Publications by authors named "Clare E M Stevenson"

56 Publications

Analysis of Protein-DNA Interactions Using Surface Plasmon Resonance and a ReDCaT Chip.

Methods Mol Biol 2021 ;2263:369-379

Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich, UK.

The recognition of specific DNA sequences by proteins is crucial to fundamental biological processes such as DNA replication, transcription, and gene regulation. The technique of surface plasmon resonance (SPR) is ideally suited for the measurement of these interactions because it is quantitative, simple to implement, reproducible, can be automated, and requires very little sample. This typically involves the direct capture of biotinylated DNA to a streptavidin (SA) chip before flowing over the protein of interest and monitoring the interaction. However, once the DNA has been immobilized on the chip, it cannot be removed without damaging the chip surface. Moreover, if the protein-DNA interaction is strong, then it may not be possible to remove the protein from the DNA without damaging the chip surface. Given that the chips are costly, this will limit the number of samples that can be tested. Therefore, we have developed a Reusable DNA Capture Technology, or ReDCaT chip, that enables a single streptavidin chip to be used multiple times making the technique simple, quick, and cost effective. The general steps to prepare the ReDCaT chip, run a simple binding experiment, and analysis of data will be described in detail. Some additional applications will also be introduced.
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http://dx.doi.org/10.1007/978-1-0716-1197-5_17DOI Listing
January 2021

The pentapeptide-repeat protein, MfpA, interacts with mycobacterial DNA gyrase as a DNA T-segment mimic.

Proc Natl Acad Sci U S A 2021 Mar;118(11)

Department of Biological Chemistry, John Innes Centre, NR4 7UH Norwich, United Kingdom;

DNA gyrase, a type II topoisomerase, introduces negative supercoils into DNA using ATP hydrolysis. The highly effective gyrase-targeted drugs, fluoroquinolones (FQs), interrupt gyrase by stabilizing a DNA-cleavage complex, a transient intermediate in the supercoiling cycle, leading to double-stranded DNA breaks. MfpA, a pentapeptide-repeat protein in mycobacteria, protects gyrase from FQs, but its molecular mechanism remains unknown. Here, we show that MfpA (MsMfpA) inhibits negative supercoiling by gyrase (Msgyrase) in the absence of FQs, while in their presence, MsMfpA decreases FQ-induced DNA cleavage, protecting the enzyme from these drugs. MsMfpA stimulates the ATPase activity of Msgyrase by directly interacting with the ATPase domain (MsGyrB47), which was confirmed through X-ray crystallography of the MsMfpA-MsGyrB47 complex, and mutational analysis, demonstrating that MsMfpA mimics a T (transported) DNA segment. These data reveal the molecular mechanism whereby MfpA modulates the activity of gyrase and may provide a general molecular basis for the action of other pentapeptide-repeat proteins.
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http://dx.doi.org/10.1073/pnas.2016705118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7980463PMC
March 2021

Assessment of Pathogen Effector Binding to Host Proteins by Surface Plasmon Resonance.

Bio Protoc 2020 Jul 5;10(13):e3676. Epub 2020 Jul 5.

Department of Biological Chemistry, John Innes Centre, Colney Lane, Norwich, NR4 7UH, UK.

The mechanisms of virulence and immunity are often governed by molecular interactions between pathogens and host proteins. The study of these interactions has major implications on understanding virulence activities, and how the host immune system recognizes the presence of pathogens to initiate an immune response. Frequently, the association between pathogen molecules and host proteins are assessed using qualitative techniques. As small differences in binding affinity can have a major biological effect, techniques that can quantitatively compare the binding between different proteins are required. However, these techniques can be manually intensive and often require large amounts of purified proteins. Here we present a simplified Surface Plasmon Resonance (SPR) protocol that allows a reproducible side-by-side quantitative comparison of the binding between different proteins, even in cases where the binding affinity cannot be confidently calculated. We used this method to assess the binding of virulence proteins (termed effectors) from the blast fungus , to a domain of a host immune receptor. This approach represents a rapid and quantitative way to study how pathogen molecules bind to host proteins, requires only limited quantities of proteins, and is highly reproducible. Although this method requires the use of an SPR instrument, these can often be accessed through shared scientific services at many institutions. Thus, this technique can be implemented in any study that aims to understand host-pathogen interactions, irrespective of the expertise of the investigator.
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http://dx.doi.org/10.21769/BioProtoc.3676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7842709PMC
July 2020

Base-pair resolution analysis of the effect of supercoiling on DNA flexibility and major groove recognition by triplex-forming oligonucleotides.

