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ABSTRACT: Arylamine N-acetyltransferase 1 (NAT1) is a phase II xenobiotic-metabolizing enzyme that plays an important role in the biotransformation of aromatic drugs and carcinogens. NAT1 activity has long been associated with susceptibility to various cancers. Evidence for a role of NAT1 in malignant progression has also been obtained, particularly for breast and prostate cancer. Cisplatin is widely used in chemotherapy against human cancers, and it is thought to act principally by forming DNA adducts. However, recent studies have suggested that some of the pharmacological and/or toxicological effects of cisplatin may be due to the direct targeting and inhibition of certain cellular enzymes. We show here that the exposure of breast cancer cells, known to express functional NAT1 enzyme, to therapeutically relevant concentrations of cisplatin impairs the catalytic activity of endogenous NAT1. Endogenous NAT1 was also found to be inactivated, in vivo, in the tissues of mice treated with cisplatin. Mechanistic studies with purified human NAT1 indicated that this inhibition resulted from the irreversible formation of a cisplatin adduct with the active-site cysteine residue of the enzyme. Kinetic studies suggested that NAT1 interacts rapidly with cisplatin, with a second-order rate inhibition constant of 700 M(-1) min(-1). This rate constant is one the highest ever reported for the reaction of cisplatin with a biological macromolecule. Few enzymes have been clearly shown to be inactivated by cisplatin. We provide here molecular and cellular evidence suggesting that NAT1 is one of the targets of cisplatin in cells.
Molecular pharmacology 07/2008; 73(6):1761-8. · 4.53 Impact Factor
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ABSTRACT: The arylamine N-acetyltransferase (NAT) enzymes are xenobiotic metabolizing enzymes that have been found in a large range of eukaryotes and prokaryotes. These enzymes catalyse the acetylation of arylamine drugs and/or pollutants. Recently, a Bacillus anthracis NAT isoform (BanatC) has been cloned and shown to acetylate the sulfonamide antimicrobial sulfamethoxazole (SMX). Subsequently, it was shown that BanatC contributes to the resistance of this bacterium to SMX. Here, the crystallization and the X-ray characterization of BanatC (Y38F mutant) are reported. The crystals belong to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 53.70, c = 172.40 A, and diffract to 1.95 A resolution on a synchrotron source.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 11/2007; 63(Pt 10):862-4. · 0.51 Impact Factor
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ABSTRACT: Muscle glycogen phosphorylase (GP) is a key enzyme in glucose metabolism, and its impairment can lead to muscle dysfunction. Tyrosine nitration of glycogen phosphorylase occurs during aging and has been suggested to be involved in progressive loss of muscle performance. Here, we show that GP (in its T and R form) is irreversibly impaired by exposure to peroxynitrite, a biological nitrogen species known to nitrate reactive tyrosine residues, and to be involved in physiological and pathological processes. Kinetic and biochemical analysis indicated that irreversible inactivation of GP by peroxynitrite is due to the fast (k(inact)=3 x 10(4) M(-1) s(-1)) nitration of a unique tyrosine residue of the enzyme. Endogenous GP was tyrosine nitrated and irreversibly inactivated in skeletal muscle cells upon exposure to peroxynitrite, with concomitant impairment of glycogen mobilization. Ligand protection assays and mass spectrometry analysis using purified GP suggested that the peroxynitrite-dependent inactivation of the enzyme could be due to the nitration of Tyr613, a key amino acid of the allosteric inhibitor site of the enzyme. Our findings suggest that GP functions may be regulated by tyrosine nitration.
Journal of Molecular Biology 10/2007; 372(4):1009-21. · 4.00 Impact Factor
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ABSTRACT: The arylamine N-acetyltransferases are important xenobiotic-metabolizing enzymes that catalyze an acetyl group transfer from acetylCoA to arylamine substrates. NAT enzymes possess an active-site loop (the active-site P-loop) involved in substrate binding and selectivity. The Gly/Ala residue present at the start of the active-site P-loop, although conserved in all NAT enzymes, is not involved in the catalytic mechanism or substrate binding. Here we show that a small amino acid (such as Gly or Ala) at this position is important not only for maintaining the functions of the active-site P-loop but, more surprisingly, also important for maintaining the overall structural integrity of NAT enzymes. Our data thus suggest that in addition to its role in substrate binding and selectivity, the active-site P-loop could play a wider structural role in NAT enzymes.
