I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[9948, 13587]
Abstract: Glycans are major nutrients available to the human gut microbiota (HGM). The Bacteroides are generalist glycan degraders and this function is mediated largely by polysaccharide utilization loci (PULs). The genomes of several Bacteroides species contain a PUL, PUL1,6-beta;-glucan, that was predicted to target mixed linked plant 1,3;1,4-beta-glucans. To test this hypothesis we characterized the proteins encoded by this locus in Bacteroides thetaiotaomicron, a member of the HGM. We show here that PUL1,6-β-glucan does not orchestrate the degradation of a plant polysaccharide but targets a fungal cell wall glycan, 1,6-beta-glucan, which is a growth substrate for the bacterium. The locus is upregulated by 1,6-beta-glucan, and encodes two enzymes, a surface endo-1,6-beta-glucanase, BT3312, and a periplasmic beta-glucosidase that targets primarily 1,6-beta-glucans. The non-catalytic proteins encoded by PUL1,6-beta-glucan target 1,6-beta-glucans and comprise a surface glycan binding protein and a SusD homologue that delivers glycans to the outer membrane transporter. We identified the central role of the endo-1,6-beta-glucanase in 1,6-beta-glucan depolymerization by deleting bt3312, which prevented the growth of B. thetaiotaomicron on 1,6-beta-glucan. The crystal structure of BT3312 in complex with β-glucosyl-1,6-deoxynojirimycin, revealed a TIM barrel catalytic domain that contains a deep substrate binding cleft tailored to accommodate the hook-like structure adopted by 1,6-beta-glucan. Specificity is driven by the complementarity of the enzyme active site cleft and the conformation of the substrate. We also noted that PUL1,6-beta-glucan is syntenic to many PULs from other Bacteroidetes suggesting that utilization of yeast and fungal cell wall 1,6-beta-glucans is a widespread adaptation within the human microbiota.
|
May 2017
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
M. Fleur
Sernee
,
Julie E.
Ralton
,
Tracy L.
Nero
,
Lukasz F.
Sobala
,
Joachim
Kloehn
,
Marcel A.
Vieira-lara
,
Simon A.
Cobbold
,
Lauren
Stanton
,
Douglas E. V.
Pires
,
Eric
Hanssen
,
Alexandra
Males
,
Tom
Ward
,
Laurence M.
Bastidas
,
Phillip L.
Van Der Peet
,
Michael W.
Parker
,
David B.
Ascher
,
Spencer J.
Williams
,
Gideon J.
Davies
,
Malcolm J.
Mcconville
Diamond Proposal Number(s):
[13587, 18598]
Open Access
Abstract: Parasitic protists belonging to the genus Leishmania synthesize the non-canonical carbohydrate reserve, mannogen, which is composed of β-1,2-mannan oligosaccharides. Here, we identify a class of dual-activity mannosyltransferase/phosphorylases (MTPs) that catalyze both the sugar nucleotide-dependent biosynthesis and phosphorolytic turnover of mannogen. Structural and phylogenic analysis shows that while the MTPs are structurally related to bacterial mannan phosphorylases, they constitute a distinct family of glycosyltransferases (GT108) that have likely been acquired by horizontal gene transfer from gram-positive bacteria. The seven MTPs catalyze the constitutive synthesis and turnover of mannogen. This metabolic rheostat protects obligate intracellular parasite stages from nutrient excess, and is essential for thermotolerance and parasite infectivity in the mammalian host. Our results suggest that the acquisition and expansion of the MTP family in Leishmania increased the metabolic flexibility of these protists and contributed to their capacity to colonize new host niches.
|
Sep 2019
|
|
I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
|
Alan
Cartmell
,
Jose
Muñoz-muñoz
,
Jonathon A.
Briggs
,
Didier A.
Ndeh
,
Elisabeth C.
Lowe
,
Arnaud
Basle
,
Nicolas
Terrapon
,
Katherine
Stott
,
Tiaan
Heunis
,
Joe
Gray
,
Li
Yu
,
Paul
Dupree
,
Pearl Z.
