N-hydroxyarylamine O-acetyltransferase Research Articles

Reversibly acetylated lysine residues play important roles in the enzymatic activity of Escherichia coli N‐hydroxyarylamine O‐acetyltransferase

CobB is a bacterial NAD(+)-dependent protein deacetylase. Although progress has been made in functional studies of this protein in recent years, its substrates and biological functions are still largely unclear. Using proteome microarray technology, potential substrates of Escherichia coli CobB were screened and nine proteins were identified, including N-hydroxyarylamine O-acetyltransferase (NhoA). In vitro acetylation/deacetylation of NhoA was verified by western blotting and mass spectrometry, and two acetylated lysine residues were identified. Site-specific mutagenesis experiments showed that mutation of each acetylated lysine decreased the acetylation level of NhoA in vitro. Further analysis showed that variant NhoA proteins carrying substitutions at the two acetylated lysine residues are involved in both the O-acetyltransferase and N-acetyltransferase activity of NhoA. Structural analyses were also performed to explore the effects of the acetylated lysine residues on the activity of NhoA. These results suggest that reversible acetylation may play a role in the activity of Escherichia coli NhoA.

Purification and biochemical properties of an N-hydroxyarylamine O-acetyltransferase from Escherichia coli

The N-hydroxyarylamine O-acetyltransferase of Escherichia coli has been expressed as a histidine tagged fusion protein and purified using immobilized nickel column chromatography. The molecular mass of the histidine tagged N-hydroxyarylamine O-acetyltransferase was estimated to be 60.0 kDa by gel filtration and 34.0 kDa by SDS–PAGE and DNA sequence, suggesting that the native enzyme exists as homo dimer. The catalytic properties were investigated using o-aminobenzoic acid as a substrate. No difference in acetyltransfer activity was observed between histidine tagged protein and untagged enzyme. Kinetic studies indicated a ping-pong bi bi mechanism of the catalysis. Inhibition by N-ethylmaleimide and salicylic acid was competitive with o-aminobenzoic acid and non-competitive with acetyl-CoA.

New O-acetyltransferase-deficient Ames Salmonella strains generated by specific gene disruption

CoASAc-dependent N-hydroxyarylamine O-acetyltransferase (OAT) is an enzyme involved in the intracellular metabolic activation of N-hydroxyarylamines derived from mutagenic nitroarenes and aromatic amines. The oat gene encoding the enzyme of S. typhimurium TA98 and TA100 was specifically disrupted and the sensitivities of the resulting strains, i.e., YG7130 and YG7126, to mutagens were compared with those of the conventional oat-deficient strains, i.e., TA98/1,8DNP6 and TA100/1,8DNP, respectively. The new oat-deficient strains and the conventional strains exhibited similar sensitivity against most of the chemicals tested: both strains YG7130 and strain TA98/1,8-DNP6 were resistant to mutagenicity by 1,8-dinitropyrene (1,8-DNP), 1-nitropyrene, 2-amino-6-methyldipyrido[1,2-α:3′,2′-d]imidazole (Glu-P-1) and 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ); neither strain YG7130 nor strain TA98/1,8-DNP6 was resistant to the mutagenicity of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2); strain YG7126 and strain TA100/1,8-DNP were refractory to the mutagenicity of 1,8-DNP. However, the order of the sensitivity against 2-nitrofluorene (2-NF) was TA98>YG7130>TA98/1,8-DNP6 and TA100>YG7126>TA100/1,8-DNP. Since the strains YG7130 and YG7126 have chloramphenicol resistance (Cmr) gene in place of the chromosomal oat gene for gene disruption, the possible involvement of chloramphenicol acetyltransferase (CAT) encoded by the Cmr gene in the activation of 2-NF was examined. Strikingly, introduction of plasmid pACYC184 carrying the Cmr gene alone substantially enhanced the sensitivity of the conventional oat-deficient strains to 2-NF. These results suggest that the new strains as well as the conventional strains are useful to assess the roles of OAT in the metabolic activation of nitroaromatics and aromatic amines in S. typhimurium, and also that CAT has the ability to activate N-hydroxy aromatic amines to mutagens.

