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48 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3, 4, 5 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I03-Macromolecular Crystallography	A marine viral halogenase that iodinates diverse substrates Danai S. Gkotsi , Hannes Ludewig , Sunil V. Sharma , Jack A. Connolly , Jagwinder Dhaliwal , Yunpeng Wang , William P. Unsworth , Richard J. K. Taylor , Matthew M. W. Mclachlan , Stephen Shanahan , James Naismith , Rebecca J. M. Goss Nature Chemistry 117 DOI: 10.1038/s41557-019-0349-z Diamond Proposal Number(s): [14980] Show Abstract ▼ Abstract: Oceanic cyanobacteria are the most abundant oxygen-generating phototrophs on our planet and are therefore important to life. These organisms are infected by viruses called cyanophages, which have recently shown to encode metabolic genes that modulate host photosynthesis, phosphorus cycling and nucleotide metabolism. Herein we report the characterization of a wild-type flavin-dependent viral halogenase (VirX1) from a cyanophage. Notably, halogenases have been previously associated with secondary metabolism, tailoring natural products. Exploration of this viral halogenase reveals it capable of regioselective halogenation of a diverse range of substrates with a preference for forming aryl iodide species; this has potential implications for the metabolism of the infected host. Until recently, a flavin-dependent halogenase that is capable of iodination in vitro had not been reported. VirX1 is interesting from a biocatalytic perspective as it shows strikingly broad substrate flexibility and a clear preference for iodination, as illustrated by kinetic analysis. These factors together render it an attractive tool for synthesis.	Oct 2019
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	The complex of ferric-enterobactin with its transporter from Pseudomonas aeruginosa suggests a two-site model Lucile Moynie , Stefan Milenkovic , Gaëtan L. A. Mislin , Véronique Gasser , Giuliano Malloci , Etienne Baco , Rory P. Mccaughan , Malcolm G. P. Page , Isabelle J. Schalk , Matteo Ceccarelli , James H. Naismith Nature Communications 10 DOI: 10.1038/s41467-019-11508-y Open Access Show Abstract ▼ Abstract: Bacteria use small molecules called siderophores to scavenge iron. Siderophore-Fe3+ complexes are recognised by outer-membrane transporters and imported into the periplasm in a process dependent on the inner-membrane protein TonB. The siderophore enterobactin is secreted by members of the family Enterobacteriaceae, but many other bacteria including Pseudomonas species can use it. Here, we show that the Pseudomonas transporter PfeA recognises enterobactin using extracellular loops distant from the pore. The relevance of this site is supported by in vivo and in vitro analyses. We suggest there is a second binding site deeper inside the structure and propose that correlated changes in hydrogen bonds link binding-induced structural re-arrangements to the structural adjustment of the periplasmic TonB-binding motif.	Aug 2019
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Preacinetobactin not acinetobactin is essential for iron uptake by the BauA transporter of the pathogen Acinetobacter baumannii Lucile Moynie , Ilaria Serra , Mariano A. Scorciapino , Emilia Oueis , Malcolm G. P. Page , Matteo Ceccarelli , James Naismith Elife 7 DOI: 10.7554/eLife.42270 Open Access Show Abstract ▼ Abstract: New strategies are urgently required to develop antibiotics. The siderophore uptake system has attracted considerable attention but rational design of siderophore antibiotic conjugates requires knowledge of recognition by the cognate outer membrane transporter. Acinetobacter baumannii is a serious pathogen, which utilizes (pre)acinetobactin to scavenge iron from the host. We report the structure of the (pre)acinetobactin transporter BauA bound to the siderophore, identifying the structural determinants of recognition. Detailed biophysical analysis confirms that BauA recognises preacinetobactin. We show that acinetobactin is not recognised by the protein thus preacinetobactin is essential for iron uptake. The structure shows and NMR confirms that under physiological conditions a molecule of acinetobactin will bind to two free coordination sites on the iron preacinetobactin complex. The ability to recognise a heterotrimeric iron preacinetobactin acinetobactin complex may rationalize contradictory reports in the literature. These results open new avenues for the design of novel antibiotic conjugates (trojan horse) antibiotics.	Dec 2018
I24-Microfocus Macromolecular Crystallography	A key role for the periplasmic PfeE esterase in iron acquisition via the siderophore Enterobactin in Pseudomonas aeruginosa Quentin Perraud , Lucile Moynie , Véronique Gasser , Mathilde Munier , Julien Godet , Françoise Hoegy , Yves Mély , Gaëtan L. A. Mislin , James H. Naismith , Isabelle J. Schalk Acs Chemical Biology DOI: 10.1021/acschembio.8b00543 Show Abstract ▼ Abstract: Enterobactin (ENT) is a siderophore (iron-chelating compound) produced by Escherichia coli in order to gain access to iron, an essential nutriment for bacterial growth. ENT is used as an exosiderophore by the opportunistic human pathogen Pseudomonas aeruginosa with transport of ferri-ENT across the bacterial outer membrane by the transporter PfeA. Next to pfeA gene on the chromosome is localized a gene encoding for an