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Rodney F. Minchin - One of the best experts on this subject based on the ideXlab platform.
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modulation of human Arylamine n acetyltransferase 1 activity by lysine acetylation role of p300 creb binding protein and sirtuins 1 and 2
Molecular Pharmacology, 2020Co-Authors: Neville J. Butcher, Rachel Burow, Rodney F. MinchinAbstract:Arylamine N-Acetyltransferase 1 (NAT1) is a phase II xenobiotic-metabolizing enzyme that also has a role in cancer cell growth and metabolism. Recently, it was reported that NAT1 undergoes lysine acetylation, an important post-translational modification that can regulate protein function. In the current study, we use site-directed mutagenesis to identify K100 and K188 as major sites of lysine acetylation in the NAT1 protein. Acetylation of ectopically expressed NAT1 in HeLa cells was decreased by C646, an inhibitor of the protein acetyltransferases p300/CREB-binding protein (CBP). Recombinant p300 directly acetylated NAT1 in vitro. Acetylation of NAT1 was enhanced by the sirtuin (SIRT) inhibitor nicotinamide but not by the histone deacetylase inhibitor trichostatin A. Cotransfection of cells with NAT1 and either SIRT 1 or 2, but not SIRT3, significantly decreased NAT1 acetylation. NAT1 activity was evaluated in cells after nicotinamide treatment to enhance acetylation or cotransfection with SIRT1 to inhibit acetylation. The results indicated that NAT1 acetylation impaired its enzyme kinetics, suggesting decreased acetyl coenzyme A binding. In addition, acetylation attenuated the allosteric effects of ATP on NAT1. Taken together, this study shows that NAT1 is acetylated by p300/CBP in situ and is deacetylated by the sirtuins SIRT1 and 2. It is hypothesized that post-translational modification of NAT1 by acetylation at K100 and K188 may modulate NAT1 effects in cells. SIGNIFICANCE STATEMENT: There is growing evidence that Arylamine N-Acetyltransferase 1 has an important cellular role in addition to xenobiotic metabolism. Here, we show that NAT1 is acetylated at K100 and K188 and that changes in protein acetylation equilibrium can modulate its activity in cells.
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Human Arylamine N-Acetyltransferase Type 1
Arylamine N-acetyltransferases in Health and Disease, 2018Co-Authors: Neville J. Butcher, Pengcheng Li, Lili Wang, Rodney F. MinchinAbstract:Human Arylamine N-Acetyltransferase 1 (NAT1) is one of the two functional NATs in humans that acetylate primary Arylamine and hydrazine xenobiotics. There is a growing body of evidence that suggests NAT1 may have an important physiological role in the cell in addition to its seemingly secondary role in xenobiotic metabolism. Specifically, roles in folate homeostasis, epigenetic regulation of gene expression, cell growth and survival, and cancer cell invasion and metastasis have been proposed. This chapter discusses the current evidence linking NAT1 to physiological processes and cancer cell biology, as well as its potential as a diagnostic/prognostic biomarker and therapeutic target.
