I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Alice R.
Cross
,
Sumita
Roy
,
Mirella
Vivoli Vega
,
Martin
Rejzek
,
Sergey A.
Nepogodiev
,
Matthew
Cliff
,
Debbie
Salmon
,
Michail N.
Isupov
,
Robert A.
Field
,
Joann L.
Prior
,
Nicholas J.
Harmer
Diamond Proposal Number(s):
[16378]
Open Access
Abstract: The sugars streptose and dihydrohydroxystreptose (DHHS) are unique to the bacteria Streptomyces griseus and Coxiella burnetii, respectively. Streptose forms the central moiety of the antibiotic streptomycin, whilst DHHS is found in the O-antigen of the zoonotic pathogen C. burnetii. Biosynthesis of these sugars has been proposed to follow a similar path to that of TDP-rhamnose, catalyzed by the enzymes RmlA, RmlB, RmlC, and RmlD, but the exact mechanism is unclear. Streptose and DHHS biosynthesis unusually requires a ring contraction step that could be performed by orthologues of RmlC or RmlD. Genome sequencing of S. griseus and C. burnetii has identified StrM and CBU1838 proteins as RmlC orthologues in these respective species. Here, we demonstrate that both enzymes can perform the RmlC 3’’,5’’ double epimerization activity necessary to support TDP-rhamnose biosynthesis in vivo. This is consistent with the ring contraction step being performed on a double epimerized substrate. We further demonstrate that proton exchange is faster at the 3’’-position than the 5’’-position, in contrast to a previously studied orthologue. We additionally solved the crystal structures of CBU1838 and StrM in complex with TDP, and show that they form an active site highly similar to those of the previously characterized enzymes RmlC, EvaD, and ChmJ. These results support the hypothesis that streptose and DHHS are biosynthesized using the TDP pathway and that an RmlD paralogue most likely performs ring contraction following double epimerization. This work will support the elucidation of the full pathways for biosynthesis of these unique sugars.
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Apr 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8889]
Open Access
Abstract: A novel transketolase has been reconstituted from two separate polypeptide chains encoded by a ‘split-gene’ identified in the genome of the hyperthermophilic bacterium, Carboxydothermus hydrogenoformans. The reconstituted active α2β2 tetrameric enzyme has been biochemically characterized and its activity has been determined using a range of aldehydes including glycolaldehyde, phenylacetaldehyde and cyclohexanecarboxaldehyde as the ketol acceptor and hydroxypyruvate as the donor. This reaction proceeds to near 100% completion due to the release of the product carbon dioxide and can be used for the synthesis of a range of sugars of interest to the pharmaceutical industry. This novel reconstituted transketolase is thermally stable with no loss of activity after incubation for 1 h at 70°C and is stable after 1 h incubation with 50% of the organic solvents methanol, ethanol, isopropanol, DMSO, acetonitrile and acetone. The X-ray structure of the holo reconstituted α2β2 tetrameric transketolase has been determined to 1.4 Å resolution. In addition, the structure of an inactive tetrameric β4 protein has been determined to 1.9 Å resolution. The structure of the active reconstituted α2β2 enzyme has been compared to the structures of related enzymes; the E1 component of the pyruvate dehydrogenase complex and D-xylulose-5-phosphate synthase, in an attempt to rationalize differences in structure and substrate specificity between these enzymes. This is the first example of a reconstituted ‘split-gene’ transketolase to be biochemically and structurally characterized allowing its potential for industrial biocatalysis to be evaluated.
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Oct 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):
[11945, 16378, 19576, 8889]
Open Access
Abstract: Two novel epoxide hydrolases (EHs), Sibe-EH and CH65-EH, were identified in the metagenomes of samples collected in hot springs in Russia and China, respectively. The two α/β hydrolase superfamily fold enzymes were cloned, over-expressed in Escherichia coli, purified and characterized. The new EHs were active toward a broad range of substrates, and in particular, Sibe-EH was excellent in the desymmetrization of cis-2,3-epoxybutane producing the (2R,3R)-diol product with ee exceeding 99%. Interestingly these enzymes also hydrolyse (4R)-limonene-1,2-epoxide with Sibe-EH being specific for the trans isomer. The Sibe-EH is a monomer in solution whereas the CH65-EH is a dimer. Both enzymes showed high melting temperatures with the CH65-EH being the highest at 85°C retaining 80% of its initial activity after 3 h thermal treatment at 70°C making it the most thermal tolerant wild type epoxide hydrolase described. The Sibe-EH and CH65-EH have been crystallized and their structures determined to high resolution, 1.6 and 1.4 Å, respectively. The CH65-EH enzyme forms a dimer via its cap domains with different relative orientation of the monomers compared to previously described EHs. The entrance to the active site cavity is located in a different position in CH65-EH and Sibe-EH in relation to other known bacterial and mammalian EHs.
