36 results on '"Dunstan, Mark S."'
Search Results
2. An automated pipeline for the screening of diverse monoterpene synthase libraries
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Leferink, Nicole G. H., Dunstan, Mark S., Hollywood, Katherine A., Swainston, Neil, Currin, Andrew, Jervis, Adrian J., Takano, Eriko, and Scrutton, Nigel S.
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- 2019
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3. The transcriptional regulator CprK detects chlorination by combining direct and indirect readout mechanisms
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Kemp, Laura R., Dunstan, Mark S., Fisher, Karl, Warwicker, Jim, and Leys, David
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- 2013
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4. Reductive dehalogenase structure suggests a mechanism for B12-dependent dehalogenation
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Payne, Karl A.P., Quezada, Carolina P., Fisher, Karl, Dunstan, Mark S., Collins, Fraser A., Sjuts, Hanno, Levy, Colin, Hay, Sam, Rigby, Stephen E.J., and Leys, David
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Catalysis -- Analysis ,Polychlorinated biphenyls -- Structure ,Halogens -- Structure ,Enzymes -- Structure -- Physiological aspects ,Environmental issues ,Science and technology ,Zoology and wildlife conservation - Abstract
Organohalide chemistry underpins many industrial and agricultural processes, and a large proportion of environmental pollutants are organohalides (1). Nevertheless, organohalide chemistry is not exclusively of anthropogenic origin, with natural abiotic and biological processes contributing to the global halide cycle (2, 3). Reductive dehalogenases are responsible for biological dehalogenation in organohalide respiring bacteria (4, 5), with substrates including polychlorinated biphenyls or dioxins (6, 7). Reductive dehalogenases form a distinct subfamily of cobalamin (B12)-dependent enzymes that are usually membrane associated and oxygen sensitive, hindering detailed studies (8, 12). Here we report the characterization of a soluble, oxygen-tolerant reductive dehalogenase and, by combining structure determination with EPR (electron paramagnetic resonance) spectroscopy and simulation, show that a direct interaction between the cobalamin cobalt and the substrate halogen underpins catalysis. In contrast to the carbon-cobalt bond chemistry catalysed by the other cobalamin-dependent subfamilies (13), we propose that reductive dehalogenases achieve reduction of the organohalide substrate via halogen-cobalt bond formation. This presents a new model in both organohalide and cobalamin (bio)chemistry that will guide future exploitation of these enzymes in bioremediation or biocatalysis., Cobalamin consists of a cobalt ion coordinated by a tetrapyrrole-derived macrocycle and readily forms a weak organometallic C-Co(III) bond (14). This bond can be homolytically or heterolytically cleaved and the [...]
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- 2015
5. Reengineering Orthogonally Selective Riboswitches
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Dixon, Neil, Duncan, John N., Geerlings, Torsten, Dunstan, Mark S., McCarthy, John E. G., Leys, David, Micklefield, Jason, and Patel, Dinshaw J.
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- 2010
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6. The structure and catalytic mechanism of a poly(ADP-ribose) glycohydrolase
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Slade, Dea, Dunstan, Mark S., Barkauskaite, Eva, Weston, Ria, Lafite, Pierre, Dixon, Neil, Ahel, Marijan, Leys, David, and Ahel, Ivan
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Catalysis -- Research ,Hydrolases -- Physiological aspects -- Research ,Enzymes -- Physiological aspects -- Research ,Microbial metabolism -- Research ,Environmental issues ,Science and technology ,Zoology and wildlife conservation - Abstract
Post-translational modification of proteins by poly(ADP-ribosyl) ation regulates many cellular pathways that are critical for genome stability, including DNA repair, chromatin structure, mitosis and apoptosis (1). Poly(ADP-ribose) (PAR) is composed of repeating ADP-ribose units linked via a unique glycosidic ribose-ribose bond, and is synthesized from NAD by PAR polymerases (1,2). PAR glycohydrolase (PARG) is the only protein capable of specific hydrolysis of the ribose-ribose bonds present in PAR chains; its deficiency leads to cell death (3,4). Here we show that filamentous fungi and a number of bacteria possess a divergent form of PARG that has all the main characteristics of the human PARG enzyme. We present the first PARG crystal structure (derived from the bacterium Thermomonospora curvata), which reveals that the PARG catalytic domain is a distant member of the ubiquitous ADP-ribose-binding macrodomain family (5,6). High-resolution structures of T. curvata PARG in complexes with ADP-ribose and the PARG inhibitor ADP-HPD, complemented by biochemical studies, allow us to propose a model for PAR binding and catalysis by PARG. The insights into the PARG structure and catalytic mechanism should greatly improve our understanding of how PARG activity controls reversible protein poly(ADP-ribosyl) ation and potentially of how the defects in this regulation are linked to human disease., Whereas the mechanism and functional aspects of PAR synthesis have been extensively characterized, the present understanding of the PAR-degradation pathways catalysed by PARG is comparatively poor. Until now, the catalytic [...]
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- 2011
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7. Prototyping of microbial chassis for the biomanufacturing of high-value chemical targets.
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Robinson, Christopher J., Tellechea-Luzardo, Jonathan, Carbonell, Pablo, Jervis, Adrian J., Cunyu Yan, Hollywood, Katherine A., Dunstan, Mark S., Currin, Andrew, Takano, Eriko, and Scrutton, Nigel S.
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INDUSTRIAL engineering ,INDUSTRIAL engineers ,EXPERIMENTAL design ,RAPID prototyping - Abstract
Metabolic engineering technologies have been employed with increasing success over the last three decades for the engineering and optimization of industrial host strains to competitively produce high-value chemical targets. To this end, continued reductions in the time taken from concept, to development, to scale-up are essential. Design-Build-Test-Learn pipelines that are able to rapidly deliver diverse chemical targets through iterative optimization of microbial production strains have been established. Biofoundries are employing in silico tools for the design of genetic parts, alongside combinatorial design of experiments approaches to optimize selection from within the potential design space of biological circuits based on multi-criteria objectives. These genetic constructs can then be built and tested through automated laboratory workflows, with performance data analysed in the learn phase to inform further design. Successful examples of rapid prototyping processes for microbially produced compounds reveal the potential role of biofoundries in leading the sustainable production of next-generation bio-based chemicals. [ABSTRACT FROM AUTHOR]
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- 2021
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8. A plasmid toolset for CRISPR‐mediated genome editing and CRISPRi gene regulation in Escherichia coli.