esterase, PfeE, whose transcription is regulated, as for pfeA, by the presence of ENT in bacterial environment. Purified PfeE hydrolyzed ferri-ENT into three molecules of 2,3 DHBS (2,3 dihydroxybenzoylserine) still complexed with ferric iron, and complete dissociation of iron from ENT chelating groups was only possible in the presence of both PfeE and an iron reducer, such as DTT. The crystal structure of PfeE and an inactive PfeE mutant complexed with ferri-ENT or a non-hydrolysable ferri-catechol complex allowed identification of the enzyme binding site and the catalytic triad. Finally, cell fractionation and fluorescence microscopy showed periplasmic localization of PfeE in P. aeruginosa cells. Thus, the molecular mechanism of iron release from ENT in P. aeruginosa differs from that previously described in E. coli. In P. aeruginosa, siderophore hydrolysis occurs in the periplasm, with ENT never reaching the bacterial cytoplasm. In E. coli, ferri-ENT crosses the inner membrane via the ABC transporter FepBCD and ferri-ENT is hydrolyzed by the esterase Fes only once it is in the cytoplasm.	Aug 2018
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	A molecular mechanism for the enzymatic methylation of nitrogen atoms within peptide bonds Haigang Song , Niels S. Van Der Velden , Sally L. Shiran , Patrick Bleiziffer , Christina Zach , Ramon Sieber , Aman S. Imani , Florian Krausbeck , Markus Aebi , Michael F. Freeman , Sereina Riniker , Markus Künzler , James H. Naismith Science Advances 4 DOI: 10.1126/sciadv.aat2720 Open Access Show Abstract ▼ Abstract: The peptide bond, the defining feature of proteins, governs peptide chemistry by abolishing nucleophilicity of the nitrogen. This and the planarity of the peptide bond arise from the delocalization of the lone pair of electrons on the nitrogen atom into the adjacent carbonyl. While chemical methylation of an amide bond uses a strong base to generate the imidate, OphA, the precursor protein of the fungal peptide macrocycle omphalotin A, self-hypermethylates amides at pH 7 using S-adenosyl methionine (SAM) as cofactor. The structure of OphA reveals a complex catenane-like arrangement in which the peptide substrate is clamped with its amide nitrogen aligned for nucleophilic attack on the methyl group of SAM. Biochemical data and computational modeling suggest a base-catalyzed reaction with the protein stabilizing the reaction intermediate. Backbone N-methylation of peptides enhances their protease resistance and membrane permeability, a property that holds promise for applications to medicinal chemistry.	Aug 2018
	Getting drugs into Gram-negative bacteria: Rational rules for permeation through general porins Silvia Acosta-gutierrez , Luana Ferrara , Monisha Pathania , Muriel Masi , Jiajun Wang , Igor Bodrenko , Michael Zahn , Mathias Winterhalter , Robert A. Stavenger , Jean-marie Pages , James H. Naismith , Bert Van Den Berg , Malcolm G. P. Page , Matteo Ceccarelli Acs Infectious Diseases DOI: 10.1021/acsinfecdis.8b00108 Show Abstract ▼ Abstract: Small, hydrophilic molecules, including most important antibiotics in clinical use, cross the Gram-negative outer membrane through the water-filled channels provided by porins. We have determined the X-ray crystal structures of the principal general porins from three species of Enterobacteriaceae, namely Enterobacter aerogenes, Enterobacter cloacae and Klebsiella pneumoniae and determined their antibiotic permeabilities as well as those of the orthologues from Escherichia coli. Starting from the structure of the porins and molecules we propose a physical mechanism underlying transport and condense it in a computationally efficient scoring function. The scoring function shows good agreement with in-vitro penetration data and will enable the screening of virtual databases to identify molecules with optimal permeability through porins and help to guide the optimization of antibiotics with poor permeation.	Jul 2018
	Catalytic and anticatalytic snapshots of a short-form ATP phosphoribosyltransferase Magnus S. Alphey , Gemma Fisher , Ying Ge , Eoin R. Gould , Teresa Guerreiro Machado , Huanting Liu , Gordon J. Florence , James H. Naismith , Rafael Guimaraes Da Silva Acs Catalysis DOI: 10.1021/acscatal.8b00867 Show Abstract ▼ Abstract: Allosteric modulation of catalysis is a common regulatory strategy of flux-controlling biosynthetic enzymes. The enzyme ATP phosphoribosyltransferase (ATPPRT) catalyses the first reaction in histidine biosynthesis, the magnesium-dependent condensation of ATP and 5-phospho--D-ribosyl-1-pyrophosphate (PRPP) to generate N1-(5-phospho--D-ribosyl)-ATP (PRATP) and pyrophosphate (PPi). ATPPRT is allosterically inhibited by the final product of the pathway, histidine. Hetero-octameric ATPPRT consists of four catalytic subunits (HisGS) and four regulatory subunits (HisZ) engaged in intricate catalytic regulation. HisZ enhances HisGS catalysis in the absence of histidine while mediating allosteric inhibition in its presence. Here we report HisGS structures for the apoenzyme and complexes with substrates (PRPP, PRPP-ATP, PRPP-ADP), product (PRATP), and inhibitor (AMP), along with ATPPRT holoenzyme structures in complexes with substrates (PRPP, PRPP-ATP, PRPP-ADP) and product (PRATP). These ten crystal structures provide an atomic view of the catalytic cycle and allosteric activation of Psychrobacter arcticus ATPPRT. In both ternary complexes