10 results on '"Ben Bdira F"'
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2. Understanding functional dynamics and conformational stability of beta-glycosidases
- Author
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Ben Bdira, F., Ubbink, M., Aerts, J.M.F.G., Overkleeft, H.S., Noteborn, M.H.M., McIntosh, L.P., Berkel, W.J.H. van, Claessens, M.M.A.E., and Leiden University
- Subjects
Glucocerebrosidase ,Paramagnetic NMR ,Activity based probes ,Retaining beta-glycosidases ,Xylanase ,Relaxation dispersion ,Endoglycoceramidase II - Abstract
Due to their central physiological roles in living organisms, retaining beta-glycosidases have been the subject of tremendous research efforts to examine their structure/function relation using numerous biophysical and biochemical approaches. Since the proposition of the hydrolysis mechanism in the late fifties by Koshland1, the fundamental research on retaining b-glycosidases has been revolutionized by the discovery of multiple reversible and irreversible inhibitors. One of the most successful class of inhibitors are mechanism based inactivators, which were extensively used to identify the nucleophilic catalytic residues and to comprehend the catalytic mechanism and substrate itinerary. Subsequently, covalent inhibitors were used as warheads to synthesize chromogenic activity based probes (ABPs), which were widely used to selectively label and discover new retaining beta-glycosidases in complex biological samples. The organic synthesis and biological applications of these ABPs has become routine. Nevertheless, their binding mechanism and influences on protein conformation and dynamics remained unexplored. Therefore, this work is aimed to establish a bridge between the two research disciplines, using ABP technology to understand functional dynamics and conformational stability of retaining bglycosidases in solution and in vivo. The research relied on standard biochemistry and advanced NMR spectroscopy research approaches.
- Published
- 2018
3. Bimodal substrate binding in the active site of the glycosidase BcX.
- Author
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Saberi M, Chikunova A, Ben Bdira F, Cramer-Blok A, Timmer M, Voskamp P, and Ubbink M
- Subjects
- Substrate Specificity, Crystallography, X-Ray, Models, Molecular, Bacillus enzymology, Bacillus genetics, Binding Sites, Bacterial Proteins metabolism, Bacterial Proteins chemistry, Bacterial Proteins genetics, Protein Binding, Endo-1,4-beta Xylanases chemistry, Endo-1,4-beta Xylanases genetics, Endo-1,4-beta Xylanases metabolism, Glycoside Hydrolases metabolism, Glycoside Hydrolases chemistry, Glycoside Hydrolases genetics, Xylose metabolism, Xylose chemistry, Kinetics, Catalytic Domain
- Abstract
Bacillus circulans xylanase (BcX) from the glycoside hydrolase family 11 degrades xylan through a retaining, double-displacement mechanism. The enzyme is thought to hydrolyze glycosidic bonds in a processive manner and has a large, active site cleft, with six subsites allowing the binding of six xylose units. Such an active site architecture suggests that oligomeric xylose substrates can bind in multiple ways. In the crystal structure of the catalytically inactive variant BcX E78Q, the substrate xylotriose is observed in the active site, as well as bound to the known secondary binding site and a third site on the protein surface. Nuclear magnetic resonance (NMR) titrations with xylose oligomers of different lengths yield nonlinear chemical shift trajectories for active site nuclei resonances, indicative of multiple binding orientations for these substrates for which binding and dissociation are in fast exchange on the NMR timescale, exchanging on the micro- to millisecond timescale. Active site binding can be modeled with a 2 : 1 model with dissociation constants in the low and high millimolar range. Extensive mutagenesis of active site residues indicates that tight binding occurs in the glycon binding site and is stabilized by Trp9 and the thumb region. Mutations F125A and W71A lead to large structural rearrangements. Binding at the glycon site is sensed throughout the active site, whereas the weak binding mostly affects the aglycon site. The interactions with the two active site locations are largely independent of each other and of binding at the secondary binding site., (© 2024 The Author(s). The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)
- Published
- 2024
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4. Tuning the Transglycosylation Reaction of a GH11 Xylanase by a Delicate Enhancement of its Thumb Flexibility.
