I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Yurong
Chen
,
Zachary
Armstrong
,
Marta
Artola
,
Bogdan I.
Florea
,
Chi-Lin
Kuo
,
Casper
De Boer
,
Mikkel S.
Rasmussen
,
Maher
Abou Hachem
,
Gijsbert A.
Van Der Marel
,
Jeroen D. C.
Codée
,
Johannes M. F. G.
Aerts
,
Gideon J.
Davies
,
Herman S.
Overkleeft
Diamond Proposal Number(s):
[18598]
Abstract: Amylases are key enzymes in the processing of starch in many kingdoms of life. They are important catalysts in industrial biotechnology where they are applied in, among others, food processing and the production of detergents. In man amylases are the first enzymes in the digestion of starch to glucose and arguably also the preferred target in therapeutic strategies aimed at the treatment of type 2 diabetes patients through down-tuning glucose assimilation. Efficient and sensitive assays that report selectively on retaining amylase activities irrespective of the nature and complexity of the biomaterial studied are of great value both in finding new and effective human amylase inhibitors and in the discovery of new microbial amylases with potentially advantageous features for biotechnological application. Activity-based protein profiling (ABPP) of retaining glycosidases is inherently suited for the development of such an assay format. We here report on the design and synthesis of 1,6-epi-cyclophellitol-based pseudodisaccharides equipped with a suite of reporter entities and their use in ABPP of retaining amylases from human saliva, murine tissue as well as secretomes from fungi grown on starch. The activity and efficiency of the inhibitors and probes are substantiated by extensive biochemical analysis, and the selectivity for amylases over related retaining endoglycosidases is validated by structural studies.
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Jan 2021
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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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I04-Macromolecular Crystallography
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Nicholas G. S.
Mcgregor
,
Marta
Artola
,
Alba
Nin-Hill
,
Daniel
Linzel
,
Mireille
Haon
,
Jos
Reijngoud
,
Arthur F. J.
Ram
,
Marie-Noelle
Rosso
,
Gijsbert A.
Van Der Marel
,
Jeroen D. C.
Codée
,
Gilles P.
Van Wezel
,
Jean-Guy
Berrin
,
Carme
Rovira
,
Herman S.
Overkleeft
,
Gideon J.
Davies
Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Identifying and characterizing the enzymes responsible for an observed activity within a complex eukaryotic catabolic system remains one of the most significant challenges in the study of biomass-degrading systems. The debranching of both complex hemicellulosic and pectinaceous polysaccharides requires the production of α-L-arabinofuranosidases among a wide variety of co-expressed carbohydrate-active enzymes. To selectively detect and identify α-L-arabinofuranosidases produced by fungi grown on complex biomass, potential covalent inhibitors and probes which mimic α-L-arabinofuranosides were sought. The conformational free energy landscapes of free α-L-arabinofuranose and several rationally designed covalent α-L-arabinofuranosidase inhibitors were analyzed. A synthetic route to these inhibitors was subsequently developed based on a key Wittig-Still rearrangement. Through a combination of kinetic measurements, intact mass spectrometry, and structural experiments, the designed inhibitors were shown to efficiently label the catalytic nucleophiles of retaining GH51 and GH54 α-L-arabinofuranosidases. Activity-based probes elaborated from an inhibitor with an aziridine warhead were applied to the identification and characterization of α-L-arabinofuranosidases within the secretome of A. niger grown on arabinan. This method was extended to the detection and identification of α-L-arabinofuranosidases produced by eight biomass-degrading basidiomycete fungi grown on complex biomass. The broad applicability of the cyclophellitol-derived activity-based probes and inhibitors presented here make them a valuable new tool in the characterization of complex eukaryotic carbohydrate-degrading systems and in the high-throughput discovery of α-L-arabinofuranosidases.
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Feb 2020
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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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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13587]
Open Access
Abstract: Gaucher disease is caused by inherited deficiency in glucocerebrosidase (GBA, a retaining β-glucosidase), and deficiency in GBA constitutes the largest known genetic risk factor for Parkinson’s disease. In the past, animal models of Gaucher disease have been generated by treatment with the mechanism-based GBA inhibitors, conduritol B epoxide (CBE), and cyclophellitol. Both compounds, however, also target other retaining glycosidases, rendering generation and interpretation of such chemical knockout models complicated. Here we demonstrate that cyclophellitol derivatives carrying a bulky hydrophobic substituent at C8 are potent and selective GBA inhibitors and that an unambiguous Gaucher animal model can be readily generated by treatment of zebrafish with these.
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Mar 2019
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Marta
Artola
,
Chi-lin
Kuo
,
Stephen
Mcmahon
,
Verena
Oehler
,
Thomas
Hansen
,
Martijn
Van Der Lienden
,
Xu
He
,
Hans
Van Den Elst
,
Bogdan I.
Florea
,
Allison R.
Kermode
,
Gijsbert A.
Van Der Marel
,
Tracey M.
Gloster
,
Jeroen D. C.
Codée
,
Herman S.
Overkleeft
,
Johannes M. F. G.
