Publications by authors named "David M Lawson"

96 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

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

Requirements for the Packaging of Geminivirus Circular Single-Stranded DNA: Effect of DNA Length and Coat Protein Sequence.

Viruses 2020 10 30;12(11). Epub 2020 Oct 30.

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

Geminivirus particles, consisting of a pair of twinned isometric structures, have one of the most distinctive capsids in the virological world. Until recently, there was little information as to how these structures are generated. To address this, we developed a system to produce capsid structures following the delivery of geminivirus coat protein and replicating circular single-stranded DNA (cssDNA) by the infiltration of gene constructs into plant leaves. The transencapsidation of cssDNA of the genus by coat protein of different geminivirus genera was shown to occur with full-length but not half-length molecules. Double capsid structures, distinct from geminate capsid structures, were also generated in this expression system. By increasing the length of the encapsidated cssDNA, triple geminate capsid structures, consisting of straight, bent and condensed forms were generated. The straight geminate triple structures generated were similar in morphology to those recorded for a potato-infecting virus from Peru. These finding demonstrate that the length of encapsidated DNA controls both the size and stability of geminivirus particles.
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http://dx.doi.org/10.3390/v12111235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7694086PMC
October 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

Diversification of DNA-Binding Specificity by Permissive and Specificity-Switching Mutations in the ParB/Noc Protein Family.

Cell Rep 2020 07;32(3):107928

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

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.
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http://dx.doi.org/10.1016/j.celrep.2020.107928DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7383237PMC
July 2020

Considerations of Dissociation, Betrayal Trauma, and Complex Trauma in the Treatment of Incest.

J Child Sex Abus 2020 Aug-Sep;29(6):677-696. Epub 2020 Jun 10.

Department of Counselor Education, Sam Houston State University , Huntsville, TX.

Child sexual abuse committed by a parent (incest) is related to particularly severe physical and psychological symptoms across the life span. Incest is associated with low self-esteem, self-loathing, feelings of contamination, worthlessness, and helplessness, as well as somatization and low self-efficacy. A child's negative constructions often are attempts to derive some meaning that justifies the incest, such as, "it is because of my badness that it happens to me." Survival often involves voluntary or involuntary disconnection from self, others, and the environment, or compartmentalization of the traumatic experiences. Dissociation with survivors of child abuse, especially when the perpetrators are from within the child's caregiver system, can be accounted for by the concept of betrayal trauma. However, with few exceptions, little appears in the literature integrating dissociation, betrayal trauma, complex trauma, and incest for the purpose of treatment. Our purpose of this review to examine the relationship between trauma-related dissociation, betrayal trauma, and complex trauma, and how understanding these concepts and their relationship can inform the treatment of incest.
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http://dx.doi.org/10.1080/10538712.2020.1751369DOI Listing
April 2021

Insecure Attachment and Therapeutic Bond as Mediators of Social, Relational, and Social Distress and Interpersonal Problems in Adult Females with Childhood Sexual Abuse History.

J Child Sex Abus 2020 Aug-Sep;29(6):659-676. Epub 2020 May 15.

Sam Houston State University , Huntsville, TX, USA.

Establishing trust is an important part of building the therapeutic relationships and achieving the goal of effective trauma treatment for individuals who have experienced childhood sexual abuse. The current study explored the associations between attachment style, therapeutic bond, distress, and interpersonal problems. This study investigated whether attachment style and therapeutic bond mediated the association between the level of early treatment emotional distress and later treatment interpersonal problems among two groups: clients reporting histories of childhood sexual abuse and clients not reporting histories of childhood sexual abuse. Research indicates that disruption of attachment security as well as the therapeutic relationship is common in survivors of childhood sexual abuse. We explored the mediating role of insecure attachment and the therapeutic bond on the predictive relationship between early treatment emotional distress and the interpersonal difficulties that one experiences in their daily life. For clients with histories of child sexual abuse, the model showed that anxious attachment and avoidant attachment mediated the associations between emotional distress and interpersonal relations. Therapeutic bond was not a significant mediator. For clients without histories of sexual abuse, results showed significant association between emotional distress and interpersonal relations, but insecure attachment or therapeutic bond did not mediate this relationship.
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http://dx.doi.org/10.1080/10538712.2020.1751368DOI Listing
April 2021

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

Structural insights into simocyclinone as an antibiotic, effector ligand and substrate.

FEMS Microbiol Rev 2018 01;42(1)

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

Simocyclinones are antibiotics produced by Streptomyces and Kitasatospora species that inhibit the validated drug target DNA gyrase in a unique way, and they are thus of therapeutic interest. Structural approaches have revealed their mode of action, the inducible-efflux mechanism in the producing organism, and given insight into one step in their biosynthesis. The crystal structures of simocyclinones bound to their target (gyrase), the transcriptional repressor SimR and the biosynthetic enzyme SimC7 reveal fascinating insight into how molecular recognition is achieved with these three unrelated proteins.
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http://dx.doi.org/10.1093/femsre/fux055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5812520PMC
January 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

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

Eliminating anti-nutritional plant food proteins: the case of seed protease inhibitors in pea.

PLoS One 2015 12;10(8):e0134634. Epub 2015 Aug 12.

Department of Metabolic Biology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom.

Several classes of seed proteins limit the utilisation of plant proteins in human and farm animal diets, while plant foods have much to offer to the sustainable intensification of food/feed production and to human health. Reduction or removal of these proteins could greatly enhance seed protein quality and various strategies have been used to try to achieve this with limited success. We investigated whether seed protease inhibitor mutations could be exploited to enhance seed quality, availing of induced mutant and natural Pisum germplasm collections to identify mutants, whilst acquiring an understanding of the impact of mutations on activity. A mutant (TILLING) resource developed in Pisum sativum L. (pea) and a large germplasm collection representing Pisum diversity were investigated as sources of mutations that reduce or abolish the activity of the major protease inhibitor (Bowman-Birk) class of seed protein. Of three missense mutations, predicted to affect activity of the mature trypsin / chymotrypsin inhibitor TI1 protein, a C77Y substitution in the mature mutant inhibitor abolished inhibitor activity, consistent with an absolute requirement for the disulphide bond C77-C92 for function in the native inhibitor. Two further classes of mutation (S85F, E109K) resulted in less dramatic changes to isoform or overall inhibitory activity. The alternative strategy to reduce anti-nutrients, by targeted screening of Pisum germplasm, successfully identified a single accession (Pisum elatius) as a double null mutant for the two closely linked genes encoding the TI1 and TI2 seed protease inhibitors. The P. elatius mutant has extremely low seed protease inhibitory activity and introgression of the mutation into cultivated germplasm has been achieved. The study provides new insights into structure-function relationships for protease inhibitors which impact on pea seed quality. The induced and natural germplasm variants identified provide immediate potential for either halving or abolishing the corresponding inhibitory activity, along with associated molecular markers for breeding programmes. The potential for making large changes to plant protein profiles for improved and sustainable food production through diversity is illustrated. The strategy employed here to reduce anti-nutritional proteins in seeds may be extended to allergens and other seed proteins with negative nutritional effects. Additionally, the novel variants described for pea will assist future studies of the biological role and health-related properties of so-called anti-nutrients.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0134634PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4534040PMC
May 2016

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