Nat Commun 2021 02 16;12(1):1053. Epub 2021 Feb 16.

School of Physics and Astronomy, University of Leeds, Leeds, UK.

In the cell, DNA is arranged into highly-organised and topologically-constrained (supercoiled) structures. It remains unclear how this supercoiling affects the detailed double-helical structure of DNA, largely because of limitations in spatial resolution of the available biophysical tools. Here, we overcome these limitations, by a combination of atomic force microscopy (AFM) and atomistic molecular dynamics (MD) simulations, to resolve structures of negatively-supercoiled DNA minicircles at base-pair resolution. We observe that negative superhelical stress induces local variation in the canonical B-form DNA structure by introducing kinks and defects that affect global minicircle structure and flexibility. We probe how these local and global conformational changes affect DNA interactions through the binding of triplex-forming oligonucleotides to DNA minicircles. We show that the energetics of triplex formation is governed by a delicate balance between electrostatics and bonding interactions. Our results provide mechanistic insight into how DNA supercoiling can affect molecular recognition, that may have broader implications for DNA interactions with other molecular species.
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http://dx.doi.org/10.1038/s41467-021-21243-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7887228PMC
February 2021

Potent DNA gyrase inhibitors bind asymmetrically to their target using symmetrical bifurcated halogen bonds.

Nat Commun 2021 01 8;12(1):150. Epub 2021 Jan 8.

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, 1000, Ljubljana, Slovenia.

Novel bacterial type II topoisomerase inhibitors (NBTIs) stabilize single-strand DNA cleavage breaks by DNA gyrase but their exact mechanism of action has remained hypothetical until now. We have designed a small library of NBTIs with an improved DNA gyrase-binding moiety resulting in low nanomolar inhibition and very potent antibacterial activity. They stabilize single-stranded cleavage complexes and, importantly, we have obtained the crystal structure where an NBTI binds gyrase-DNA in a single conformation lacking apparent static disorder. This directly proves the previously postulated NBTI mechanism of action and shows that they stabilize single-strand cleavage through asymmetric intercalation with a shift of the scissile phosphate. This crystal stucture shows that the chlorine forms a halogen bond with the backbone carbonyls of the two symmetry-related Ala68 residues. To the best of our knowledge, such a so-called symmetrical bifurcated halogen bond has not been identified in a biological system until now.
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http://dx.doi.org/10.1038/s41467-020-20405-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7794245PMC
January 2021

Exploring the Chemical Space of Benzothiazole-Based DNA Gyrase B Inhibitors.

ACS Med Chem Lett 2020 Dec 15;11(12):2433-2440. Epub 2020 Oct 15.

University of Ljubljana, Faculty of Pharmacy, Aškerčeva cesta 7, 1000 Ljubljana, Slovenia.

We designed and synthesized a series of inhibitors of the bacterial enzymes DNA gyrase and DNA topoisomerase IV, based on our recently published benzothiazole-based inhibitor bearing an oxalyl moiety. To improve the antibacterial activity and retain potent enzymatic activity, we systematically explored the chemical space. Several strategies of modification were followed: varying substituents on the pyrrole carboxamide moiety, alteration of the central scaffold, including variation of substitution position and, most importantly, modification of the oxalyl moiety. Compounds with acidic, basic, and neutral properties were synthesized. To understand the mechanism of action and binding mode, we have obtained a crystal structure of compound , bearing a primary amino group, in complex with the N-terminal domain of gyrase B (24 kDa) (PDB: ). Compound , with a low molecular weight of 383 Da, potent inhibitory activity on gyrase (IC = 9.5 nM), potent antibacterial activity on (MIC = 3.13 μM), and efflux impaired strain (MIC = 0.78 μM), is an important contribution for the development of novel gyrase and topoisomerase IV inhibitors in Gram-negative bacteria.
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http://dx.doi.org/10.1021/acsmedchemlett.0c00416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7734788PMC
December 2020

Structural and mechanistic analysis of ATPase inhibitors targeting mycobacterial DNA gyrase.

