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Instruments / Technical Area	Publication Details	Published
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	New thermophilic α/β class epoxide hydrolases found in metagenomes from hot environments Erica Elisa Ferrandi , Christopher Sayer , Simone Antonio De Rose , Elisa Guazzelli , Carlotta Marchesi , Vahid Saneei , Misha N. Isupov , Jennifer A. Littlechild , Daniela Monti Frontiers In Bioengineering And Biotechnology 6 DOI: 10.3389/fbioe.2018.00144 Diamond Proposal Number(s): [11945, 16378, 19576, 8889] Open Access Show Abstract ▼ 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.	Oct 2018
B21-High Throughput SAXS I04-Macromolecular Crystallography	Molecular mechanism for the subversion of the retromer coat by the Legionella effector RidL Miguel Romano-moreno , Adriana L. Rojas , Chad D. Williamson , David C. Gershlick , María Lucas , Michail N. Isupov , Juan S. Bonifacino , Matthias P. Machner , Aitor Hierro Proceedings Of The National Academy Of Sciences 27 DOI: 10.1073/pnas.1715361115 Open Access Show Abstract ▼ 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.	Dec 2017
I03-Macromolecular Crystallography	Structural and biochemical characterisation of Archaeoglobus fulgidus esterase reveals a bound CoA molecule in the vicinity of the active site Christopher Sayer , William Finnigan , Michail N. Isupov , Mark Levisson , Servé W. M. Kengen , John Van Der Oost , Nicholas Harmer , Jennifer Littlechild Scientific Reports 6 DOI: 10.1038/srep25542 Diamond Proposal Number(s): [8889, 11945] Open Access Show Abstract ▼ 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.	May 2016
I04-1-Macromolecular Crystallography (fixed wavelength)	Discovery and Characterization of a Thermostable and Highly Halotolerant GH5 Cellulase from an Icelandic Hot Spring Isolate 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 Plos One 11 DOI: 10.1371/journal.pone.0146454 PMID: 26741138 Diamond Proposal Number(s): [8889, 11945] Open Access Show Abstract ▼ 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.	Jan 2016
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Unraveling the B. pseudomallei Heptokinase WcbL: From Structure to Drug Discovery Mirella Vivoli , Misha Isupov , Rebecca Nicholas , Andrew Hill , Andrew e. Scott , Paul Kosma , Joann l. Prior , Nicholas Harmer Chemistry & Biology 22, 1622 - 1632 DOI: 10.1016/j.chembiol.2015.10.015 PMID: 26687481 Diamond Proposal Number(s): [1087, 6851, 8889] Open Access Show Abstract ▼ 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.	Dec 2015
I04-1-Macromolecular Crystallography (fixed wavelength)	The oxygenating constituent of 3,6-diketocamphane monooxygenase from the CAM plasmid of Pseudomonas putida: the first crystal structure of a type II Baeyer-Villiger monooxygenase 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 Acta Crystallographica Section D Biological Crystallography 71 (11), 2344 - 2353 DOI: 10.1107/S1399004715017939 PMID: 26527149 Diamond Proposal Number(s): [8889] Open Access Show Abstract ▼ 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.	Nov 2015
I04-1-Macromolecular Crystallography (fixed wavelength)	The Structure of a Novel Thermophilic Esterase from the Planctomycetes Species, Thermogutta terrifontis Reveals an Open Active Site Due to a Minimal ‘Cap’ Domain Christopher Sayer , Zalan Szabo , Michail N. Isupov , Colin Ingham , Jennifer A Littlechild Frontiers In Microbiology 6 DOI: 10.3389/fmicb.2015.01294 PMID: 26635762 Diamond Proposal Number(s): [8889, 11945] Open Access Show Abstract ▼ 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.	Nov 2015
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Structural studies of a thermophilic esterase from a new Planctomycetes species, Thermogutta terrifontis Christopher Sayer , Misha Isupov , Elizaveta Bonch Osmolovskaya , Jennifer Littlechild Febs Journal 282 (15), 2846-2857 DOI: 10.1111/febs.13326 PMID: 26011036 Diamond Proposal Number(s): [8889] Show Abstract ▼ 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.	May 2015
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Discovery and characterisation of thermophilic limonene-1,2-epoxide hydrolases from hot spring metagenomic libraries Erica Elisa Ferrandi , Christopher Sayer , Michail Isupov , Celeste Annovazzi , Carlotta Marchesi , Gianluca Iacobone , Xu Peng , Elizaveta Bonch- Osmolovskaya , Roland Wohlgemuth , Jennifer Littlechild , Daniela Monti Febs Journal 282 (15), 2879-2894 DOI: 10.1111/febs.13328 PMID: 26032250 Diamond Proposal Number(s): [8889] Show Abstract ▼ Abstract: The epoxide hydrolases (EHs) represent an attractive option for the synthe- sis of chiral epoxides and 1,2-diols which are valuable building blocks for the synthesis of several pharmaceutical compounds. A metagenomic approach has been used to identify two new members of the atypical EH limonene-1,2-epoxide hydrolase (LEH) family of enzymes. These two LEHs (Tomsk-LEH and CH55-LEH) show EH activities towards different epox- ide substrates, differing in most cases from those previously identified for Rhodococcus erythropolis (Re-LEH) in terms of stereoselectivity. Tomsk- LEH and CH55-LEH, both from thermophilic sources, have higher opti- mal temperatures and apparent melting temperatures than Re-LEH. The new LEH enzymes have been crystallized and their structures solved to high resolution in the native form and in complex with the inhibitor valpr- omide for Tomsk-LEH and poly(ethylene glycol) for CH55-LEH. The structural analysis has provided insights into the LEH mechanism, sub- strate specificity and stereoselectivity of these new LEH enzymes, which has been supported by mutagenesis studies.	May 2015
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	The structure of a tetrameric -carbonic anhydrase from Thermovibrio ammonificans reveals a core formed around intermolecular disulfides that contribute to its thermostability Paul James , Misha Isupov , Christopher Sayer , Vahid Saneei , Svein Berg , Maria Lioliou , Hans Kristian Kotlar , Jennifer Littlechild Acta Crystallographica Section D Biological Crystallography 70, 2607 - 2618 DOI: 10.1107/S1399004714016526 PMID: 25286845 Diamond Proposal Number(s): [6851, 8889] Show Abstract ▼ Abstract: Carbonic anhydrase enzymes catalyse the reversible hydration of carbon dioxide to bicarbonate. A thermophilic Thermovibrio ammonificans -carbonic anhydrase (TaCA) has been expressed in Escherichia coli and structurally and biochemically characterized. The crystal structure of TaCA has been determined in its native form and in two complexes with bound inhibitors. The tetrameric enzyme is stabilized by a unique core in the centre of the molecule formed by two intersubunit disulfides and a single lysine residue from each monomer that is involved in intersubunit ionic interactions. The structure of this core protects the intersubunit disulfides from reduction, whereas the conserved intrasubunit disulfides are not formed in the reducing environment of the E. coli host cytosol. When oxidized to mimic the environment of the periplasmic space, TaCA has increased thermostability, retaining 90% activity after incubation at 70°C for 1 h, making it a good candidate for industrial carbon-dioxide capture. The reduction of all TaCA cysteines resulted in dissociation of the tetrameric molecule into monomers with lower activity and reduced thermostability. Unlike other characterized -carbonic anhydrases, TaCA does not display esterase activity towards p-nitrophenyl acetate, which appears to result from the increased rigidity of its protein scaffold.	Oct 2014

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