13 results on '"Sim, Edith"'
Search Results
2. Identification of NAD(P)H Quinone Oxidoreductase Activity in Azoreductases from P. aeruginosa: Azoreductases and NAD(P)H Quinone Oxidoreductases Belong to the Same FMN-Dependent Superfamily of Enzymes.
- Author
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Ryan, Ali, Kaplan, Elise, Nebel, Jean-Christophe, Polycarpou, Elena, Crescente, Vincenzo, Lowe, Edward, Preston, Gail M., and Sim, Edith
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QUINONE ,OXIDOREDUCTASES ,REDUCTASES ,PSEUDOMONAS aeruginosa ,HOST-parasite relationships ,PROTEIN structure - Abstract
Water soluble quinones are a group of cytotoxic anti-bacterial compounds that are secreted by many species of plants, invertebrates, fungi and bacteria. Studies in a number of species have shown the importance of quinones in response to pathogenic bacteria of the genus Pseudomonas. Two electron reduction is an important mechanism of quinone detoxification as it generates the less toxic quinol. In most organisms this reaction is carried out by a group of flavoenzymes known as NAD(P)H quinone oxidoreductases. Azoreductases have previously been separate from this group, however using azoreductases from Pseudomonas aeruginosa we show that they can rapidly reduce quinones. Azoreductases from the same organism are also shown to have distinct substrate specificity profiles allowing them to reduce a wide range of quinones. The azoreductase family is also shown to be more extensive than originally thought, due to the large sequence divergence amongst its members. As both NAD(P)H quinone oxidoreductases and azoreductases have related reaction mechanisms it is proposed that they form an enzyme superfamily. The ubiquitous and diverse nature of azoreductases alongside their broad substrate specificity, indicates they play a wide role in cellular survival under adverse conditions. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
3. From Arylamine N-Acetyltransferase to Folate-Dependent Acetyl CoA Hydrolase: Impact of Folic Acid on the Activity of (HUMAN)NAT1 and Its Homologue (MOUSE)NAT2.
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Laurieri, Nicola, Dairou, Julien, Egleton, James E., Stanley, Lesley A., Russell, Angela J., Dupret, Jean-Marie, Sim, Edith, and Rodrigues-Lima, Fernando
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ARYLAMINE N-acetyltransferase ,ACETYLCOENZYME A ,AROMATIC amines ,ACETYLATION ,FOLIC acid ,HYDROLASES ,LABORATORY mice - Abstract
Acetyl Coenzyme A-dependent N-, O- and N,O-acetylation of aromatic amines and hydrazines by arylamine N-acetyltransferases is well characterised. Here, we describe experiments demonstrating that human arylamine N-acetyltransferase Type 1 and its murine homologue (Type 2) can also catalyse the direct hydrolysis of acetyl Coenzyme A in the presence of folate. This folate-dependent activity is exclusive to these two isoforms; no acetyl Coenzyme A hydrolysis was found when murine arylamine N-acetyltransferase Type 1 or recombinant bacterial arylamine N-acetyltransferases were incubated with folate. Proton nuclear magnetic resonance spectroscopy allowed chemical modifications occurring during the catalytic reaction to be analysed in real time, revealing that the disappearance of acetyl CH
3 from acetyl Coenzyme A occurred concomitantly with the appearance of a CH3 peak corresponding to that of free acetate and suggesting that folate is not acetylated during the reaction. We propose that folate is a cofactor for this reaction and suggest it as an endogenous function of this widespread enzyme. Furthermore, in silico docking of folate within the active site of human arylamine N-acetyltransferase Type 1 suggests that folate may bind at the enzyme’s active site, and facilitate acetyl Coenzyme A hydrolysis. The evidence presented in this paper adds to our growing understanding of the endogenous roles of human arylamine N-acetyltransferase Type 1 and its mouse homologue and expands the catalytic repertoire of these enzymes, demonstrating that they are by no means just xenobiotic metabolising enzymes but probably also play an important role in cellular metabolism. These data, together with the characterisation of a naphthoquinone inhibitor of folate-dependent acetyl Coenzyme A hydrolysis by human arylamine N-acetyltransferase Type 1/murine arylamine N-acetyltransferase Type 2, open up a range of future avenues of exploration, both for elucidating the developmental role of these enzymes and for improving chemotherapeutic approaches to pathological conditions including estrogen receptor-positive breast cancer. [ABSTRACT FROM AUTHOR]- Published
- 2014
- Full Text
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4. Arylamine N-acetyltransferases: From Structure to Function.