J Antimicrob Chemother 2020 10;75(10):2835-2842

Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.

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.
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http://dx.doi.org/10.1093/jac/dkaa286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556816PMC
October 2020

Structure of the Mycobacterium smegmatis α-maltose-1-phosphate synthase GlgM.

Acta Crystallogr F Struct Biol Commun 2020 Apr 3;76(Pt 4):175-181. Epub 2020 Apr 3.

Biological Chemistry Department, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom.

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.
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http://dx.doi.org/10.1107/S2053230X20004343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7137382PMC
April 2020

Structural basis of cycloaddition in biosynthesis of iboga and aspidosperma alkaloids.

Nat Chem Biol 2020 04 17;16(4):383-386. Epub 2020 Feb 17.

Max Planck Institute of Chemical Ecology, Department of Natural Product Biosynthesis, Jena, Germany.

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.
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http://dx.doi.org/10.1038/s41589-019-0460-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7104359PMC
April 2020

New insights into the binding mode of pyridine-3-carboxamide inhibitors of E. coli DNA gyrase.

Bioorg Med Chem 2019 08 14;27(16):3546-3550. Epub 2019 Jun 14.

School of Chemistry and the Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK. Electronic address:

Previously we have reported on a series of pyridine-3-carboxamide inhibitors of DNA gyrase and DNA topoisomerase IV that were designed using a computational de novo design approach and which showed promising antibacterial properties. Herein we describe the synthesis of additional examples from this series aimed specifically at DNA gyrase, along with crystal structures confirming the predicted mode of binding and in vitro ADME data which describe the drug-likeness of these compounds.
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http://dx.doi.org/10.1016/j.bmc.2019.06.015DOI Listing
August 2019

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.

Proteins 2019 10 6;87(10):885-892. Epub 2019 Jun 6.

Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich, UK.

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.
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http://dx.doi.org/10.1002/prot.25745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6771811PMC
October 2019

Architecture of Microcin B17 Synthetase: An Octameric Protein Complex Converting a Ribosomally Synthesized Peptide into a DNA Gyrase Poison.

Mol Cell 2019 02 17;73(4):749-762.e5. Epub 2019 Jan 17.

Centre for Life Sciences, Skolkovo Institute of Science and Technology, 143026 Moscow, Russia; Institute of Gene Biology of the Russian Academy of Sciences, 119334 Moscow, Russia; Waksman Institute for Microbiology, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA. Electronic address:

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 BCD 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.
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http://dx.doi.org/10.1016/j.molcel.2018.11.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6395948PMC
February 2019

Uncoupled activation and cyclization in catmint reductive terpenoid biosynthesis.

Nat Chem Biol 2019 01 10;15(1):71-79. Epub 2018 Dec 10.

The John Innes Centre, Department of Biological Chemistry, Norwich Research Park, Norwich, UK.

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.
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http://dx.doi.org/10.1038/s41589-018-0185-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6513753PMC
January 2019

Unravelling the Specificity of Laminaribiose Phosphorylase from Paenibacillus sp. YM-1 towards Donor Substrates Glucose/Mannose 1-Phosphate by Using X-ray Crystallography and Saturation Transfer Difference NMR Spectroscopy.

Chembiochem 2019 01 4;20(2):181-192. Epub 2018 Jul 4.

Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK.

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. YM-1 (PsLBP), we have demonstrated an enzymatic synthesis of disaccharide 1 (β-d-mannopyranosyl-(1→3)-d-glucopyranose) by using a natural acceptor glucose and noncognate donor substrate α-mannose 1-phosphate (Man1P). To investigate how the enzyme recognises different sugar 1-phosphates, the X-ray crystal structures of PsLBP in complex with Glc1P and Man1P have been solved, providing the first molecular detail of the recognition of a noncognate donor substrate by GPs, which revealed the importance of hydrogen bonding between the active site residues and hydroxy groups at C2, C4, and C6 of sugar 1-phosphates. Furthermore, we used saturation transfer difference NMR spectroscopy to support crystallographic studies on the sugar 1-phosphates, as well as to provide further insights into the PsLBP recognition of the acceptors and disaccharide products.
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http://dx.doi.org/10.1002/cbic.201800260DOI Listing
January 2019

Discovery of a Short-Chain Dehydrogenase from Catharanthus roseus that Produces a New Monoterpene Indole Alkaloid.