Fernandes
,
Sayali
Shah
,
Spencer J.
Williams
,
Aurore
Labourel
,
Matthias
Trost
,
Bernard
Henrissat
,
Harry J.
Gilbert
Diamond Proposal Number(s):
[1960, 7854, 9948]
Abstract: Glycans are major nutrients for the human gut microbiota (HGM). Arabinogalactan proteins (AGPs) comprise a heterogenous group of plant glycans in which a β1,3-galactan backbone and β1,6-galactan side chains are conserved. Diversity is provided by the variable nature of the sugars that decorate the galactans. The mechanisms by which nutritionally relevant AGPs are degraded in the HGM are poorly understood. Here we explore how the HGM organism Bacteroides thetaiotaomicron metabolizes AGPs. We propose a sequential degradative model in which exo-acting glycoside hydrolase (GH) family 43 β1,3-galactanases release the side chains. These oligosaccharide side chains are depolymerized by the synergistic action of exo-acting enzymes in which catalytic interactions are dependent on whether degradation is initiated by a lyase or GH. We identified two GHs that establish two previously undiscovered GH families. The crystal structures of the exo-β1,3-galactanases identified a key specificity determinant and departure from the canonical catalytic apparatus of GH43 enzymes. Growth studies of Bacteroidetes spp. on complex AGP revealed 3 keystone organisms that facilitated utilization of the glycan by 17 recipient bacteria, which included B. thetaiotaomicron. A surface endo-β1,3-galactanase, when engineered into B. thetaiotaomicron, enabled the bacterium to utilize complex AGPs and act as a keystone organism.
|
Nov 2018
|
|
I02-Macromolecular Crystallography
|
Yi
Jin
,
Marija
Petricevic
,
Alan
John
,
Lluís
Raich
,
Huw
Jenkins
,
Leticia
Portela De Souza
,
Fiona
Cuskin
,
Harry J.
Gilbert
,
Carme
Rovira
,
Ethan D.
Goddard-borger
,
Spencer J.
Williams
,
Gideon J.
Davies
Diamond Proposal Number(s):
[9948]
Abstract: The enzymatic cleavage of β-1,4-mannans is achieved by endo-β-1,4-mannanases, enzymes involved in germination of seeds and microbial hemicellulose degradation, and which have increasing industrial and consumer product applications. β-Mannanases occur in a range of families of the CAZy sequence-based glycoside hydrolase (GH) classification scheme including families 5, 26, and 113. In this work we reveal that β-mannanases of the newly described GH family 134 differ from other mannanase families in both their mechanism and tertiary structure. A representative GH family 134 endo-β-1,4-mannanase from a Streptomyces sp. displays a fold closely related to that of hen egg white lysozyme but acts with inversion of stereochemistry. A Michaelis complex with mannopentaose, and a product complex with mannotriose, reveal ligands with pyranose rings distorted in an unusual inverted chair conformation. Ab initio quantum mechanics/molecular mechanics metadynamics quantified the energetically accessible ring conformations and provided evidence in support of a 1C4 → 3H4‡ → 3S1 conformational itinerary along the reaction coordinate. This work, in concert with that on GH family 124 cellulases, reveals how the lysozyme fold can be co-opted to catalyze the hydrolysis of different polysaccharides in a mechanistically distinct manner.
|
Nov 2016
|
|
I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[13587]
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.
|
Jul 2017
|
|
I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
|
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.
|
Apr 2020
|
|
I24-Microfocus Macromolecular Crystallography
|
Ratana
Charoenwattanasatien
,
Salila
Pengthaisong
,
Imogen
Breen
,
Risa
Mutoh
,
Sompong
Sansenya
,
Yanling
Hua
,
Anupong
Tankrathok
,
Liang
Wu
,
Chomphunuch
Songsiriritthigul
,
Hideaki
Tanaka
,
Spencer J.