Aerts
Diamond Proposal Number(s):
[14980]
Open Access
Abstract: Cyclophellitol aziridines are potent irreversible inhibitors of retaining glycosidases and versatile intermediates in the synthesis of activity‐based glycosidase probes (ABPs). Direct 3‐amino‐2‐(trifluoromethyl)quinazolin‐4(3H)‐one‐mediated aziridination of l‐ido‐configured cyclohexene has enabled the synthesis of new covalent inhibitors and ABPs of α‐l‐iduronidase, deficiency of which underlies the lysosomal storage disorder mucopolysaccharidosis type I (MPS I). The iduronidase ABPs react covalently and irreversibly in an activity‐based manner with human recombinant α‐l‐iduronidase (rIDUA, Aldurazyme®). The structures of IDUA when complexed with the inhibitors in a non‐covalent transition state mimicking form and a covalent enzyme‐bound form provide insights into its conformational itinerary. Inhibitors 1–3 adopt a half‐chair conformation in solution (4H3 and 3H4), as predicted by DFT calculations, which is different from the conformation of the Michaelis complex observed by crystallographic studies. Consequently, 1–3 may need to overcome an energy barrier in order to switch from the 4H3 conformation to the transition state (2, 5B) binding conformation before reacting and adopting a covalent 5S1 conformation. rIDUA can be labeled with fluorescent Cy5 ABP 2, which allows monitoring of the delivery of therapeutic recombinant enzyme to lysosomes, as is intended in enzyme replacement therapy for the treatment of MPS I patients.
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Nov 2018
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I03-Macromolecular Crystallography
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Sybrin P.
Schröder
,
Liang
Wu
,
Marta
Artola
,
Thomas
Hansen
,
Wendy A.
Offen
,
Maria J.
Ferraz
,
Kah-yee
Li
,
Johannes M. F. G.
Aerts
,
Gijsbert A.
Van Der Marel
,
Jeroen D. C.
Codée
,
Gideon J.
Davies
,
Herman S.
Overkleeft
Diamond Proposal Number(s):
[13587]
Abstract: Gluco-azoles competitively inhibit glucosidases by transition-state mimicry and their ability to interact with catalytic acid residues in glucosidase active sites. We noted that no azole-type inhibitors described, to date, possess a protic nitrogen characteristic for 1H-imidazoles. Here, we present gluco-1H-imidazole, a gluco-azole bearing a 1H-imidazole fused to a glucopyranose-configured cyclitol core, and three close analogues as new glucosidase inhibitors. All compounds inhibit human retaining β-glucosidase, GBA1, with the most potent ones inhibiting this enzyme (deficient in Gaucher disease) on a par with glucoimidazole. None inhibit glucosylceramide synthase, cytosolic β-glucosidase GBA2 or α-glucosidase GAA. Structural, physical and computational studies provide first insights into the binding mode of this conceptually new class of retaining β-glucosidase inhibitors.
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Mar 2018
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I02-Macromolecular Crystallography
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Sybrin P.
Schröder
,
Jasper W.
Van De Sande
,
Wouter W.
Kallemeijn
,
Chi-lin
Kuo
,
Marta
Artola
,
Eva J.
Van Rooden
,
Jianbing
Jiang
,
Thomas J. M.
Beenakker
,
Bogdan I.
Florea
,
Wendy
Offen
,
Gideon
Davies
,
Adriaan J.
Minnaard
,
Johannes M. F. G.
Aerts
,
Jeroen D. C.
Codée
,
Gijsbert A.
Van Der Marel
,
Herman S.
Overkleeft
Diamond Proposal Number(s):
[9948]
Open Access
Abstract: Activity-based protein profiling has emerged as a powerful tool for visualizing glycosidases in complex biological samples. Several configurational cyclophellitol isomers have been shown to display high selectivity as probes for glycosidases processing substrates featuring the same configuration. Here, a set of deoxygenated cyclophellitols are presented which enable inter-class profiling of β-glucosidases and β-galactosidases.
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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
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Liang
Wu
,
Jianbing
Jiang
,
Yi
Jin
,
Wouter W.
Kallemeijn
,
Chi-lin
Kuo
,
Marta
Artola
,
Wei
Dai
,
Cas
Van Elk
,
Marco
Van Eijk
,
Gijsbert A.
Van Der Marel
,
Jeroen D. C.
Codee
,
Bogdan I.
Florea
,
Johannes M. F. G.
Aerts
,
Herman S.
Overkleeft
,
Gideon J.
Davies
Diamond Proposal Number(s):
[9948, 13587]
Abstract: Humans express at least two distinct β-glucuronidase enzymes that are involved in disease: exo-acting β-glucuronidase (GUSB), whose deficiency gives rise to mucopolysaccharidosis type VII, and endo-acting heparanase (HPSE), whose overexpression is implicated in inflammation and cancers. The medical importance of these enzymes necessitates reliable methods to assay their activities in tissues. Herein, we present a set of β-glucuronidase-specific activity-based probes (ABPs) that allow rapid and quantitative visualization of GUSB and HPSE in biological samples, providing a powerful tool for dissecting their activities in normal and disease states. Unexpectedly, we find that the supposedly inactive HPSE proenzyme proHPSE is also labeled by our ABPs, leading to surprising insights regarding structural relationships between proHPSE, mature HPSE, and their bacterial homologs. Our results demonstrate the application of β-glucuronidase ABPs in tracking pathologically relevant enzymes and provide a case study of how ABP-driven approaches can lead to discovery of unanticipated structural and biochemical functionality.
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Jun 2017
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