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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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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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[7864, 9948]
Open Access
Abstract: A dominant human gut microbe, the well studied symbiont Bacteroides thetaiotaomicron (Bt), is a glyco-specialist that harbors a large repertoire of genes devoted to carbohydrate processing. Despite strong similarities among them, many of the encoded enzymes have evolved distinct substrate specificities, and through the clustering of cognate genes within operons termed polysaccharide-utilization loci (PULs) enable the fulfilment of complex biological roles. Structural analyses of two glycoside hydrolase family 92 α-mannosidases, BT3130 and BT3965, together with mechanistically relevant complexes at 1.8–2.5 Å resolution reveal conservation of the global enzyme fold and core catalytic apparatus despite different linkage specificities. Structure comparison shows that Bt differentiates the activity of these enzymes through evolution of a highly variable substrate-binding region immediately adjacent to the active site. These observations unveil a genetic/biochemical mechanism through which polysaccharide-processing bacteria can evolve new and specific biochemical activities from otherwise highly similar gene products.
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May 2018
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Glyn
Hemsworth
,
Andrew J.
Thompson
,
Judith
Stepper
,
Lukasz
Sobala
,
Travis
Coyle
,
Johan
Larsbrink
,
Oliver
Spadiut
,
Ethan D.
Goddard-Borger
,
Keith A.
Stubbs
,
Harry
Brumer
,
Gideon
Grogan
Diamond Proposal Number(s):
[7864]
Open Access
Abstract: The human gastrointestinal tract harbours myriad bacterial species, collectively termed the microbiota, that strongly influence human health. Symbiotic members of our microbiota play a pivotal role in the digestion of complex carbohydrates that are otherwise recalcitrant to assimilation. Indeed, the intrinsic human polysaccharide-degrading enzyme repertoire is limited to various starch-based substrates; more complex polysaccharides demand microbial degradation. Select Bacteroidetes are responsible for the degradation of the ubiquitous vegetable xyloglucans (XyGs), through the concerted action of cohorts of enzymes and glycan-binding proteins encoded by specific xyloglucan utilization loci (XyGULs). Extending recent (meta)genomic, transcriptomic and biochemical analyses, significant questions remain regarding the structural biology of the molecular machinery required for XyG saccharification. Here, we reveal the three-dimensional structures of an α-xylosidase, a β-glucosidase, and two α-l-arabinofuranosidases from the Bacteroides ovatus XyGUL. Aided by bespoke ligand synthesis, our analyses highlight key adaptations in these enzymes that confer individual specificity for xyloglucan side chains and dictate concerted, stepwise disassembly of xyloglucan oligosaccharides. In harness with our recent structural characterization of the vanguard endo-xyloglucanse and cell-surface glycan-binding proteins, the present analysis provides a near-complete structural view of xyloglucan recognition and catalysis by XyGUL proteins.
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Jul 2016
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: TFIIB-related factor 2 (Brf2) is a member of the family of TFIIB-like core transcription factors. Brf2 recruits RNA polymerase (Pol) III to type III gene-external promoters, including the U6 spliceosomal RNA and selenocysteine tRNA genes. Found only in vertebrates, Brf2 has been linked to tumorigenesis but the underlying mechanisms remain elusive. We have solved crystal structures of a human Brf2-TBP complex bound to natural promoters, obtaining a detailed view of the molecular interactions occurring at Brf2-dependent Pol III promoters and highlighting the general structural and functional conservation of human Pol II and Pol III pre-initiation complexes. Surprisingly, our structural and functional studies unravel a Brf2 redox-sensing module capable of specifically regulating Pol III transcriptional output in living cells. Furthermore, we establish Brf2 as a central redox-sensing transcription factor involved in the oxidative stress pathway and provide a mechanistic model for Brf2 genetic activation in lung and breast cancer.
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Dec 2015
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Andrew J.
Thompson
,
Gaetano
Speciale
,
Javier
Iglesias-Fernández
,
Zalihe
Hakki
,
Tyson
Belz
,
Alan
Cartmell
,
Richard J.
Spears
,
Emily
Chandler
,
Max J.
Temple
,
Judith
Stepper
,
Harry J.
Gilbert
,
Carme
Rovira
,
Spencer J.
Williams
,
Gideon J.
Davies
Diamond Proposal Number(s):
[7864, 9948]
Abstract: carbohydrates;computational chemistry;conformational analysis;enzymatic mechanisms;glycosidase inhibitors
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Mar 2015
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[1221]
Open Access
Abstract: The large bowel microbiota, a complex ecosystem resident
within the gastrointestinal tract of all human beings and large
mammals, functions as an essential, nonsomatic metabolic
organ, hydrolysing complex dietary polysaccharides and
modulating the host immune system to adequately tolerate
ingested antigens. A significant member of this community,
Bacteroides thetaiotaomicron, has evolved a complex system
for sensing and processing a wide variety of natural
glycoproducts in such a way as to provide maximum benefit
to itself, the wider microbial community and the host. The
immense ability of B. thetaiotaomicron as a glycan specialist
resides in its enormous array of carbohydrate-active enzymes,
many of which are arranged into polysaccharide-utilization
loci (PULs) that are able to degrade sugar polymers that are
often inaccessible to other gut residents, notably -mannan.
