I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Marta
Artola
,
Christinne
Hedberg
,
Rhianna J.
Rowland
,
Lluís
Raich
,
Kassiani
Kytidou
,
Liang
Wu
,
Amanda
Schaaf
,
Maria Joao
Ferraz
,
Gijsbert A.
Van Der Marel
,
Jeroen D. C.
Codée
,
Carme
Rovira
,
Johannes M. F. G.
Aerts
,
Gideon J.
Davies
,
Herman S.
Overkleeft
Diamond Proposal Number(s):
[13587]
Open Access
Abstract: Fabry disease is an inherited lysosomal storage disorder that is characterized by a deficiency in lysosomal α-D-galactosidase activity. One current therapeutic strategy involves enzyme replacement therapy, in which patients are treated with a recombinant enzyme. Co-treatment with enzyme active-site stabilizers is advocated to increase treatment efficacy, a strategy that requires effective and selective enzyme stabilizers. Here, we describe the design and development of an α-D-gal-cyclophellitol cyclosulfamidate as a new class of neutral, conformationally constrained competitive glycosidase inhibitors that act by mimicry of the Michaelis complex conformation. We found that D-galactose-configured α-cyclosulfamidate 4 effectively stabilizes recombinant human α-D-galactosidase (agalsidase beta, Fabrazyme®) both in vitro and in cellulo.
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Aug 2019
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Sybrin P.
Schröder
,
Casper
De Boer
,
Nicholas G. S.
Mcgregor
,
Rhianna J.
Rowland
,
Olga
Moroz
,
Elena
Blagova
,
Jos
Reijngoud
,
Mark
Arentshorst
,
David
Osborn
,
Marc D.
Morant
,
Eric
Abbate
,
Mary A.
Stringer
,
Kristian B. R. M.
Krogh
,
Lluís
Raich
,
Carme
Rovira
,
Jean-Guy
Berrin
,
Gilles P.
Van Wezel
,
Arthur F. J.
Ram
,
Bogdan I.
Florea
,
Gijsbert A.
Van Der Marel
,
Jeroen D. C.
Codée
,
Keith S.
Wilson
,
Liang
Wu
,
Gideon J.
Davies
,
Herman S.
Overkleeft
Diamond Proposal Number(s):
[13587]
Abstract: Plant polysaccharides represent a virtually unlimited feedstock for the generation of biofuels and other commodities. However, the extraordinary recalcitrance of plant polysaccharides toward breakdown necessitates a continued search for enzymes that degrade these materials efficiently under defined conditions. Activity-based protein profiling provides a route for the functional discovery of such enzymes in complex mixtures and under industrially relevant conditions. Here, we show the detection and identification of β-xylosidases and endo-β-1,4-xylanases in the secretomes of Aspergillus niger, by the use of chemical probes inspired by the β-glucosidase inhibitor cyclophellitol. Furthermore, we demonstrate the use of these activity-based probes (ABPs) to assess enzyme–substrate specificities, thermal stabilities, and other biotechnologically relevant parameters. Our experiments highlight the utility of ABPs as promising tools for the discovery of relevant enzymes useful for biomass breakdown.
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May 2019
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I03-Macromolecular Crystallography
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Matthew K.
Bilyard
,
Henry J.
Bailey
,
Lluís
Raich
,
Maria A.
Gafitescu
,
Takuya
Machida
,
Javier
Iglésias-Fernández
,
Seung Seo
Lee
,
Christopher D.
Spicer
,
Carme
Rovira
,
Wyatt W.
Yue
,
Benjamin G.
Davis
Abstract: Biosynthesis of glycogen, the essential glucose (and hence energy) storage molecule in humans, animals and fungi1, is initiated by the glycosyltransferase enzyme, glycogenin (GYG). Deficiencies in glycogen formation cause neurodegenerative and metabolic disease2,3,4, and mouse knockout5 and inherited human mutations6 of GYG impair glycogen synthesis. GYG acts as a ‘seed core’ for the formation of the glycogen particle by catalysing its own stepwise autoglucosylation to form a covalently bound gluco-oligosaccharide chain at initiation site Tyr 195. Precise mechanistic studies have so far been prevented by an inability to access homogeneous glycoforms of this protein, which unusually acts as both catalyst and substrate. Here we show that unprecedented direct access to different, homogeneously glucosylated states of GYG can be accomplished through a palladium-mediated enzyme activation ‘shunt’ process using on-protein C–C bond formation. Careful mimicry of GYG intermediates recapitulates catalytic activity at distinct stages, which in turn allows discovery of triphasic kinetics and substrate plasticity in GYG’s use of sugar substrates. This reveals a tolerant but ‘proof-read’ mechanism that underlies the precision of this metabolic process. The present demonstration of direct, chemically controlled access to intermediate states of active enzymes suggests that such ligation-dependent activation could be a powerful tool in the study of mechanism.