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Jervis, Adrian J., Hanko, Erik K.R., Dunstan, Mark S., Robinson, Christopher J., Takano, Eriko, and Scrutton, Nigel S.
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GENETIC regulation ,CRISPRS ,ESCHERICHIA coli ,GENOME editing ,GENOMES ,DNA - Abstract
Summary: CRISPR technologies have become standard laboratory tools for genetic manipulations across all kingdoms of life. Despite their origins in bacteria, the development of CRISPR tools for engineering bacteria has been slower than for eukaryotes; nevertheless, their function and application for genome engineering and gene regulation via CRISPR interference (CRISPRi) has been demonstrated in various bacteria, and adoption has become more widespread. Here, we provide simple plasmid‐based systems for genome editing (gene knockouts/knock‐ins, and genome integration of large DNA fragments) and CRISPRi in E. coli using a CRISPR‐Cas12a system. The described genome engineering protocols allow markerless deletion or genome integration in just seven working days with high efficiency (> 80% and 50%, respectively), and the CRISPRi protocols allow robust transcriptional repression of target genes (> 90%) with a single cloning step. The presented minimized plasmids and their associated design and experimental protocols provide efficient and effective CRISPR‐Cas12 genome editing, genome integration and CRISPRi implementation. These simple‐to‐use systems and protocols will allow the easy adoption of CRISPR technology by any laboratory. [ABSTRACT FROM AUTHOR]
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- 2021
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9. Engineering Escherichia coli towards de novo production of gatekeeper (2S)-flavanones: naringenin, pinocembrin, eriodictyol and homoeriodictyol.
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Dunstan, Mark S, Robinson, Christopher J, Jervis, Adrian J, Yan, Cunyu, Carbonell, Pablo, Hollywood, Katherine A, Currin, Andrew, Swainston, Neil, Feuvre, Rosalind Le, Micklefield, Jason, Faulon, Jean-Loup, Breitling, Rainer, Turner, Nicholas, Takano, Eriko, and Scrutton, Nigel S
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ESCHERICHIA coli , *FLAVANONES , *DIETARY supplements , *GLYCERIN , *ENZYMES - Abstract
Natural plant-based flavonoids have drawn significant attention as dietary supplements due to their potential health benefits, including anti-cancer, anti-oxidant and anti-asthmatic activities. Naringenin, pinocembrin, eriodictyol and homoeriodictyol are classified as (2 S)-flavanones, an important sub-group of naturally occurring flavonoids, with wide-reaching applications in human health and nutrition. These four compounds occupy a central position as branch point intermediates towards a broad spectrum of naturally occurring flavonoids. Here, we report the development of Escherichia coli production chassis for each of these key gatekeeper flavonoids. Selection of key enzymes, genetic construct design and the optimization of process conditions resulted in the highest reported titers for naringenin (484 mg/l), improved production of pinocembrin (198 mg/l) and eriodictyol (55 mg/l from caffeic acid), and provided the first example of in vivo production of homoeriodictyol directly from glycerol (17 mg/l). This work provides a springboard for future production of diverse downstream natural and non-natural flavonoid targets. [ABSTRACT FROM AUTHOR]
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- 2020
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10. SelProm: A Queryable and Predictive Expression Vector Selection Tool for Escherichia coli.
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Jervis, Adrian J., Carbonell, Pablo, Taylor, Sandra, Sung, Rehana, Dunstan, Mark S., Robinson, Christopher J., Breitling, Rainer, Takano, Eriko, and Scrutton, Nigel S.
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- 2019
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11. Machine Learning of Designed Translational Control Allows Predictive Pathway Optimization in Escherichia coli.
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Jervis, Adrian J., Carbonell, Pablo, Vinaixa, Maria, Dunstan, Mark S., Hollywood, Katherine A., Robinson, Christopher J., Rattray, Nicholas J. W., Cunyu Yan, Neil Swainston, Currin, Andrew, Sung, Rehana, Toogood, Helen, Taylor, Sandra, Faulon, Jean-Loup, Breitling, Rainer, Takano, Eriko, and Scrutton, Nigel S.
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- 2019
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12. Structure and Biocatalytic Scope of Coclaurine N‐Methyltransferase.
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Bennett, Matthew R., Thompson, Mark L., Shepherd, Sarah A., Dunstan, Mark S., Herbert, Abigail J., Smith, Duncan R. M., Cronin, Victoria A., Menon, Binuraj R. K., Levy, Colin, and Micklefield, Jason
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METHYLTRANSFERASES ,BIOCATALYSIS ,CRYSTAL structure ,TRANSFERASES ,CATALYSIS - Abstract
Abstract: Benzylisoquinoline alkaloids (BIAs) are a structurally diverse family of plant secondary metabolites, which have been exploited to develop analgesics, antibiotics, antitumor agents, and other therapeutic agents. Biosynthesis of BIAs proceeds via a common pathway from tyrosine to (S)‐reticulene at which point the pathway diverges. Coclaurine N‐methyltransferase (CNMT) is a key enzyme in the pathway to (S)‐reticulene, installing the N‐methyl substituent that is essential for the bioactivity of many BIAs. In this paper, we describe the first crystal structure of CNMT which, along with mutagenesis studies, defines the enzymes active site architecture. The specificity of CNMT was also explored with a range of natural and synthetic substrates as well as co‐factor analogues. Knowledge from this study could be used to generate improved CNMT variants required to produce BIAs or synthetic derivatives. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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13. Adenylation Activity of Carboxylic Acid Reductases Enables the Synthesis of Amides.