Chembiochem 2018 05 22;19(9):940-948. Epub 2018 Mar 22.

Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK.

Plant monoterpene indole alkaloids, a large class of 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 plant producers of monoterpene indole alkaloids (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 catalyse. 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.
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http://dx.doi.org/10.1002/cbic.201700621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6003104PMC
May 2018

The Origins of Specificity in the Microcin-Processing Protease TldD/E.

Structure 2017 10 21;25(10):1549-1561.e5. Epub 2017 Sep 21.

Centre for Data-Intensive Biomedicine and Biotechnology, Skolkovo Institute of Science and Technology, 143026 Moscow, Russia; Bionano Institute, Peter the Great Saint Petersburg State Polytechnical University, Saint Petersburg 195251, Russia; Waksman Institute for Microbiology, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.str.2017.08.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5810440PMC
October 2017

Cellodextrin phosphorylase from Ruminiclostridium thermocellum: X-ray crystal structure and substrate specificity analysis.

Carbohydr Res 2017 Nov 21;451:118-132. Epub 2017 Jul 21.

Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK. Electronic address:

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.
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http://dx.doi.org/10.1016/j.carres.2017.07.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5667895PMC
November 2017

One ligand, two regulators and three binding sites: How KDPG controls primary carbon metabolism in Pseudomonas.

PLoS Genet 2017 Jun 28;13(6):e1006839. Epub 2017 Jun 28.

John Innes Centre, Norwich Research Park, Colney Lane, Norwich, United Kingdom.

Effective regulation of primary carbon metabolism is critically important for bacteria to successfully adapt to different environments. We have identified an uncharacterised transcriptional regulator; RccR, that controls this process in response to carbon source availability. Disruption of rccR in the plant-associated microbe Pseudomonas fluorescens inhibits growth in defined media, and compromises its ability to colonise the wheat rhizosphere. Structurally, RccR is almost identical to the Entner-Doudoroff (ED) pathway regulator HexR, and both proteins are controlled by the same ED-intermediate; 2-keto-3-deoxy-6-phosphogluconate (KDPG). Despite these similarities, HexR and RccR control entirely different aspects of primary metabolism, with RccR regulating pyruvate metabolism (aceEF), the glyoxylate shunt (aceA, glcB, pntAA) and gluconeogenesis (pckA, gap). RccR displays complex and unusual regulatory behaviour; switching repression between the pyruvate metabolism and glyoxylate shunt/gluconeogenesis loci depending on the available carbon source. This regulatory complexity is enabled by two distinct pseudo-palindromic binding sites, differing only in the length of their linker regions, with KDPG binding increasing affinity for the 28 bp aceA binding site but decreasing affinity for the 15 bp aceE site. Thus, RccR is able to simultaneously suppress and activate gene expression in response to carbon source availability. Together, the RccR and HexR regulators enable the rapid coordination of multiple aspects of primary carbon metabolism, in response to levels of a single key intermediate.
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http://dx.doi.org/10.1371/journal.pgen.1006839DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5489143PMC
June 2017

Identification of new DNA i-motif binding ligands through a fluorescent intercalator displacement assay.

Org Biomol Chem 2017 Jul 1;15(27):5669-5673. Epub 2017 Jun 1.

School of Pharmacy, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK. and Centre for Molecular and Structural Biochemistry, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK.

i-Motifs are quadruplex DNA structures formed from sequences rich in cytosine and held together by intercalated, hemi-protonated cytosine-cytosine base pairs. These sequences are prevalent in gene promoter regions and may play a role in gene transcription. Targeting these structures with ligands could provide a novel way to target genetic disease but there are very few ligands which have been shown to interact with i-motif DNA. Fluorescent intercalator displacement (FID) assays are a simple way to screen ligands against DNA secondary structures. Here we characterise how thiazole orange interacts with i-motif DNA and assess its ability for use in a FID assay. Additionally, we report FID-based ligand screening using thiazole orange against the i-motif forming sequence from the human telomere to reveal new i-motif binding compounds which have the potential for further development.
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http://dx.doi.org/10.1039/c7ob00710hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5708337PMC
July 2017

Mitoxantrone and Analogues Bind and Stabilize i-Motif Forming DNA Sequences.