Mutagenic analysis of 2,3-diaminophenazine and 2-amino-3-hydroxyphenazine in Salmonella strains expressing different levels of O-acetyltransferase with and without plant and mammalian activation

2,3-Diaminophenazine (DAP) and 2-amino-3-hydroxyphenazine (AHP) are products generated from oxidative-type phenylenediamine hair dyes and are also present in pesticide formulations as contaminants. Earlier studies demonstrated that DAP and AHP were mutagenic in Salmonella typhimurium strains after mammalian microsomal activation. Plant systems can activate structurally similar arylamines. S. typhimurium strains have been developed that express elevated levels of acetyl-CoA: N-hydroxyarylamine O-acetyltransferase (OAT). O-acetyltransferase expression is necessary for the generation of the ultimate arylamine promutagen after plant activation. A number of arylamines including 2-aminofluorene, benzidine and 4-aminobiphenyl were activated by plant cells into mutagens in the OAT over-expressing S. typhimurium strain, YG1024. The objectives of this research were to examine the mutagenicity of DAP and AHP with mammalian or plant activation in Salmonella strains with different acetyltransferase activities. The hypothesis tested was whether and to what degree a metabolite of DAP or AHP could serve as a substrate for bacterial O-acetyltransferase and induce mutation in Salmonella. DAP and AHP without activation induced both frameshift and base pair substitution mutations in S. typhimurium strains that exhibited elevated levels of O-acetyltransferase activity. The mutagenicity of DAP and AHP were greatly enhanced with mammalian hepatic microsomal activation resulting in a preferential induction of frameshift mutations. With the hisD3052 allele as the gene target, S9-activated DAP induced frameshift mutations in YG1024 and TA98 as well as the OAT deficient strain TA98/1,8-DNP 6. S9-activated AHP induced mutation only in the OAT over-expressing strain, YG1024. With the hisG46 allele, O-acetyltransferase activity was necessary for the metabolism of DAP and AHP to products that induce base pair substitution mutations. An intriguing finding of this work was the

N-hydroxyarylamine O-acetyltransferase of Salmonella typhimurium: proposal for a common catalytic mechanism of arylamine acetyltransferase enzymes.

Acetyl-CoA:N-hydroxyarylamine O-acetyltransferase is an enzyme involved in the metabolic activation of N-hydroxyarylamines derived from mutagenic and carcinogenic aromatic amines and nitroarenes. The O-acetyltransferase gene of Salmonella typhimurium has been cloned, and new Ames tester substrains highly sensitive to mutagenic aromatic amines and nitroarenes have been established in our laboratory. The nucleotide sequence of the O-acetyltransferase gene was determined. There was an open reading frame of 843 nucleotides coding for a protein with a calculated molecular weight of 32,177, which was close to the molecular weight of the O-acetyltransferase protein determined by using the maxicell technique. Only the residue of Cys69 in O-acetyltransferase of S. typhimurium and its corresponding residue (Cys68) in N-acetyltransferase of higher organisms were conserved in all acetyltransferase enzymes sequenced so far. The amino acid sequence Arg-Gly-Gly-X-Cys, including the Cys69, was highly conserved. A mutant O-acetyltransferase of S. typhimurium, which contained Ala69 instead of Cys69, no longer showed the activities of O- and N-acetyltransferase. These results suggest that the Cys69 of S. typhimurium and the corresponding cysteine residues of the higher organisms are essential for the enzyme activities as an acetyl-CoA binding site. We propose a new catalytic model of acetyltransferase for S. typhimurium and the higher organisms.

