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Enhancing the Catalytic Specificity of the β-Glucuronidase At GUS from Aspergillus terreus Li-20 by Site-Directed Mutagenesis on Loop 8
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Licorice, a natural medicine derived from the roots and rhizomes of Glycyrrhiza species, possesses a wide range of therapeutic applications, including antiviral properties. Glycyrrhizic acid (GL) and glycyrrhetinic acid (GA) are the most important active ingredients in licorice. Glycyrrhetinic acid 3-O-mono-β-d-glucuronide (GAMG) is the active metabolite of GL. GL and its metabolites have a wide range of antiviral activities against viruses, such as, the hepatitis virus, herpes virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and so on. Although their antiviral activity has been widely reported, the specific mechanism of action involving multiple links such as the virus itself, cells, and immunity are not clearly established. In this review, we will give an update on the role of GL and its metabolites as antiviral agents, and detail relevant evidence on the potential use and mechanisms of actions. Analyzing antivirals, their signaling, and the impacts of tissue and autoimmune protection may provide promising new therapeutic strategies.
Wild‐type, BaGH5‐WT and mutant, BaGH5‐UV2 (aspartate residue mutated to glycine), endoglucanases belonging to glycoside hydrolase family 5 (GH5), from wild‐type, and UV2 mutant strain of Bacillus amyloliquefaciens SS35, respectively, were earlier cloned in pHTP0 cloning vector. In this study, genes encoding BaGH5‐WT or BaGH5‐UV2 were cloned into pET28a(+) expression‐vector and expressed in Escherichia coli BL‐21(DE3)pLysS cells. BaGH5‐UV2 showed 10‐fold (43.6 U/mg) higher specific activity against carboxymethylcellulose sodium salt (CMC‐Na), higher optimal temperature by 10°C at 65°C, and 22‐fold higher catalytic efficiency against CMC‐Na, than BaGH5‐WT. BaGH5‐UV2 showed stability in wider acidic pH range (5.0–7.0) unlike BaGH5‐WT in narrow basic pH range (7.0–7.5). BaGH5‐UV2 displayed a mutation, Asp256Gly in L11 loop, connecting β6‐sheet with α6‐helix, near active site toward the domain surface of (α/β)8‐TIM barrel fold. Molecular dynamics simulation studies showed more stable structure, accessibility of substrate for a catalytic site, and increased flexibility of loop L11 of BaGH5‐UV2 than the wild type, suggesting enhanced catalysis by BaGH5‐UV2. Molecular docking analysis displayed enhanced hydrogen bond interactions of cello‐oligosaccharides with BaGH5‐UV2, unlike BaGH5‐WT. Thus, Gly256 residue of loop L11 plays an important role in enhancing catalytic efficiency, and pH stability of GH5 endoglucanase. Therefore, these results help in protein engineering of GH5 endoglucanase for improved biochemical properties.
The biological conversion of lignocellulosic matter into high-value chemicals or biofuels is of increasing industrial importance as the sector slowly transitions away from nonrenewable sources. Many industrial processes involve the use of cellulolytic enzyme cocktails – a selection of glycoside hydrolases and, increasingly, polysaccharide oxygenases – to break down recalcitrant plant polysaccharides. ORFs from the genome of Teredinibacter turnerae, a symbiont hosted within the gills of marine shipworms, were identified in order to search for enzymes with desirable traits. Here, a putative T. turnerae glycoside hydrolase from family 8, hereafter referred to as TtGH8, is analysed. The enzyme is shown to be active against β-1,4-xylan and mixed-linkage (β-1,3,β-1,4) marine xylan. Kinetic parameters, obtained using high-performance anion-exchange chromatography with pulsed amperometric detection and 3,5-dinitrosalicyclic acid reducing-sugar assays, show that TtGH8 catalyses the hydrolysis of β-1,4-xylohexaose with a kcat/Km of 7.5 × 10⁷ M⁻¹ min⁻¹ but displays maximal activity against mixed-linkage polymeric xylans, hinting at a primary role in the degradation of marine polysaccharides. The three-dimensional structure of TtGH8 was solved in uncomplexed and xylobiose-, xylotriose- and xylohexaose-bound forms at approximately 1.5 Å resolution; the latter was consistent with the greater kcat/Km for hexasaccharide substrates. A 2,5B boat conformation observed in the −1 position of bound xylotriose is consistent with the proposed conformational itinerary for this class of enzyme. This work shows TtGH8 to be effective at the degradation of xylan-based substrates, notably marine xylan, further exemplifying the potential of T. turnerae for effective and diverse biomass degradation.