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Wood, Alexander J. L., Weise, Nicholas J., Frampton, Joseph D., Dunstan, Mark S., Hollas, Michael A., Derrington, Sasha R., Lloyd, Richard C., Quaglia, Daniela, Parmeggiani, Fabio, Leys, David, Turner, Nicholas J., and Flitsch, Sabine L.
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CARBOXYLIC acids ,AMIDE synthesis ,ADENYLATION (Biochemistry) ,ADENOSINE triphosphate ,NICOTINAMIDE adenine dinucleotide phosphate ,BIOCATALYSIS - Abstract
Carboxylic acid reductases (CARs) catalyze the reduction of a broad range of carboxylic acids to aldehydes using the cofactors adenosine triphosphate and nicotinamide adenine dinucleotide phosphate, and have become attractive biocatalysts for organic synthesis. Mechanistic understanding of CARs was used to expand reaction scope, generating biocatalysts for amide bond formation from carboxylic acid and amine. CARs demonstrated amidation activity for various acids and amines. Optimization of reaction conditions, with respect to pH and temperature, allowed for the synthesis of the anticonvulsant ilepcimide with up to 96 % conversion. Mechanistic studies using site-directed mutagenesis suggest that, following initial enzymatic adenylation of substrates, amidation of the carboxylic acid proceeds by direct reaction of the acyl adenylate with amine nucleophiles. [ABSTRACT FROM AUTHOR]
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- 2017
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14. Structure and biocatalytic scope of thermophilic flavin-dependent halogenase and flavin reductase enzymes.
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Menon, Binuraj R. K., Latham, Jonathan, Dunstan, Mark S., Brandenburger, Eileen, Klemstein, Ulrike, Leys, David, Karthikeyan, Chinnan, Greaney, Michael F., Shepherd, Sarah A., and Micklefield, Jason
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- 2016
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15. Single-Biocatalyst Synthesis of Enantiopure d-Arylalanines Exploiting an Engineered d-Amino Acid Dehydrogenase.
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Parmeggiani, Fabio, Ahmed, Syed T., Thompson, Matthew P., Weise, Nicholas J., Galman, James L., Gahloth, Deepankar, Dunstan, Mark S., Leys, David, and Turner, Nicholas J.
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BIOCATALYSIS ,AMINO acids ,DEHYDROGENASES ,RACEMIC mixtures ,DEAMINASES ,ASYMMETRIC synthesis ,OXIDOREDUCTASES - Abstract
A practical and efficient biocatalytic synthesis of aromatic d-amino acids has been developed, based on the reductive amination of the corresponding α-keto acids via a recombinant whole cell system composed of an engineered dehydrogenase and cofactor recycling apparatus. The reaction was shown to give excellent enantioselectivity (≥98%) and good yields at the preparative scale across a broad range of substrates. Additionally, the structure of the variant enzyme was solved to allow rationalisation of the observed reaction rates. The engineered whole cell catalyst was also used to mediate the production of d-phenylalanine derivatives from racemic mixtures and cheaper l-amino acids by combining it with an enantiocomplementary deaminase. [ABSTRACT FROM AUTHOR]
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- 2016
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16. Structures of the methyltransferase component of Desulfitobacterium hafniense DCB-2 O-demethylase shed light on methyltetrahydrofolate formation.
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Sjuts, Hanno, Dunstan, Mark S., Fisher, Karl, and Leys, David
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METHYLTRANSFERASES , *DEMETHYLASE , *METHYL ether , *VITAMIN B12 , *PROTON transfer reactions - Abstract
O-Demethylation by acetogenic or organohalide-respiring bacteria leads to the formation of methyltetrahydrofolate from aromatic methyl ethers. O-Demethylases, which are cobalamin-dependent, three-component enzyme systems, catalyse methyl-group transfers from aromatic methyl ethers to tetrahydrofolate via methylcobalamin intermediates. In this study, crystal structures of the tetrahydrofolate-binding methyltransferase module from a Desulfitobacterium hafniense DCB-2 O-demethylase were determined both in complex with tetrahydrofolate and the product methyltetrahydrofolate. While these structures are similar to previously determined methyltransferase structures, the position of key active-site residues is subtly altered. A strictly conserved Asn is displaced to establish a putative proton-transfer network between the substrate N5 and solvent. It is proposed that this supports the efficient catalysis of methyltetrahydrofolate formation, which is necessary for efficient O-demethylation. [ABSTRACT FROM AUTHOR]
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- 2015
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17. Modular Riboswitch Toolsets for Synthetic Genetic Control in Diverse Bacterial Species.
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Robinson, Christopher J., Vincent, Helen A., Ming-Cheng Wu, Lowe, Phillip T., Dunstan, Mark S., Leys, David, and Micklefield, Jason
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- 2014
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18. Structure of the cobalamin-binding protein of a putative O-demethylase from Desulfitobacterium hafniense DCB-2.
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Sjuts, Hanno, Dunstan, Mark S., Fisher, Karl, and Leys, David
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STRUCTURAL analysis (Science) , *VITAMIN B12 , *N-terminal residues , *C-terminal residues , *ELECTRON paramagnetic resonance spectroscopy , *METHYLTRANSFERASES - Abstract
This study describes the identification and the structural and spectroscopic analysis of a cobalamin-binding protein (termed CobDH) implicated in O-demethylation by the organohalide-respiring bacterium Desulfitobacterium hafniense DCB-2. The 1.5 Å resolution crystal structure of CobDH is presented in the cobalamin-bound state and reveals that the protein is composed of an N-terminal helix-bundle domain and a C-terminal Rossmann-fold domain, with the cobalamin coordinated in the base-off/His-on conformation similar to other cobalamin-binding domains that catalyse methyl-transfer reactions. EPR spectroscopy of CobDH confirms cobalamin binding and reveals the presence of a cob(III)alamin superoxide, indicating binding of oxygen to the fully oxidized cofactor. These data provide the first structural insights into the methyltransferase reactions that occur during O-demethylation by D. hafniense. [ABSTRACT FROM AUTHOR]
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- 2013
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19. Novel Inhibitors of NRH:Quinone Oxidoreductase 2 (NQO2): Crystal Structures, Biochemical Activity, and Intracellular Effects of Imidazoacridin-6-ones.