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Sim, Edith, Walters, Kylie, and Boukouvala, Sotiria
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ACETYLTRANSFERASES , *ENZYMES , *XENOBIOTICS , *CATALYSTS , *PHARMACOGENOMICS , *ISOENZYMES , *MYCOBACTERIA , *PROKARYOTES - Abstract
Arylamine N-acetyltransferases (NATs) are cytosolic conjugating enzymes which transfer an acetyl group from acetylCoenzyme A to a xenobiotic acceptor substrate. The enzyme has an active site cysteine as part of a catalytic triad with histidine and aspartate. NATs have had an important role in pharmacogenetics. Polymorphism in acetylation (and inactivation) of the anti-tubercular agent isoniazid resides in human NAT2, one of two polymorphic human NATs. In humans there is also a third pseudogene and in rodents there are three isozymes. Comparison of human and rodent NAT enzymes and their genes is aiding our understanding of the roles of the individual isoenzymes. This may have clinical importance since human NAT1 is overexpressed in a sub-population of breast cancers and control of expression of the NAT genes is ripe for investigation. The mammalian NAT enzymes are involved in metabolism of drugs and carcinogens but there is growing evidence, including from transgenic mice, that human NAT1 has an endogenous role in folate degradation. Structural studies and intracellular tracking of polymorphic NAT variants, is contributing to appreciation of how individual mutations result in loss of NAT activity. Genome analyses have identified NAT homologues in bacteria including Mycobacterium tuberculosis, in which the NAT enzyme metabolises inactivation of isoniazid. More intriguingly, deletion of the nat gene in mycobacteria, leads to deficits in cell wall synthesis. Structural comparisons of NATs from prokaryotes and eukaryotes, particularly in relation to CoA binding, provide a platform for understanding how the unique NAT protein fold may lend itself to a wide range of functions. [ABSTRACT FROM AUTHOR]
- Published
- 2008
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5. Cloning, functional expression and characterization of Mesorhizobium loti arylamine N-acetyltransferases: rhizobial symbiosis supplies leguminous plants with the xenobiotic N-acetylation pathway.
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Rodrigues-Lima, Fernando, Dairou, Julien, Diaz, Clara L., Rubio, Maria C., Sim, Edith, Spaink, Herman P., and Dupret, Jean-Marie
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AROMATIC amines ,ACETYLTRANSFERASES ,XENOBIOTICS ,RHIZOBIUM ,METABOLIC conjugation ,ENZYMES ,SYMBIOSIS - Abstract
Arylamine N-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes involved in the detoxification of numerous aromatic chemicals. The NAT-dependent N-acetylation pathway has not previously been detected in plants. We demonstrate here the occurrence of the NAT-dependent pathway in leguminous plants, due to symbiosis with Mesorhizobium loti. We cloned two NAT enzymes from M. loti and showed that these two recombinant enzymes catalysed the N-acetylation of several known NAT substrates, including aniline-derived pesticide residues. We also demonstrate the existence of a functional NAT-dependent acetylation pathway in the root nodules of Lotus japonicus inoculated with M. loti. M. loti is the first non-eukaryotic organism shown to express two catalytically active NAT isoforms. This work also provides the first evidence for acquisition of a xenobiotic detoxification pathway by a plant through symbiosis with a soil microbe. [ABSTRACT FROM AUTHOR]
- Published
- 2006
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6. Characterization of the putative operon containing arylamine N-acetyltransferase ( nat) in Mycobacterium bovis BCG.