Biochemical and Biophysical Research Communications 10/2007; 361(1):256-62. · 2.48 Impact Factor
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ABSTRACT: The arylamine N-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes that catalyze the N-acetylation of arylamines and their N-hydroxylated metabolites. These enzymes play a key role in detoxication of numerous drugs and xenobiotics. We report here the cloning, functional expression, and characterization of three new NAT genes (termed banatA, banatB, and banatC) from the pathogen Bacillus anthracis. The sequences of the corresponding proteins are approximately 30% identical with those of characterized eukaryotic and prokaryotic NAT enzymes, and the proteins were recognized by an anti-NAT antibody. The three genes were endogenously expressed in B. anthracis, and NAT activity was found in cell extracts. The three NAT homologues exhibited distinct structural and enzymatic properties, some of which have not previously been observed with other NAT enzymes. Recombinant BanatC displayed strong NAT activity toward several prototypic NAT substrates, including the sulfonamide antibiotic sulfamethoxazole (SMX). As opposed to BanatC, BanatB also had acetyl-CoA (AcCoA) and p-nitrophenyl acetate (PNPA) hydrolysis activity in the absence of arylamine substrates, indicating that it may act as an AcCoA hydrolase. BanatA was devoid of NAT or AcCoA/PNPA hydrolysis activities, suggesting that it may be a new bacterial NAT-like protein with unknown function. Expression of BanatC in Escherichia coli afforded higher-than-normal resistance to SMX in the recombinant bacteria, whereas an inactive mutant of the enzyme did not. These data indicate that BanatC could contribute to the resistance of B. anthracis to SMX.
Biochemistry 07/2007; 46(23):7069-78. · 3.42 Impact Factor
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ABSTRACT: We have recently focused on the interaction between hyperhomocysteinemia, defined by high plasma homocysteine levels, and paraoxonase-1 expression and found a reduced activity of paraoxonase-1 associated with a reduced gene expression in the liver of cystathionine beta synthase (CBS) deficient mice, a murine model of hyperhomocysteinemia. As it has been demonstrated that polyphenolic compounds could modulate the expression level of the paraoxonase-1 gene in vitro, we have investigated the possible effect of flavonoid supplementation on the impaired paraoxonase-1 gene expression and activity induced by hyperhomocysteinemia and have evaluated the link with homocysteine metabolism. High-methionine diet significantly increased serum homocysteine levels, decreased hepatic CBS activity, and down-regulated paraoxonase-1 mRNA and its activity. However, chronic administration of catechin but not quercetin significantly reduced plasma homocysteine levels, attenuated the reduction of the hepatic CBS activity, and restored the decreased paraoxonase-1 gene expression and activity induced by chronic hyperhomocysteinemia. These data suggest that catechin could act on the homocysteine levels by increasing the rate of catabolism of homocysteine.
Biochemical and Biophysical Research Communications 04/2007; 355(1):221-7. · 2.48 Impact Factor
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ABSTRACT: Arylamine N-acetyltransferase I (NAT1) is a phase II enzyme that acetylates a wide range of arylamine and hydrazine substrates. The NAT1 gene is located on chromosome 8 and shares homology to NAT genes found in most mammalian species. Gene expression occurs from at least two promoters and a number of tissue-specific transcripts have been identified. The gene is polymorphic with most mutations identified to date producing an unstable protein that is subject to polyubiquitination. The NAT1 protein contains a catalytic triad similar to a number of cysteine proteases and transglutaminases. NAT1 is widely distributed in the body, but the only endogenous substrate identified to date is the folate catabolite p-aminobenzoylglutamate. Recent links between NAT1 genotypes and susceptibility to spina bifida suggests that the enzyme has an important role in folate homeostasis.
The International Journal of Biochemistry & Cell Biology 01/2007; 39(11):1999-2005. · 4.63 Impact Factor
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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.