The B. thetaiotaomicron genome encodes ten putative
-mannanases spread across various PULs; however, little is
known about the activity of these enzymes or the wider
implications of -mannan metabolism for the health of both
the microbiota and the host. In this study, SAD phasing of a
selenomethionine derivative has been used to investigate the
structure of one such B. thetaiotaomicron enzyme, BT2949,
which belongs to the GH76 family of -mannanases. BT2949
presents a classical (/)6-barrel structure comprising a large
extended surface cleft common to other GH76 family
members. Analysis of the structure in conjunction with
sequence alignments reveals the likely location
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Feb 2015
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[7864]
Abstract: Glycoside hydrolase family 99 (GH99) was created to categorize sequence-related glycosidases possessing endo-α-mannosidase activity: the cleavage of mannosidic linkages within eukaryotic N-glycan precursors (Glc1–3Man9GlcNAc2), releasing mono-, di- and triglucosylated-mannose (Glc1–3-1,3-Man). GH99 family members have recently been implicated in the ability of Bacteroides spp., present within the gut microbiota, to metabolize fungal cell wall α-mannans, releasing α-1,3-mannobiose by hydrolysing αMan-1,3-αMan1,2-αMan-1,2-αMan sequences within branches off the main α-1,6-mannan backbone. We report the development of a series of substrates and inhibitors, which we use to kinetically and structurally characterise this novel endo-α-1,2-mannanase activity of bacterial GH99 enzymes from Bacteroides thetaiotaomicron and xylanisolvens. These data reveal an approximate 5 kJ mol−1 preference for mannose-configured substrates in the −2 subsite (relative to glucose), which inspired the development of a new inhibitor, α-mannopyranosyl-1,3-isofagomine (ManIFG), the most potent (bacterial) GH99 inhibitor reported to date. X-ray structures of ManIFG or a substrate in complex with wild-type or inactive mutants, respectively, of B. xylanisolvens GH99 reveal the structural basis for binding to D-mannose- rather than D-glucose-configured substrates.
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Jan 2015
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I03-Macromolecular Crystallography
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Fiona
Cuskin
,
Elisabeth C.
Lowe
,
Max J.
Temple
,
Yanping
Zhu
,
Elizabeth A.
Cameron
,
Nicholas A.
Pudlo
,
Nathan T.
Porter
,
Karthik
Urs
,
Andrew J.
Thompson
,
Alan
Cartmell
,
Artur
Rogowski
,
Brian S.
Hamilton
,
Rui
Chen
,
Thomas J.
Tolbert
,
Kathleen
Piens
,
Debby
Bracke
,
Wouter
Vervecken
,
Zalihe
Hakki
,
Gaetano
Speciale
,
Jose L.
Munoz-Munoz
Diamond Proposal Number(s):
[7864]
Abstract: Yeasts, which have been a component of the human diet for at least 7,000 years, possess an elaborate cell wall α-mannan. The influence of yeast mannan on the ecology of the human microbiota is unknown. Here we show that yeast α-mannan is a viable food source for the Gram-negative bacterium Bacteroides thetaiotaomicron, a dominant member of the microbiota. Detailed biochemical analysis and targeted gene disruption studies support a model whereby limited cleavage of α-mannan on the surface generates large oligosaccharides that are subsequently depolymerized to mannose by the action of periplasmic enzymes. Co-culturing studies showed that metabolism of yeast mannan by B. thetaiotaomicron presents a selfish model for the catabolism of this difficult to breakdown polysaccharide. Genomic comparison with B. thetaiotaomicron in conjunction with cell culture studies show that a cohort of highly successful members of the microbiota has evolved to consume sterically-restricted yeast glycans, an adaptation that may reflect the incorporation of eukaryotic microorganisms into the human diet.
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Jan 2015
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[7864]
Open Access
Abstract: The degradation of plant biomass by saprophytes is an ecologically important part of the global carbon cycle, which has also inspired a vast diversity of industrial enzyme applications. The xyloglucans (XyGs) constitute a family of ubiquitous and abundant plant cell wall polysaccharides, yet the enzymology of XyG saccharification is poorly studied. Here, we present the identification and molecular characterization of a complex genetic locus that is required for xyloglucan utilization by the model saprophyte Cellvibrio japonicus. In harness, transcriptomics, reverse genetics, enzyme kinetics, and structural biology indicate that the encoded cohort of an α-xylosidase, a β-galactosidase, and an α-l-fucosidase is specifically adapted for efficient, concerted saccharification of dicot (fucogalacto)xyloglucan oligosaccharides following import into the periplasm via an associated TonB-dependent receptor. The data support a biological model of xyloglucan degradation by C. japonicus with striking similarities – and notable differences – to the complex polysaccharide utilization loci of the Bacteroidetes.
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Oct 2014
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