Glycyrrhizin (GL), the principal sweet-tasting bioactive ingredient of licorice (root of Glycyrrhiza glabra), shows poor oral absorption and gut microbial transformation of GL to glycyrrhetinic acid (GA) plays a major role for its multiple pharmacological effects. Co-administration of GL-hydrolyzing bacteria appears to be a feasible strategy to enhance GA exposure. This study reported a gut bacterial strain Staphylococcus pasteuri 3I10 which exhibited moderate p-nitrophenyl-β-D-glucuronide (PNPG)-hydrolyzing activity but low GL deglucuronidation activity in its crude lysate. The gus gene encoding S. pasteuri 3I10 β-glucuronidase was successfully cloned and overexpressed in Escherichia coli BL21(DE3). The purified β-glucuronidase (SpasGUS) was 71 kDa and showed optimal pH and temperature at 6.0 and 50 °C, respectively. Comparing to E. coli β-glucuronidase (EcoGUS), SpasGUS displayed lower velocity and affinity to PNPG hydrolysis (Vmax 16.1 ± 0.9 vs 140.0 ± 4.1 μmolmin⁻¹ mg⁻¹; Km 469.4 ± 73.4 vs 268.0 ± 25.8 μM), but could selectively convert GL to GA at much higher efficiency (Vmax 0.41 ± 0.011 vs 0.005 ± 0.002 μmolmin⁻¹ mg⁻¹; Km 116.9 ± 15.4 vs 53.4 ± 34.8 μM). Molecular docking studies suggested SpasGUS formed hydrogen bond interactions with the glucuronic acids at Asn414, Glu415 and Leu450, and Val159, Tyr475, Ala368, and Phe367 provided a hydrophobic environment for enhanced activity. Two special substrate interaction loops near the binding pocket of SpasGUS (loop 1 β-glucuronidase) may account for the selective and efficient bioconversion of GL to GA, predicting that loop 1 β-glucuronidases show high possibility in processing GL than mini-loop 1 and loop 2 β-glucuronidases. These findings support potential applications of SpasGUS in cleaving GL to facilitate GA production in vivo or in pharmaceutical industry.
Compared to chemical methods, the biotransformation of glycyrrhizin (GL) into glycyrrhetinic acid 3- O -mono- β - d -glucuronide (GAMG), which has a higher sweetness and stronger pharmacological activity than those of GL, via catalysis by β -glucuronidase is an environmentally friendly approach due to the mild reaction conditions and the high yield of GAMG. However, currently available GUSs show low substrate specificity toward GL and further hydrolyze GAMG to glycyrrhetinic acid (GA) as a by-product, increasing the difficulty of subsequent separation and purification. In the present study, we succeeded in isolating a novel β -glucuronidase (named TpGUS79A) from Talaromyces pinophilus Li-93 that specifically hydrolyzes GL to GAMG without the formation of GA. TpGUS79A also shows higher activity on GL than those of the previously characterized GUSs. Moreover, the gene for TpGUS79A was cloned and its function verified by heterologous expression in P. pastoris . Therefore, TpGUS79A can serve as a powerful biocatalyst for the cost-effective production of GAMG through GL transformation.
Background
Cellulases of glycosyl hydrolase (GH) family 5 share a (β/α)8 TIM-barrel fold structure with eight βα loops surrounding the catalytic pocket. These loops exposed on the surface play a vital role in protein functions, primarily due to the interactions of some key amino acids with solvent and ligand molecules. It has been reported that motions of these loops facilitate substrate access and product release, and loops 6 and 7 located at the substrate entrance of the binding pocket promote proton transfer reaction at the catalytic site motions. However, the role of these flexible loops in catalysis of GH5 cellulase remains to be explored.
Results
In the present study, an acidic, mesophilic GH5 cellulase (with optimal activity at pH 4.0 and 70 °C), GtCel5, was identified in Gloeophyllum trabeum CBS 900.73. The specific activities of GtCel5 toward CMC-Na, barley β-glucan, and lichenan were 1117 ± 43, 6257 ± 26 and 5318 ± 54 U/mg, respectively. Multiple sequence alignment indicates that one amino acid residue at position 233 on the loop 6 shows semi-conservativeness and might contribute to the great catalytic performance. Saturation mutagenesis at position 233 was then conducted to reveal the vital roles of this position in enzyme properties. In comparison to the wild type, variants N233A and N233G showed decreased optimal temperature (− 10 °C) but increased activities (27 and 70%) and catalytic efficiencies (kcat/Km; 45 and 52%), respectively. The similar roles of position 233 in catalytic performance were also verified in the other two GH5 homologs, TeEgl5A and PoCel5, by reverse mutation. Further molecular dynamics simulations suggested that the substitution of asparagine with alanine or glycine may introduce more hydrogen bonds, increase the flexibility of loop 6, enhance the interactions between enzyme and substrate, and thus improve the substrate affinity and catalytic efficiency.