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Oct 2018
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B21-High Throughput SAXS
I04-Macromolecular Crystallography
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Open Access
Abstract: Microbial pathogens employ sophisticated virulence strategies to cause infections in humans. The intracellular pathogen Legionella pneumophila encodes RidL to hijack the host scaffold protein VPS29, a component of retromer and retriever complexes critical for endosomal cargo recycling. Here, we determined the crystal structure of L. pneumophila RidL in complex with the human VPS29–VPS35 retromer subcomplex. A hairpin loop protruding from RidL inserts into a conserved pocket on VPS29 that is also used by cellular ligands, such as Tre-2/Bub2/Cdc16 domain family member 5 (TBC1D5) and VPS9-ankyrin repeat protein for VPS29 binding. Consistent with the idea of molecular mimicry in protein interactions, RidL outcompeted TBC1D5 for binding to VPS29. Furthermore, the interaction of RidL with retromer did not interfere with retromer dimerization but was essential for association of RidL with retromer-coated vacuolar and tubular endosomes. Our work thus provides structural and mechanistic evidence into how RidL is targeted to endosomal membranes.
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Dec 2017
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8889, 11945]
Open Access
Abstract: A new carboxyl esterase, AF-Est2, from the hyperthermophilic archaeon Archaeoglobus fulgidus has been cloned, over-expressed in Escherichia coli and biochemically and structurally characterized. The enzyme has high activity towards short- to medium-chain p-nitrophenyl carboxylic esters with optimal activity towards the valerate ester. The AF-Est2 has good solvent and pH stability and is very thermostable, showing no loss of activity after incubation for 30 min at 80 °C. The 1.4 Å resolution crystal structure of AF-Est2 reveals Coenzyme A (CoA) bound in the vicinity of the active site. Despite the presence of CoA bound to the AF-Est2 this enzyme has no CoA thioesterase activity. The pantetheine group of CoA partially obstructs the active site alcohol pocket suggesting that this ligand has a role in regulation of the enzyme activity. A comparison with closely related α/β hydrolase fold enzyme structures shows that the AF-Est2 has unique structural features that allow CoA binding. A comparison of the structure of AF-Est2 with the human carboxyl esterase 1, which has CoA thioesterase activity, reveals that CoA is bound to different parts of the core domain in these two enzymes and approaches the active site from opposite directions.
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May 2016
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Dimitra
Zarafeta
,
Dimitrios
Kissas
,
Christopher
Sayer
,
Sóley R.
Gudbergsdottir
,
Efthymios
Ladoukakis
,
Misha
Isupov
,
Aristotelis
Chatziioannou
,
Xu
Peng
,
Jennifer
Littlechild
,
Georgios
Skretas
,
Fragiskos N.
Kolisis
,
Maria
Gasset
Diamond Proposal Number(s):
[8889, 11945]
Open Access
Abstract: With the ultimate goal of identifying robust cellulases for industrial biocatalytic conversions,
we have isolated and characterized a new thermostable and very halotolerant GH5 cellulase.
This new enzyme, termed CelDZ1, was identified by bioinformatic analysis from the
genome of a polysaccharide-enrichment culture isolate, initiated from material collected
from an Icelandic hot spring. Biochemical characterization of CelDZ1 revealed that it is a
glycoside hydrolase with optimal activity at 70°C and pH 5.0 that exhibits good thermostability,
high halotolerance at near-saturating salt concentrations, and resistance towards metal
ions and other denaturing agents. X-ray crystallography of the new enzyme showed that
CelDZ1 is the first reported cellulase structure that lacks the defined sugar-binding 2 subsite
and revealed structural features which provide potential explanations of its biochemical
characteristics.
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Jan 2016
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[1087, 6851, 8889]
Open Access
Abstract: Gram-negative bacteria utilize heptoses as part of
their repertoire of extracellular polysaccharide virulence
determinants. Disruption of heptose biosynthesis
offers an attractive target for novel antimicrobials.