Molecular Microbiology 05/2006; 60(2):505-12. · 5.01 Impact Factor
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ABSTRACT: The arylamine N-acetyltransferases (NAT; EC 2.3.1.5) are xenobiotic-metabolizing enzymes (XME) that catalyze the transfer of an acetyl group from acetylCoA (Ac-CoA) to arylamine, hydrazines and their N-hydroxylated metabolites. Eukaryotes may have up to three NAT isoforms, but Mesorhizobium loti is the only prokaryote with two functional NAT isoforms (MLNAT1 and MLNAT2). The three-dimensional structure of MLNAT1 has been determined (Holton, S.J., Dairou, J., Sandy, J., Rodrigues-Lima, F., Dupret, J.M., Noble, M.E.M. and Sim, E. (2005) Structure of Mesorhizobium loti arylamine N-acetyltransferase 1. Acta Cryst, F61, 14-16). No MLNAT2 crystals have yet been produced, despite the production of sufficient quantities of pure protein. Using purified recombinant MLNAT1 and MLNAT2, we showed here that MLNAT1 was intrinsically more stable than MLNAT2. To test whether different structural features could explain these differences in intrinsic stability, we constructed a high-quality homology model for MLNAT2 based on far UV-CD data. Despite low levels of sequence identity with other prokaryotic NAT enzymes ( approximately 28% identity), this model suggests that MLNAT2 adopts the characteristic three-domain NAT fold. More importantly, molecular dynamics simulations on the structures of MLNAT1 and MLNAT2 suggested that MLNAT2 was less stable than MLNAT1 due to differences in amino-acid sequence/structure features in the alpha/beta lid domain.
FEBS Letters 04/2006; 580(7):1780-8. · 3.54 Impact Factor
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ABSTRACT: Reactive nitrogen species and their by-products, such as peroxynitrite, modulate many physiological functions of skeletal muscle. Peroxynitrite generation occuring under specific conditions, such as inflammation, may also lead to skeletal muscle dysfunction and pathologies. Arylamine N-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes (XMEs) involved in the detoxification and/or metabolic activation of several drugs and chemicals. In addition to other XMEs, such as gluthatione S-transferases or cytochromes P450, NAT enzymes are expressed in skeletal muscle. We show here that functional NAT1 and NAT2 isoforms are expressed in mouse myotubes and that peroxynitrite may impair their activity in these cells. We show that this inactivation is likely due to the irreversible modification of NATs catalytic cysteine residue in vivo. Our results suggest that peroxynitrite-dependent inactivation of muscle XMEs such as NATs may contribute to muscle dysfunction by impairing the biotransformation activity of this key cellular defense enzyme system.
FEBS Letters 09/2005; 579(21):4719-23. · 3.54 Impact Factor
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ABSTRACT: The human arylamine N-acetyltransferases NAT1 and NAT2 are important xenobiotic-metabolizing enzymes involved in the detoxification and metabolic activation of numerous drugs and chemicals. NAT activity depends on genetic polymorphisms and on environmental factors. It has been shown that low NAT-acetylation activity could increase the risk of age-dependent cataract, suggesting that NAT detoxification function may be important for lens cells homeostasis. We report here that the NAT acetylation pathway may occur in human lens epithelial (HLE) cells. Functional NAT1 enzyme was readily detected in HLE cells by reverse transcription-polymerase chain reaction, Western blotting, and enzyme activity assays. NAT2 mRNA and enzymic activity were also detected. We investigated whether oxidants, known to be produced in HLE cells during oxidative stresses and involved in age-dependent cataract formation, decreased endogenous NAT1 and NAT2 activity. The exposure of HLE cells to peroxynitrite led to the dose-dependent irreversible inactivation of both NAT isoforms. Exposing HLE cells to continuously generated H(2)O(2) gave a dose-dependent inactivation of NAT1 and NAT2, reversible on addition of high concentrations of reducing agents. UVB irradiation also induced the reversible dose-dependent inactivation of endogenous NAT1 and NAT2, reversible on addition of reducing agents. Thus, our data suggest that functional NAT1 and NAT2 are present in HLE cells and may be impaired by oxidants produced during oxidative and photooxidative stresses. Oxidative-dependent inhibition of NATs in these cells may increase exposure of lens to the harmful effects of toxic chemicals that could contribute to cataractogenesis over time.