Conclusion
This study proposed a novel cellulase with potentials for industrial application. A specific position was identified to play key roles in cellulase–substrate interactions and enzyme catalysis. It is of great importance for understanding the binding mechanism of GH5 cellulases, and provides an effective strategy to improve the catalytic performance of cellulases.
Electronic supplementary material
The online version of this article (10.1186/s13068-018-1080-5) contains supplementary material, which is available to authorized users.
Glycoside hydrolases (GHs) have attracted special attention in research aimed at modifying natural products by partial removal of sugar moieties to manipulate their solubility and efficacy. However, these modifications are challenging to control because the low substrate specificity of most GHs often generates undesired by-products. We previously identified a GH2-type fungal β-glucuronidase from Aspergillus oryzae (PGUS) exhibiting promiscuous substrate specificity in hydrolysis of triterpenoid saponins. Here, we present the PGUS structure, representing the first structure of a fungal β-glucuronidase, and of an inactive PGUS mutant in complex with the native substrate glycyrrhetic acid 3-O-mono-β- glucuronide (GAMG). PGUS displayed a homotetramer structure, with each monomer comprising three distinct domains: a sugarbinding, an immunoglobulin-like β-sandwich, and a TIM barrel domain. Two catalytic residues, Glu-414 and Glu-505, acted as acid/base and nucleophile, respectively. Structural and mutational analyses indicated that the GAMG glycan moiety is recognized by polar interactions with nine residues (Asp-162, His-332, Asp-414, Tyr-469, Tyr-473, Asp-505, Arg-563, Asn-567, and Lys-569) and that the aglycone moiety is recognized by aromatic stacking and by a π interaction with the four aromatic residues Tyr- 469, Phe-470, Trp-472, and Tyr-473. Finally, structure-guided mutagenesis to precisely manipulate PGUS substrate specificity in the biotransformation of glycyrrhizin into GAMG revealed that two amino acids, Ala-365 and Arg- 563, are critical for substrate specificity. Moreover, we obtained several mutants with dramatically improved GAMG yield (>95%). Structural analysis suggested that modulating the interaction of β-glucuronidase simultaneously toward glycan and aglycone moieties is critical for tuning its substrate specificity toward triterpenoid saponins.
Objective:
To isolate and characterize the kinetics of variants of E. coli β-glucuronidase (GUS) having altered substrate specificity.
Results:
Two small combinatorial libraries of E. coli GUS variants were constructed and screened for improved activities towards the substrate p-nitrophenyl-β-D-galactoside (pNP-gal). Nine of the most active variants were purified and their kinetic parameters were determined. These variants show up to 134-fold improved kcat/KM value towards pNP-gal compared to wild-type GUS, up to 9 × 10(8)-fold shift in specificity from p-nitrophenyl-β-D-glucuronide (pNP-glu) to pNP-gal compared to wild-type, and 10(3)-fold increase in specificity shift compared to a previously evolved GUS variant.
Conclusions:
The kinetic data collected for nine new GUS variants is invaluable for training computational protein design models that better predict amino acid substitutions which improve activity of enzyme variants having altered substrate specificity.
Importance:
Loop structures play critical roles in the substrate specificity and catalytic hydrolysis of GH12 enzymes. Three typical loops exist in these enzymes: Loop 1 and 2 are recognized as the catalytic loops, and closely related to the substrate specificity and catalytic efficiency. Loop 3 locates in the -1 or +1 subsites and varies a lot in amino acid composition may play a role in catalysis. In this study, two GH12 glucanases, NfEG12A and EG, which were mutated by introducing or deleting partial loop 3 sequences FN and/or STTQA, were selected to identify the function of loop 3. It revealed that longer loop 3 of GH12 glucanases may strengthen the hydrogen network interactions between the substrate and protein, and consequently increased the turnover rate (kcat). This study proposes a strategy to increase the catalytic efficiency of GH12 glucanases by improving the hydrogen network between substrates and catalytic loops.