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Dunstan, Mark S., Barnes, John, Humphries, Matthew, Whitehead, Roger C., Bryce, Richard A., Leys, David, Stratford, Ian J., and Nolan, Karen A.
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QUINONE , *MOLECULAR structure , *BIOCHEMISTRY , *IMIDAZOPYRIDINES , *ENZYME inhibitors , *MOLECULAR models , *QSAR models , *CANCER cells , *CARRIER proteins - Abstract
Imidazoacridin-6-ones are shown to be potent nanomolar inhibitors of the enzyme NQO2. By use of computational molecular modeling, a reliable QSAR was established, relating inhibitory potency with calculated binding affinity. Further, crystal structures of NQO2 containing two of the imidazoacridin-6-ones have been solved. To generate compounds with reduced off-target (DNA binding) effects, an N-oxide moiety was introduced into the tertiary aminoalkyl side chain of the imidazoacridin-6-ones. This resulted in substantially less toxicity in a panel of eight cancer cell lines, decreased protein binding, and reduced DNA binding and nuclear accumulation. Finally, one of the N-oxides showed potent ability to inhibit the enzymatic function of NQO2 in cells, and therefore, it may be useful as a pharmacological probe to study the properties of the enzyme in vitro and in vivo. [ABSTRACT FROM AUTHOR]
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- 2011
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20. Structure of the Thiostrepton Resistance Methyltransferase•S-Adenosyl-L-methionine Complex and Its Interaction with Ribosomal RNA.
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Dunstan, Mark S., Hang, Pei C., ZeIinskaya, Natalia V., Honek, John F., and Conn, Graeme L.
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METHYLTRANSFERASES , *RIBOSOMAL DNA , *ADENOSYLMETHIONINE , *NUCLEOTIDES , *RNA synthesis , *PROTEIN binding , *ENZYME regulation , *X-rays - Abstract
The x-ray crystal structure of the thiostrepton resistance RNA methyltransferase (Tsr)∙S-adenosy1-L-methionine (AdoMet) complex was determined at 2.45-Å resolution. Tsr is definitively confirmed as a Class IV methyltransferase of the SpoU family with an N-terminal "L30-like" putative target recognition domain. The structure and our in vitro analysis of the interaction of Tsr with its target domain from 23 S ribosomal RNA (rRNA) demonstrate that the active biological unit is a Tsr homodimer. In vitro methylation assays show that Tsr activity is optimal against a 29-nucleotide hairpin rRNA though the full 58-nucleotide Lu-binding domain and intact 23 S rRNA are also effective substrates. Molecular docking experiments predict that TsrrRNA binding is dictated entirely by the sequence and structure of the rRNA hairpin containing the A1067 target nucleotide and is most likely driven primarily by large complementary electrostatic surfaces. One L30-like domain is predicted to bind the target loop and the other is near an internal loop more distant from the target site where a nucleotide change (U1061 to A) also decreases methylation by Tsr. Furthermore, a predicted interaction with this internal loop by Tsr amino acid Phe-88 was confirmed by mutagenesis and RNA binding experiments. We therefore propose that Tsr achieves its absolute target specificity using the N-terminal domains of each monomer in combination to recognize the two distinct structural elements of the target rRNA hairpin such that both Tsr subunits contribute directly to the positioning of the target nucleotide on the enzyme. [ABSTRACT FROM AUTHOR]
- Published
- 2009
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21. Coevolution of Protein and RNA Structures within a Highly Conserved Ribosomal Domain
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Dunstan, Mark S., GuhaThakurta, Debraj, Draper, David. E., and Conn, Graeme L.
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RNA , *PROTEINS , *RIBOSOMES , *HYDROGEN bonding - Abstract
Summary: The X-ray crystal structure of a ribosomal L11-rRNA complex with chloroplast-like mutations in both protein and rRNA is presented. The global structure is almost identical to that of the wild-type (bacterial) complex, with only a small movement of the protein α helix away from the surface of the RNA required to accommodate the altered protein residue. In contrast, the specific hydrogen bonding pattern of the mutated residues is substantially different, and now includes a direct interaction between the protein side chain and an RNA base edge and a water-mediated contact. Comparison of the two structures allows the observations of sequence variation and relative affinities of wild-type and mutant complexes to be clearly rationalized, but reinforces the concept that there is no single simple code for protein-RNA recognition. [Copyright &y& Elsevier]
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- 2005
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22. Visualization of poly(ADP-ribose) bound to PARG reveals inherent balance between exo- and endo-glycohydrolase activities
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Barkauskaite, Eva, Brassington, Amy, Tan, Edwin S., Warwicker, Jim, Dunstan, Mark S., Banos, Benito, Lafite, Pierre, Ahel, Marijan, Mitchison, Timothy J., Ahel, Ivan, and Leys, David
- Abstract
Poly-ADP-ribosylation is a post-translational modification that regulates processes involved in genome stability. Breakdown of the poly(ADP-ribose) (PAR) polymer is catalysed by poly(ADP-ribose) glycohydrolase (PARG), whose endo-glycohydrolase activity generates PAR fragments. Here we present the crystal structure of PARG incorporating the PAR substrate. The two terminal ADP-ribose units of the polymeric substrate are bound in exo-mode. Biochemical and modelling studies reveal that PARG acts predominantly as an exo-glycohydrolase. This preference is linked to Phe902 (human numbering), which is responsible for low-affinity binding of the substrate in endo-mode. Our data reveal the mechanism of poly-ADP-ribosylation reversal, with ADP-ribose as the dominant product, and suggest that the release of apoptotic PAR fragments occurs at unusual PAR/PARG ratios.
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- 2013
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23. Highly multiplexed, fast and accurate nanopore sequencing for verification of synthetic DNA constructs and sequence libraries.