- Author
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Marneth K, van den Elst H, Cramer-Blok A, Codee J, Overkleeft HS, Aerts JMFG, Ubbink M, and Ben Bdira F
- Subjects
- Bacillus enzymology, Bacterial Proteins chemistry, Bacterial Proteins genetics, Binding Sites, Catalytic Domain, Endo-1,4-beta Xylanases chemistry, Endo-1,4-beta Xylanases genetics, Glycosylation, Molecular Dynamics Simulation, Mutagenesis, Site-Directed, Nuclear Magnetic Resonance, Biomolecular, Transition Temperature, Bacterial Proteins metabolism, Endo-1,4-beta Xylanases metabolism
- Abstract
Glycoside hydrolases (GHs) are attractive tools for multiple biotechnological applications. In conjunction with their hydrolytic function, GHs can perform transglycosylation under specific conditions. In nature, oligosaccharide synthesis is performed by glycosyltransferases (GTs); however, the industrial use of GTs is limited by their instability in solution. A key difference between GTs and GHs is the flexibility of their binding site architecture. We have used the xylanase from Bacillus circulans (BCX) to study the interplay between active-site flexibility and transglycosylation. Residues of the BCX "thumb" were substituted to increase the flexibility of the enzyme binding site. Replacement of the highly conserved residue P116 with glycine shifted the balance of the BCX enzymatic reaction toward transglycosylation. The effects of this point mutation on the structure and dynamics of BCX were investigated by NMR spectroscopy. The P116G mutation induces subtle changes in the configuration of the thumb and enhances the millisecond dynamics of the active site. Based on our findings, we propose the remodelling of the GH enzymes glycon site flexibility as a strategy to improve the transglycosylation efficiency of these biotechnologically important catalysts., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)
- Published
- 2021
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5. Dynamics of Ligand Binding to a Rigid Glycosidase*.
- Author
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Ben Bdira F, Waudby CA, Volkov AN, Schröder SP, Ab E, Codée JDC, Overkleeft HS, Aerts JMFG, van Ingen H, and Ubbink M
- Subjects
- Hydrolysis, Kinetics, Ligands, Models, Molecular, Protein Binding, Glycoside Hydrolases metabolism
- Abstract
The single-domain GH11 glycosidase from Bacillus circulans (BCX) is involved in the degradation of hemicellulose, which is one of the most abundant renewable biomaterials in nature. We demonstrate that BCX in solution undergoes minimal structural changes during turnover. NMR spectroscopy results show that the rigid protein matrix provides a frame for fast substrate binding in multiple conformations, accompanied by slow conversion, which is attributed to an enzyme-induced substrate distortion. A model is proposed in which the rigid enzyme takes advantage of substrate flexibility to induce a conformation that facilitates the acyl formation step of the hydrolysis reaction., (© 2020 The Authors. Published by Wiley-VCH GmbH.)
- Published
- 2020
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6. Distinguishing the differences in β-glycosylceramidase folds, dynamics, and actions informs therapeutic uses.
- Author
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Ben Bdira F, Artola M, Overkleeft HS, Ubbink M, and Aerts JMFG
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- Animals, Cerebrosides metabolism, Gaucher Disease metabolism, Glycoconjugates metabolism, Glycolipids metabolism, Humans, Lactase-Phlorizin Hydrolase metabolism, Parkinson Disease metabolism, Glucosylceramidase chemistry, Glucosylceramidase metabolism
- Abstract
Glycosyl hydrolases (GHs) are carbohydrate-active enzymes that hydrolyze a specific β-glycosidic bond in glycoconjugate substrates; β-glucosidases degrade glucosylceramide, a ubiquitous glycosphingolipid. GHs are grouped into structurally similar families that themselves can be grouped into clans. GH1, GH5, and GH30 glycosidases belong to clan A hydrolases with a catalytic (β/α)
8 TIM barrel domain, whereas GH116 belongs to clan O with a catalytic (α/α)6 domain. In humans, GH abnormalities underlie metabolic diseases. The lysosomal enzyme glucocerebrosidase (family GH30), deficient in Gaucher disease and implicated in Parkinson disease etiology, and the cytosol-facing membrane-bound glucosylceramidase (family GH116) remove the terminal glucose from the ceramide lipid moiety. Here, we compare enzyme differences in fold, action, dynamics, and catalytic domain stabilization by binding site occupancy. We also explore other glycosidases with reported glycosylceramidase activity, including human cytosolic β-glucosidase, intestinal lactase-phlorizin hydrolase, and lysosomal galactosylceramidase. Last, we describe the successful translation of research to practice: recombinant glycosidases and glucosylceramide metabolism modulators are approved drug products (enzyme replacement therapies). Activity-based probes now facilitate the diagnosis of enzyme deficiency and screening for compounds that interact with the catalytic pocket of glycosidases. Future research may deepen the understanding of the functional variety of these enzymes and their therapeutic potential., (Copyright © 2018 Ben Bdira et al.)- Published
- 2018
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7. Repurposing ciclopirox as a pharmacological chaperone in a model of congenital erythropoietic porphyria.