In this study, we proposed a loop transplant strategy to improve the thermostability of Penicillium purpurogenum Li-3 β-glucuronidase expressed in Escherichia coli (abbreviated to PGUS-E). Firstly, three unstable surface loops of PGUS-E to be replaced were identified with regards to B-factor values and in-depth structure analysis: loops 205-211, 258-263, and 25-31. Then, based on B-factor analysis, eight stable loops for substitution were selected from two typical thermophilic glycosidases which had low homology with PGUS-E (less than 25 %). By analyzing the common features of these stable loops, it was found that they shared a common residue skeleton DXXTX(X)R, based on this, three chimera loops were also manually designed: RSQTSND, RSSTQRD, and DDQTSR. All these loops were introduced to replace the unstable loops of PGUS-E by homology structure modeling, and only mutants with increased hydrogen bonds number and good compatibility with the local mutated region were further subjected to experimental verification. By using this strategy, 10 mutants were experimentally generated, among which three mutants, M1, M3, and M8, were obtained which showed 11.8, 3.3, and 9.4 times higher half-life at 70 °C than that of wild-type (8.5 min). Finally, the MD simulation indicated that the increased hydrogen bonds, decreased flexibility of N-terminal, and increased π-π stacking interaction were responsible for the improved thermostability.
This study provides the scientific basis for the anti-inflammatory effects of licorice extract in a t-BHP (tert-butyl hydrogen peroxide)-induced liver damage model and the effects of its ingredients, glycyrrhizic acid (GA), liquiritin (LQ) and liquiritigenin (LG), in a lipopolysaccharide (LPS)-stimulated microglial cell model. The GA, LQ and LG inhibited the LPS-stimulated elevation of pro-inflammatory mediators, such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and interleukin (IL)-6 in BV2 (mouse brain microglia) cells. Furthermore, licorice extract inhibited the expression levels of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) in the livers of t-BHP-treated mice models. This result suggested that mechanistic-based evidence substantiating the traditional claims of licorice extract and its three bioactive components can be applied for the treatment of inflammation-related disorders, such as oxidative liver damage and inflammation diseases.
Three fungi with different types of transformation of glycyrrhizin (GL) were isolated from the soil samples of glycyrrhiza glabra planting area in China. According to their morphologies and 18 S rDNA gene sequence analysis, the three fungi were identified and named as Penicillium purpurogenum Li-3, Aspergillus terreus Li-20 and Aspergillus ustus Li-62. Transforming products analysis by TLC and HPLC-MS indicated that P. purpurogenum Li-3, A. terreus Li-20 and A. ustus Li-62 could stably transform GL into GAMG, GAMG and GA, and GA, respectively. P. purpurogenum Li-3 was especially valuable to directly prepare GAMG for applications in the pharmaceutical industry.
We cloned the β-glucuronidase gene (AtGUS) from Aspergillus terreus Li-20 encoding 657 amino acids (aa), which can transform glycyrrhizin into glycyrrhetinic acid monoglucuronide (GAMG) and glycyrrhetinic acid (GA). Based on sequence alignment, the C-terminal non-conservative sequence showed low identity with those of other species; thus, the partial sequence AtGUS(-3t) (1-592 aa) was amplified to determine the effects of the non-conservative sequence on the enzymatic properties. AtGUS and AtGUS(-3t) were expressed in E. coli BL21, producing AtGUS-E and AtGUS(-3t)-E, respectively. At the similar optimum temperature (55°C) and pH (AtGUS-E, 6.6; AtGUS(-3t)-E, 7.0) conditions, the thermal stability of AtGUS(-3t)-E was enhanced at 65°C, and the metal ions Co(2+), Ca(2+) and Ni(2+) showed opposite effects on AtGUS-E and AtGUS(-3t)-E, respectively. Furthermore, Km of AtGUS(-3t)-E (1.95 mM) was just nearly one-seventh that of AtGUS-E (12.9 mM), whereas the catalytic efficiency of AtGUS(-3t)-E was 3.2 fold higher than that of AtGUS-E (7.16 vs. 2.24 mM s(-1)), revealing that the truncation of non-conservative sequence can significantly improve the catalytic efficiency of AtGUS. Conformational analysis illustrated significant difference in the secondary structure between AtGUS-E and AtGUS(-3t)-E by circular dichroism (CD). The results showed that the truncation of the non-conservative sequence could preferably alter and influence the stability and catalytic efficiency of enzyme.
