I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I23-Long wavelength MX
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Diamond Proposal Number(s):
[18598]
Open Access
Abstract: α-L-Arabinofuranosidases from glycoside hydrolase family 51 use a stereochemically retaining hydrolytic mechanism to liberate nonreducing terminal α-L-arabinofuranose residues from plant polysaccharides such as arabinoxylan and arabinan. To date, more than ten fungal GH51 α-L-arabinofuranosidases have been functionally characterized, yet no structure of a fungal GH51 enzyme has been solved. In contrast, seven bacterial GH51 enzyme structures, with low sequence similarity to the fungal GH51 enzymes, have been determined. Here, the crystallization and structural characterization of MgGH51, an industrially relevant GH51 α-L-arabinofuranosidase cloned from Meripilus giganteus, are reported. Three crystal forms were grown in different crystallization conditions. The unliganded structure was solved using sulfur SAD data collected from a single crystal using the I23 in vacuo diffraction beamline at Diamond Light Source. Crystal soaks with arabinose, 1,4-dideoxy-1,4-imino-L-arabinitol and two cyclophellitol-derived arabinose mimics reveal a conserved catalytic site and conformational itinerary between fungal and bacterial GH51 α-L-arabinofuranosidases.
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Nov 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Lukasz F.
Sobala
,
Pearl Z.
Fernandes
,
Zalihe
Hakki
,
Andrew J.
Thompson
,
Jonathon D.
Howe
,
Michelle
Hill
,
Nicole
Zitzmann
,
Scott
Davies
,
Zania
Stamataki
,
Terry D.
Butters
,
Dominic S.
Alonzi
,
Spencer J.
Williams
,
Gideon
Davies
Diamond Proposal Number(s):
[1221, 12587, 18598]
Open Access
Abstract: Mammalian protein N-linked glycosylation is critical for glycoprotein folding, quality control, trafficking, recognition, and function. N-linked glycans are synthesized from Glc3Man9GlcNAc2 precursors that are trimmed and modified in the endoplasmic reticulum (ER) and Golgi apparatus by glycoside hydrolases and glycosyltransferases. Endo-α-1,2-mannosidase (MANEA) is the sole endo-acting glycoside hydrolase involved in N-glycan trimming and is located within the Golgi, where it allows ER-escaped glycoproteins to bypass the classical N-glycosylation trimming pathway involving ER glucosidases I and II. There is considerable interest in the use of small molecules that disrupt N-linked glycosylation as therapeutic agents for diseases such as cancer and viral infection. Here we report the structure of the catalytic domain of human MANEA and complexes with substrate-derived inhibitors, which provide insight into dynamic loop movements that occur on substrate binding. We reveal structural features of the human enzyme that explain its substrate preference and the mechanistic basis for catalysis. These structures have inspired the development of new inhibitors that disrupt host protein N-glycan processing of viral glycans and reduce the infectivity of bovine viral diarrhea and dengue viruses in cellular models. These results may contribute to efforts aimed at developing broad-spectrum antiviral agents and help provide a more in-depth understanding of the biology of mammalian glycosylation.
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Nov 2020
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598]
Abstract: A synthetic heparan sulfate disaccharide has been assessed as a fluorogenic heparanase substrate, enabling enzyme turnover and inhibition kinetics measurements despite slow turnover. Crystal structures with human heparanase also provide the first ever observation of a substrate in an activated 1S3 conformation, highlighting previously unknown interactions involved in enzymatic processing. Our data provide insights into the heparanase catalytic mechanism, and will inform the design of improved heparanase substrates and inhibitors.
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Oct 2020
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Patrick
Weber
,
Martin
Thonhofer
,
Summer
Averill
,
Gideon J.
Davies
,
Andres Gonzalez
Santana
,
Philipp
Müller
,
Seyed A.
Nasseri
,
Wendy A.
Offen
,
Bettina M.
Pabst
,
Eduard
Paschke
,
Michael
Schalli
,
Ana
Torvisco
,
Marion
Tschernutter
,
Christina
Tysoe
,
Werner
Windischhofer
,
Stephen G.
Withers
,
Andreas
Wolfsgruber
,
Tanja M.
Wrodnigg
,
Arnold E.
Stütz
Open Access
Abstract: Glycosidase inhibitors have shown great potential as pharmacological chaperones for lysosomal storage diseases. In light of this, a series of new cyclopentanoid β-galactosidase inhibitors were prepared and their inhibitory and pharmacological chaperoning activities determined and compared with those of lipophilic analogs of the potent β-d-galactosidase inhibitor 4-epi-isofagomine. Structure-activity relationships were investigated by X-ray crystallography as well as by alterations in the cyclopentane moiety such as deoxygenation and replacement by fluorine of a “strategic” hydroxyl group. New compounds have revealed highly promising activities with a range of β-galactosidase-compromised human cell lines and may serve as leads towards new pharmacological chaperones for GM1-gangliosidosis and Morquio B disease.