- Author
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Urquiza P, Laín A, Sanz-Parra A, Moreno J, Bernardo-Seisdedos G, Dubus P, González E, Gutiérrez-de-Juan V, García S, Eraña H, San Juan I, Macías I, Ben Bdira F, Pluta P, Ortega G, Oyarzábal J, González-Muñiz R, Rodríguez-Cuesta J, Anguita J, Díez E, Blouin JM, de Verneuil H, Mato JM, Richard E, Falcón-Pérez JM, Castilla J, and Millet O
- Subjects
- Allosteric Site, Animals, Biophysical Phenomena, Cell Line, Ciclopirox pharmacokinetics, Disease Models, Animal, Homeostasis, Mice, Phenotype, Porphyria, Erythropoietic enzymology, Porphyria, Erythropoietic pathology, Uroporphyrinogen III Synthetase antagonists & inhibitors, Uroporphyrinogen III Synthetase chemistry, Uroporphyrinogen III Synthetase metabolism, Ciclopirox therapeutic use, Drug Repositioning, Porphyria, Erythropoietic drug therapy
- Abstract
Congenital erythropoietic porphyria is a rare autosomal recessive disease produced by deficient activity of uroporphyrinogen III synthase, the fourth enzyme in the heme biosynthetic pathway. The disease affects many organs, can be life-threatening, and currently lacks curative treatments. Inherited mutations most commonly reduce the enzyme's stability, altering its homeostasis and ultimately blunting intracellular heme production. This results in uroporphyrin by-product accumulation in the body, aggravating associated pathological symptoms such as skin photosensitivity and disfiguring phototoxic cutaneous lesions. We demonstrated that the synthetic marketed antifungal ciclopirox binds to the enzyme, stabilizing it. Ciclopirox targeted the enzyme at an allosteric site distant from the active center and did not affect the enzyme's catalytic role. The drug restored enzymatic activity in vitro and ex vivo and was able to alleviate most clinical symptoms of congenital erythropoietic porphyria in a genetic mouse model of the disease at subtoxic concentrations. Our findings establish a possible line of therapeutic intervention against congenital erythropoietic porphyria, which is potentially applicable to most of deleterious missense mutations causing this devastating disease., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)
- Published
- 2018
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8. Stabilization of Glucocerebrosidase by Active Site Occupancy.
- Author
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Ben Bdira F, Kallemeijn WW, Oussoren SV, Scheij S, Bleijlevens B, Florea BI, van Roomen CPAA, Ottenhoff R, van Kooten MJFM, Walvoort MTC, Witte MD, Boot RG, Ubbink M, Overkleeft HS, and Aerts JMFG
- Subjects
- Animals, Binding Sites, Enzyme Stability drug effects, Imino Pyranoses chemistry, Imino Pyranoses pharmacology, Liver drug effects, Liver enzymology, Mice, Molecular Structure, Temperature, Catalytic Domain physiology, Enzyme Stability physiology, Glucosylceramidase chemistry, Glucosylceramidase metabolism, Imino Pyranoses metabolism
- Abstract
Glucocerebrosidase (GBA) is a lysosomal β-glucosidase that degrades glucosylceramide. Its deficiency results in Gaucher disease (GD). We examined the effects of active site occupancy of GBA on its structural stability. For this, we made use of cyclophellitol-derived activity-based probes (ABPs) that bind irreversibly to the catalytic nucleophile (E340), and for comparison, we used the potent reversible inhibitor isofagomine. We demonstrate that cyclophellitol ABPs improve the stability of GBA in vitro, as revealed by thermodynamic measurements (T
m increase by 21 °C), and introduce resistance to tryptic digestion. The stabilizing effect of cell-permeable cyclophellitol ABPs is also observed in intact cultured cells containing wild-type GBA, N370S GBA (labile in lysosomes), and L444P GBA (exhibits impaired ER folding): all show marked increases in lysosomal forms of GBA molecules upon exposure to ABPs. The same stabilization effect is observed for endogenous GBA in the liver of wild-type mice injected with cyclophellitol ABPs. Stabilization effects similar to those observed with ABPs were also noted at high concentrations of the reversible inhibitor isofagomine. In conclusion, we provide evidence that the increase in cellular levels of GBA by ABPs and by the reversible inhibitor is in part caused by their ability to stabilize GBA folding, which increases the resistance of GBA against breakdown by lysosomal proteases. These effects are more pronounced in the case of the amphiphilic ABPs, presumably due to their high lipophilic potential, which may promote further structural compactness of GBA through hydrophobic interactions. Our study provides further rationale for the design of chaperones for GBA to ameliorate Gaucher disease.- Published
- 2017
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9. Hydrophobic Interactions Contribute to Conformational Stabilization of Endoglycoceramidase II by Mechanism-Based Probes.