The dose-limiting side effect of the common colon cancer chemotherapeutic CPT-11 is severe diarrhea caused by symbiotic bacterial
β-glucuronidases that reactivate the drug in the gut. We sought to target these enzymes without killing the commensal bacteria
essential for human health. Potent bacterial β-glucuronidase inhibitors were identified by high-throughput screening and shown
to have no effect on the orthologous mammalian enzyme. Crystal structures established that selectivity was based on a loop
unique to bacterial β-glucuronidases. Inhibitors were highly effective against the enzyme target in living aerobic and anaerobic
bacteria, but did not kill the bacteria or harm mammalian cells. Finally, oral administration of an inhibitor protected mice
from CPT-11–induced toxicity. Thus, drugs may be designed to inhibit undesirable enzyme activities in essential microbial
symbiotes to enhance chemotherapeutic efficacy.
Human beta-glucuronidase (hGUSB) is a member of family 2 glycosylhydrolases that cleaves beta-D-glucuronic acid residues from the nonreducing termini of glycosaminoglycans. Amino acid sequence and structural homology of hGUSB and Escherichia coli beta-galactosidase active sites led us to propose that residues Glu(451), Glu(540), and Tyr(504) in hGUSB are involved in catalysis, Glu(451) being the acid-base residue and Glu(540) the nucleophile. To test this hypothesis, we introduced mutations in these residues and determined their effects on enzymes expressed in COS cells and GUSB-deficient fibroblasts. The extremely low activity in cells expressing Glu(451), Glu(540), and Tyr(504) hGUSBs supported their roles in catalysis. For kinetic analysis, wild type and mutant enzymes were produced in baculovirus and purified to homogeneity by affinity chromatography. The k(cat)/K(m) values (mM(-1).s(-1)) of the E540A, E451A, and Y504A enzymes were 34,000-, 9100-, and 830-fold lower than that of wild type hGUSB, respectively. High concentrations of azide stimulated the activity of the E451A mutant enzyme, supporting the role of Glu(451) as the acid-base catalyst. We conclude that, like their homologues in E. coli beta-galactosidase, Glu(540) is the nucleophilic residue, Glu(451) the acid-base catalyst, and Tyr(504) is also important for catalysis, although its role is unclear. All three residues are located in the active site cavity previously determined by structural analysis of hGUSB.
Protein engineers have widely adopted directed evolution as a design algorithm, but practitioners have not come to a consensus about the best method to evolve protein molecular recognition. We previously used DNA shuffling to direct the evolution of Escherichia coli beta-glucuronidase (GUS) variants with increased beta-galactosidase activity. Epistatic (synergistic) mutations in amino acids 557, 566, and 568, which are part of an active site loop, were identified in that experiment (Matsumura, I., and Ellington, A. D. (2001) J. Mol. Biol. 305, 331-339). Here we show that site saturation mutagenesis of these residues, overexpression of the resulting library in E. coli, and high throughput screening led to the rapid evolution of clones exhibiting increased activity in reactions with p-nitrophenyl-beta-d-xylopyranoside (pNP-xyl). The xylosidase activities of the 14 fittest clones were 30-fold higher on average than that of the wild-type GUS. The 14 corresponding plasmids were pooled, amplified by long PCR, self-ligated with T4 DNA ligase, and transformed into E. coli. Thirteen clones exhibiting an average of 80-fold improvement in xylosidase activity were isolated in a second round of screening. One of the evolved proteins exhibited a approximately 200-fold improvement over the wild type in reactivity (k(cat)/K(m)) with pNP-xyl, with a 290,000-fold inversion of specificity. Sequence analysis of the 13 round 2 isolates suggested that all were products of intermolecular recombination events that occurred during whole plasmid PCR. Further rounds of evolution using DNA shuffling and staggered extension process (StEP) resulted in modest improvement. These results underscore the importance of epistatic interactions and demonstrate that they can be optimized through variations of the facile whole plasmid PCR technique.