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Trimodal distribution of Arylamine N-Acetyltransferase 1 mRNA in breast cancer tumors: association with overall survival and drug resistance
'Springer Science and Business Media LLC', 2018Co-Authors: Rodney F. Minchin, Neville J. ButcherAbstract:Abstract Background Arylamine N-Acetyltransferase 1 (NAT1) is a drug metabolizing enzyme that has been associated with cancer cell proliferation in vitro and with survival in vivo. NAT1 expression has been associated with the estrogen receptor and it has been proposed as a prognostic marker for estrogen receptor positive cancers. However, little is known about the distribution of NAT1 mRNA across an entire patient population or its effects on outcomes. To address this, gene expression data from breast cancer patient cohorts were investigated to identify sub-populations based on the level of NAT1 expression. Patient survival and drug response was examined to determine whether NAT1 mRNA levels influenced any of these parameters. Results NAT1 expression showed a trimodal distribution in breast cancer samples (n = 1980) but not in tumor tissue from ovarian, prostate, cervical or colorectal cancers. In breast cancer, NAT1 mRNA in each sub-population correlated with a separate set of genes suggesting different mechanisms of NAT1 gene regulation. Kaplan-Meier plots showed significantly better survival in patients with highest NAT1 mRNA compared to those with intermediate or low expression. While NAT1 expression was elevated in estrogen receptor-positive patients, it did not appear to be dependent on estrogen receptor expression. Overall survival was analyzed in patients receiving no treatment, hormone therapy or chemotherapy. NAT1 expression correlated strongly with survival in the first 5 years in those patients receiving chemotherapy but did not influence survival in the other two groups. This suggests that low NAT1 expression is associated with chemo-resistance. The sensitivity of NAT1 mRNA levels as a single parameter to identify non-responders to chemotherapy was 0.58 at a log(2)
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Arylamine N-Acetyltransferase 1 protects against reactive oxygen species during glucose starvation: Role in the regulation of p53 stability
2018Co-Authors: Lili Wang, Rodney F. Minchin, Neville J. ButcherAbstract:Human Arylamine N-Acetyltransferase 1 (NAT1) has been associated with cancer cell growth and invasion, but the underlying molecular mechanisms remain unknown. NAT1 is located on the short arm of chromosome 8 (8p21), a region that is commonly deleted in colon cancer. Previously, it was reported that HT-29 colon cancer cells, which have a large deletion at 8p21-22, show marked morphological changes, increased E-cadherin expression and altered cell-cell contact inhibition following down-regulation of NAT1 with shRNA. By contrast, no effects on growth were observed in HeLa cells. In the present study, cellular changes following knockout of NAT1 with CRISPR/Cas9 in HT-29 and HeLa cells were compared in the presence and absence of glucose. Cell growth decreased in both cell-lines during glucose starvation, but it was enhanced in HT-29 cells following NAT1 deletion. This was due to an increase in ROS production that induced cell apoptosis. Both ROS production and cell death were prevented by the glutathione precursor N-acetylcysteine. NAT1 knockout also resulted in a loss of the gain-of-function p53 protein in HT-29 cells. When p53 expression was inhibited with siRNA in parental HT-29 cells, ROS production and apoptosis increased to levels seen in the NAT1 knockout cells. The loss of p53 may explain the decreased colony formation and increased contact inhibition previously reported following NAT1 down-regulation in these cells. In conclusion, NAT1 is important in maintaining intracellular ROS, especially during glucose starvation, by stabilizing gain-of-function p53 in HT-29 cells. These results suggest that NAT1 may be a novel target to decrease intracellular gain-of -function p53.
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the role of lysine 100 in the binding of acetylcoenzyme a to human Arylamine n acetyltransferase 1 implications for other acetyltransferases
Biochemical Pharmacology, 2015Co-Authors: Rodney F. Minchin, Neville J. ButcherAbstract:1. IntroductionAcetyltransferases are a diverse superfamily of enzymesinvolved in the modification of small drug molecules, xenobio-tics, peptide and proteins. They are found in all prokaryoticand eukaryotic species studied to date and are essential fornumerous intracellular pathways. While the acetyl acceptorvaries considerablybetweendifferentacetyltransferases,theyallshare a common acetyl donor, acetylcoenzyme A (AcCoA). TheArylamine N-Acetyltransferases(NATs; EC 2.3.1.5)are xenobioticmetabolizing enzymes widelydistributedin the animal kingdom[1]. They are distinguished by the presence of a conservedcatalytic triad that prefers aromatic amine and hydrazinesubstrates [2]. In humans, there are 2 NATs (NAT1 and NAT2)and their crystal structure and catalytic function have beendescribed in detail [3–6]. Both NAT1 and NAT2 are geneticallypolymorphic, which impacts on the pharmacology of manytherapeutic agents that are metabolized by these enzymes [7].Moreover, recent studies have shown a relationship betweenNAT1 and cancer cell proliferation and survival suggesting thatthis protein is a potentialdrug target [7,8]. There have alsobeena number of reports on the development of small moleculeinhibitors for human and non-human NATs [9–12].The NATs catalyze the acetylation of small molecules via adouble displacement or ping pong bi bi reaction [13]. An in-depthunderstanding of the catalytic mechanism of the mammalianNAT’s was provided by Wang et al. who examined the acetylationof various substrates by the hamster homolog of NAT1 usingBronsted plot analyses, kinetic solvent isotope effects and pH-dependence studies [14,15]. This work showed that the formationof a thiolate-imidazolium ion pair by Cys
Neville J. Butcher - One of the best experts on this subject based on the ideXlab platform.