Williams
,
Gideon
Davies
,
Genji
Kurisu
,
James R. Ketudat
Cairns
Abstract: Human glucosylcerebrosidase 2 (GBA2) of the CAZy family GH116 is responsible for the breakdown of
glycosphingolipids on the cytoplasmic face of the endoplasmic reticulum and Golgi apparatus. Genetic defects in GBA2 result in
spastic paraplegia and cerebellar ataxia, while cross-talk between GBA2 and GBA1 glucosylceramidases may affect Gaucher
disease. Here, we report the first three-dimensional structure for any GH116 enzyme, Thermoanaerobacterium xylanolyticum
TxGH116 β-glucosidase, alone and in complex with diverse ligands. These structures allow identification of the glucoside binding
and active site residues, which are shown to be conserved with GBA2. Mutagenic analysis of TxGH116 and structural modeling
of GBA2 provide a detailed structural and functional rationale for pathogenic missense mutations of GBA2.
|
May 2016
|
|
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
|
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.
|
May 2018
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
|
Rohan J.
Williams
,
Javier
Iglesias-fernández
,
Judith
Stepper
,
Adam
Jackson
,
Andrew
Thompson
,
Elisabeth C.
Lowe
,
John
White
,
Harry J.
Gilbert
,
Carme
Rovira
,
Gideon J.
Davies
,
Spencer J.
Williams
Diamond Proposal Number(s):
[7864]
Abstract: Mannosidases catalyze the hydrolysis of a diverse range of polysaccharides and glycoconjugates, and the various sequence-based mannosidase families have evolved ingenious strategies to overcome the stereoelectronic challenges of mannoside chemistry. Using a combination of computational chemistry, inhibitor design and synthesis, and X-ray crystallography of inhibitor/enzyme complexes, it is demonstrated that mannoimidazole-type inhibitors are energetically poised to report faithfully on mannosidase transition-state conformation, and provide direct evidence for the conformational itinerary used by diverse mannosidases, including β-mannanases from families GH26 and GH113. Isofagomine-type inhibitors are poor mimics of transition-state conformation, owing to the high energy barriers that must be crossed to attain mechanistically relevant conformations, however, these sugar-shaped heterocycles allow the acquisition of ternary complexes that span the active site, thus providing valuable insight into active-site residues involved in substrate recognition.
|
Jan 2014
|
|
I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[13587]
Abstract: Conformational analysis of enzyme-catalyzed mannoside hydrolysis has revealed two predominant conformational itineraries through B2,5 or 3H4 transition-state (TS) conformations. A prominent unassigned catalytic itinerary is that of exo-1,6-α-mannosidases belonging to CAZy family 125. A published complex of Clostridium perfringens GH125 enzyme with a nonhydrolyzable 1,6-α-thiomannoside substrate mimic bound across the active site revealed an undistorted 4C1 conformation and provided no insight into the catalytic pathway of this enzyme. We show through a purely computational approach (QM/MM metadynamics) that sulfur-for-oxygen substitution in the glycosidic linkage fundamentally alters the energetically accessible conformational space of a thiomannoside when bound within the GH125 active site. Modeling of the conformational free energy landscape (FEL) of a thioglycoside strongly favors a mechanistically uninformative 4C1 conformation within the GH125 enzyme active site, but the FEL of corresponding O-glycoside substrate reveals a preference for a Michaelis complex in an OS2 conformation (consistent with catalysis through a B2,5 TS). This prediction was tested experimentally by determination of the 3D X-ray structure of the pseudo-Michaelis complex of an inactive (D220N) variant of C. perfringens GH125 enzyme in complex with 1,6-α-mannobiose. This complex revealed unambiguous distortion of the −1 subsite mannoside to an OS2 conformation, matching that predicted by theory and supporting an OS2 → B2,5 → 1S5 conformational itinerary for GH125 α-mannosidases. This work highlights the power of the QM/MM approach and identified shortcomings in the use of nonhydrolyzable substrate analogues for conformational analysis of enzyme-bound species.
|
Jan 2017
|
|