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Currin, Andrew, Swainston, Neil, Dunstan, Mark S, Jervis, Adrian J, Mulherin, Paul, Robinson, Christopher J, Taylor, Sandra, Carbonell, Pablo, Hollywood, Katherine A, Yan, Cunyu, Takano, Eriko, Scrutton, Nigel S, and Breitling, Rainer
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SINGLE nucleotide polymorphisms ,NANOPORES ,NUCLEOTIDE sequencing ,SYNTHETIC biology ,POLYMERASE chain reaction - Abstract
Synthetic biology utilizes the Design–Build–Test–Learn pipeline for the engineering of biological systems. Typically, this requires the construction of specifically designed, large and complex DNA assemblies. The availability of cheap DNA synthesis and automation enables high-throughput assembly approaches, which generates a heavy demand for DNA sequencing to verify correctly assembled constructs. Next-generation sequencing is ideally positioned to perform this task, however with expensive hardware costs and bespoke data analysis requirements few laboratories utilize this technology in-house. Here a workflow for highly multiplexed sequencing is presented, capable of fast and accurate sequence verification of DNA assemblies using nanopore technology. A novel sample barcoding system using polymerase chain reaction is introduced, and sequencing data are analyzed through a bespoke analysis algorithm. Crucially, this algorithm overcomes the problem of high-error rate nanopore data (which typically prevents identification of single nucleotide variants) through statistical analysis of strand bias, permitting accurate sequence analysis with single-base resolution. As an example, 576 constructs (6 × 96 well plates) were processed in a single workflow in 72 h (from Escherichia coli colonies to analyzed data). Given our procedure's low hardware costs and highly multiplexed capability, this provides cost-effective access to powerful DNA sequencing for any laboratory, with applications beyond synthetic biology including directed evolution, single nucleotide polymorphism analysis and gene synthesis. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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24. Heterologous expression, purification and cofactor reconstitution of the reductive dehalogenase PceA from Dehalobacter restrictus
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Sjuts, Hanno, Fisher, Karl, Dunstan, Mark S., Rigby, Stephen E., and Leys, David
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GENE expression , *ANAEROBIC bacteria , *PROTEINS , *PROTEOLYSIS , *DEHALOGENASES , *ORGANIC compounds , *VITAMIN B12 , *AFFINITY chromatography , *BACTERIA - Abstract
Abstract: Organohalide respiration is used by a limited set of anaerobic bacteria to derive energy from the reduction of halogenated organic compounds. The enzymes that catalyze the reductive dehalogenation reaction, the reductive dehalogenases, represent a novel and distinct class of cobalamin and Fe-S cluster dependent enzymes. Until now, biochemical studies on reductive dehalogenases have been hampered by the lack of a reliable protein source. Here we present an efficient and robust heterologous production system for the reductive dehalogenase PceA from Dehalobacter restrictus. Large quantities of Strep-tagged PceA fused to a cold-shock induced trigger factor could be obtained from Escherichia coli. The recombinant enzyme was conveniently purified in milligram quantities under anaerobic conditions by StrepTactin affinity chromatography, and the trigger factor could be removed through limited proteolysis. Characterization of the purified PceA by UV–Vis and electron paramagnetic resonance (EPR) spectroscopy reveal that the recombinant protein binds methylcobalamin in the base-on form after proteolytic cleavage of the trigger factor, and that 4Fe-4S clusters can be chemically reconstituted under anoxic conditions. This study demonstrates a novel PceA production platform that allows further study of this new enzyme class. [Copyright &y& Elsevier]
- Published
- 2012
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25. Ribosomal Protein L11 Selectively Stabilizes a Tertiary Structure of the GTPase Center rRNA Domain.
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Welty, Robb, Rau, Michael, Pabit, Suzette, Dunstan, Mark S., Conn, Graeme L., Pollack, Lois, and Hall, Kathleen B.
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TERTIARY structure , *GUANOSINE triphosphatase , *RIBOSOMAL RNA , *BACTERIAL RNA , *RIBOSOMAL proteins , *CRYSTAL structure - Abstract
The GTPase Center (GAC) RNA domain in bacterial 23S rRNA is directly bound by ribosomal protein L11, and this complex is essential to ribosome function. Previous cocrystal structures of the 58-nucleotide GAC RNA bound to L11 revealed the intricate tertiary fold of the RNA domain, with one monovalent and several divalent ions located in specific sites within the structure. Here, we report a new crystal structure of the free GAC that is essentially identical to the L11-bound structure, which retains many common sites of divalent ion occupation. This new structure demonstrates that RNA alone folds into its tertiary structure with bound divalent ions. In solution, we find that this tertiary structure is not static, but rather is best described as an ensemble of states. While L11 protein cannot bind to the GAC until the RNA has adopted its tertiary structure, new experimental data show that L11 binds to Mg2+-dependent folded states, which we suggest lie along the folding pathway of the RNA. We propose that L11 stabilizes a specific GAC RNA tertiary state, corresponding to the crystal structure, and that this structure reflects the functionally critical conformation of the rRNA domain in the fully assembled ribosome. Image 1 • GAC RNA structures in the crystal and cocrystals are the same. • L11 stabilizes the functional GAC tertiary fold. • L11 binds RNA during Mg2+-dependent folding trajectory. • L11 prevents misfolding of tertiary structure. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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26. Epoxyqueuosine Reductase Structure Suggests a Mechanism for Cobalamin-dependent tRNA Modification.