Construction of Syrian Hamster Lines Congenic at the Polymorphic Acetyltransferase Locus ( NAT2): Acetylator Genotype-Dependent N- and O-Acetylation of Arylamine Carcinogens

Congenic Bio. l.5/H- NAT2 Syrian hamster lines were constructed by introducing the NAT2 r gene from MHA/SsLak inbred hamsters into a background BIO 1.5 Syrian inbred hamster line. Genetic identity of the Bio. 1.5/H- NAT2 congenic lines and nonidentity with the previously constructed Bio. 82.73/H- Pat congenic lines were determined by "DNA fingerprints" of genomic DNA derived from the different hamster lines. The N-acetylation capacity of the Bio. 1.5/H- NAT2 congenic hamster lines was clearly NAT2-dependent both in vivo and in vitro, with highest levels expressed in Bio. 1.5/H- NAT2 r homozygous rapid acetylators, intermediate levels in Bio. l.5/H- NAT2 r/ NAT2 s heterozygous acetylators, and lowest levels in Bio. 1.5/ H- NAT2 s homozygous slow acetylators. The NAT2-dependent expression of N-acetyltransferase activity was evident toward p-aminobenzoic acid, 4-aminophenol, 2-aminofluorene, 4-aminobiphenyl, β-naphthylamine, and 3,2′-dimethyl-4-amino-biphenyl in liver, kidney, colon, lung, and urinary bladder cytosols. The polymorphic acetyltransferase (NAT2) and the monomorphic acetyltransferase (NAT1) were isolated from hepatic cytosols and tested separately for their ability to catalyze arylamine N-acetyltransferase and N-hydroxyarylamine O-acetyltransferase activities. Both arylamine N-acetylation and N-hydroxyarylamine O-acetylation were clearly acetylator genotype-dependent when catalyzed by NAT2, and both were clearly acetylator genotype-independent when catalyzed by NAT1. NAT2/NAT1 activity ratios varied with the particular arylamine substrate acetylated. These studies show an important role for NAT2 acetylator genotype in Syrian hamster carcinogenic arylamine metabolism and confirm its role in the metabolic activation of N-hydroxyarylamines. The Bio. 1.5/H- NAT2 congenic lines provide a new model for investigating the preciserole of the NAT2 gene locus in arylamine metabolism and toxicity.

Characterization of stable high molecular weight mutagenic product(s) of plant-activated m-phenylenediamine

Monocyclic aromatic amines are environmental contaminants and many are promutagens and procarcinogens. Cultured tobacco cells, strain TX1, activated m-phenylenediamine into a frameshift mutagen that reverted the hisD3052 allele in Salmonella typhimurium strains TA98 and YG1024. However, the plant-activated products were refractory in strain TA98/1,8-DNP 6. This indicated that these plant-activated products were substrates for bacterial acetyl-CoA: N-hydroxyarylamine O-acetyltransferase. A stable, high molecular weight ( > 300 kDa) proximal mutagen was isolated by molecular ultrafiltration membranes. No parent compound was associated with the isolated mutagenic fraction. The high molecular weight fraction induced mutation in S. typhimurium strains TA98, YG1021 and YG1024. From these data we propose a model for the plant-activation of aromatic amine promutagens.

Comparison of the sensitivity of Salmonella typhimurium strains YG1024 and YG1012 for detecting the mutagenicity of aromatic amines and nitroarenes

Salmonella typhimurium YG1024 is a derivative of S. typhimurium TA98 with a high level of N-hydroxyarylamine O-acetyltransferase (OAT) activity. We have demonstrated that this strain is highly sensitive to the mutagenic actions of N-hydroxyarylamines derived from aromatic amines and nitroarenes. In this paper, we compared the sensitivities of YG1024 with those of S. typhimurium YG1012, which has about 4 times higher OAt activity than YG1024 but lacks plasmid pKM101. It turned out that YG1024 was more sensitive to the mutagenic actions of 1-aminonaphthalene, 1-nitropyrene, 1,8-dinitropyrene and 2-nitronaphthalene than YG1012 and showed comparable sensitivity to 2-hydroxyacetylaminofluorene, 2-aminoanthracene and 2-amino-6-methyldipyridol[1,2-α:3′,2′-d]imidazole (Glu-P-1) to YG1012. These results suggested that YG1024 is more suitable than YG1012 for the efficient detection of mutagenic aromatic amines and nitroarenes.