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modulation of human Arylamine n acetyltransferase 1 activity by lysine acetylation role of p300 creb binding protein and sirtuins 1 and 2
Molecular Pharmacology, 2020Co-Authors: Neville J. Butcher, Rachel Burow, Rodney F. MinchinAbstract:Arylamine N-Acetyltransferase 1 (NAT1) is a phase II xenobiotic-metabolizing enzyme that also has a role in cancer cell growth and metabolism. Recently, it was reported that NAT1 undergoes lysine acetylation, an important post-translational modification that can regulate protein function. In the current study, we use site-directed mutagenesis to identify K100 and K188 as major sites of lysine acetylation in the NAT1 protein. Acetylation of ectopically expressed NAT1 in HeLa cells was decreased by C646, an inhibitor of the protein acetyltransferases p300/CREB-binding protein (CBP). Recombinant p300 directly acetylated NAT1 in vitro. Acetylation of NAT1 was enhanced by the sirtuin (SIRT) inhibitor nicotinamide but not by the histone deacetylase inhibitor trichostatin A. Cotransfection of cells with NAT1 and either SIRT 1 or 2, but not SIRT3, significantly decreased NAT1 acetylation. NAT1 activity was evaluated in cells after nicotinamide treatment to enhance acetylation or cotransfection with SIRT1 to inhibit acetylation. The results indicated that NAT1 acetylation impaired its enzyme kinetics, suggesting decreased acetyl coenzyme A binding. In addition, acetylation attenuated the allosteric effects of ATP on NAT1. Taken together, this study shows that NAT1 is acetylated by p300/CBP in situ and is deacetylated by the sirtuins SIRT1 and 2. It is hypothesized that post-translational modification of NAT1 by acetylation at K100 and K188 may modulate NAT1 effects in cells. SIGNIFICANCE STATEMENT: There is growing evidence that Arylamine N-Acetyltransferase 1 has an important cellular role in addition to xenobiotic metabolism. Here, we show that NAT1 is acetylated at K100 and K188 and that changes in protein acetylation equilibrium can modulate its activity in cells.
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Human Arylamine N-Acetyltransferase Type 1
Arylamine N-acetyltransferases in Health and Disease, 2018Co-Authors: Neville J. Butcher, Pengcheng Li, Lili Wang, Rodney F. MinchinAbstract:Human Arylamine N-Acetyltransferase 1 (NAT1) is one of the two functional NATs in humans that acetylate primary Arylamine and hydrazine xenobiotics. There is a growing body of evidence that suggests NAT1 may have an important physiological role in the cell in addition to its seemingly secondary role in xenobiotic metabolism. Specifically, roles in folate homeostasis, epigenetic regulation of gene expression, cell growth and survival, and cancer cell invasion and metastasis have been proposed. This chapter discusses the current evidence linking NAT1 to physiological processes and cancer cell biology, as well as its potential as a diagnostic/prognostic biomarker and therapeutic target.