Licorice (Glycyrrhiza glabra L., Leguminosae) is frequently used in traditional medicine to treat inflammatory and allergic diseases. In this study, the main components (glycyrrhizin, 18beta-glycyrrhetinic acid, isoliquiritin, and liquiritigenin) were isolated from licorice, and their anti-allergic effects, such as antiscratching behavior and IgE production-inhibitory activity, were evaluated both in vitro and in vivo. Liquiritigenin and 18beta-glycyrrhetinic acid most potently inhibited the degranulation of RBL-2H3 cells induced by IgE with the antigen (DNP-HSA) and rat peritoneal mast cells induced by compound 48/80. Liquiritigenin and 18beta-glycyrrhetinic acid potently inhibited the passive cutaneous anaphylactic reaction as well as the scratching behavior in mice induced by compound 48/80. These components inhibited the production of IgE in ovalbumin-induced asthma mice but liquiritigenin had little effect. This suggests that the antiallergic effects of licorice are mainly due to glycyrrhizin, 18beta-glycyrrhetinic acid, and liquiritigenin, which can relieve IgE-induced allergic diseases such as dermatitis and asthma.
Licorice (Glycyrrhiza glabra) is a well-known natural herb used to treat different ailments since ancient times. Glycyrrhizin (GL), which is the primary triterpenoid compound of licorice extract, has been known to have broad-spectrum pharmacological effects. GL is cleaved into glucuronide and the aglycone, glycyrrhetinic acid (GA), which exists in two stereoisomeric forms: 18α- and 18β-GA. It is well documented that GL and GA have great potential as anti-inflammatory, anti-cancer, antiviral, anti-diabetic, antioxidant, and hepatoprotective agents. Studies undertaken during the coronavirus disease 2019 pandemic suggest that GL is effective at inhibiting the viral replication of severe acute respiratory syndrome coronavirus 2. The anti-cancer effects of GL and GA involve modulating various signaling pathways, such as the phosphatase and tensin homolog/phosphatidylinositol 3-kinase/protein kinase B pathway, the mitogen-activated protein kinase, and the mammalian target of rapamycin/signal transducer and activator of transcription 3, which are mainly involved in regulating cancer cell death, oxidative stress, and inflammation. The potential of GL and GA in preventing cancer development and suppressing the growth and invasion of different cancer types has been reviewed in this paper. This review also provides molecular insights on the mechanism of action for the oncopreventive and oncotherapeutic effects of GL and its derivative, GA, which could help develop more specific forms of these agents for clinical use.
Glucuronidated drug metabolites can be quantified from urine samples by first hydrolyzing conjugates with β-glucuronidase (β-GUS) and then separating free drug molecules by liquid chromatography and mass spectrometry detection (LC-MS). To improve the activity and specificity of various β-GUS, we designed enzyme chimeras and generated site-saturation variants based on structural analyses, then screened them for improved activity on drug metabolites important to clinical and forensic drug-testing laboratories. Often, an increase of activity on one substrate of interest was countered by loss of activity against another, and there was no strong correlation of activity on standard β-glucuronidase substrates to activity on recalcitrant drug glucuronides. However, we discovered a chimera of two enzymes from different species of Aspergillus that displays a 27% increase in activity on morphine-3-glucuronide than the parent proteins. Furthermore, mutations in the M-loop, which is a loop near the active site, resulted in numerous variants with dramatically increased rates of hydrolysis on drug glucuronides. Specifically, the M-loop variant Q451D/A452E of a β-GUS from Brachyspira pilosicoli has a 50-fold and 25-fold increase in activity on the recalcitrant substrates codeine-6-glucuronide and dihydrocodeine-6-glucuronide, respectively, compared to the parent enzyme.
Glycyrrhetinic acid monoglucuronide (GAMG) is an innovative functional sweetener with higher sweetness and stronger pharmacological activity than glycyrrhizin (GL). A novel β-glucuronidase (cg-GUS) was firstly screened from plant endophytic fungus Chaetomium globosum DX-THS3. The cg-GUS demonstrated the specify and highly transform glycyrrhizin (GL) to generate GAMG, and the maximum activity of β-glucuronidase at 45 °C and pH 6.0, displaying excellent thermostability and pH-stability. The Km and Vmax values of cg-GUS were 0.134 mM and 236.42 mM/min/mg, respectively, which showed the high chemical bond selectivity and biotransformation efficiency of cg-GUS. Meanwhile, the cg-GUS gene (1896 bp) was analyzed, and Gly-345, Ser-539, Gly-563, Ala-579, Ser-581 and Glu-619 in GH2 catalytic domain of cg-GUS are potential mutation position for result in high-efficient and substrate-specify of cg-GUS. Our results were indicated that cg-GUS is a biocatalyst for production of GAMG and potent application in food and medicinal industry.