Sci Rep 2016 12 22;6:39456. Epub 2016 Dec 22.

School of Pharmacy, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK.

There are hundreds of ligands which can interact with G-quadruplex DNA, yet very few which target i-motif. To appreciate an understanding between the dynamics between these structures and how they can be affected by intervention with small molecule ligands, more i-motif binding compounds are required. Herein we describe how the drug mitoxantrone can bind, induce folding of and stabilise i-motif forming DNA sequences, even at physiological pH. Additionally, mitoxantrone was found to bind i-motif forming sequences preferentially over double helical DNA. We also describe the stabilisation properties of analogues of mitoxantrone. This offers a new family of ligands with potential for use in experiments into the structure and function of i-motif forming DNA sequences.
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http://dx.doi.org/10.1038/srep39456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5177923PMC
December 2016

The Production and Utilization of GDP-glucose in the Biosynthesis of Trehalose 6-Phosphate by Streptomyces venezuelae.

J Biol Chem 2017 01 30;292(3):945-954. Epub 2016 Nov 30.

From the Biological Chemistry Department, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom and

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 utilize 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 pseudoglycosyltransferase enzyme. Comparison of the donor binding sites reveals that the amino acids associated with the binding of diphosphoribose are almost all identical in these three enzymes. By contrast, the amino acids associated with binding guanine in VldE (Asn, Thr, and Val) are similar in S. venezuelae OtsA (Asp, Ser, and Phe, respectively) but not conserved 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 when grown on galactose. To determine how the GDP-glucose is generated, we characterized 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 utilize GDP-glucose in the biosynthesis of trehalose 6-phosphate.
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http://dx.doi.org/10.1074/jbc.M116.758664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5247666PMC
January 2017

Structural characterization of EasH (Aspergillus japonicus) - an oxidase involved in cycloclavine biosynthesis.

Chem Commun (Camb) 2016 Dec;52(99):14306-14309

Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.

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.
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http://dx.doi.org/10.1039/c6cc08438aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5317212PMC
December 2016

Substrate-Assisted Catalysis in Polyketide Reduction Proceeds via a Phenolate Intermediate.

Cell Chem Biol 2016 Sep 8;23(9):1091-1097. Epub 2016 Sep 8.

Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK. Electronic address:

SimC7 is a polyketide ketoreductase involved in biosynthesis of the angucyclinone moiety of the gyrase inhibitor simocyclinone D8 (SD8). SimC7, which belongs to the short-chain dehydrogenase/reductase (SDR) superfamily, catalyzes reduction of the C-7 carbonyl of the angucyclinone, and the resulting hydroxyl is essential for antibiotic activity. SimC7 shares little sequence similarity with characterized ketoreductases, suggesting it might have a distinct mechanism. To investigate this possibility, we determined the structures of SimC7 alone, with NADP(+), and with NADP(+) and the substrate 7-oxo-SD8. These structures show that SimC7 is distinct from previously characterized polyketide ketoreductases, lacking the conserved catalytic triad, including the active-site tyrosine that acts as central acid-base catalyst in canonical SDR proteins. Taken together with functional analyses of active-site mutants, our data suggest that SimC7 catalyzes a substrate-assisted, two-step reaction for reduction of the C-7 carbonyl group involving intramolecular transfer of a substrate-derived proton to generate a phenolate intermediate.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5039031PMC
http://dx.doi.org/10.1016/j.chembiol.2016.07.018DOI Listing
September 2016

Ligand-bound Structures and Site-directed Mutagenesis Identify the Acceptor and Secondary Binding Sites of Streptomyces coelicolor Maltosyltransferase GlgE.

J Biol Chem 2016 Oct 16;291(41):21531-21540. Epub 2016 Aug 16.

From the Biological Chemistry Department, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom

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.
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http://dx.doi.org/10.1074/jbc.M116.748160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5076824PMC
October 2016

Structural investigation of heteroyohimbine alkaloid synthesis reveals active site elements that control stereoselectivity.