Involvement of Cys69 residue in the catalytic mechanism of N-hydroxyarylamine O-acetyltransferase of Salmonella typhimurium. Sequence similarity at the amino acid level suggests a common catalytic mechanism of acetyltransferase for S. typhimurium and higher organisms.

Acetyl-coenzyme A:N-hydroxyarylamine O-acetyltransferase is ubiquitous in species ranging from bacteria to mammals and is involved in the metabolic activation of N-hydroxyarylamines derived from mutagenic and carcinogenic aromatic amines and nitroarenes. The nucleotide sequence of the gene that encodes O-acetyltransferase of Salmonella typhimurium was determined, and its deduced amino acid sequence was compared with those of arylamine N-acetyltransferases (EC 2.3.1.5) of higher organisms. The gene of S. typhimurium encoded a protein with a calculated molecular weight of 32,177. Chromosome DNA of S. typhimurium TA1538/1,8-DNP, an O-acetyltransferase-deficient strain, had a -1 frameshift mutation of CCC to CC at the coding region. To date, 11 genes encoding N-acetyltransferase have been cloned from human, rabbit, hamster, and chicken. The N-terminal region of O-acetyltransferase of S. typhimurium with about 170 amino acids showed 25-33% homology with the corresponding region of N-acetyltransferase of the higher organisms. Of the 5 cysteine residues of O-acetyltransferase of S. typhimurium, Cys69 was the only residue that was conserved in all N-acetyltransferases of the higher organisms. The amino acid sequence of Arg-Gly-Gly-X-Cys, including the Cys69, was highly conserved. The mutant O-acetyltransferase of S. typhimurium, which contained Ala69 instead of Cys69, no longer showed the activities of O- and N-acetyltransferase. These results suggest that the Cys69 of S. typhimurium and its corresponding cysteine residues of the higher organisms are essential for the enzyme activities as acetyl-coenzyme A-binding sites.

Mutagenicity of 30 chemicals in Salmonella typhimurium strains possessing different nitroreductase or O-acetyltransferase activities

Salmonella typhimurium YG1021, YG1024, YG1026 and YG1029 are new derivatives of the Ames tester strains TA98 and TA100, with elevated ‘classical’ nitroreductase or acetyl-CoA: N-hydroxyarylamine O-acetyltransferase level. Thirty mutagens with different structures were tested using these strains and the sensitivities were compared with those of the conventional strains and of the enzyme-deficient strains. Elevated O-acetyltransferase activity of the indicator strains specifically increased their ability to detect the mutagenicity of aromatic nitro, amino and hydroxylamino compounds, whereas the strains with high nitroreductase activity were very sensitive to some nitroaromatics. The combined use of the isogenic tester strains with different metabolic capacities was quite useful to assess the intracellular metabolic activation and detoxification mechanisms of chemical mutagens.

Sensitive method for the detection of mutagenic nitroarenes and aromatic amines: new derivatives of Salmonella typhimurium tester strains possessing elevated O-acetyltransferase levels

Acetyl-CoA: N-hydroxyarylamine O-acetyltransferase is an enzyme involved in the intracellular metabolic activation of arylhydroxylamines derived from mutagenic nitroarenes and aromatic amines. The acetyltransferase gene of Salmonella typhimurium TA1538 was cloned into pBR322 and the plasmids harboring the gene were introduced into TA98 and TA100. The resulting strains (YG1024 and YG1029) had about 100 times higher 2-hydroxyamino-6-methyldipyrido[1,2- a: 3′, 2′- d]-imidazole ( N-hydroxy-Glu-P-1) O-acetyltransferase activity than TA1538 containing pBR322, and were extremely sensitive to the mutagenic actions of 2-nitrofluorene, 1-nitropyrene, 1,8-dinitropyrene, 2-amino-6-methyldipyrido[1,2- a: 3′, 2- d]-imidazole (Glu-P-1), 2-aminofluorene and 2-aminoanthracene. These results indicate that the new strains permit the efficient detection of the mutagenicity of environmental nitroarenes and aromatic amines.