A critical step in the synthesis of heptoses
is their 1-O phosphorylation, mediated by kinases
such as HldE or WcbL. Here, we present the structure
of WcbL from Burkholderia pseudomallei. We report
that WcbL operates through a sequential ordered Bi-
Bi mechanism, loading the heptose first and then
ATP. We show that dimeric WcbL binds ATP anticooperatively
in the absence of heptose, and cooperatively
in its presence. Modeling of WcbL suggests
that heptose binding causes an elegant switch in the
hydrogen-bonding network, facilitating the binding
of a second ATP molecule. Finally, we screened a
library of drug-like fragments, identifying hits that
potently inhibit WcbL. Our results provide a novel
mechanism for control of substrate binding and
emphasize WcbL as an attractive anti-microbial
target for Gram-negative bacteria.
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Dec 2015
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Mishail N.
Isupov
,
Ewald
Schröder
,
Robert P.
Gibson
,
Jean
Beecher
,
Giuliana
Donadio
,
Vahid
Saneei
,
Stephlina A.
Dcunha
,
Emma J.
Mcghie
,
Christopher
Sayer
,
Colin F.
Davenport
,
Peter C.
Lau
,
Yoshie
Hasegawa
,
Hiroaki
Iwaki
,
Maria
Kadow
,
Kathleen
Balke
,
Uwe T.
Bornscheuer
,
Gleb
Bourenkov
,
Jennifer
Littlechild
Diamond Proposal Number(s):
[8889]
Open Access
Abstract: The three-dimensional structures of the native enzyme and the FMN complex of the overexpressed form of the oxygenating component of the type II Baeyer-Villiger 3,6-diketocamphane monooxygenase have been determined to 1.9 Å resolution. The structure of this dimeric FMN-dependent enzyme, which is encoded on the large CAM plasmid of Pseudomonas putida, has been solved by a combination of multiple anomalous dispersion from a bromine crystal soak and molecular replacement using a bacterial luciferase model. The orientation of the isoalloxazine ring of the FMN cofactor in the active site of this TIM-barrel fold enzyme differs significantly from that previously observed in enzymes of the bacterial luciferase-like superfamily. The Ala77 residue is in a cis conformation and forms a -bulge at the C-terminus of -strand 3, which is a feature observed in many proteins of this superfamily.
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Nov 2015
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[8889, 11945]
Open Access
Abstract: A carboxyl esterase (TtEst2) has been identified in a novel thermophilic bacterium, Thermogutta terrifontis from the phylum Planctomycetes and has been cloned and over-expressed in Escherichia coli. The enzyme has been characterised biochemically and shown to have activity towards small p-nitrophenyl (pNP) carboxylic esters with optimal activity for pNP-acetate.The enzyme shows moderate thermostability retaining 75% activity after incubation for 30 minutes at 70°C. The crystal structures have been determined for the native TtEst2 and its complexes with the carboxylic acid products propionate, butyrate and valerate. TtEst2 differs from most enzymes of the α/β-hydrolase family 3 as it lacks the majority of the ‘cap’ domain and its active site cavity is exposed to the solvent. The bound ligands have allowed the identification of the carboxyl pocket in the enzyme active site. Comparison of TtEst2 with structurally related enzymes has given insight into how differences in their substrate preference can be rationalised based upon the properties of their active site pockets.
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Nov 2015
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8889]
Abstract: A carboxyl esterase (TtEst) has been identified in a novel thermophilic bacterium, Thermogutta terrifontis from the phylum Planctomycetes and has been cloned and over-expressed in Escherichia coli. The enzyme has been characterised biochemically and shown to have activity towards small p-nitrophenyl (pNP) carboxylic esters with optimal activity for pNP-propionate. The enzyme retained 95% activity after incubation for 1 hour at 80°C. The crystal structures of the native TtEst and its complexes with the substrate analogue, D-malate and the product acetate have been determined to high resolution. The bound ligands have allowed the identification of the carboxyl and alcohol binding pockets in the enzyme active site. Comparison of TtEst with structurally related enzymes has given insight into how differences in their catalytic activity can be rationalised based upon the properties of the amino acid residues in their active site pockets. The mutant enzymes L37A and L251A have been constructed to extend the substrate range of TtEst towards the larger butyrate and valerate pNP-esters. These mutant enzymes have also shown a significant increase in activity towards acetate and propionate pNP esters. A crystal structure of the L37A mutant was determined with the butyrate product bound in the carboxyl pocket of the active site. The mutant structure shows an expansion of the pocket that binds the substrate carboxyl group, which is consistent with the observed increase in activity towards pNP-butyrate.
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May 2015
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