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Trimodal distribution of Arylamine N-Acetyltransferase 1 mRNA in breast cancer tumors: association with overall survival and drug resistance
'Springer Science and Business Media LLC', 2018Co-Authors: Rodney F. Minchin, Neville J. ButcherAbstract:Abstract Background Arylamine N-Acetyltransferase 1 (NAT1) is a drug metabolizing enzyme that has been associated with cancer cell proliferation in vitro and with survival in vivo. NAT1 expression has been associated with the estrogen receptor and it has been proposed as a prognostic marker for estrogen receptor positive cancers. However, little is known about the distribution of NAT1 mRNA across an entire patient population or its effects on outcomes. To address this, gene expression data from breast cancer patient cohorts were investigated to identify sub-populations based on the level of NAT1 expression. Patient survival and drug response was examined to determine whether NAT1 mRNA levels influenced any of these parameters. Results NAT1 expression showed a trimodal distribution in breast cancer samples (n = 1980) but not in tumor tissue from ovarian, prostate, cervical or colorectal cancers. In breast cancer, NAT1 mRNA in each sub-population correlated with a separate set of genes suggesting different mechanisms of NAT1 gene regulation. Kaplan-Meier plots showed significantly better survival in patients with highest NAT1 mRNA compared to those with intermediate or low expression. While NAT1 expression was elevated in estrogen receptor-positive patients, it did not appear to be dependent on estrogen receptor expression. Overall survival was analyzed in patients receiving no treatment, hormone therapy or chemotherapy. NAT1 expression correlated strongly with survival in the first 5 years in those patients receiving chemotherapy but did not influence survival in the other two groups. This suggests that low NAT1 expression is associated with chemo-resistance. The sensitivity of NAT1 mRNA levels as a single parameter to identify non-responders to chemotherapy was 0.58 at a log(2)
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Arylamine N-Acetyltransferase 1 protects against reactive oxygen species during glucose starvation: Role in the regulation of p53 stability
2018Co-Authors: Lili Wang, Rodney F. Minchin, Neville J. ButcherAbstract:Human Arylamine N-Acetyltransferase 1 (NAT1) has been associated with cancer cell growth and invasion, but the underlying molecular mechanisms remain unknown. NAT1 is located on the short arm of chromosome 8 (8p21), a region that is commonly deleted in colon cancer. Previously, it was reported that HT-29 colon cancer cells, which have a large deletion at 8p21-22, show marked morphological changes, increased E-cadherin expression and altered cell-cell contact inhibition following down-regulation of NAT1 with shRNA. By contrast, no effects on growth were observed in HeLa cells. In the present study, cellular changes following knockout of NAT1 with CRISPR/Cas9 in HT-29 and HeLa cells were compared in the presence and absence of glucose. Cell growth decreased in both cell-lines during glucose starvation, but it was enhanced in HT-29 cells following NAT1 deletion. This was due to an increase in ROS production that induced cell apoptosis. Both ROS production and cell death were prevented by the glutathione precursor N-acetylcysteine. NAT1 knockout also resulted in a loss of the gain-of-function p53 protein in HT-29 cells. When p53 expression was inhibited with siRNA in parental HT-29 cells, ROS production and apoptosis increased to levels seen in the NAT1 knockout cells. The loss of p53 may explain the decreased colony formation and increased contact inhibition previously reported following NAT1 down-regulation in these cells. In conclusion, NAT1 is important in maintaining intracellular ROS, especially during glucose starvation, by stabilizing gain-of-function p53 in HT-29 cells. These results suggest that NAT1 may be a novel target to decrease intracellular gain-of -function p53.
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the role of lysine 100 in the binding of acetylcoenzyme a to human Arylamine n acetyltransferase 1 implications for other acetyltransferases
Biochemical Pharmacology, 2015Co-Authors: Rodney F. Minchin, Neville J. ButcherAbstract:1. IntroductionAcetyltransferases are a diverse superfamily of enzymesinvolved in the modification of small drug molecules, xenobio-tics, peptide and proteins. They are found in all prokaryoticand eukaryotic species studied to date and are essential fornumerous intracellular pathways. While the acetyl acceptorvaries considerablybetweendifferentacetyltransferases,theyallshare a common acetyl donor, acetylcoenzyme A (AcCoA). TheArylamine N-Acetyltransferases(NATs; EC 2.3.1.5)are xenobioticmetabolizing enzymes widelydistributedin the animal kingdom[1]. They are distinguished by the presence of a conservedcatalytic triad that prefers aromatic amine and hydrazinesubstrates [2]. In humans, there are 2 NATs (NAT1 and NAT2)and their crystal structure and catalytic function have beendescribed in detail [3–6]. Both NAT1 and NAT2 are geneticallypolymorphic, which impacts on the pharmacology of manytherapeutic agents that are metabolized by these enzymes [7].Moreover, recent studies have shown a relationship betweenNAT1 and cancer cell proliferation and survival suggesting thatthis protein is a potentialdrug target [7,8]. There have alsobeena number of reports on the development of small moleculeinhibitors for human and non-human NATs [9–12].The NATs catalyze the acetylation of small molecules via adouble displacement or ping pong bi bi reaction [13]. An in-depthunderstanding of the catalytic mechanism of the mammalianNAT’s was provided by Wang et al. who examined the acetylationof various substrates by the hamster homolog of NAT1 usingBronsted plot analyses, kinetic solvent isotope effects and pH-dependence studies [14,15]. This work showed that the formationof a thiolate-imidazolium ion pair by Cys
Edith Sim - One of the best experts on this subject based on the ideXlab platform.
