LQBG8 were characterized using cellobiose as substrate, with Km and Vmax values of 28 ± 1.9 mg/mL and 55 ± 3.2 μmol/min/mg, respectively. Furthermore, the introduction of LQBG8 (at a concentration of 0.03 mg/mL) into a conventional cellulase reaction system led to an impressive 43.7% augmentation in glucose yield from corn stover over a 24-h period. Molecular dynamics simulations offered valuable insights into LQBG8’s thermophilic nature, attributing its robust stability to reduced fluctuations, conformational changes, and heightened structural rigidity in comparison to mesophilic β-glucosidases.
Discussion
In summation, its thermophilic, thermostable, and glucose-tolerant attributes, render LQBG8 ripe for potential applications across diverse domains encompassing food, feed, and the production of lignocellulosic ethanol.
... ThBg2 has superior glucose tolerance than many previously reported beta-glucosidases, like those of metagenomic origin such as Unbgl1A [48], Tt-BGL from Thermotoga thermarum DSM 5069T [49], and DturβGlu from Dictyoglomus turgidum [50]. The superior glucose tolerance of ThBg2 may be related to the shape and electrostatic properties of the entrance to the active site [51], where the composition of amino acid residues improves glucose tolerance by enhancing the mobility of flexible loops around the active site [52]. In addition, its synergy with commercial cellulase also provides an alternative source for lignocellulose degradation. ...
β-glucosidase is a key enzyme in the degradation of lignocellulosic biomass, which is responsible for the conversion of oligosaccharides from cellulose hydrolysates to glucose. However, its required high temperatures and the presence of inhibitors have limited its use in industry. In this study, a new β-glucosidase gene, named thbg2, was obtained from the metagenome Ruidian Hot Spring, Tengchong City, Yunnan Province, southwestern China. The gene was synthesized, cloned, heterologously expressed, and enzymatically characterized. Its optimum temperature and pH were 60 °C and pH 5.6, respectively. ThBg2 exhibited more than 60% relative activity in temperatures ranging from 40 °C to 70 °C and across a pH of 4.0–6.6. It maintained 100% relative activity after incubation at either 50 °C for 24 h or 60 °C for 12 h and more than 80% residual activity after incubation at pH 4.0–6.0 for 24 h. Moreover, it maintained more than 80% relative activity in the presence of heavy metal ions, ethanol, SDS etc. Furthermore, glucose yields from corn stalks increased by 20% after ThBg2 (0.05 mg/mL) was added to the commercial cellulase reaction system. Overall, this work identified a thermophilic and inhibitor-tolerant β-glucosidase with potential applications in commercial lignocellulose utilization and the bioenergy industry.
... In structural analysis, glucose tolerance is related to the active site accessibility of enzymes. The active site of GH1 -glucosidases has been determined to be deeper and narrower than the enzymes in the GH3 family (de Giuseppe et al., 2014). Therefore, considering the presence of glucose in the digestive system, -glucosidases in the GH1 family would be more suitable as animal feed additives (Kaushal et al., 2021). ...
Background: Feed additives are used for different purposes and include different substances. Recently, the feed additive potential
of aromatic plants has been frequently investigated. -glucosidases are used as feed additive enzymes to increase nutritional value.
This study was carried out to determine the biochemical properties of Oregano onites L. -glucosidase in terms of expected biochemical
properties from feed additive enzymes.
Methods: Oregano β-glucosidase was purified by hydrophobic interaction chromatography. The purified enzyme was checked the
purity on SDS-PAGE. The biochemical properties (optimal pH and temperature, thermal stability, glucose and alcohol tolerance) of
the purified enzyme were determined using para-Nitrophenyl--D-glucopyranoside as a substrate.
Result: Origanum onites L. -glucosidase was purified to electrophoretic homojenity with 23.15-fold with a yield of 8.2% and it was
visualised as a single band at 65.7 kDa on SDS-PAGE. The enzyme had maximum activity at pH 4.0 and 45C and retained over 50%
activity at pH 4-7. The enzyme maintained up to 50% of its activity after 60 minutes of incubation at 40C, 50C, and 60C. The
purified enzyme showed a high tolerance to glucose and was also found to be tolerant to alcohol. The enzyme’s biochemical
properties similar to exogenous enzymes recommended as feed additives. Therefore, the results of this study supports the use of
oregano as a feed additive.
Key words: Aromatic plant, β-glucosidase, Enzyme characterisation, Feed additive, Origanum onites L.