In this work, steam explosion (SE) was exploited as a green and facile process to deconstruct liquorice's structure and deglycosylate glycyrrhizic acid (GL) to improve conversion and diffusion efficacy of GL and its hydrolyzed products. Results showed SE induced auto-hydrolysis of GL into glycyrrhetic acid 3-O-mono-β-D-glucuronide (GAMG) and glycyrrhetinic acid (GA), by which 30.71% of GL conversion, 5.24% and 21.47% of GAMG and GA formation were obtained. GL hydrolytic pathways were revealed by reaction kinetics and thermodynamics, which possessed complex consecutive and parallel reactions with endothermic, non-spontaneous and entropy-decreasing features. SE referred to cause cleavage of the β-1,3 glycosidic bond in GL which was hydrolyzed to GA as a main product and GAMG and glucuronic acids as minor products. Diffusion of hydrolyzed products was accelerated by raising the diffusion coefficient and shortening the equilibrium time by over 90%. This work provides a sustainable and efficient route for product conversion and function enhancement of bioactive components.
β‐Glucuronidase (GUS) enzymes in the gastrointestinal tract are involved in maintaining mammalian‐microbial symbiosis and can play key roles in drug efficacy and toxicity. Parabacteroides merdae GUS was identified as an abundant mini‐Loop 2 (mL2) type GUS enzyme in the Human Microbiome Project gut metagenomic database. Here, we report the crystal structure of P. merdae GUS and highlight the differences between this enzyme and extant structures of gut microbial GUS proteins. We find that P. merdae GUS exhibits a distinct tetrameric quaternary structure and that the mL2 motif traces a unique path within the active site, which also includes two arginines distinctive to this GUS. We observe two states of the P. merdae GUS active site; a loop repositions itself by more than 50 Å to place a functionally‐relevant residue into the enzyme's catalytic site. Finally, we find that P. merdae GUS is able to bind to homo‐ and heteropolymers of the polysaccharide alginic acid. Together, these data broaden our understanding of the structural and functional diversity in the GUS family of enzymes present in the human gut microbiome and point to specialization as an important feature of microbial GUS orthologs.
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Glycyrrhetic acid 3‐O‐mono‐β‐d‐glucuronide (GAMG) is an important derivative of glycyrrhizin (GL) and has attracted considerable attention, especially in the food and pharmaceutical industries, due to its natural high sweetness and strong biological activities. The biotransformation process is becoming an efficient route for GAMG production with the advantages of mild reaction conditions, environmentally friendly process, and high production efficiency. Recent studies showed that several β‐glucuronidases (β‐GUS) are key GAMG‐producing enzymes, displaying a high potential to convert GL directly into the more valuable GAMG and providing new insights into the generation of high‐value compounds. This review provides details of the structural properties, health benefits, and potential applications of GAMG. The progress in the development of the biotransformation processes and fermentation strategies to improve the yield of GAMG is also discussed. This work further summarizes recent advances in the enzymatic synthesis of GAMG using β‐GUS with emphasis on the physicochemical and biological properties, molecular modifications, and enzymatic strategies to improve β‐GUS biocatalytic efficiencies. This information contributes to a better framework to explore production and application of bioactive GAMG.
Endo-polygalacturonases (PGs) of glycosyl hydrolase (GH) family 28 share a right handed parallel β-helical structure with a cleft formed by loops T1 and T3. To reveal the effect of non-active-site residues on endo-PG catalysis, Thr113 of Achaetomium sp. endo-PG (PG8fn) located on the T3 loop was substituted. The experimental results indicated that the catalytic efficiency of PG8fn depends on the side-chain structure of residue at position 113, following the order of Glu (negatively charged) <Gly, Ala and Ile (nonpolar) <Ser, Gln and Thr (polar) <Arg and Lys (positively charged). Void pathway analysis demonstrated that substitution with Arg caused the increased occupancy rates of void pathways T2 and T3 and resulted in the improvement of catalytic efficiency by increasing the kinetics of substrate access (kon) or product release (koff). Further molecular dynamics (MD) simulations illustrated that the substitution enhanced the flexibility of T3 loop and influenced the interaction between enzyme and substrate. The functional role of Arg113 was also verified in another GH28 endo-PG by increasing the catalytic efficiency of ∼2.4-fold. This study proposes a novel strategy, i.e. selective modification of the non-active site residues, to improve the catalytic performance of GH28 PGs.