Molecular Pharmacology 05/2005; 67(4):1299-306. · 4.88 Impact Factor
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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 A was collected from a single crystal at 100 K. The crystal belongs to the orthorhombic spacegroup P2(1)2(1)2(1), 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.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 02/2005; 61(Pt 1):14-6. · 0.51 Impact Factor
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ABSTRACT: Arylamine N-acetyltransferases (NAT) are xenobiotic-metabolizing enzymes responsible for N-acetylation of many arylamines. They are also important for O-acetylation of N-hydroxylated heterocyclic amines. These enzymes play thus an important role in the detoxification and activation of numerous therapeutic drugs and carcinogens. Two closely related polymorphic isoforms (NAT1 and NAT2) have been described in humans and interindividual variations in NAT genes have been shown to be a potential source of adverse drug reaction. In addition, NAT1 and/or NAT2 phenotypes may modulate the risk of certain cancers in people exposed to aromatic amine carcinogens. Recent advances on the regulation of human NAT1 activity has shown that hydroxylamine and/or nitroso intermediates of NAT1 substrates inhibit the enzyme through direct irreversible interaction with its catalytic cysteine residue. Oxidative molecules such as hydrogen peroxide, S-nitrosothiols and peroxynitrite have also been shown to inactivate reversibly or irreversibly the enzyme in a similar manner. In this review, after summarizing the general background on human NAT enzymes, we focus on the recent developments on the regulation of the activity of these drug-metabolizing enzymes by substrate-intermediates and by oxidant molecules. The recent findings reviewed here provide possible mechanisms by which these non genetic determinants inhibit NAT1 activity and thereby may affect drug efficacy/toxicity.
Current Medicinal Chemistry 02/2005; 12(3):311-8. · 4.86 Impact Factor
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ABSTRACT: Arylamine N-acetyltransferases (NAT) are xenobiotic-metabolizing enzymes responsible for the acetylation of many arylamine and heterocyclic amines. They therefore play an important role in the detoxification and activation of numerous drugs and carcinogens. Two closely related isoforms (NAT1 and NAT2) have been described in humans. NAT2 is present mainly in the liver and intestine, whereas NAT1 is found in a wide range of tissues. Interindividual variations in NAT genes have been shown to be a potential source of pharmacological and/or pathological susceptibility. Evidence now shows that redox conditions may also contribute to overall NAT activity. This chapter summarizes current knowledge on human NAT1 regulation by reactive oxygen and nitrogen species.
Methods in Enzymology 02/2005; 400:215-29. · 2.04 Impact Factor
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ABSTRACT: The gene for NAT (arylamine N-acetyltransferase) from Pseudomonas aeruginosa (panat) has been cloned from genomic DNA, and the gene product (PANAT) expressed as an N-terminal histidine-tagged protein in Escherichia coli and purified via nickel ion affinity chromatography. The specific activities of PANAT against a broad range of substrates have been investigated and compared with those of other prokaryotic NAT enzymes. For most arylamine substrates identified, PANAT exhibits in vitro specific activities typically one order of magnitude greater than those of recombinant NAT enzymes from Mycobacterium smegmatis or Salmonella typhimurium. Among the substrates of PANAT so far identified are the anti-tubercular drug isoniazid, 5-aminosalicylate (a drug used in the treatment of inflammatory bowel disease), as well as important environmental pollutants such as 3,4-dichloroaniline and 2-aminofluorene. As well as acetylating common NAT substrates, PANAT is unique among the prokaryotic NATs so far studied in acetylating the folate precursor 4-aminobenzoic acid and the folate catabolite 4-aminobenzoylglutamate. The recombinant protein has been expressed in sufficient quantity to allow protein crystallization, and we have subsequently determined the 1.95 A structure of PANAT by X-ray crystallography.