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Payne, Karl A. P., Fisher, Karl, Sjuts, Hanno, Dunstan, Mark S., Bellina, Bruno, Johannissen, Linus, Barran, Perdita, Hay, Sam, Rigby, Stephen E. J., and Leys, David
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QUEUOSINE , *REDUCTASES , *VITAMIN B12 , *TRANSFER RNA , *BIOSYNTHESIS , *DEHALOGENASES , *STREPTOCOCCUS thermophilus - Abstract
Queuosine (Q) is a hypermodified RNA base that replaces guanine in the wobble positions of 5'-GUN-3' tRNA molecules. Q is exclusively made by bacteria, and the corresponding queuine base is a micronutrient salvaged by eukaryotic species. The final step in Q biosynthesis is the reduction of the epoxide precursor, epoxyqueuosine, to yield the Q cyclopentene ring. The epoxyqueuosine reductase responsible, QueG, shares distant homology with the cobalamin-dependent reductive dehalogenase (RdhA), however the role played by cobalamin in QueG catalysis has remained elusive.Wereport the solution and structural characterization of Streptococcus thermophilus QueG, revealing the enzyme harbors a redox chain consisting of two [4Fe-4S] clusters and a cob(II)alamin in the base-off form, similar to RdhAs. In contrast to the shared redox chain architecture, the QueG active site shares little homology with RdhA, with the notable exception of a conserved Tyr that is proposed to function as a proton donor during reductive dehalogenation. Docking of an epoxyqueuosine substrate suggests the QueG active site places the substrate cyclopentane moiety in close proximity of the cobalt. Both the Tyr and a conserved Asp are implicated as proton donors to the epoxide leaving group. This suggests that, in contrast to the unusual carbon-halogen bond chemistry catalyzed by RdhAs, QueG acts via Co-C bond formation. Our study establishes the common features of Class III cobalamindependent enzymes, and reveals an unexpected diversity in the reductive chemistry catalyzed by these enzymes. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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27. SpeedyGenesXL: an Automated, High-Throughput Platform for the Preparation of Bespoke Ultralarge Variant Libraries for Directed Evolution.
- Author
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Sadler JC, Swainston N, Dunstan MS, Currin A, and Kell DB
- Subjects
- Gene Library, Directed Molecular Evolution methods, Proteins
- Abstract
Directed evolution of proteins is a highly effective strategy for tailoring biocatalysts to a particular application, and is capable of engineering improvements such as k
cat, thermostability and organic solvent tolerance. It is recognized that large and systematic libraries are required to navigate a protein's vast and rugged sequence landscape effectively, yet their preparation is nontrivial and commercial libraries are extremely costly. To address this, we have developed SpeedyGenesXL, an automated, high-throughput platform for the production of wild-type genes, Boolean OR, combinatorial, or combinatorial-OR-type libraries based on the SpeedyGenes methodology. Together this offers a flexible platform for library synthesis, capable of generating many different bespoke, diverse libraries simultaneously., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)- Published
- 2022
- Full Text
- View/download PDF
28. Structure and Biocatalytic Scope of Coclaurine N-Methyltransferase.
- Author
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Bennett MR, Thompson ML, Shepherd SA, Dunstan MS, Herbert AJ, Smith DRM, Cronin VA, Menon BRK, Levy C, and Micklefield J
- Subjects
- Alkaloids chemistry, Benzylisoquinolines chemistry, Benzylisoquinolines metabolism, Biocatalysis, Catalytic Domain, Coptis enzymology, Crystallography, X-Ray, Kinetics, Methyltransferases chemistry, Methyltransferases genetics, Mutagenesis, Site-Directed, Plant Proteins chemistry, Plant Proteins genetics, Substrate Specificity, Alkaloids biosynthesis, Methyltransferases metabolism, Plant Proteins metabolism
- Abstract
Benzylisoquinoline alkaloids (BIAs) are a structurally diverse family of plant secondary metabolites, which have been exploited to develop analgesics, antibiotics, antitumor agents, and other therapeutic agents. Biosynthesis of BIAs proceeds via a common pathway from tyrosine to (S)-reticulene at which point the pathway diverges. Coclaurine N-methyltransferase (CNMT) is a key enzyme in the pathway to (S)-reticulene, installing the N-methyl substituent that is essential for the bioactivity of many BIAs. In this paper, we describe the first crystal structure of CNMT which, along with mutagenesis studies, defines the enzymes active site architecture. The specificity of CNMT was also explored with a range of natural and synthetic substrates as well as co-factor analogues. Knowledge from this study could be used to generate improved CNMT variants required to produce BIAs or synthetic derivatives., (© 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)
- Published
- 2018
- Full Text
- View/download PDF
29. Engineering the "Missing Link" in Biosynthetic (-)-Menthol Production: Bacterial Isopulegone Isomerase.
- Author
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Currin A, Dunstan MS, Johannissen LO, Hollywood KA, Vinaixa M, Jervis AJ, Swainston N, Rattray NJW, Gardiner JM, Kell DB, Takano E, Toogood HS, and Scrutton NS
- Abstract
The realization of a synthetic biology approach to microbial (1 R ,2 S ,5 R )-( - )-menthol ( 1 ) production relies on the identification of a gene encoding an isopulegone isomerase (IPGI), the only enzyme in the Mentha piperita biosynthetic pathway as yet unidentified. We demonstrate that Δ5-3-ketosteroid isomerase (KSI) from Pseudomonas putida can act as an IPGI, producing ( R )-(+)-pulegone (( R )- 2 ) from (+)- cis -isopulegone ( 3 ). Using a robotics-driven semirational design strategy, we identified a key KSI variant encoding four active site mutations, which confer a 4.3-fold increase in activity over the wild-type enzyme. This was assisted by the generation of crystal structures of four KSI variants, combined with molecular modeling of 3 binding to identify key active site residue targets. The KSI variant was demonstrated to function efficiently within cascade biocatalytic reactions with downstream Mentha enzymes pulegone reductase and (-)-menthone:(-)-menthol reductase to generate 1 from 3 . This study introduces the use of a recombinant IPGI, engineered to function efficiently within a biosynthetic pathway for the production of 1 in microorganisms., Competing Interests: The authors declare the following competing financial interest(s): A.C., H.S.T., and N.S.S. are named inventors on a submitted patent describing the work in this paper.