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Oct 2018
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I03-Macromolecular Crystallography
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Matilde
De Las Rivas
,
Earnest James
Paul Daniel
,
Helena
Coelho
,
Erandi
Lira-Navarrete
,
Lluis
Raich
,
Ismael
Compañón
,
Ana
Diniz
,
Laura
Lagartera
,
Jesús
Jiménez-Barbero
,
Henrik
Clausen
,
Carme
Rovira
,
Filipa
Marcelo
,
Francisco
Corzana
,
Thomas A.
Gerken
,
Ramon
Hurtado-Guerrero
Diamond Proposal Number(s):
[10121]
Open Access
Abstract: Mucin-type O-glycosylation is initiated by a family of polypeptide GalNAc-transferases (GalNAc-Ts) which are type-II transmembrane proteins that contain Golgi luminal catalytic and lectin domains that are connected by a flexible linker. Several GalNAc-Ts, including GalNAc-T4, show both long-range and short-range prior glycosylation specificity, governed by their lectin and catalytic domains, respectively. While the mechanism of the lectin-domain-dependent glycosylation is well-known, the molecular basis for the catalytic-domain-dependent glycosylation of glycopeptides is unclear. Herein, we report the crystal structure of GalNAc-T4 bound to the diglycopeptide GAT*GAGAGAGT*TPGPG (containing two α-GalNAc glycosylated Thr (T*), the PXP motif and a “naked” Thr acceptor site) that describes its catalytic domain glycopeptide GalNAc binding site. Kinetic studies of wild-type and GalNAc binding site mutant enzymes show the lectin domain GalNAc binding activity dominates over the catalytic domain GalNAc binding activity and that these activities can be independently eliminated. Surprisingly, a flexible loop protruding from the lectin domain was found essential for the optimal activity of the catalytic domain. This work provides the first structural basis for the short-range glycosylation preferences of a GalNAc-T.
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Sep 2018
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[15304]
Abstract: Glycolipids play a central role in a variety of important biological processes in all living organisms. PatA is a membrane acyltransferase involved in the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs), key structural elements and virulence factors of Mycobacterium tuberculosis. PatA catalyzes the transfer of a palmitoyl moiety from palmitoyl-CoA to the 6-position of the mannose ring linked to the 2-position of inositol in PIM1/PIM2. We report here the crystal structure of PatA in the presence of 6-O-palmitoyl-α-D-mannopyranoside, unraveling the acceptor binding mechanism. The acceptor mannose ring localizes in a cavity at the end of a surface-exposed, long groove where the active site is located, whereas the palmitate moiety accommodates into a hydrophobic pocket deeply buried in the α/β core of the protein. Both fatty acyl chains of the PIM2 acceptor are essential for the reaction to take place, highlighting their critical role in the generation of a competent active site. By the use of combined structural and quantum-mechanics/molecular-mechanics (QM/MM) metadynamics we unravel the catalytic mechanism of PatA at the atomic-electronic level. Our study provides a detailed structural rationale for a stepwise reaction, with the generation of a tetrahedral transition state for the rate-determining step. Finally, the crystal structure of PatA in the presence of β-D-mannopyranose and palmitate suggest an inhibitory mechanism for the enzyme, providing exciting possibilities for inhibitor design and the discovery of chemotherapeutic agents against this major human pathogen.
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Nov 2017
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Marta
Artola
,
Liang
Wu
,
Maria J.
Ferraz
,
Chi-Lin
Kuo
,
Lluís
Raich
,
Imogen Z.
Breen
,
Wendy A.
Offen
,
Jeroen D. C.
Codée
,
Gijsbert A.
Van Der Marel
,
Carme
Rovira
,
Johannes M. F. G.
Aerts
,
Gideon J.
Davies
,
Herman S.
Overkleeft
Diamond Proposal Number(s):
[13587]
Open Access
Abstract: The essential biological roles played by glycosidases, coupled to the diverse therapeutic benefits of pharmacologically targeting these enzymes, provide considerable motivation for the development of new inhibitor classes. Cyclophellitol epoxides and aziridines are recently established covalent glycosidase inactivators. Inspired by the application of cyclic sulfates as electrophilic equivalents of epoxides in organic synthesis, we sought to test whether cyclophellitol cyclosulfates would similarly act as irreversible glycosidase inhibitors. Here we present the synthesis, conformational analysis, and application of novel 1,6-cyclophellitol cyclosulfates. We show that 1,6-epi-cyclophellitol cyclosulfate (α-cyclosulfate) is a rapidly reacting α-glucosidase inhibitor whose 4C1 chair conformation matches that adopted by α-glucosidase Michaelis complexes. The 1,6-cyclophellitol cyclosulfate (β-cyclosulfate) reacts more slowly, likely reflecting its conformational restrictions. Selective glycosidase inhibitors are invaluable as mechanistic probes and therapeutic agents, and we propose cyclophellitol cyclosulfates as a valuable new class of carbohydrate mimetics for application in these directions.