- Author
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Ben Bdira F, Jiang J, Kallemeijn W, de Haan A, Florea BI, Bleijlevens B, Boot R, Overkleeft HS, Aerts JM, and Ubbink M
- Subjects
- Amino Acid Sequence, Bacterial Proteins genetics, Bacterial Proteins metabolism, Catalytic Domain, Cyclohexanols chemistry, Enzyme Stability, Gaucher Disease enzymology, Glucosylceramidase chemistry, Glucosylceramidase genetics, Glucosylceramidase metabolism, Glycoside Hydrolases genetics, Glycoside Hydrolases metabolism, Humans, Hydrophobic and Hydrophilic Interactions, Models, Molecular, Molecular Probes chemistry, Protein Conformation, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Rhodococcus enzymology, Rhodococcus genetics, Structural Homology, Protein, Bacterial Proteins chemistry, Glycoside Hydrolases chemistry
- Abstract
Small compound active site interactors receive considerable attention for their ability to positively influence the fold of glycosidases. Endoglycoceramidase II (EGCII) from Rhodococcus sp. is an endo-β-glucosidase releasing the complete glycan from ceramide in glycosphingolipids. Cleavage of the β-glycosidic linkage between glucose and ceramide is also catalyzed by glucocerebrosidase (GBA), the exo-β-glucosidase deficient in Gaucher disease. We demonstrate that established β-glucoside-configured cyclophellitol-type activity-based probes (ABPs) for GBA also are effective, mechanism-based, and irreversible inhibitors of EGCII. The stability of EGCII is markedly enhanced by formation of covalent complexes with cyclophellitol ABPs substituted with hydrophobic moieties, as evidenced by an increased melting temperature, resistance against tryptic digestion, changes in (15)N-(1)H transverse relaxation optimized spectroscopy spectra of the [(15)N]Leu-labeled enzyme, and relative hydrophobicity as determined by 8-anilino-1-naphthalenesulfonic acid fluorescence. The stabilization of EGCII conformation correlates with the shape and hydrophobicity of the substituents of the ABPs. We conclude that the amphipathic active site binders with aliphatic moieties act as a "hydrophobic zipper" on the flexible EGCII protein structure.
- Published
- 2016
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10. Tuning intracellular homeostasis of human uroporphyrinogen III synthase by enzyme engineering at a single hotspot of congenital erythropoietic porphyria.
- Author
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ben Bdira F, González E, Pluta P, Laín A, Sanz-Parra A, Falcon-Perez JM, and Millet O
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- Amino Acid Substitution, Catalysis, Enzyme Activation, Enzyme Stability, Humans, Intracellular Space metabolism, Kinetics, Models, Molecular, Mutation, Porphyria, Erythropoietic enzymology, Porphyria, Erythropoietic genetics, Protein Conformation, Uroporphyrinogen III Synthetase chemistry, Uroporphyrinogen III Synthetase genetics, Homeostasis, Porphyria, Erythropoietic metabolism, Protein Engineering, Uroporphyrinogen III Synthetase metabolism
- Abstract
Congenital erythropoietic porphyria (CEP) results from a deficiency in uroporphyrinogen III synthase enzyme (UROIIIS) activity that ultimately stems from deleterious mutations in the uroS gene. C73 is a hotspot for these mutations and a C73R substitution, which drastically reduces the enzyme activity and stability, is found in almost one-third of all reported CEP cases. Here, we have studied the structural basis, by which mutations in this hotspot lead to UROIIIS destabilization. First, a strong interdependency is observed between the volume of the side chain at position 73 and the folded protein. Moreover, there is a correlation between the in vitro half-life of the mutated proteins and their expression levels in eukaryotic cell lines. Molecular modelling was used to rationalize the results, showing that the mutation site is coupled to the hinge region separating the two domains. Namely, mutations at position 73 modulate the inter-domain closure and ultimately affect protein stability. By incorporating residues capable of interacting with R73 to stabilize the hinge region, catalytic activity was fully restored and a moderate increase in the kinetic stability of the enzyme was observed. These results provide an unprecedented rationale for a destabilizing missense mutation and pave the way for the effective design of molecular chaperones as a therapy against CEP., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)
- Published
- 2014
- Full Text
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