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31 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-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
I02-Macromolecular Crystallography	Cellodextrin phosphorylase from Ruminiclostridium thermocellum : X-ray crystal structure and substrate specificity analysis Ellis C. O'neill , Giulia Pergolizzi , Clare E. M. Stevenson , David M. Lawson , Sergey A. Nepogodiev , Robert A. Field Carbohydrate Research DOI: 10.1016/j.carres.2017.07.005 Diamond Proposal Number(s): [7641] Open Access Show Abstract ▼ Abstract: The GH94 glycoside hydrolase cellodextrin phosphorylase (CDP, EC 2.4.1.49) produces cellodextrin oligomers from short β-1→4-glucans and α-D-glucose 1-phosphate. Compared to cellobiose phosphorylase (CBP), which produces cellobiose from glucose and α-D-glucose 1-phosphate, CDP is biochemically less well characterised. Herein, we investigate the donor and acceptor substrate specificity of recombinant CDP from Ruminiclostridium thermocellum and we isolate and characterise a glucosamine addition product to the cellobiose acceptor with the non-natural donor α-D-glucosamine 1-phosphate. In addition, we report the first X-ray crystal structure of CDP, along with comparison to the available structures from CBPs and other closely related enzymes, which contributes to understanding of the key structural features necessary to discriminate between monosaccharide (CBP) and oligosaccharide (CDP) acceptor substrates.	Jul 2017
I04-1-Macromolecular Crystallography (fixed wavelength)	The Production and Utilization of GDP-glucose in the Biosynthesis of Trehalose 6-Phosphate by Streptomyces venezuelae Matías D. Asención Diez , Farzana Miah , Clare E. M. Stevenson , David M. Lawson , Alberto A. Iglesias , Stephen Bornemann Journal Of Biological Chemistry 292, 945 - 954 DOI: 10.1074/jbc.M116.758664 Diamond Proposal Number(s): [7641] Open Access Show Abstract ▼ Abstract: Trehalose-6-phosphate synthase OtsA from streptomycetes is unusual in that it uses GDP-glucose as the donor substrate rather than the more commonly used UDP-glucose. We now confirm that OtsA from Streptomyces venezuelae has such a preference for GDP-glucose and can utilise ADP-glucose to some extent too. A crystal structure of the enzyme shows that it shares twin Rossmann-like domains with the UDP-glucose-specific OtsA from Escherichia coli. However, it is structurally more similar to Streptomyces hygroscopicus VldE, a GDP-valienol-dependent pseudo-glycosyltransferase enzyme. Comparison of the donor binding sites reveals that the amino acids associated with the binding of diphosphoribose are almost completely conserved between these three enzymes. By contrast, the amino acids associated with binding guanine in VldE (Asn, Thr and Val) are functionally conserved in S. venezuelae OtsA (Asp, Ser and Phe, respectively) but not in E. coli OtsA (His, Leu and Asp, respectively), providing a rationale for the purine base-specificity of S. venezuelae OtsA. To establish which donor is used in vivo, we generated an otsA null mutant in S. venezuelae. The mutant had a cell-density-dependent growth phenotype and accumulated galactose-1-phosphate, glucose-1-phosphate and GDP-glucose. To determine how the GDP-glucose is generated, we characterised three candidate GDP-glucose pyrophosphorylases. SVEN_3027 is a UDP-glucose pyrophosphorylase, SVEN_3972 is an unusual ITP-mannose pyrophosphorylase, and SVEN_2781 is a pyrophosphorylase that is capable of generating GDP-glucose as well as GDP-mannose. We have therefore established how S. venezuelae can make and utilise GDP-glucose in the biosynthesis of trehalose-6-phosphate.	Jan 2017
I02-Macromolecular Crystallography	Structural characterization of EasH (Aspergillus japonicus) – an oxidase involved in cycloclavine biosynthesis Dorota Jakubczyk , Lorenzo Caputi , Clare E. M. Stevenson , David M. Lawson , Sarah E. O'connor Chem. Commun. 52, 14306 - 14309 DOI: 10.1039/C6CC08438A Diamond Proposal Number(s): [9475] Open Access Show Abstract ▼ Abstract: Aj_EasH is a non-heme iron- and α-keto-glutarate-dependent oxidase that is responsible for an unusual cyclopropyl ring formation in the biosynthesis of the fungal ergot alkaloid cycloclavine. The three dimensional structure of Aj_EasH (2.2 Å resolution) reported here provides insight into the mechanism of this unusual and complex reaction.	Nov 2016
I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	Ligand-bound Structures and Site-directed Mutagenesis Identify the Acceptor and Secondary Binding Sites of Streptomyces coelicolor Maltosyltransferase GlgE Karl Syson , Clare E. M. Stevenson , Farzana Miah , J. Elaine Barclay , Minhong Tang , Andrii Gorelik , Abdul M. Rashid , David M. Lawson , Stephen Bornemann Journal Of Biological Chemistry 291, 21531 - 21540 DOI: 10.1074/jbc.M116.748160 Diamond Proposal Number(s): [1219, 9475] Open Access Show Abstract ▼ Abstract: GlgE is a maltosyltransferase involved in α-glucan biosynthesis in bacteria that has been genetically validated as a target for tuberculosis therapies. Crystals of the Mycobacterium tuberculosis enzyme diffract at low resolution so most structural studies have been with the very similar Streptomyces coelicolor GlgE isoform 1. Although the donor binding site for α-maltose 1-phosphate had been previously structurally defined, the acceptor site had not. Using mutagenesis, kinetics, and protein crystallography of the S. coelicolor enzyme, we have now identified the +1 to +6 subsites of the acceptor/product, which overlap with the known cyclodextrin binding site. The sugar residues in the acceptor subsites +1 to +5 are oriented such that they disfavor the binding of malto-oligosaccharides that bear branches at their 6-positions, consistent with the known acceptor chain specificity of GlgE. A secondary binding site remote from the catalytic center was identified that is distinct from one reported for the M. tuberculosis enzyme. This new site is capable of binding a branched α-glucan and is most likely involved in guiding acceptors toward the donor site because its disruption kinetically compromises the ability of GlgE to extend polymeric substrates. However, disruption of this site, which is conserved in the Streptomyces venezuelae GlgE enzyme, did not affect the growth of S. venezuelae or the structure of the polymeric product. The acceptor subsites +1 to +4 in the S. coelicolor enzyme are well conserved in the M. tuberculosis enzyme so their identification could help inform the design of inhibitors with therapeutic potential.	Oct 2016

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