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Jul 2017
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13587]
Open Access
Abstract: The non-hydrolyzable S-linked azasugars, 1,6-α-mannosylthio- and 1,6-α-mannobiosylthioisofagomine, were synthesized and shown to bind with high affinity to a family 76 endo-1,6-α-mannanase from Bacillus circulans. X-ray crystallography showed an atypical interaction of the isofagomine nitrogen with the catalytic acid/base. Molecular dynamics simulations reveal that the atypical binding results from sulfur perturbing the most stable form away from the nucleophile interaction preferred for the O-linked congener.
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Jul 2017
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Abstract: SNi-like mechanisms, which involve front-face leaving group departure and nucleophile approach, have been observed experimentally and computationally in chemical and enzymatic substitution at α-glycosyl electrophiles. Since SNi-like, SN1 and SN2 substitution pathways can be energetically comparable, engineered switching could be feasible. Here, engineering of Sulfolobus solfataricus β-glycosidase, which originally catalyzed double SN2 substitution, changed its mode to SNi-like. Destruction of the first SN2 nucleophile through E387Y mutation created a β-stereoselective catalyst for glycoside synthesis from activated substrates, despite lacking a nucleophile. The pH profile, kinetic and mutational analyses, mechanism-based inactivators, X-ray structure and subsequent metadynamics simulations together suggest recruitment of substrates by π–sugar interaction and reveal a quantum mechanics–molecular mechanics (QM/MM) free-energy landscape for the substitution reaction that is similar to those of natural, SNi-like glycosyltransferases. This observation of a front-face mechanism in a β-glycosyltransfer enzyme highlights that SNi-like pathways may be engineered in catalysts with suitable environments and suggests that 'β-SNi' mechanisms may be feasible for natural glycosyltransfer enzymes.
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Jun 2017
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[13587]
Open Access
Abstract: The varied yet family-specific conformational pathways utilized by individual glycoside hydrolases (GHs) offer a tantalising prospect for the design of tight binding and specific enzyme inhibitors. A cardinal example of a GH family specific inhibitor, and one that finds widespread practical use, is the natural product kifunensine, which is a low nanomolar inhibitor selective for GH family 47 inverting a-mannosidases. Here we show, through quantum mechanical approaches, that kifunensine is restrained to a 'ring-flipped' 1C4 conformation with another accessible, but higher-energy, region around the 1,4B conformation. The conformations of kifunensine in complex with a range of GH47 enzymes including an atomic level (1 Å) resolution structure of kifunensine with Caulobacter sp. CkGH47 reported herein, and on GH family 38 and 92 a-mannosidases, were mapped onto the kifunensine free energy landscape. These studies revealed that kifunensine has the ability to mimic the product state of GH47 enzymes but cannot mimic any conformational states relevant to the reaction coordinate of mannosidases from other families.
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May 2017
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I02-Macromolecular Crystallography
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Thomas J. M.
Beenakker
,
Dennis P. A.
Wander
,
Wendy
Offen
,
Marta
Artola
,
Lluís
Raich
,
Maria J.
Ferraz
,
Kah-Yee
Li
,
Judith H. P. M.
Houben
,
Erwin R.
Van Rijssel
,
Thomas
Hansen
,
Gijsbert A.
Van Der Marel
,
Jeroen D. C.
Codée
,
Johannes M. F. G.
Aerts
,
Carme
Rovira
,
Gideon J.
Davies
,
Herman S.
Overkleeft
Diamond Proposal Number(s):
[13587]
Open Access
Abstract: The conformational analysis of glycosidases affords a route to their specific inhibition through transition-state mimicry. Inspired by the rapid reaction rates of cyclophellitol and cyclophellitol aziridine—both covalent retaining β-glucosidase inhibitors—we postulated that the corresponding carba “cyclopropyl” analogue would be a potent retaining β-glucosidase inhibitor for those enzymes reacting through the 4H3 transition-state conformation. Ab initio metadynamics simulations of the conformational free energy landscape for the cyclopropyl inhibitors show a strong bias for the 4H3 conformation, and carba-cyclophellitol, with an N-(4-azidobutyl)carboxamide moiety, proved to be a potent inhibitor (Ki = 8.2 nM) of the Thermotoga maritima TmGH1 β-glucosidase. 3-D structural analysis and comparison with unreacted epoxides show that this compound indeed binds in the 4H3 conformation, suggesting that conformational strain induced through a cyclopropyl unit may add to the armory of tight-binding inhibitor designs.
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May 2017
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