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

Instruments / Technical Area	Publication Details	Published
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Structural and mechanistic analysis of ATPase inhibitors targeting mycobacterial DNA gyrase Sara R. Henderson , Clare E. M. Stevenson , Brandon Malone , Yelyzaveta Zholnerovych , Lesley A Mitchenall , Mark Pichowicz , David H Mcgarry , Ian R Cooper , Cedric Charrier , Anne-marie Salisbury , David M. Lawson , Anthony Maxwell Journal Of Antimicrobial Chemotherapy 22 DOI: 10.1093/jac/dkaa286 Diamond Proposal Number(s): [18565] Open Access Show Abstract ▼ Abstract: Objectives: To evaluate the efficacy of two novel compounds against mycobacteria and determine the molecular basis of their action on DNA gyrase using structural and mechanistic approaches. Methods: Redx03863 and Redx04739 were tested in antibacterial assays, and also against their target, DNA gyrase, using DNA supercoiling and ATPase assays. X-ray crystallography was used to determine the structure of the gyrase B protein ATPase sub-domain from Mycobacterium smegmatis complexed with the aminocoumarin drug novobiocin, and structures of the same domain from Mycobacterium thermoresistibile complexed with novobiocin, and also with Redx03863. Results: Both compounds, Redx03863 and Redx04739, were active against selected Gram-positive and Gram-negative species, with Redx03863 being the more potent, and Redx04739 showing selectivity against M. smegmatis. Both compounds were potent inhibitors of the supercoiling and ATPase reactions of DNA gyrase, but did not appreciably affect the ATP-independent relaxation reaction. The structure of Redx03863 bound to the gyrase B protein ATPase sub-domain from M. thermoresistibile shows that it binds at a site adjacent to the ATP- and novobiocin-binding sites. We found that most of the mutations that we made in the Redx03863-binding pocket, based on the structure, rendered gyrase inactive. Conclusions: Redx03863 and Redx04739 inhibit gyrase by preventing the binding of ATP. The fact that the Redx03863-binding pocket is distinct from that of novobiocin, coupled with the lack of activity of resistant mutants, suggests that such compounds could have potential to be further exploited as antibiotics.	Jul 2020
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Diversification of DNA-binding specificity by permissive and specificity-switching mutations in the ParB/Noc protein family Adam S. B. Jalal , Ngat T. Tran , Clare E. Stevenson , Elliot W. Chan , Rebecca Lo , Xiao Tan , Agnes Noy , David M. Lawson , Tung B. K. Le Cell Reports 32 DOI: 10.1016/j.celrep.2020.107928 Diamond Proposal Number(s): [18565] Open Access Show Abstract ▼ Abstract: Specific interactions between proteins and DNA are essential to many biological processes. Yet, it remains unclear how the diversification in DNA-binding specificity was brought about, and the mutational paths that led to changes in specificity are unknown. Using a pair of evolutionarily related DNA-binding proteins, each with a different DNA preference (ParB [Partitioning Protein B] and Noc [Nucleoid Occlusion Factor], which both play roles in bacterial chromosome maintenance), we show that specificity is encoded by a set of four residues at the protein-DNA interface. Combining X-ray crystallography and deep mutational scanning of the interface, we suggest that permissive mutations must be introduced before specificity-switching mutations to reprogram specificity and that mutational paths to new specificity do not necessarily involve dual-specificity intermediates. Overall, our results provide insight into the possible evolutionary history of ParB and Noc and, in a broader context, might be useful for understanding the evolution of other classes of DNA-binding proteins.	Jul 2020
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Structure of the Mycobacterium smegmatis α-maltose-1-phosphate synthase GlgM Karl Syson , Clare Stevenson , David M. Lawson , Stephen Bornemann Acta Crystallographica Section F Structural Biology Communications 76, 175 - 181 DOI: 10.1107/S2053230X20004343 Diamond Proposal Number(s): [13467] Open Access Show Abstract ▼ Abstract: Mycobacterium tuberculosis produces glycogen (also known as α-glucan) to help evade human immunity. This pathogen uses the GlgE pathway to generate glycogen rather than the more well known glycogen synthase GlgA pathway, which is absent in this bacterium. Thus, the building block for this glucose polymer is α-maltose-1-phosphate rather than an NDP-glucose donor. One of the routes to α-maltose-1-phosphate is now known to involve the GlgA homologue GlgM, which uses ADP-glucose as a donor and α-glucose-1-phosphate as an acceptor. To help compare GlgA (a GT5 family member) with GlgM enzymes (GT4 family members), the X-ray crystal structure of GlgM from Mycobacterium smegmatis was solved to 1.9 Å resolution. While the enzymes shared a GT-B fold and several residues responsible for binding the donor substrate, they differed in some secondary-structural details, particularly in the N-terminal domain, which would be expected to be largely responsible for their different acceptor-substrate specificities.	Apr 2020