- Published
- 2018
- Full Text
- View/download PDF
30. Multifragment DNA Assembly of Biochemical Pathways via Automated Ligase Cycling Reaction.
- Author
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Robinson CJ, Dunstan MS, Swainston N, Titchmarsh J, Takano E, Scrutton NS, and Jervis AJ
- Subjects
- Computer Simulation, DNA metabolism, Escherichia coli metabolism, Ligases metabolism, Plasmids metabolism, Synthetic Biology methods, Workflow, Biosynthetic Pathways, DNA genetics, Escherichia coli genetics, Ligases genetics, Plasmids genetics, Software
- Abstract
The microbial production of commodity, fine, and specialty chemicals is a driving force in biotechnology. An essential requirement is to introduce biosynthetic pathways to the target compound(s) into chassis organisms. First suitable enzymes must be selected and characterized, and then genetic pathways must be designed and assembled into suitable expression vectors. The design of these pathways is crucial for balancing the pathway for efficient in vivo activity. This can be achieved through optimization of the pathway regulation by altering transcription and translation rates. The possible permutations of a multigene pathway create a vast design space which is intractable to explore using traditional time-consuming and laborious pathway assembly methods. The advent of multifragment DNA assembly technologies has enabled simultaneous, multiplexed pathway construction allowing an increased capability to sample the design space. Furthermore, the implementation of laboratory automation allows error-reduced, high-throughput (HTP) construction of pathways. In this chapter, we present a workflow that combines automated in silico design of DNA parts followed by pathway assembly using the ligase cycling reaction on robotics platforms, to allow multiplexed assembly of plasmid-borne gene pathways with high efficiency. Details and considerations in designing DNA parts for expression bacterial chassis are discussed followed by laboratory protocols for HTP pathway assembly and screening using robotics platforms. This workflow is employed in the SYNBIOCHEM Synthetic Biology Research Center, providing the capability to assemble over 96 plasmids simultaneously, with over 40% of clones from each assembly harboring the correctly assembled plasmids. This workflow is easy to modify for use in other laboratories and will help to accelerate synthetic biology projects with diverse applications., (© 2018 Elsevier Inc. All rights reserved.)
- Published
- 2018
- Full Text
- View/download PDF
31. Adenylation Activity of Carboxylic Acid Reductases Enables the Synthesis of Amides.
- Author
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Wood AJL, Weise NJ, Frampton JD, Dunstan MS, Hollas MA, Derrington SR, Lloyd RC, Quaglia D, Parmeggiani F, Leys D, Turner NJ, and Flitsch SL
- Abstract
Carboxylic acid reductases (CARs) catalyze the reduction of a broad range of carboxylic acids to aldehydes using the cofactors adenosine triphosphate and nicotinamide adenine dinucleotide phosphate, and have become attractive biocatalysts for organic synthesis. Mechanistic understanding of CARs was used to expand reaction scope, generating biocatalysts for amide bond formation from carboxylic acid and amine. CARs demonstrated amidation activity for various acids and amines. Optimization of reaction conditions, with respect to pH and temperature, allowed for the synthesis of the anticonvulsant ilepcimide with up to 96 % conversion. Mechanistic studies using site-directed mutagenesis suggest that, following initial enzymatic adenylation of substrates, amidation of the carboxylic acid proceeds by direct reaction of the acyl adenylate with amine nucleophiles., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2017
- Full Text
- View/download PDF
32. Zymophore identification enables the discovery of novel phenylalanine ammonia lyase enzymes.
- Author
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Weise NJ, Ahmed ST, Parmeggiani F, Galman JL, Dunstan MS, Charnock SJ, Leys D, and Turner NJ
- Subjects
- Amino Acid Sequence, Bacterial Proteins genetics, Bacterial Proteins metabolism, Biocatalysis, Drug Discovery, Enzyme Stability, Phenylalanine Ammonia-Lyase chemistry, Protein Conformation, Sequence Analysis methods, Structure-Activity Relationship, Phenylalanine Ammonia-Lyase genetics, Phenylalanine Ammonia-Lyase metabolism
- Abstract
The suite of biological catalysts found in Nature has the potential to contribute immensely to scientific advancements, ranging from industrial biotechnology to innovations in bioenergy and medical intervention. The endeavour to obtain a catalyst of choice is, however, wrought with challenges. Herein we report the design of a structure-based annotation system for the identification of functionally similar enzymes from diverse sequence backgrounds. Focusing on an enzymatic activity with demonstrated synthetic and therapeutic relevance, five new phenylalanine ammonia lyase (PAL) enzymes were discovered and characterised with respect to their potential applications. The variation and novelty of various desirable traits seen in these previously uncharacterised enzymes demonstrates the importance of effective sequence annotation in unlocking the potential diversity that Nature provides in the search for tailored biological tools. This new method has commercial relevance as a strategy for assaying the 'evolvability' of certain enzyme features, thus streamlining and informing protein engineering efforts.
- Published
- 2017
- Full Text
- View/download PDF
33. Structures of carboxylic acid reductase reveal domain dynamics underlying catalysis.
- Author
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Gahloth D, Dunstan MS, Quaglia D, Klumbys E, Lockhart-Cairns MP, Hill AM, Derrington SR, Scrutton NS, Turner NJ, and Leys D
- Subjects
- Models, Molecular, Molecular Structure, Substrate Specificity, Catalytic Domain physiology, Oxidoreductases chemistry
- Abstract
Carboxylic acid reductase (CAR) catalyzes the ATP- and NADPH-dependent reduction of carboxylic acids to the corresponding aldehydes. The enzyme is related to the nonribosomal peptide synthetases, consisting of an adenylation domain fused via a peptidyl carrier protein (PCP) to a reductase termination domain. Crystal structures of the CAR adenylation-PCP didomain demonstrate that large-scale domain motions occur between the adenylation and thiolation states. Crystal structures of the PCP-reductase didomain reveal that phosphopantetheine binding alters the orientation of a key Asp, resulting in a productive orientation of the bound nicotinamide. This ensures that further reduction of the aldehyde product does not occur. Combining crystallography with small-angle X-ray scattering (SAXS), we propose that molecular interactions between initiation and termination domains are limited to competing PCP docking sites. This theory is supported by the fact that (R)-pantetheine can support CAR activity for mixtures of the isolated domains. Our model suggests directions for further development of CAR as a biocatalyst.