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Sep 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Tessa
Keenan
,
Fabio
Parmeggiani
,
Julien
Malassis
,
Clement Q.
Fontenelle
,
Jean-baptiste
Vendeville
,
Wendy
Offen
,
Peter
Both
,
Kun
Huang
,
Andrea
Marchesi
,
Alex
Heyam
,
Carl
Young
,
Simon J.
Charnock
,
Gideon J.
Davies
,
Bruno
Linclau
,
Sabine L.
Flitsch
,
Martin A.
Fascione
Diamond Proposal Number(s):
[13587, 18598]
Abstract: Fluorinated sugar-1-phosphates are of emerging importance as intermediates in the chemical and biocatalytic synthesis of modified oligosaccharides, as well as probes for chemical biology. Here we present a systematic study of the activity of a wide range of anomeric sugar kinases (galacto- and N-acetylhexosamine kinases) against a panel of fluorinated monosaccharides, leading to the first examples of polyfluorinated substrates accepted by this class of enzymes. We have discovered four new N-acetylhexosamine kinases with a different substrate scope, thus expanding the number of homologs available in this subclass of kinases. Lastly, we have solved the crystal structure of a galactokinase in complex with 2-deoxy-2-fluorogalactose, giving insight into changes in the active site that may account for the specificity of the enzyme toward certain substrate analogs.
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Jul 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598]
Abstract: α-mannoside β-1,6-N-acetylglucosaminyltransferase V (MGAT5) is a mammalian glycosyltransferase involved in complex N-glycan formation, which strongly drives cancer when overexpressed. Despite intense interest, the catalytic mechanism of MGAT5 is not known in detail, precluding therapeutic exploitation. We solved structures of MGAT5 complexed to glycosyl donor and acceptor ligands, revealing an unforeseen role for donor induced loop rearrangements in controlling acceptor substrate engagement. QM/MM metadynamics simulations of MGAT5 catalysis highlight the key assisting role of Glu297, and reveal considerable conformational distortions imposed upon the glycosyl donor during transfer. Detailed mechanistic characterization of MGAT5 will aid inhibitor development to correct cancer associated N-glycosylation.
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Jul 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Zachary
Armstrong
,
Chi-lin
Kuo
,
Daniël
Lahav
,
Bing
Liu
,
Rachel
Johnson
,
Thomas J. M.
Beenakker
,
Casper
De Boer
,
Chung-sing
Wong
,
Erwin R.
Van Rijssel
,
Marjoke F.
Debets
,
Bogdan I.
Florea
,
Colin
Hissink
,
Rolf G.
Boot
,
Paul P.
Geurink
,
Huib
Ovaa
,
Mario
Van Der Stelt
,
Gijsbert M.
Van Der Marel
,
Jeroen D. C.
Codée
,
Johannes M. F. G.
Aerts
,
Liang
Wu
,
Herman S.
Overkleeft
,
Gideon
Davies
Diamond Proposal Number(s):
[18598]
Abstract: Golgi mannosidase II (GMII) catalyzes the sequential hydrolysis of two mannosyl residues from GlcNAcMan5GlcNAc2 to produce GlcNAcMan3GlcNAc2, the precursor for all complex N-glycans, including the branched N-glycans associated with cancer. Inhibitors of GMII are potential cancer therapeutics, but their usefulness is limited by off-target effects, which produce α-mannosidosis-like symptoms. Despite many structural and mechanistic studies of GMII, we still lack a potent and selective inhibitor of this enzyme. Here, we synthesized manno-epi-cyclophellitol epoxide and aziridines and demonstrate their covalent modification and time-dependent inhibition of GMII. Application of fluorescent manno-epi-cyclophellitol aziridine derivatives enabled activity-based protein profiling of α-mannosidases from both human cell lysate and mouse tissue extracts. Synthesized probes also facilitated a fluorescence polarization-based screen for dGMII inhibitors. We identified seven previously unknown inhibitors of GMII from a library of over 350 iminosugars and investigated their binding modalities through X-ray crystallography. Our results reveal previously unobserved inhibitor binding modes and promising scaffolds for the generation of selective GMII inhibitors.
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Jul 2020
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I03-Macromolecular Crystallography
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Casper
De Boer
,
Nicholas G. S.
Mcgregor
,
Evert
Peterse
,
Sybrin P.
Schröder
,
Bogdan I.