I03-Macromolecular Crystallography	Structural basis of cycloaddition in biosynthesis of iboga and aspidosperma alkaloids Lorenzo Caputi , Jakob Franke , Kate Bussey , Scott C. Farrow , Ivo Jose Curcino Vieira , Clare E. M. Stevenson , David N. Lawson , Sarah E. O’connor Nature Chemical Biology 23 DOI: 10.1038/s41589-019-0460-x Diamond Proposal Number(s): [13467] Show Abstract ▼ Abstract: Cycloaddition reactions generate chemical complexity in a single step. Here we report the crystal structures of three homologous plant-derived cyclases involved in the biosynthesis of iboga and aspidosperma alkaloids. These enzymes act on the same substrate, named angryline, to generate three distinct scaffolds. Mutational analysis reveals how these highly similar enzymes control regio- and stereo-selectivity.	Feb 2020
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	The structure of a GH149 β‐(1 → 3) glucan phosphorylase reveals a new surface oligosaccharide binding site and additional domains that are absent in the disaccharide‐specific GH94 glucose‐β‐(1 → 3)‐glucose (laminaribiose) phosphorylase Sakonwan Kuhaudomlarp , Clare Stevenson , David M. Lawson , Robert Field Proteins: Structure, Function And Bioinformatics DOI: 10.1002/prot.25745 Diamond Proposal Number(s): [13467] Open Access Show Abstract ▼ Abstract: Glycoside phosphorylases (GPs) with specificity for β‐(1 → 3)‐gluco‐oligosaccharides are potential candidate biocatalysts for oligosaccharide synthesis. GPs with this linkage specificity are found in two families thus far—glycoside hydrolase family 94 (GH94) and the recently discovered glycoside hydrolase family 149 (GH149). Previously, we reported a crystallographic study of a GH94 laminaribiose phosphorylase with specificity for disaccharides, providing insight into the enzyme's ability to recognize its' sugar substrate/product. In contrast to GH94, characterized GH149 enzymes were shown to have more flexible chain length specificity, with preference for substrate/product with higher degree of polymerization. In order to advance understanding of the specificity of GH149 enzymes, we herein solved X‐ray crystallographic structures of GH149 enzyme Pro_7066 in the absence of substrate and in complex with laminarihexaose (G6). The overall domain organization of Pro_7066 is very similar to that of GH94 family enzymes. However, two additional domains flanking its catalytic domain were found only in the GH149 enzyme. Unexpectedly, the G6 complex structure revealed an oligosaccharide surface binding site remote from the catalytic site, which, we suggest, may be associated with substrate targeting. As such, this study reports the first structure of a GH149 phosphorylase enzyme acting on β‐(1 → 3)‐gluco‐oligosaccharides and identifies structural elements that may be involved in defining the specificity of the GH149 enzymes.	May 2019
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Architecture of microcin B17 synthetase: an octameric protein complex converting a ribosomally synthesized peptide into a DNA gyrase poison Dmitry Ghilarov , Clare E. M. Stevenson , Dmitrii Y. Travin , Julia Piskunova , Marina Serebryakova , Anthony Maxwell , David M. Lawson , Konstantin Severinov Molecular Cell DOI: 10.1016/j.molcel.2018.11.032 Diamond Proposal Number(s): [9475, 13467] Open Access Show Abstract ▼ Abstract: The introduction of azole heterocycles into a peptide backbone is the principal step in the biosynthesis of numerous compounds with therapeutic potential. One of them is microcin B17, a bacterial topoisomerase inhibitor whose activity depends on the conversion of selected serine and cysteine residues of the precursor peptide to oxazoles and thiazoles by the McbBCD synthetase complex. Crystal structures of McbBCD reveal an octameric B4C2D2 complex with two bound substrate peptides. Each McbB dimer clamps the N-terminal recognition sequence, while the C-terminal heterocycle of the modified peptide is trapped in the active site of McbC. The McbD and McbC active sites are distant from each other, which necessitates alternate shuttling of the peptide substrate between them, while remaining tethered to the McbB dimer. An atomic-level view of the azole synthetase is a starting point for deeper understanding and control of biosynthesis of a large group of ribosomally synthesized natural products.	Jan 2019
I03-Macromolecular Crystallography	Uncoupled activation and cyclization in catmint reductive terpenoid biosynthesis Benjamin R. Lichman , Mohamed O. Kamileen , Gabriel R. Titchiner , Gerhard Saalbach , Clare E. M. Stevenson , David M. Lawson , Sarah E. O'connor Nature Chemical Biology 15, 71 - 79 DOI: 10.1038/s41589-018-0185-2 Diamond Proposal Number(s): [13467] Show Abstract ▼ Abstract: Terpene synthases typically form complex molecular scaffolds by concerted activation and cyclization of linear starting materials in a single enzyme active site. Here we show that iridoid synthase, an atypical reductive terpene synthase, catalyzes the activation of its substrate 8-oxogeranial into a reactive enol intermediate, but does not catalyze the subsequent cyclization into nepetalactol. This discovery led us to identify a class of nepetalactol-related short-chain dehydrogenase enzymes (NEPS) from catmint (Nepeta mussinii) that capture this reactive intermediate and catalyze the stereoselective cyclisation into distinct nepetalactol stereoisomers. Subsequent oxidation of nepetalactols by NEPS1 provides nepetalactones, metabolites that are well known for both insect-repellent activity and euphoric effects in cats. Structural characterization of the NEPS3 cyclase reveals that it binds to NAD+ yet does not utilize it chemically for a non-oxidoreductive formal [4 + 2] cyclization. These discoveries will complement metabolic reconstructions of iridoid and monoterpene indole alkaloid biosynthesis.	Dec 2018