- Author
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Anderton, Matthew C., Bhakta, Sanjib, Besra, Gurdyal S., Jeavons, Peter, Eltis, Lindsay D., and Sim, Edith
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RESEARCH ,ACETYLTRANSFERASES ,MYCOBACTERIUM bovis ,MYCOBACTERIUM tuberculosis ,MYCOBACTERIUM ,ENZYMES ,GENES ,OPERONS ,LIPIDS ,CELLS - Abstract
Mycobacterium bovis BCG and Mycobacterium tuberculosis possess a single arylamine N-acetyltransferase whose gene is predicted to occur within a six-gene operon. Deletion of the nat gene caused an extended lag phase in M. bovis BCG and a cell morphology associated with an altered pattern of cell wall mycolates. Analysis of cDNA from M. bovis BCG shows that during in vitro growth all the genes in the putative nat operon are expressed and the open reading frames are contiguous, supporting the existence of an operon. Two genes in the operon, Mb3599c and Mb3600c, are predicted to encode homologues of enzymes annotated as a 2,3-dihydroxybiphenyl 1,2-dioxygenase ( bphC5) and a 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase ( bphD2), respectively, in Rhodococcus RHA1. As predicted, M. bovis BCG cell lysates metabolized the BphC substrate 2,3-dihydroxybiphenyl (2,3-DHB) to 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA), a BphD substrate, which was subsequently hydrolysed. Immunoprecipitation of the BphD homologue from these lysates led to an accumulation of HOPDA. M. bovis BCG growth on both solid and liquid media was inhibited with either 2,3-DHB or an inhibitor of BphC, 3-chlorocatechol (3-CC). In addition, incubation with 2,3-DHB affects the lipid composition of the cell wall resulting in a diminished level of mycolates and an altered cell morphology similar to the Δ nat strain. We propose the enzymes encoded by the putative operon have a similar endogenous role to that of the NAT enzyme and are part of a pathway important for cell wall synthesis. [ABSTRACT FROM AUTHOR]
- Published
- 2006
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7. Structure of arylamine N-acetyltransferase reveals a catalytic triad.
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Sinclair, John C., Sandy, James, Delgoda, Rupika, Sim, Edith, and Noble, Martin E.M.
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ENZYMES ,ACETYLTRANSFERASES ,ESCHERICHIA coli ,ACETYLATION ,HYDRAZINE - Abstract
Enzymes of the arylamine N-acetyltransferase (NAT) family are found in species ranging from Escherichia coli to humans. In humans they are known to be responsible for the acetylation of a number of arylamine and hydrazine drugs, and they are strongly linked to the carcinogenic potentiation of certain foreign substances. In prokaryotes their substrate specificities may vary and members of the gene family have been linked to pathways including amide synthesis during rifamycin production. Here we report the crystal structure at 2.8 Å resolution of a representative member of this family from Salmonella typhimurium in the presence and absence of a covalently bound product analog. The structure reveals surprising mechanistic information including the presence of a Cys-His-Asp catalytic triad. The fold can be described in terms of three domains of roughly equal length with the second and third domains linked by an interdomain helix. The first two domains, a helical bundle and a β-barrel, make up the catalytic triad using a structural motif identical to that of the cysteine protease superfamily. [ABSTRACT FROM AUTHOR]