β-glucosidases (EC. 3.2.1.21) are enzymes that hydrolyze glucosidic bonds of oligosaccharides, in special disaccharides, such as cellobiose, realizing glucose at the end of the process. They are highly used in second-generation biofuel production.
A program for evaluating the solution scattering from macromolecules with known atomic structure is presented. The program uses multipole expansion for fast calculation of the spherically averaged scattering pattern and takes into account the hydration shell. Given the atomic coordinates (e.g. from the Brookhaven Protein Data Bank) it can either predict the solution scattering curve or fit the experimental scattering curve using only two free parameters, the average displaced solvent volume per atomic group and the contrast of the hydration layer. The program runs on IBM PCs and on the major UNIX platforms.
A program suite for one-dimensional small-angle scattering data processing running on IBM-compatible PCs under Windows 9x/NT/2000/XP is presented. The main program, PRIMUS, has a menu-driven graphical user interface calling computational modules to perform data manipulation and analysis. Experimental data in binary OTOKO format can be reduced by calling the program SAPOKO, which includes statistical analysis of time frames, averaging and scaling. Tools to generate the angular axis and detector response files from diffraction patterns of calibration samples, as well as binary to ASCII transformation programs, are available. Several types of ASCII files can be directly imported into PRIMUS, in particular, sasCIF or ILL-type files are read without modification. PRIMUS provides basic data manipulation functions (averaging, background subtraction, merging of data measured in different angular ranges, extrapolation to zero sample concentration, etc.) and computes invariants from Guinier and Porod plots. Several external modules coupled with PRIMUSvia pop-up menus enable the user to evaluate the characteristic functions by indirect Fourier transformation, to perform peak analysis for partially ordered systems and to find shape approximations in terms of three-parametric geometrical bodies. For the analysis of mixtures, PRIMUS enables model-independent singular value decomposition or linear fitting if the scattering from the components is known. An interface is also provided to the general non-linear fitting program MIXTURE, which is designed for quantitative analysis of multicomponent systems represented by simple geometrical bodies, taking shape and size polydispersity as well as interparticle interference effects into account.
Residue depth accurately measures burial and parameterizes local protein environment. Depth is the distance of any atom/residue
to the closest bulk water. We consider the non-bulk waters to occupy cavities, whose volumes are determined using a Voronoi
procedure. Our estimation of cavity sizes is statistically superior to estimates made by CASTp and VOIDOO, and on par with
McVol over a data set of 40 cavities. Our calculated cavity volumes correlated best with the experimentally determined destabilization
of 34 mutants from five proteins. Some of the cavities identified are capable of binding small molecule ligands. In this study,
we have enhanced our depth-based predictions of binding sites by including evolutionary information. We have demonstrated
that on a database (LigASite) of ∼200 proteins, we perform on par with ConCavity and better than MetaPocket 2.0. Our predictions,
while less sensitive, are more specific and precise. Finally, we use depth (and other features) to predict pKas of GLU, ASP, LYS and HIS residues. Our results produce an average error of just <1 pH unit over 60 predictions. Our simple
empirical method is statistically on par with two and superior to three other methods while inferior to only one. The DEPTH
server (http://mspc.bii.a-star.edu.sg/depth/) is an ideal tool for rapid yet accurate structural analyses of protein structures.
The β-glucosidase encoded by the td2f2 gene was isolated from a compost microbial metagenomic library by functional screening. The protein was identified to be
a member of the glycoside hydrolase family 1 and was overexpressed in Escherichia coli, purified, and biochemically characterized. The recombinant β-glucosidase, Td2F2, exhibited enzymatic activity with β-glycosidic
substrates, with preferences for glucose, fucose, and galactose. Hydrolysis occurred at the nonreducing end and in an exo
manner. The order of catalytic efficiency for glucodisaccharides and cellooligosaccharides was sophorose > cellotetraose >
cellotriose > laminaribiose > cellobiose > cellopentaose > gentiobiose, respectively. Intriguingly, the p-nitrophenyl-β-d-glucopyranoside hydrolysis activity of Td2F2 was activated by various monosaccharides and sugar alcohols. At a d-glucose concentration of 1000 mm, enzyme activity was 6.7-fold higher than that observed in the absence of d-glucose. With 31.3 mm d-glucose, Td2F2 catalyzed transglycosylation to generate sophorose, laminaribiose, cellobiose, and gentiobiose. Transglycosylation
products were detected under all activated conditions, suggesting that the activity enhancement induced by monosaccharides
and sugar alcohols may be due to the transglycosylation activity of the enzyme. These results show that Td2F2 obtained from
a compost microbial metagenome may be a potent candidate for industrial applications.