with PRPP-ATP, the adenine ring is found in an anticatalytic orientation, rotated 180° from the catalytic rotamer. Arg32 interacts with phosphate groups of ATP and PRPP, bringing the substrates in proximity for catalysis. The negative charge repulsion is further attenuated by a magnesium ion sandwiched between the - and -phosphate groups of both substrates. HisZ binding to form the hetero-octamer brings HisGS subunits closer together in a tighter dimer in the Michaelis complex, which poises Arg56 from the adjacent HisGS molecule for cross-subunit stabilisation of the PPi leaving group at the transition state. The more electrostatically pre-organised active site of the holoenzyme likely minimises the reorganisation energy required to accommodate the transition state. This provides a structural basis for allosteric activation in which chemistry is accelerated by facilitating leaving group departure.	May 2018
I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Characterization of the fast and promiscuous macrocyclase from plant PCY1 enables the use of simple substrates Hannes Ludewig , Clarissa M. Czekster , Emilia Oueis , Elizabeth S. Munday , Mohammed Arshad , Silvia A. Synowsky , Andrew F. Bent , James H. Naismith Acs Chemical Biology DOI: 10.1021/acschembio.8b00050 Show Abstract ▼ Abstract: Cyclic ribosomally derived peptides possess diverse bioactivities and are currently of major interest in drug development. However, it can be chemically challenging to synthesize these molecules, hindering the diversification and testing of cyclic peptide leads. Enzymes used in vitro offer a solution to this, however peptide macrocyclization remains the bottleneck. PCY1, involved in the biosynthesis of plant orbitides, belongs to the class of prolyl oligopeptidases and natively displays substrate promiscuity. PCY1 is a promising candidate for in vitro utilization but its substrates require an 11 to 16 residue C-terminal recognition tail. We have characterized PCY1 both kinetically and structurally with multiple substrate complexes revealing for the molecular basis of recognition and catalysis. Using these insights, we have identified a three residue C-terminal extension that replaces the natural recognition tail permitting PCY1 to operate on synthetic substrates. We demonstrate that PCY1 can macrocyclize a variety of substrates with this short tail, including unnatural amino acids and non-amino acids, highlighting PCY1's potential in biocatalysis.	Jan 2018
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Characterization of a dual function macrocyclase enables design and use of efficient macrocyclization substrates Clarissa M. Melo Czekster , Hannes Ludewig , Stephen Mcmahon , James Naismith Nature Communications 8 DOI: 10.1038/s41467-017-00862-4 Open Access Show Abstract ▼ Abstract: Peptide macrocycles are promising therapeutic molecules because they are protease resistant, structurally rigid, membrane permeable, and capable of modulating protein–protein interactions. Here, we report the characterization of the dual function macrocyclase-peptidase enzyme involved in the biosynthesis of the highly toxic amanitin toxin family of macrocycles. The enzyme first removes 10 residues from the N-terminus of a 35-residue substrate. Conformational trapping of the 25 amino-acid peptide forces the enzyme to release this intermediate rather than proceed to macrocyclization. The enzyme rebinds the 25 amino-acid peptide in a different conformation and catalyzes macrocyclization of the N-terminal eight residues. Structures of the enzyme bound to both substrates and biophysical analysis characterize the different binding modes rationalizing the mechanism. Using these insights simpler substrates with only five C-terminal residues were designed, allowing the enzyme to be more effectively exploited in biotechnology.	Oct 2017
I03-Macromolecular Crystallography	Rift Valley fever phlebovirus NSs protein core domain structure suggests molecular basis for nuclear filaments Michal Barski , Benjamin Brennan , Ona K. Miller , Jane A. Potter , Swetha Vijayakrishnan , David Bhella , James H. Naismith , Richard M. Elliot , Ulrich Schwarz-linek Elife DOI: 10.7554/eLife.29236 Diamond Proposal Number(s): [10071] Open Access Show Abstract ▼ Abstract: Rift Valley fever phlebovirus (RVFV) is a clinically and economically important pathogen increasingly likely to cause widespread epidemics. RVFV virulence depends on the interferon antagonist non-structural protein (NSs), which remains poorly characterized. We identified a stable core domain of RVFV NSs (residues 83–248), and solved its crystal structure, a novel all-helical fold organized into highly ordered fibrils. A hallmark of RVFV pathology is NSs filament formation in infected cell nuclei. Recombinant virus encoding the NSs core domain induced intranuclear filaments, suggesting it contains all essential determinants for nuclear translocation and filament formation. Mutations of key crystal fibril interface residues in viruses encoding full-length NSs completely abrogated intranuclear filament formation in infected cells. We propose the fibrillar arrangement of the NSs core domain in crystals reveals the molecular basis of assembly of this key virulence factor in cell nuclei. Our findings have important implications for fundamental understanding of RVFV virulence.	Sep 2017

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