- Published
- 2000
8. Structure of Mesorhizobium loti arylamine N-acetyltransferase 1.
- Author
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Holton, Simon J., Dairou, Julien, Sandy, James, Rodrigues-Lima, Fernando, Dupret, Jean-Marie, Noble, Martin E. M., and Sim, Edith
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ACETYLTRANSFERASES ,ACYLTRANSFERASES ,EUKARYOTIC cells ,PROTISTA ,COENZYMES ,ENZYMES - Abstract
The arylamine N-acetyltransferase (NAT) enzymes have been found in a broad range of both eukaryotic and prokaryotic organisms. The NAT enzymes catalyse the transfer of an acetyl group from acetyl Co-enzyme A onto the terminal nitrogen of a range of arylamine, hydrazine and arylhydrazine compounds. Recently, several NAT structures have been reported from different prokaryotic sources including Salmonella typhimurium, Mycobacterium smegmatis and Pseudomonas aeruginosa. Bioinformatics analysis of the Mesorhizobium loti genome revealed two NAT paralogues, the first example of multiple NAT isoenzymes in a eubacterial organism. The M. loti NAT 1 enzyme was recombinantly expressed and purified for X-ray crystallographic studies. The purified enzyme was crystallized in 0.5 M Ca(OAc)
2 , 16% PEG 3350, 0.1 M Tris-HCl pH 8.5 using the sitting-drop vapour-diffusion method. A data set diffracting to 2.0 Å was collected from a single crystal at 100 K. The crystal belongs to the orthorhombic spacegroup P21 21 21 , with unit-cell parameters a = 53.2, b = 97.3, c = 114.3 Å. The structure was refined to a final free-R factor of 24.8%. The structure reveals that despite low sequence homology, M. loti NAT1 shares the common fold as reported in previous NAT structures and exhibits the same catalytic triad of residues (Cys-His-Asp) in the active site. [ABSTRACT FROM AUTHOR]- Published
- 2005
- Full Text
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9. Small molecule colorimetric and fluorescent probes for specific protein detection
- Author
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Egleton, James Edward, Russell, Angela, and Sim, Edith
- Subjects
547 ,Organic chemistry ,Chemical biology ,Enzymes ,Pharmacology ,colorimetric probe ,fluorescent probe ,protein detection ,arylamine N-acetyltransferase ,breast cancer - Abstract
This thesis describes the design, synthesis, analysis, mechanistic evaluation and optimisation of small molecule probes for the specific detection of proteins, focusing on the target protein human arylamine N-acetyltransferase type 1 (HUMAN(NAT1)) and its murine homologue, mouse arylamine N-acetyltransferase type 2 (MOUSE(NAT2)). The HUMAN(NAT1) gene is reported to be one of the most highly overexpressed genes in estrogen-receptor-positive (ER+) breast tumours, leading to its potential use as both a novel diagnostic biomarker and a novel therapeutic target for this disease. Chapter 1 reviews the literature on optical methods for the specific detection of a protein target, exploring strategies both based on biosensors and on chemical probes, before introducing the arylamine N-acetyltransferases as a family of enzymes. In Chapter 2, a family of naphthoquinone inhibitors of HUMAN(NAT1) are introduced, which undergo a colour change from red to blue upon binding specifically to the enzyme. The mechanism of this colour change, a proton transfer-mediated process, is discussed via the synthesis, pharmacological and colorimetric evaluation of close analogues of the hit compound lacking a key acidic sulfonamide-NH proton. During these studies, it was found that direct O-methylation of a sulfonamide is possible under certain conditions; such a reaction has not previously been reported. Furthermore, upon heating in polar solvents the O-methylated sulfonamide was observed to undergo rearrangement, and the mechanism of this process is investigated via NMR and kinetic studies. In Chapter 3, the design, synthesis and evaluation of HUMAN(NAT1) inhibitors with improved pharmacological and colorimetric profiles over the initial hit are described. From this optimisation, structure-activity relationships and an in silico model of interactions between the inhibitors and enzyme are evaluated. Testing of these compounds in cellular environments, however, exposes some limitations of this approach, notably the lack of sensitivity of the probes when dosed at low concentrations in cellular samples. In order to overcome this limitation, in Chapter 4 fluorescent analogues of the hit compound are designed and synthesised. Initial compounds developed in this series possess promising properties, but each compound generated suffers from either a low fluorescent intensity, lack of a pH-dependent switch in fluorescence or a low fluorescence excitation wavelength, which overlaps with those of tryptophan or tyrosine residues in proteins. Insights into the mechanism of molecular fluorescence and application of some simple quantum mechanical principles, however, lead to the design of a species which possesses all the required properties. The fluorescent emission intensity of this probe correlates linearly with [MOUSE(NAT2)] in E. coli cell extracts, and can quantify as little as 0.64% MOUSE(NAT2) in the samples; furthermore, the probe is capable of unambiguously detecting HUMAN(NAT1) within a cell extract from the ER+ breast cancer cell line ZR-75-1; future work on this probe may therefore enable its clinical use in improved early diagnosis of breast tumours. This study also represents, to the best of our knowledge, the first ever example of a small molecule, non-covalent probe capable of quantifying the concentration of a target protein in cellular extracts. In Chapter 5, the series of naphthoquinone probes is further optimised in order to study the roles of HUMAN(NAT1) in a cellular environment. Firstly, structure-activity relationships are utilised to design inhibitors with improved physical properties such as aqueous solubility and cell membrane permeability, in order to test the effect of HUMAN(NAT1) inhibitors in tumour cell models, which could have implications for the future use of a HUMAN(NAT1) inhibitor as a therapeutic agent in oncology. Secondly, the effect of the cofactor folic acid on the function and activity of HUMAN(NAT1) is explored. Finally, in Chapter 6, the conclusions of this study are outlined and a hypothesis as to how the concepts developed in this thesis might be applied to alternative, more ubiquitous biological targets is discussed, paving the way for future investigations.