Nat Commun 2016 07 15;7:12116. Epub 2016 Jul 15.

The John Innes Centre, Department of Biological Chemistry, Norwich NR4 7UH, UK.

Plants produce an enormous array of biologically active metabolites, often with stereochemical variations on the same molecular scaffold. These changes in stereochemistry dramatically impact biological activity. Notably, the stereoisomers of the heteroyohimbine alkaloids show diverse pharmacological activities. We reported a medium chain dehydrogenase/reductase (MDR) from Catharanthus roseus that catalyses formation of a heteroyohimbine isomer. Here we report the discovery of additional heteroyohimbine synthases (HYSs), one of which produces a mixture of diastereomers. The crystal structures for three HYSs have been solved, providing insight into the mechanism of reactivity and stereoselectivity, with mutation of one loop transforming product specificity. Localization and gene silencing experiments provide a basis for understanding the function of these enzymes in vivo. This work sets the stage to explore how MDRs evolved to generate structural and biological diversity in specialized plant metabolism and opens the possibility for metabolic engineering of new compounds based on this scaffold.
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http://dx.doi.org/10.1038/ncomms12116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4947188PMC
July 2016

Structural determinants of reductive terpene cyclization in iridoid biosynthesis.

Nat Chem Biol 2016 Jan 9;12(1):6-8. Epub 2015 Nov 9.

The John Innes Centre, Department of Biological Chemistry, Norwich Research Park, Norwich NR4 7UH, UK.

The carbon skeleton of ecologically and pharmacologically important iridoid monoterpenes is formed in a reductive cyclization reaction unrelated to canonical terpene cyclization. Here we report the crystal structure of the recently discovered iridoid cyclase (from Catharanthus roseus) bound to a mechanism-inspired inhibitor that illuminates substrate binding and catalytic function of the enzyme. Key features that distinguish iridoid synthase from its close homolog progesterone 5β-reductase are highlighted.
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http://dx.doi.org/10.1038/nchembio.1955DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4685742PMC
January 2016

Structural Dissection of the Maltodextrin Disproportionation Cycle of the Arabidopsis Plastidial Disproportionating Enzyme 1 (DPE1).

J Biol Chem 2015 Dec 26;290(50):29834-53. Epub 2015 Oct 26.

From the Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom and

The degradation of transitory starch in the chloroplast to provide fuel for the plant during the night requires a suite of enzymes that generate a series of short chain linear glucans. However, glucans of less than four glucose units are no longer substrates for these enzymes, whereas export from the plastid is only possible in the form of either maltose or glucose. In order to make use of maltotriose, which would otherwise accumulate, disproportionating enzyme 1 (DPE1; a 4-α-glucanotransferase) converts two molecules of maltotriose to a molecule of maltopentaose, which can now be acted on by the degradative enzymes, and one molecule of glucose that can be exported. We have determined the structure of the Arabidopsis plastidial DPE1 (AtDPE1), and, through ligand soaking experiments, we have trapped the enzyme in a variety of conformational states. AtDPE1 forms a homodimer with a deep, long, and open-ended active site canyon contained within each subunit. The canyon is divided into donor and acceptor sites with the catalytic residues at their junction; a number of loops around the active site adopt different conformations dependent on the occupancy of these sites. The "gate" is the most dynamic loop and appears to play a role in substrate capture, in particular in the binding of the acceptor molecule. Subtle changes in the configuration of the active site residues may prevent undesirable reactions or abortive hydrolysis of the covalently bound enzyme-substrate intermediate. Together, these observations allow us to delineate the complete AtDPE1 disproportionation cycle in structural terms.
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http://dx.doi.org/10.1074/jbc.M115.682245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4705983PMC
December 2015

Bacterial rotary export ATPases are allosterically regulated by the nucleotide second messenger cyclic-di-GMP.

J Biol Chem 2015 Oct 11;290(40):24470-83. Epub 2015 Aug 11.