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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
2016Co-Authors: Nicola Laurieri, Edith Sim, Julien Dairou, James E Egleton, Lesley A Stanley, Angela J Russell, Marie Dupret, O Rodrigues-limaAbstract: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 CH3 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 probabl
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Exploration of Piperidinols as Potential Antitubercular Agents
Molecules (Basel Switzerland), 2014Co-Authors: Areej Abuhammad, Sanjib Bhakta, Angela J Russell, Elizabeth Fullam, Garrett M. Morris, Paul W. Finn, Edith SimAbstract:Novel drugs to treat tuberculosis are required and the identification of potential targets is important. Piperidinols have been identified as potential antimycobacterial agents (MIC < 5 μg/mL), which also inhibit mycobacterial Arylamine N-Acetyltransferase (NAT), an enzyme essential for mycobacterial survival inside macrophages. The NAT inhibition involves a prodrug-like mechanism in which activation leads to the formation of bioactive phenyl vinyl ketone (PVK). The PVK fragment selectively forms an adduct with the cysteine residue in the active site. Time dependent inhibition of the NAT enzyme from Mycobacterium marinum (M. marinum) demonstrates a covalent binding mechanism for all inhibitory piperidinol analogues. The structure activity relationship highlights the importance of halide substitution on the piperidinol benzene ring. The structures of the NAT enzymes from M. marinum and M. tuberculosis, although 74% identical, have different residues in their active site clefts and allow the effects of amino acid substitutions to be assessed in understanding inhibitory potency. In addition, we have used the piperidinol 3-dimensional shape and electrostatic properties to identify two additional distinct chemical scaffolds as inhibitors of NAT. While one of the scaffolds has anti-tubercular activity, both inhibit NAT but through a non-covalent mechanism.
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Characterisation of a putative AraC transcriptional regulator from Mycobacterium smegmatis
Tuberculosis, 2014Co-Authors: Dimitrios Evangelopoulos, Antima Gupta, Nathan A. Lack, Arundhati Maitra, Annemieke Ten Bokum, Sharon L. Kendall, Edith Sim, Sanjib BhaktaAbstract:MSMEG_0307 is annotated as a transcriptional regulator belonging to the AraC protein family and is located adjacent to the Arylamine N-Acetyltransferase (nat) gene in Mycobacterium smegmatis, in a gene cluster, conserved in most environmental mycobacterial species. In order to elucidate the function of the AraC protein from the nat operon in M. smegmatis, two conserved palindromic DNA motifs were identified using bioinformatics and tested for protein binding using electrophoretic mobility shift assays with a recombinant form of the AraC protein. We identified the formation of a DNA:AraC protein complex with one of the motifs as well as the presence of this motif in 20 loci across the whole genome of M. smegmatis, supporting the existence of an AraC controlled regulon. To characterise the effects of AraC in the regulation of the nat operon genes, as well as to gain further insight into its function, we generated a ΔaraC mutant strain where the araC gene was replaced by a hygromycin resistance marker. The level of expression of the nat and MSMEG_0308 genes was down-regulated in the ΔaraC strain when compared to the wild type strain indicating an activator effect of the AraC protein on the expression of the nat operon genes.
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design synthesis and structure activity relationships of 3 5 diaryl 1h pyrazoles as inhibitors of Arylamine n acetyltransferase
ChemInform, 2013Co-Authors: Elizabeth Fullam, Angela J Russell, Isaac M Westwood, Stephen G Davies, James Talbot, Areej Abuhammed, Edith SimAbstract:The synthesis and inhibitory potencies of a novel series of 3,5-diaryl-1H-pyrazoles as specific inhibitors of prokaryotic Arylamine N-Acetyltransferase enzymes is described. The series is based on hit compound 1 3,5-diaryl-1H-pyrazole identified from a high-throughout screen that has been carried out previously and found to inhibit the growth of Mycobacterium tuberculosis.