- Published
- 2017
- Full Text
- View/download PDF
34. Structure and mechanism of a canonical poly(ADP-ribose) glycohydrolase.
- Author
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Dunstan MS, Barkauskaite E, Lafite P, Knezevic CE, Brassington A, Ahel M, Hergenrother PJ, Leys D, and Ahel I
- Subjects
- Glycoside Hydrolases classification, Glycoside Hydrolases metabolism, Humans, Phylogeny, Protein Structure, Secondary, Protein Structure, Tertiary, Tetrahymena thermophila enzymology, Glycoside Hydrolases chemistry
- Abstract
Poly(ADP-ribosyl)ation is a reversible post-translational protein modification involved in the regulation of a number of cellular processes including DNA repair, chromatin structure, mitosis, transcription, checkpoint activation, apoptosis and asexual development. The reversion of poly(ADP-ribosyl)ation is catalysed by poly(ADP-ribose) (PAR) glycohydrolase (PARG), which specifically targets the unique PAR (1''-2') ribose-ribose bonds. Here we report the structure and mechanism of the first canonical PARG from the protozoan Tetrahymena thermophila. In addition, we reveal the structure of T. thermophila PARG in a complex with a novel rhodanine-containing mammalian PARG inhibitor RBPI-3. Our data demonstrate that the protozoan PARG represents a good model for human PARG and is therefore likely to prove useful in guiding structure-based discovery of new classes of PARG inhibitors.
- Published
- 2012
- Full Text
- View/download PDF
35. In silico screening reveals structurally diverse, nanomolar inhibitors of NQO2 that are functionally active in cells and can modulate NF-κB signaling.
- Author
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Nolan KA, Dunstan MS, Caraher MC, Scott KA, Leys D, and Stratford IJ
- Subjects
- Animals, Aziridines pharmacology, Aziridines toxicity, Breast Neoplasms metabolism, Cell Line, Tumor, Female, Humans, Leukemia, Myelogenous, Chronic, BCR-ABL Positive metabolism, Macrophages, Mice, Proto-Oncogene Proteins c-bcl-2 metabolism, Quinone Reductases genetics, RNA Interference, RNA, Small Interfering, Signal Transduction, Structure-Activity Relationship, Transcription, Genetic drug effects, Tumor Necrosis Factor-alpha metabolism, NF-kappa B metabolism, Quinone Reductases antagonists & inhibitors, Quinone Reductases metabolism
- Abstract
The National Cancer Institute chemical database has been screened using in silico docking to identify novel nanomolar inhibitors of NRH:quinone oxidoreductase 2 (NQO2). The inhibitors identified from the screen exhibit a diverse range of scaffolds and the structure of one of the inhibitors, NSC13000 cocrystalized with NQO2, has been solved. This has been used to aid the generation of a structure-activity relationship between the computationally derived binding affinity and experimentally measured enzyme inhibitory potency. Many of the compounds are functionally active as inhibitors of NQO2 in cells at nontoxic concentrations. To show this, advantage was taken of the NQO2-mediated toxicity of the chemotherapeutic drug CB1954. The toxicity of this drug is substantially reduced when the function of NQO2 is inhibited, and many of the compounds achieve this in cells at nanomolar concentrations. The NQO2 inhibitors also attenuated TNFα-mediated, NF-кB-driven transcriptional activity. The link between NQO2 and the regulation of NF-кB was confirmed by using short interfering RNA to NQO2 and by the observation that NRH, the cofactor for NQO2 enzyme activity, could regulate NF-кB activity in an NQO2-dependent manner. NF-кB is a potential therapeutic target and this study reveals an underlying mechanism that may be usable for developing new anticancer drugs., (©2011 AACR.)
- Published
- 2012
- Full Text
- View/download PDF
36. Synthesis and biological evaluation of coumarin-based inhibitors of NAD(P)H: quinone oxidoreductase-1 (NQO1).
- Author
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Nolan KA, Doncaster JR, Dunstan MS, Scott KA, Frenkel AD, Siegel D, Ross D, Barnes J, Levy C, Leys D, Whitehead RC, Stratford IJ, and Bryce RA
- Subjects
- 4-Hydroxycoumarins chemistry, 4-Hydroxycoumarins toxicity, Animals, Cattle, Cell Line, Tumor, Crystallography, X-Ray, Enzyme Inhibitors chemistry, Enzyme Inhibitors toxicity, Humans, Inhibitory Concentration 50, Models, Molecular, Molecular Conformation, NAD(P)H Dehydrogenase (Quinone) chemistry, Quantitative Structure-Activity Relationship, 4-Hydroxycoumarins chemical synthesis, 4-Hydroxycoumarins pharmacology, Enzyme Inhibitors chemical synthesis, Enzyme Inhibitors pharmacology, NAD(P)H Dehydrogenase (Quinone) antagonists & inhibitors
- Abstract
The synthesis is reported here of two novel series of inhibitors of human NAD(P)H quinone oxidoreductase-1 (NQO1), an enzyme overexpressed in several types of tumor cell. The first series comprises substituted symmetric dicoumarol analogues; the second series contains hybrid compounds where one 4-hydroxycoumarin system is replaced by a different aromatic moiety. Several compounds show equivalent or improved NQO1 inhibition over dicoumarol, both in the presence and in the absence of added protein. Further, correlation is demonstrated between the ability of these agents to inhibit NQO1 and computed binding affinity. We have solved the crystal structure of NQO1 complexed to a hybrid compound and find good agreement with the in silico model. For both MIA PaCa-2 pancreatic tumor cells and HCT116 colon cancer cells, dicoumarol shows the greatest toxicity of all compounds. Thus, we provide a computational, synthetic, and biological platform to generate competitive NQO1 inhibitors with superior pharmacological properties to dicoumarol. This will allow a more definitive study of NQO1 activity in cells, in particular, its drug activating/detoxifying properties and ability to modulate oncoprotein stability.
- Published
- 2009
- Full Text
- View/download PDF
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