β-Glucuronidase (GUS) has been widely used to hydrolyze β-linked glucuronides to generate various valuable derivatives. In this study, the GUS from three fungi (PGUS from P. purpurogenum Li-3, AtGUS from A. terreus Li-20 and AuGUS from A. ustus Li-62) with significantly different types of glycyrrhizin (GL) hydrolysis were comparatively investigated. The Km values of PGUS, AtGUS and AuGUS were 0.328, 3.61 and 0.429 mM respectively. These results indicated that AtGUS showed the lowest affinity for GL among the three kinds of GUS, while PGUS and AuGUS had a similar affinity for GL. Nevertheless, the Vmax/Km values showed that AuGUS had the highest catalytic efficiency for GL hydrolysis, so it was supposed to be an efficient biocatalysis for GL biotransformation. The sequence properties analysis demonstrated that the three GUS had some special different sequence characteristics, but that is not the key reason for the discrepancy in catalytic type. The homologous modeling analysis indicated that various GL transformation types of PGUS, AtGUS and AuGUS were likely caused by the different positions of bacterial loops surrounding their aglycone binding pocket. These results can not only help us to better understand the catalytic diversity of GUS, but also give us an important guide to redesign the catalytic diversity of GUS.
The X-ray structure of the homotetrameric lysosomal acid hydrolase, human -glucuronidase (332,000 M
r), has been determined at 2.6 Å resolution. The tetramer has approximate dihedral symmetry and each protomer consists of three structural domains with topologies similar to a jelly roll barrel, an immunoglobulin constant domain and a TIM barrel respectively. Residues 179−204 form a -hairpin motif similar to the putative lysosomal targeting motif of cathepsin D, supporting the view that lysosomal targeting has a structural basis. The active site of the enzyme is formed from a large cleft at the interface of two monomers. Residues Glu 451 and Glu 540 are proposed to be important for catalysis. The structure establishes a framework for understanding mutations that lead to the human genetic disease mucopolysaccharidosis VII, and for using the enzyme in anti-cancer therapy.
One fungus, tentatively named Penicillium sp. Li-3, was screened to biosynthesize β-d-mono-glucuronide-glycyrrhizin (GAMG), directly. Using glycyrrhizin as elicitor and the sole carbon source, this strain was capable of expressing β-d-glucuronidase, one intracellular enzyme with high substrate specificity. And glycyrrhizin was hydrolyzed directly into GAMG by enzyme from Penicillium sp. Li-3 with high production. It was found that the mol conversion ratio of this reaction was up to 88.45%. Research about kinetics of β-d-glucuronidase production showed that the cell growth and enzyme production of this strain was partial coupled. During the expressing of target enzyme, carbon catabolite repression existed, so only glycyrrhizin was the best carbon source as well as the elicitor. It was found that the surfactant (Tween 80 0.12%) could improve the ability of enzyme production markedly. Under the condition of initial pH 4.8 of the medium and 32 °C of the culture temperature, the maximum enzyme activity of 181.53 U ml−1 was obtained.
To improve the taste profile of glycyrrhizin (1, the saponin of licorice root, relative sweetness to sucrose: x170), a variety of 3-O-glycosides of glycyrrhetic acid were prepared and their sweetness evaluated. It was found that a significant enhancement of sweetness was observed for the 3-O-beta-D-xyloside and the 3-O-beta-D-glucuronide (MGGR). Especially, MGGR had a high sweetness relative to sucrose; x941, and would appear to be a new potent sweetener.
The relationship between the metabolites of glycyrrhizin (18beta-glycyrrhetinic acid-3-O-beta-D-glucuronopyranosyl-(1-->2)-beta-D-glucuronide, GL) and their biological activities was investigated. By human intestinal microflora, GL was metabolized to 18beta-glycyrrhetinic acid (GA) as a main product and to 18beta-glycyrrhetinic acid-3-O-beta-D-glucuronide (GAMG) as a minor product. The former reaction was catalyzed by Eubacterium L-8 and the latter was by Streptococcus LJ-22. Among GL and its metabolites, GA and GAMG had more potent in vitro anti-platelet aggregation activity than GL. GA also showed the most potent cytotoxicity against tumor cell lines and the potent inhibitory activity on rotavirus infection as well as growth of Helicobacter pylori. GAMG, the minor metabolite of GL, was the sweetest.
Recent studies on triosephosphate isomerase (TIM)-barrel enzymes highlight the remarkable versatility of the TIM-barrel scaffold. At least 15 distinct enzyme families use this framework to generate the appropriate active site geometry, always at the C-terminal end of the eight parallel beta-strands of the barrel. Sequence and structure comparisons now suggest that many of the TIM-barrel enzymes are evolutionarily related. Common structural properties of TIM-barrel enzymes are discussed.