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structure of Arylamine n acetyltransferase from mycobacterium tuberculosis determined by cross seeding with the homologous protein from m marinum triumph over adversity
Acta Crystallographica Section D-biological Crystallography, 2013Co-Authors: Areej Abuhammad, Edith Sim, Edward D Lowe, Michael A Mcdonough, Patrick Shaw D Stewart, Stefan A Kolek, Elspeth F GarmanAbstract:Arylamine N-Acetyltransferase from Mycobacterium tuberculosis (TBNAT) plays an important role in the intracellular survival of the microorganism inside macrophages. Medicinal chemistry efforts to optimize inhibitors of the TBNAT enzyme have been hampered by the lack of a three-dimensional structure of the enzyme. In this paper, the first structure of TBNAT, determined using a lone crystal produced using cross-seeding with the homologous protein from M. marinum, is reported. Despite the similarity between the two enzymes (74% sequence identity), they show distinct physical and biochemical characteristics. The structure elegantly reveals the characteristic features of the protein surface as well as details of the active site of TBNAT relevant to drug-discovery efforts. The crystallographic analysis of the diffraction data presented many challenges, since the crystal was twinned and the habit possessed pseudo-translational symmetry.
Jeanmarie Dupret - One of the best experts on this subject based on the ideXlab platform.
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Xenobiotic-metabolizing enzymes in Bacillus anthracis: molecular and functional analysis of a truncated Arylamine N-Acetyltransferase isozyme
British Journal of Pharmacology, 2017Co-Authors: Xavier Kubiak, Jeanmarie Dupret, Benjamin Pluvinage, Romain Duval, Alain F. Chaffotte, Fernando Rodrigues-limaAbstract:BACKGROUND AND PURPOSE The Arylamine N-Acetyltransferases (NATs) are xenobiotic-metabolizing enzymes that play an important role in the detoxification and/or bioactivation of Arylamine drugs and xenobiotics. In bacteria, NATs may contribute to the resistance against antibiotics such as isoniazid or sulfamides through their acetylation, which makes this enzyme family a possible drug target. Bacillus anthracis, a bacterial species of clinical significance, expresses three NAT isozymes with distinct structural and enzymatic properties, including an inactive isozyme ((BACAN) NAT3). (BACAN) NAT3 features both a non-canonical Glu residue in its catalytic triad and a truncated C-terminus domain. However, the role these unusual characteristics play in the lack of activity of the (BACAN) NAT3 isozyme remains unclear. EXPERIMENTAL APPROACH Protein engineering, recombinant expression, enzymatic analyses with aromatic amine substrates and phylogenetic analysis approaches were conducted. KEY RESULTS The deletion of guanine 580 (G580) in the nat3 gene was shown to be responsible for the expression of a truncated (BACAN) NAT3 isozyme. Artificial re-introduction of G580 in the nat3 gene led to a functional enzyme able to acetylate several Arylamine drugs displaying structural characteristics comparable with its functional Bacillus cereus homologue ((BACCR) NAT3). Phylogenetic analysis of the nat3 gene in the B. cereus group further indicated that nat3 may constitute a pseudogene of the B. anthracis species. CONCLUSION AND IMPLICATIONS The existence of NATs with distinct properties and evolution in Bacillus species may account for their adaptation to their diverse chemical environments. A better understanding of these isozymes is of importance for their possible use as drug targets.
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SPET Journals on Septem
2016Co-Authors: Nilusha Ragunathan, Jeanmarie Dupret, Julien Dairou, Marta Martins, Benjamin Pluvinage, Emile Petit, Nathalie Janel, O Rodrigues-limaAbstract:Identification of the xenobiotic-metabolizing enzyme Arylamine N-Acetyltransferase 1 (NAT1) as a new target of cisplatin in breast cancer cells: molecular and cellular mechanisms of inhibitio
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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
PLOS ONE, 2014Co-Authors: Nicola Laurieri, Jeanmarie Dupret, Julien Dairou, James E Egleton, Lesley A Stanley, Angela J Russell, Fernando RodrigueslimaAbstract: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 CH3 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.