Biochemical Journal 02/2005; 385(Pt 2):605-12. · 4.90 Impact Factor
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ABSTRACT: Xenobiotic-metabolizing enzymes (XME) are responsible for the biotransformation of a vast range of compounds. Arylamine N-acetyltransferases (NAT) are XME responsible for the acetylation of many arylamine and heterocyclic amines. They therefore, play an important role in the detoxification and activation of numerous drugs and carcinogens. Two closely related isoforms (NAT1 and NAT2) have been described in humans. NAT2 is present mainly in the liver and gut whereas NAT1 is found in a wide range of tissues. Interindividual variations in NAT genes have been shown to be a potential source of pharmacological and / or pathological susceptibility. In addition, there is now evidence that non genetic factors, such as substrate-dependent inhibition or redox conditions, may also contribute to overall NAT1 activity. This mini-review summarizes current knowledge on human NATs. Recent aspects of NAT gene expression, regulation and structural properties are discussed.
Current Pharmacogenomics 11/2004; 2(4):333-338.
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ABSTRACT: Arylamine N-acetyltransferases (NATs) play an important role in the detoxification and metabolic activation of a variety of aromatic xenobiotics, including numerous carcinogens. Both of the human isoforms, NAT1 and NAT2, display interindividual variations, and associations between NAT genotypes and cancer risk have been established. Contrary to NAT2, NAT1 has a ubiquitous tissue distribution and has been shown to be expressed in cancer cells. Given that the activity of NAT1 depends on a reactive cysteine that can be a target for oxidants, we studied whether peroxynitrite, a highly reactive nitrogen species involved in human carcinogenesis, could inhibit the activity of endogenous NAT1 in MCF7 breast cancer cells. We show here that exposure of MCF7 cells to physiological concentrations of peroxynitrite and to a peroxynitrite generator (3-morpholinosydnonimine N-ethylcarbamide, or SIN1) leads to the irreversible inactivation of NAT1 in cells. Further kinetic and mechanistic analyses using recombinant NAT1 showed that the enzyme is rapidly (k(inact) = 5 x 10(4) m(-1).s(-1)) and irreversibly inactivated by peroxynitrite. This inactivation is due to oxidative modification of the catalytic cysteine. We conclude that the reducing cellular environment of MCF7 cells does not sufficiently protect NAT1 from peroxynitrite-dependent inactivation and that only high concentrations of reduced glutathione could significantly protect NAT1. Thus, cellular generation of peroxynitrite may contribute to carcinogenesis and tumor progression by weakening key cellular defense enzymes such as NAT1.
Journal of Biological Chemistry 03/2004; 279(9):7708-14. · 4.77 Impact Factor
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ABSTRACT: Arylamine N-acetyltransferases (NATs) play an important role in the detoxification and metabolic activation of a variety of aromatic
xenobiotics, including numerous carcinogens. Both of the human isoforms, NAT1 and NAT2, display interindividual variations,
and associations between NAT genotypes and cancer risk have been established. Contrary to NAT2, NAT1 has a ubiquitous tissue
distribution and has been shown to be expressed in cancer cells. Given that the activity of NAT1 depends on a reactive cysteine
that can be a target for oxidants, we studied whether peroxynitrite, a highly reactive nitrogen species involved in human
carcinogenesis, could inhibit the activity of endogenous NAT1 in MCF7 breast cancer cells. We show here that exposure of MCF7
cells to physiological concentrations of peroxynitrite and to a peroxynitrite generator (3-morpholinosydnonimine N-ethylcarbamide, or SIN1) leads to the irreversible inactivation of NAT1 in cells. Further kinetic and mechanistic analyses
using recombinant NAT1 showed that the enzyme is rapidly (kinact = 5 × 104 m–1·s–1) and irreversibly inactivated by peroxynitrite. This inactivation is due to oxidative modification of the catalytic cysteine.
We conclude that the reducing cellular environment of MCF7 cells does not sufficiently protect NAT1 from peroxynitrite-dependent
inactivation and that only high concentrations of reduced glutathione could significantly protect NAT1. Thus, cellular generation
of peroxynitrite may contribute to carcinogenesis and tumor progression by weakening key cellular defense enzymes such as
NAT1.
Journal of Biological Chemistry 02/2004; 279(9):7708-7714. · 4.77 Impact Factor
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ABSTRACT: 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.
Current Pharmaceutical Design 02/2004; 10(20):2519-24. · 3.87 Impact Factor
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Progrès en Urologie 12/2003; 13(5 Suppl 2):1194-6. · 0.58 Impact Factor