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Unravelling the relaxed specificity of laminaribiose phosphorylase from Paenibacillus sp. strain YM-1 towards mannose 1-phosphate Robert Andrew Field , Sakonwan Kuhaudomlarp , Samuel Walpole , Clare Stevenson , Sergey Nepogodiev , David Lawson , Jesus Angulo Chembiochem DOI: 10.1002/cbic.201800260 Diamond Proposal Number(s): [13467] Show Abstract ▼ Abstract: Glycoside phosphorylases (GPs) carry out a reversible phosphorolysis of carbohydrates into oligosaccharide acceptors and the corresponding sugar 1‐phosphates. The reversibility of the reaction enables the use of GPs as biocatalysts for carbohydrate synthesis. Glycosyl hydrolase family 94 (GH94), which only comprises GPs, is one of the most studied GP families that have been used as biocatalysts for carbohydrate synthesis, in academic research and in industrial production. Understanding the mechanism of GH94 enzymes is a crucial step towards enzyme engineering to improve and expand the applications of these enzymes in synthesis. In this work with a GH94 laminaribiose phosphorylase from Paenibacillus sp. YM1 (PsLBP), we have demonstrated an enzymatic synthesis of disaccharide 1 using natural acceptor glucose and non‐cognate donor substrate ‐mannose 1‐phosphate (Man1P). To investigate how the enzyme recognizes different sugar 1‐phosphates, we solved the X‐ray crystal structures of PsLBP in complex with Glc1P and Man1P, providing the first molecular detail of the recognition of a non‐cognate donor substrate by GPs, which revealed the importance of hydrogen bonding between the active site residues and hydroxyl groups at C2, C4 and C6 of sugar 1‐phosphates. Furthermore, we used STD NMR to support the crystallographic studies on the sugar 1‐phosphates, as well as to provide further insights into the PsLBP recognition of the acceptors and the disaccharide products.	Jun 2018
I02-Macromolecular Crystallography	Discovery of a short chain dehydrogenase from Catharanthus roseus that produces a novel monoterpene indole alkaloid Anna K. Stavrinides , Evangelos Tatsis , Thu-thuy Dang , Lorenzo Caputi , Clare Stevenson , David Lawson , Bernd Schneider , Sarah O'connor Chembiochem DOI: 10.1002/cbic.201700621 Diamond Proposal Number(s): [9475] Show Abstract ▼ Abstract: Monoterpene indole alkaloids, a large class of plant natural products, derive from the biosynthetic intermediate strictosidine aglycone. Strictosidine aglycone, which can exist as a variety of isomers, can be reduced to form numerous different structures. We have discovered a short chain alcohol dehydrogenase (SDR) from monoterpene indole alkaloid producing plants (Catharanthus roseus and Rauvolfia serpentina) that reduce strictosidine aglycone and produce an alkaloid that does not correspond to any previously reported compound. Here we report the structural characterization of this product, which we have named vitrosamine, as well as the crystal structure of the SDR. This discovery highlights the structural versatility of the strictosidine aglycone biosynthetic intermediate and expands the range of enzymatic reactions that SDRs can catalyze. This discovery further highlights how a sequence-based gene mining discovery approach in plants can reveal cryptic chemistry that would not be uncovered by classical natural product chemistry approaches.	Feb 2018
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	The Origins of Specificity in the Microcin-Processing Protease TldD/E Dmitry Ghilarov , Marina Serebryakova , Clare E. M. Stevenson , Stephen J. Hearnshaw , Dmitry Volkov , Anthony Maxwell , David M. Lawson , Konstantin Severinov Structure DOI: 10.1016/j.str.2017.08.006 Diamond Proposal Number(s): [9475] Open Access Show Abstract ▼ Abstract: TldD and TldE proteins are involved in the biosynthesis of microcin B17 (MccB17), an Escherichia coli thiazole/oxazole-modified peptide toxin targeting DNA gyrase. Using a combination of biochemical and crystallographic methods we show that E. coli TldD and TldE interact to form a heterodimeric metalloprotease. TldD/E cleaves the N-terminal leader sequence from the modified MccB17 precursor peptide, to yield mature antibiotic, while it has no effect on the unmodified peptide. Both proteins are essential for the activity; however, only the TldD subunit forms a novel metal-containing active site within the hollow core of the heterodimer. Peptide substrates are bound in a sequence-independent manner through β sheet interactions with TldD and are likely cleaved via a thermolysin-type mechanism. We suggest that TldD/E acts as a “molecular pencil sharpener”: unfolded polypeptides are fed through a narrow channel into the active site and processively truncated through the cleavage of short peptides from the N-terminal end.	Sep 2017

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