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human Arylamine n acetyltransferase 1 a drug metabolizing enzyme and a drug target
Current Drug Targets, 2010Co-Authors: Fernando Rodrigueslima, Julien Dairou, Florent Busi, Jeanmarie DupretAbstract:Human Arylamine N-Acetyltransferase 1 (NAT1) is a phase II xenobiotic-metabolizing enzyme (XME) involved in the biotransformation of many aromatic and heterocyclic amines. This XME plays key roles in both the detoxification and/or bioactivation of numerous drugs and carcinogens. NAT1 is polymorphic and displays a large tissue distribution. NAT1 activity has been extensively studied because of its potential role in the biotransformation of important carcinogens. Several recent studies suggest that NAT1 may have a role in breast cancer progression. Indeed, this XME has been shown to affect the growth and drug resistance of breast cancer cells and appears as a marker in human estrogen receptor positive breast cancer. In addition, it has been shown that this enzyme is inhibited in vivo by cancer drugs such as cisplatin or tamoxifen. Recent published data suggest that NAT1 could be of therapeutic interest for cancer. We provide here an overview on the putative involvement of NAT1 in cancer and its possible role as a drug target.
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functional and structural characterization of the Arylamine n acetyltransferase from the opportunistic pathogen nocardia farcinica
Journal of Molecular Biology, 2008Co-Authors: Marta Martins, Jeanmarie Dupret, Julien Dairou, Benjamin Pluvinage, Ines Li De La Sierragallay, Florent Barbault, Fernando RodrigueslimaAbstract:Arylamine N-Acetyltransferase (NAT) enzymes are found in a broad range of eukaryotes and prokaryotes. There is increasing evidence that NAT enzymes could contribute to antibiotic resistance in pathogenic bacteria such as Mycobacterium tuberculosis. Nocardia farcinica is an opportunistic human pathogen that causes pulmonary infections (nocardiosis) with clinical manifestations that resemble tuberculosis. While the genomic sequence of this prokaryote has been determined, studies of N. farcinica proteins remain almost nonexistent. In particular, N. farcinica proteins putatively involved in antibiotic resistance mechanisms have not been described structurally or functionally. Here, we have characterized a new NAT enzyme (NfNAT) from N. farcinica at the structural and functional level. NfNAT is the first N. farcinica protein for which a 3D structure is reported. We showed that this novel prokaryotic isoform is structurally and functionally related to the mycobacterial NAT enzymes. In particular, NfNAT was found to display high N-Acetyltransferase activity towards several known NAT substrates including the antitubercular drug isoniazid. Interestingly, isoniazid is not used for the treatment of nocardiosis and has been shown to be poorly active against several nocardial species. On the contrary, NfNAT was found to be poorly active towards sulfamethoxazole, a sulfonamide drug considered as the treatment of choice for the treatment of nocardiosis. The functional and structural data reported in this study will help to develop our understanding of the role of NAT enzymes in nocardia and mycobacteria and may help in the rational design of NAT antagonists for a range of clinical applications.
Fernando Rodrigueslima - One of the best experts on this subject based on the ideXlab platform.
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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
PLOS ONE, 2014Co-Authors: Nicola Laurieri, Jeanmarie Dupret, Julien Dairou, James E Egleton, Lesley A Stanley, Angela J Russell, Fernando RodrigueslimaAbstract: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 CH3 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.
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human Arylamine n acetyltransferase 1 a drug metabolizing enzyme and a drug target
Current Drug Targets, 2010Co-Authors: Fernando Rodrigueslima, Julien Dairou, Florent Busi, Jeanmarie DupretAbstract:Human Arylamine N-Acetyltransferase 1 (NAT1) is a phase II xenobiotic-metabolizing enzyme (XME) involved in the biotransformation of many aromatic and heterocyclic amines. This XME plays key roles in both the detoxification and/or bioactivation of numerous drugs and carcinogens. NAT1 is polymorphic and displays a large tissue distribution. NAT1 activity has been extensively studied because of its potential role in the biotransformation of important carcinogens. Several recent studies suggest that NAT1 may have a role in breast cancer progression. Indeed, this XME has been shown to affect the growth and drug resistance of breast cancer cells and appears as a marker in human estrogen receptor positive breast cancer. In addition, it has been shown that this enzyme is inhibited in vivo by cancer drugs such as cisplatin or tamoxifen. Recent published data suggest that NAT1 could be of therapeutic interest for cancer. We provide here an overview on the putative involvement of NAT1 in cancer and its possible role as a drug target.