From the Molecular Microbiology Department and the School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom

The widespread second messenger molecule cyclic di-GMP (cdG) regulates the transition from motile and virulent lifestyles to sessile, biofilm-forming ones in a wide range of bacteria. Many pathogenic and commensal bacterial-host interactions are known to be controlled by cdG signaling. Although the biochemistry of cyclic dinucleotide metabolism is well understood, much remains to be discovered about the downstream signaling pathways that induce bacterial responses upon cdG binding. As part of our ongoing research into the role of cdG signaling in plant-associated Pseudomonas species, we carried out an affinity capture screen for cdG binding proteins in the model organism Pseudomonas fluorescens SBW25. The flagella export AAA+ ATPase FliI was identified as a result of this screen and subsequently shown to bind specifically to the cdG molecule, with a KD in the low micromolar range. The interaction between FliI and cdG appears to be very widespread. In addition to FliI homologs from diverse bacterial species, high affinity binding was also observed for the type III secretion system homolog HrcN and the type VI ATPase ClpB2. The addition of cdG was shown to inhibit FliI and HrcN ATPase activity in vitro. Finally, a combination of site-specific mutagenesis, mass spectrometry, and in silico analysis was used to predict that cdG binds to FliI in a pocket of highly conserved residues at the interface between two FliI subunits. Our results suggest a novel, fundamental role for cdG in controlling the function of multiple important bacterial export pathways, through direct allosteric control of export ATPase proteins.
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http://dx.doi.org/10.1074/jbc.M115.661439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4591828PMC
October 2015

Crystal structure of a novel two domain GH78 family α-rhamnosidase from Klebsiella oxytoca with rhamnose bound.

Proteins 2015 Sep 6;83(9):1742-9. Epub 2015 Aug 6.

Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich, Nr4 7UH, United Kingdom.

The crystal structure of the GH78 family α-rhamnosidase from Klebsiella oxytoca (KoRha) has been determined at 2.7 Å resolution with rhamnose bound in the active site of the catalytic domain. Curiously, the putative catalytic acid, Asp 222, is preceded by an unusual non-proline cis-peptide bond which helps to project the carboxyl group into the active centre. This KoRha homodimeric structure is significantly smaller than those of the other previously determined GH78 structures. Nevertheless, the enzyme displays α-rhamnosidase activity when assayed in vitro, suggesting that the additional structural domains found in the related enzymes are dispensible for function.
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http://dx.doi.org/10.1002/prot.24807DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4690510PMC
September 2015

Structural insight into how Streptomyces coelicolor maltosyl transferase GlgE binds α-maltose 1-phosphate and forms a maltosyl-enzyme intermediate.

Biochemistry 2014 Apr 11;53(15):2494-504. Epub 2014 Apr 11.

Department of Biological Chemistry, John Innes Centre, Norwich Research Park , Norwich NR4 7UH, United Kingdom.

GlgE (EC 2.4.99.16) is an α-maltose 1-phosphate:(1→4)-α-d-glucan 4-α-d-maltosyltransferase of the CAZy glycoside hydrolase 13_3 family. It is the defining enzyme of a bacterial α-glucan biosynthetic pathway and is a genetically validated anti-tuberculosis target. It catalyzes the α-retaining transfer of maltosyl units from α-maltose 1-phosphate to maltooligosaccharides and is predicted to use a double-displacement mechanism. Evidence of this mechanism was obtained using a combination of site-directed mutagenesis of Streptomyces coelicolor GlgE isoform I, substrate analogues, protein crystallography, and mass spectrometry. The X-ray structures of α-maltose 1-phosphate bound to a D394A mutein and a β-2-deoxy-2-fluoromaltosyl-enzyme intermediate with a E423A mutein were determined. There are few examples of CAZy glycoside hydrolase family 13 members that have had their glycosyl-enzyme intermediate structures determined, and none before now have been obtained with a 2-deoxy-2-fluoro substrate analogue. The covalent modification of Asp394 was confirmed using mass spectrometry. A similar modification of wild-type GlgE proteins from S. coelicolor and Mycobacterium tuberculosis was also observed. Small-angle X-ray scattering of the M. tuberculosis enzyme revealed a homodimeric assembly similar to that of the S. coelicolor enzyme but with slightly differently oriented monomers. The deeper understanding of the structure-function relationships of S. coelicolor GlgE will aid the development of inhibitors of the M. tuberculosis enzyme.
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http://dx.doi.org/10.1021/bi500183cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4048318PMC
April 2014