A new assay for N-hydroxyarylamine O-acetyltransferase: Reduction of N-hydroxyarylamines through N-acetoxyarylamines

A simple fluorometric assay for N-hydroxyarylamine O-acetyltransferase is described and compared with a nucleic acid-binding assay. The assay method is based on the finding that the highly mutagenic 2-hydroxyamino-6-methyldipyrido[1,2- a:3′,2′- d]imidazole (N-OH-Glu-P-1) was reduced to the corresponding amine, 2-amino-6-methyldipyrido[1,2- a:3′,2′- d]imidazole (Glu-P-1), through N-acetoxy-Glu-P-1 as the reactive intermediate in the presence of N-hydroxyarylamine O-acetyltransferase, acetyl-CoA, and a sulfhydryl compound. The formation of Glu-P-1 was determined by its characteristic fluorescence intensity at 445 nm with excitation wavelength at 376 nm. The reductive reaction was inhibited by the addition of tRNA, DNA, and poly(G), to which the enzymatic product, N-acetoxy Glu-P-1, bound effectively due to its electrophilic nature. Since the fluorometric assay for the O-acetyltransferase is rapid, simple, and sensitive as compared with the nucleic acid-binding method using radioisotope-labeled N-hydroxyarylamine, this method is applicable to the general assay for the formation of reactive N-acetoxy-Glu-P-1.

Metabolic activation of mutagenic N-hydroxyarylamines by O-acetyltransferase in Salmonella typhimurium TA98

A new enzymatic activation of mutagenic N-hydroxyarylamines is described. An acetyl-CoA dependent enzyme that can activate 2-hydroxyamino-6-methyldipyrido[1,2- a: 3′,2′- d]imidazole ( N-OH-Glu-P-1) to a reactive species capable of binding to nucleic acid was found in a cell-free extract of Salmonella typhimurium TA98 but not in that of TA98/1,8-DNP 6, which shows low sensitivity to the mutagenic activity of N-OH-Glu-P-1. The enzyme was partially purified by streptomycin treatment, ammonium sulfate precipitation, DEAE-cellulose chromatography, and gel filtration chromatography of Sephadex G-150. Its molecular weight was estimated to be approximately 48,000. The directly mutagenic N-hydroxyarylamines, such as N-OH-Glu-P-1, 3-hydroxyamino-1-methyl-5 H-pyrido[4,3- b] indole ( N-OH-Trp-P-2), and N-hydroxy-2-aminofluorene ( N-OH-AF), were transformed to reactive derivatives by partially purified enzyme in the presence of acetyl-CoA. The K m value for acetyl-CoA was calculated to be 3.3 μ m. No acetyl residue, however, was incorporated into nucleic acid adducts. The enzymatic product of N-OH-Glu-P-1 bound most efficiently to polyguanylic acid among four polynucleotides. The enzyme did not show the N,O-acetyltransfer activity of N-hydroxyacetylaminofluorene ( N-OH-AAF). These results indicate that the enzymatic product of N-hydroxyarylamine is N-acetoxyarylamine, and that this enzyme can be called acetyl-CoA: N-hydroxyarylamine O-acetyltransferase. O-Acetyltransferase activity was inhibited by SH-blocking agents, several phenolic compounds, such as pentachlorophenol and 1-nitro-2-naphthol, and an antibiotic thiolactomycin. S. typhimurium mutation studies suggested that the O-acetyltransferase functions as an enzyme activating certain N-hydroxyarylamines within bacterial cells and is involved in the formation of mutants.