Alkyl glycosides are attractive surfactants because of their high surface activity and good biodegradability and can be produced
from renewable resources. Through enzymatic catalysis, one can obtain well-defined alkyl glycosides, something that is very
difficult to do using conventional chemistry. However, there is a need for better enzymes to get a commercially feasible process.
A thermostable β-glucosidase from the well-studied glycoside hydrolase family 1 from Thermotoga neapolitana, TnBgl1A, was mutated in an attempt to improve its value for synthesis of alkyl glycosides. This was done by rational design
using prior knowledge from structural homologues together with a recently generated model of the enzyme in question. Three
out of four studied mutations increased the hydrolytic reaction rate in an aqueous environment, while none displayed this
property in the presence of an alcohol acceptor. This shows that even if the enzyme resides in a separate aqueous phase, the
presence of an organic solvent has a great influence. We could also show that a single amino acid replacement in a less studied
part of the aglycone subsite, N220F, improves the specificity for transglycosylation 7-fold and thereby increases the potential
yield of alkyl glycoside from 17% to 58%.
Scitation is the online home of leading journals and conference proceedings from AIP Publishing and AIP Member Societies
A method is proposed for the determination of the optimum value of the regularization parameter (Lagrange multiplier) when applying indirect transform techniques in small-angle scattering data analysis. The method is based on perceptual criteria of what is the best solution. A set of simple criteria is used to construct a total estimate describing the quality of the solution. Maximization of the total estimate is straightforward. Model computations show the effectiveness of the technique. The method is implemented in the program GNOM [Svergun, Semenyuk & Feigin (1988). Acta Cryst. A44, 244–250].
Scattering patterns from geometrical bodies with different shapes and anisometry (solid and hollow spheres, cylinders, prisms) are computed and the shapes are reconstructed ab initio using envelope function and bead modelling methods. A procedure is described to analyze multiple solutions provided by bead modeling methods and to estimate stability and reliability of the shape reconstruction. It is demonstrated that flat shapes are more difficult to restore than elongated ones and types of shapes are indicated, which require additional information for reliable shape reconsrtuction from the scattering data.
Five black Aspergillus strains (A. aculeatus, A. foetidus, A. japonicus, A. niger, and A. tubingensis) were cultivated on crude wheat arabinoxylan as the carbon source under defined pH, temperature, and oxygen conditions. Protein and beta-glucosidase content differed remarkably within the obtained culture filtrates, of which eleven beta -glucosidases were isolated. Seven beta -glucosidases were purified to apparent electrophoretic homogeneity using anion-exchange and gel-permeation chromatography. They were found to be acidic proteins and most of them appeared to be glycoproteins with a molecular mass between 93 and 142 kDa. Classification of the beta -glucosidases into four groups (I-A, I-B, II, and III) is suggested according to their physicochemical and biocatalytic properties. The major beta -glucosidases were assigned to groups I-A and I-B, the minor beta -glucosidases to groups II and III, comprising acid-tolerant and glucose-tolerant enzymes, respectively.
Glycoside hydrolase family 3 (GH3) beta-glucosidases (BGLs) from filamentous fungi have been widely and commercially used for supplementation of cellulases. BGL from the fungus Aspergillus aculeatus (AaBGL1) belongs to GH3 and shows high activity toward cellooligosaccharides up to high degree of polymerization. Here we determined the crystal structure of AaBGL1. In addition to the substrate-free structure, complex structures with glucose and various inhibitors were determined. The AaBGL1 structure is highly glycosylated with 88 monosaccharides (18 N-glycan chains) in the dimer. The largest N-glycan chain comprises 10 monosaccharides and is one of the largest glycans ever observed in protein crystal structures. A prominent insertion region exists in a fibronectin type III domain, and this region extends to cover a wide surface area of the enzyme. The subsite +1 of AaBGL1 is highly hydrophobic. Three aromatic residues are involved in the subsite +1 and located in short loop regions that are uniquely present in this enzyme. There is a long cleft extending from the subsite +1, which appears to be suitable for binding long cellooligosaccharides. The crystal structures of AaBGL1 will provide an important structural basis for the technical improvement of enzymatic cellulosic biomass conversion.