- Published
- 2015
10. A Novel Mechanism for Azoreduction
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Ryan, Ali, Laurieri, Nicola, Westwood, Isaac, Wang, Chan-Ju, Lowe, Edward, and Sim, Edith
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AZO dyes , *ANTI-inflammatory agents , *PRODRUGS , *ENZYMES , *PSEUDOMONAS aeruginosa , *RECOMBINANT proteins , *MOLECULAR structure , *X-ray crystallography - Abstract
Abstract: Azoreductases are important due to their ability to activate anti-inflammatory azo pro-drugs and to detoxify azo dyes. Three genes encoding azoreductases have been identified in Pseudomonas aeruginosa. We describe here a comparison of the three enzymes. The pure recombinant proteins each have a distinct substrate specificity profile against a range of azo substrates. Using the structure of P. aeruginosa azoreductase (paAzoR) 1 and the homology models of paAzoR2 and paAzoR3, we have identified residues important for substrate specificity. We have defined a novel flavin mononucleotide binding cradle, which is a recurrent motif in many flavodoxin-like proteins. A novel structure of paAzoR1 with the azo pro-drug balsalazide bound within the active site was determined by X-ray crystallography and demonstrates that the substrate is present in a hydrazone tautomer conformation. We propose that the structure with balsalazide bound represents an enzyme intermediate and, together with the flavin mononucleotide binding cradle, we propose a novel catalytic mechanism. [Copyright &y& Elsevier]
- Published
- 2010
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11. Mouse N-acetyltransferase type 2, the homologue of human N-acetyltransferase type 1
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Kawamura, Akane, Westwood, Isaac, Wakefield, Larissa, Long, Hilary, Zhang, Naixia, Walters, Kylie, Redfield, Christina, and Sim, Edith
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ACETYLTRANSFERASES , *BREAST cancer , *NUCLEAR magnetic resonance , *ENZYMES - Abstract
Abstract: There is increasing evidence that human arylamine N-acetyltransferase type 1 (NAT1, EC 2.3.1.5), although first identified as a homologue of a drug-metabolising enzyme, appears to be a marker in human oestrogen receptor positive breast cancer. Mouse Nat2 is the mouse equivalent of human NAT1. The development of mouse models of breast cancer is important, and it is essential to explore the biological role of mouse Nat2. We have therefore produced mouse Nat2 as a recombinant protein and have investigated its substrate specificity profile in comparison with human NAT1. In addition, we have tested the effects of inhibitors on mouse Nat2, including compounds which are endogenous and exogenous steroids. We show that tamoxifen, genistein and diethylstilbestrol inhibit mouse Nat2. The steroid analogue, bisphenol A, also inhibits mouse Nat2 enzymic activity and is shown by NMR spectroscopy, through shifts in proton peaks, to bind close to the active site. A three-dimensional structure for human NAT1 has recently been released, and we have used this crystal structure to generate a model of the mouse Nat2 structure. We propose that a conformational change in the structure is required in order for ligands to bind to the active site of the protein. [Copyright &y& Elsevier]
- Published
- 2008
- Full Text
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12. Molecular Cloning, Characterisation and Ligand-bound Structure of an Azoreductase from Pseudomonas aeruginosa
- Author
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Wang, Chan-Ju, Hagemeier, Christoph, Rahman, Nawreen, Lowe, Edward, Noble, Martin, Coughtrie, Michael, Sim, Edith, and Westwood, Isaac