Florea
,
Jianbing
Jiang
,
Jos
Reijngoud
,
Arthur F. J.
Ram
,
Gilles P.
Van Wezel
,
Gijsbert A.
Van Der Marel
,
Jeroen D. C.
Codée
,
Herman S.
Overkleeft
,
Gideon
Davies
Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Cellulases and related β-1,4-glucanases are essential components of lignocellulose-degrading enzyme mixtures. The detection of β-1,4-glucanase activity typically relies on monitoring the breakdown of purified lignocellulose-derived substrates or synthetic chromogenic substrates, limiting the activities which can be detected and complicating the tracing of activity back to specific components within complex enzyme mixtures. As a tool for the rapid detection and identification of β-1,4-glucanases, a series of glycosylated cyclophellitol inhibitors mimicking β-1,4-glucan oligosaccharides have been synthesised. These compounds are highly efficient inhibitors of HiCel7B, a well-known GH7 endo-β-1,4-glucanase. An elaborated activity-based probe facilitated the direct detection and identification of β-1,4-glucanases within a complex fungal secretome without any detectable cross-reactivity with β-D-glucosidases. These probes and inhibitors add valuable new capacity to the growing toolbox of cyclophellitol-derived probes for the activity-based profiling of biomass-degrading enzymes.
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Jul 2020
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598]
Open Access
Abstract: The lysosomal glycoside hydrolase β-glucocerebrosidase (GBA; sometimes called GBA1 or GCase) catalyses the hydrolysis of glycosphingolipids. Inherited deficiencies in GBA cause the lysosomal storage disorder Gaucher disease (GD). Consequently, GBA is of considerable medical interest, with continuous advances in the development of inhibitors, chaperones and activity-based probes. The development of new GBA inhibitors requires a source of active protein; however, the majority of structural and mechanistic studies of GBA today rely on clinical enzyme-replacement therapy (ERT) formulations, which are incredibly costly and are often difficult to obtain in adequate supply. Here, the production of active crystallizable GBA in insect cells using a baculovirus expression system is reported, providing a nonclinical source of recombinant GBA with comparable activity and biophysical properties to ERT preparations. Furthermore, a novel crystal form of GBA is described which diffracts to give a 0.98 Å resolution unliganded structure. A structure in complex with the inactivator 2,4-dinitrophenyl-2-deoxy-2-fluoro-β-D-glucopyranoside was also obtained, demonstrating the ability of this GBA formulation to be used in ligand-binding studies. In light of its purity, stability and activity, the GBA production protocol described here should circumvent the need for ERT formulations for structural and biochemical studies and serve to support GD research.
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Jun 2020
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Lukasz F.
Sobala
,
Gaetano
Speciale
,
Sha
Zhu
,
Lluı́s
Raich
,
Natalia
Sannikova
,
Andrew J.
Thompson
,
Zalihe
Hakki
,
Dan
Lu
,
Saeideh
Shamsi Kazem Abadi
,
Andrew R.
Lewis
,
Vı́ctor
Rojas-cervellera
,
Ganeko
Bernardo-seisdedos
,
Yongmin
Zhang
,
Oscar
Millet
,
Jesús
Jiménez-barbero
,
Andrew J.
Bennet
,
Matthieu
Sollogoub
,
Carme
Rovira
,
Gideon J.
Davies
,
Spencer J.
Williams
Diamond Proposal Number(s):
[9948, 13587]
Open Access
Abstract: Retaining glycoside hydrolases cleave their substrates through stereochemical retention at the anomeric position. Typically, this involves two-step mechanisms using either an enzymatic nucleophile via a covalent glycosyl enzyme intermediate or neighboring-group participation by a substrate-borne 2-acetamido neighboring group via an oxazoline intermediate; no enzymatic mechanism with participation of the sugar 2-hydroxyl has been reported. Here, we detail structural, computational, and kinetic evidence for neighboring-group participation by a mannose 2-hydroxyl in glycoside hydrolase family 99 endo-α-1,2-mannanases. We present a series of crystallographic snapshots of key species along the reaction coordinate: a Michaelis complex with a tetrasaccharide substrate; complexes with intermediate mimics, a sugar-shaped cyclitol β-1,2-aziridine and β-1,2-epoxide; and a product complex. The 1,2-epoxide intermediate mimic displayed hydrolytic and transfer reactivity analogous to that expected for the 1,2-anhydro sugar intermediate supporting its catalytic equivalence. Quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar via a transition state in an unusual flattened, envelope (E3) conformation. Kinetic isotope effects (kcat/KM) for anomeric-2H and anomeric-13C support an oxocarbenium ion-like transition state, and that for C2-18O (1.052 ± 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymatic mechanism.
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Apr 2020
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