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functional and structural characterization of the Arylamine n acetyltransferase from the opportunistic pathogen nocardia farcinica
Journal of Molecular Biology, 2008Co-Authors: Marta Martins, Jeanmarie Dupret, Julien Dairou, Benjamin Pluvinage, Ines Li De La Sierragallay, Florent Barbault, Fernando RodrigueslimaAbstract:Arylamine N-Acetyltransferase (NAT) enzymes are found in a broad range of eukaryotes and prokaryotes. There is increasing evidence that NAT enzymes could contribute to antibiotic resistance in pathogenic bacteria such as Mycobacterium tuberculosis. Nocardia farcinica is an opportunistic human pathogen that causes pulmonary infections (nocardiosis) with clinical manifestations that resemble tuberculosis. While the genomic sequence of this prokaryote has been determined, studies of N. farcinica proteins remain almost nonexistent. In particular, N. farcinica proteins putatively involved in antibiotic resistance mechanisms have not been described structurally or functionally. Here, we have characterized a new NAT enzyme (NfNAT) from N. farcinica at the structural and functional level. NfNAT is the first N. farcinica protein for which a 3D structure is reported. We showed that this novel prokaryotic isoform is structurally and functionally related to the mycobacterial NAT enzymes. In particular, NfNAT was found to display high N-Acetyltransferase activity towards several known NAT substrates including the antitubercular drug isoniazid. Interestingly, isoniazid is not used for the treatment of nocardiosis and has been shown to be poorly active against several nocardial species. On the contrary, NfNAT was found to be poorly active towards sulfamethoxazole, a sulfonamide drug considered as the treatment of choice for the treatment of nocardiosis. The functional and structural data reported in this study will help to develop our understanding of the role of NAT enzymes in nocardia and mycobacteria and may help in the rational design of NAT antagonists for a range of clinical applications.
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structure of mesorhizobium loti Arylamine n acetyltransferase 1
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2005Co-Authors: S Holton, Jeanmarie Dupret, Fernando Rodrigueslima, Julien Dairou, James Sandy, M E M NobleAbstract: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 A was collected from a single crystal at 100 K. The crystal belongs to the orthorhombic spacegroup P212121, with unit-cell parameters a = 53.2, b = 97.3, c = 114.3 A. 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.
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regulation of the activity of the human drug metabolizing enzyme Arylamine n acetyltransferase 1 role of genetic and non genetic factors
Current Pharmaceutical Design, 2004Co-Authors: Fernando Rodrigueslima, Jeanmarie DupretAbstract:Human Arylamine N-Acetyltransferases 1 and 2 (NAT1 and NAT2) are polymorphic phase II xenobiotic-metabolising enzymes (XME) that acetylate Arylamine compounds. They therefore play an important role in the detoxication and/or metabolic activation of certain therapeutic drugs, occupational chemicals and carcinogens. Although the use of the term "xenobiotic" implies that XME form a separate and distinct class of enzymes, possible endogenous substrates may exist. Unlike NAT2, NAT1 is produced in most tissues. Polymorphism at the NAT1 locus has been associated with the existence of at least 26 allelic variants, generating phenotypic variations in terms of NAT1 catalytic activity. This genetic variation affects the acetylator status of individuals, leading to interindividual differences in drug response and predisposition to disease in humans. Recent studies have shown that non-genetic factors may also regulate NAT1 activity at the posttranslational level, with potentially important consequences for drug toxicity. In this mini-review, we summarise what is currently known about the regulation of NAT1 activity by non-genetic factors, including substrates and oxidative stress. Recent findings presented here may account for the genotype/phenotype relationship for the NAT1 locus being less clear-cut than that for human NAT2.