- Subjects
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MOLECULAR cloning , *PSEUDOMONAS aeruginosa , *PHOSPHODIESTERASES , *ENZYMES - Abstract
Abstract: The gene PA0785 from Pseudomonas aeruginosa strain PAO1, which is annotated as a probable acyl carrier protein phosphodiesterase (acpD), has been cloned and heterologously overexpressed in Escherichia coli. The purified recombinant enzyme exhibits activity corresponding to that of azoreductase but not acpD. Each recombinant protein molecule has an estimated molecular mass of 23,050 Da and one non-covalently bound FMN as co-factor. This enzyme, now identified as azoreductase 1 from Pseudomonas aeruginosa (paAzoR1), is a flavodoxin-like protein with an apparent molecular mass of 110 kDa as determined by gel-filtration chromatography, indicating that the protein is likely to be tetrameric in solution. The three-dimensional structure of paAzoR1, in complex with the substrate methyl red, was solved at a resolution of 2.18 Å by X-ray crystallography. The protein exists as a dimer of dimers in the crystal lattice, with two spatially separated active sites per dimer, and the active site of paAzoR1 was shown to be a well-conserved hydrophobic pocket formed between two monomers. The paAzoR1 enzyme is able to reduce different classes of azo dyes and activate several azo pro-drugs used in the treatment of inflammatory bowel disease (IBD). During azo reduction, FMN serves as a redox centre in the electron-transferring system by mediating the electron transfer from NAD(P)H to the azo substrate. The spectral properties of paAzoR1 demonstrate the hydrophobic interaction between FMN and the active site in the protein. The structure of the ligand-bound protein also highlights the π-stacking interactions between FMN and the azo substrate. [Copyright &y& Elsevier]
- Published
- 2007
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13. Eukaryotic arylamine N-acetyltransferase: Investigation of substrate specificity by high-throughput screening
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Kawamura, Akane, Graham, James, Mushtaq, Adeel, Tsiftsoglou, Stefanos A., Vath, Gregory M., Hanna, Patrick E., Wagner, Carston R., and Sim, Edith
- Subjects
- *
ACETYLTRANSFERASES , *XENOBIOTICS , *CARCINOGENS , *ENZYMES - Abstract
Abstract: Arylamine N-acetyltransferases (NAT; EC 2.3.1.5) catalyse the transfer of acetyl groups from acetylCoA to xenobiotics, including drugs and carcinogens. The enzyme is found extensively in both eukaryotes and prokaryotes, yet the endogenous roles of NATs are still unclear. In order to study the properties of eukaryotic NATs, high-throughput substrate and inhibitor screens have been developed using pure soluble recombinant Syrian hamster NAT2 (shNAT2) protein. The assay can be used with a wide range of compounds and was used to determine substrate specificity of shNAT2. We describe the expression and characterisation of shNAT2 and also purified recombinant human NAT1 and NAT2, including the use of the assay to explore the substrate specificities of each of the enzymes. Hamster NAT2 has similar substrate specificity to human NAT1, acetylating para-aminobenzoate but not arylhydrazine and hydralazine compounds. The overlapping but distinct substrate-specific activity profiles of human NAT1 and NAT2 were clearly observed from the screen. Naturally occurring compounds were tested as substrates or inhibitors of shNAT2 and succinylCoA was found to be a potent inhibitor of shNAT2. [Copyright &y& Elsevier]
- Published
- 2005
- Full Text
- View/download PDF
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