Study on Genotyping Polymorphism and Sequencing of N-Acetyltransferase 2 (NAT2) among Al-Ahsa Population.

One of the well-studied phase II drug metabolizing enzymes is N-acetyltransferase 2 (NAT2) which has an essential role in the detoxification and metabolism of several environmental toxicants and many therapeutic drugs like isoniazid (antituberculosis, TB) and antimicrobial sulfonamides. According to the variability in the acetylation rate among different ethnic groups, individuals could be classified into slow, intermediate, and fast acetylators; these variabilities in the acetylation rate are a result of single nucleotide polymorphisms (SNPs) in the coding sequence of NAT2. The variety of NAT2 acetylation status is associated with some diseases such as bladder cancer, colorectal cancer, rheumatoid arthritis, and diabetes mellitus. The main objectives of this research are to describe the genetic profile of NAT2 gene among the people of the Al-Ahsa region, to detect the significant SNPs of this gene, to determine the frequency of major NAT2 alleles and genotypes, and then categorize them into fast, intermediate, and slow acetylators. Blood samples were randomly collected from 96 unrelated people from Al-Ahsa population, followed by DNA extraction then amplifying the NAT2 gene by polymerase chain reaction (PCR); finally, functional NAT2 gene (exon 2) was sequenced using the Sanger sequencing method. The well-known seven genetic variants of NAT2 gene are 191G>A, 282C>T, 341T>C, 481C>T, 590G>A, 803A>G, and 857G>A were detected with allele frequencies 1%, 35.4%, 42.7%, 41.1%, 29.2%, 51%, and 5.7%, respectively. The most common NAT2 genetic variant among Al-Ahsa population was 803A>G with a high frequency 0.510 (95% confidence interval 0.44-0.581) followed by 341T>C 0.427 (95% confidence interval 0.357-0.497). The most frequent two haplotypes of NAT2 were NAT2∗6C (25.00%) and NAT2∗5A (22.92%) which were classified as a slow acetylators. According to trimodal distribution of acetylation activity, the predicted phenotype of Al-Ahsa population was found to be 5.21% rapid acetylators, 34.38% intermediate acetylators, and 60.42% were slow acetylators. In addition, this study found four novel haplotypes NAT2∗5TB, NAT2∗5AB, NAT2∗5ZA, and NAT2∗6W which were slow acetylators. This study revealed a high frequency of the NAT2 gene with slow acetylators (60.42%) in Al-Ahsa population, which might alter the drug's efficacy and vulnerability to some diseases.

The actinobacterium Tsukamurella paurometabola has a functionally divergent arylamine N-acetyltransferase (NAT) homolog.

Actinobacteria in the Tsukamurella genus are aerobic, high-GC, Gram-positive mycolata, considered as opportunistic pathogens and isolated from various environmental sources, including sites contaminated with oil, urban or industrial waste and pesticides. Although studies look into xenobiotic biotransformation by Tsukamurella isolates, the relevant enzymes remain uncharacterized. We investigated the arylamine N-acetyltransferase (NAT) enzyme family, known for its role in the xenobiotic metabolism of prokaryotes and eukaryotes. Xenobiotic sensitivity of Tsukamurella paurometabola type strain DSM 20162T was assessed, followed by cloning, recombinant expression and functional characterization of its single NAT homolog (TSUPD)NAT1. The bacterium appeared quite robust against chloroanilines, but more sensitive to 4-anisidine and 2-aminophenol. However, metabolic activity was not evident towards those compounds, presumably due to mechanisms protecting cells from xenobiotic entry. Of the pharmaceutical arylhydrazines tested, hydralazine was toxic, but the bacterium was less sensitive to isoniazid, a drug targeting mycolic acid biosynthesis in mycobacteria. Although (TSUPD)NAT1 protein has an atypical Cys-His-Glu (instead of the expected Cys-His-Asp) catalytic triad, it is enzymatically active, suggesting that this deviation is likely due to evolutionary adaptation potentially serving a different function. The protein was indeed found to use malonyl-CoA, instead of the archetypal acetyl-CoA, as its preferred donor substrate. Malonyl-CoA is important for microbial biosynthesis of fatty acids (including mycolic acids) and polyketide chains, and the corresponding enzymatic systems have common evolutionary histories, also linked to xenobiotic metabolism. This study adds to accummulating evidence suggesting broad phylogenetic and functional divergence of microbial NAT enzymes that goes beyond xenobiotic metabolism and merits investigation.

Molecular and Functional Characterization of N-Acetyltransferases NAT1 and NAT2 in Cynomolgus Macaque.

Arylamine N-acetyltransferases (NATs) are drug-metabolizing enzymes essential for the metabolism of endogenous substrates and xenobiotics, and their molecular characteristics have been extensively investigated in humans, but not in cynomolgus macaques, nonhuman primate species important for drug metabolism studies. In this study, cynomolgus NAT1 and NAT2 cDNAs were isolated from livers. NAT1 and NAT2 were characterized by molecular analyses and drug-metabolizing assays. A total of 9 transcript variants were found for cynomolgus NAT1, similar to human NAT1, and contained 1-4 exons with the coding region largely conserved with human NAT1. Genomic organization was similar between cynomolgus macaques and humans. Cynomolgus NAT1 and NAT2 amino acid sequences showed high sequence homology (95% and 89%, respectively) and showed close relationships with human NAT1 and NAT2 in a phylogenetic tree. Cynomolgus NAT2 mRNA was predominantly expressed in liver among the 10 different tissues analyzed, followed by kidney and jejunum. In contrast, cynomolgus NAT1 mRNA showed more ubiquitous expression with relatively more abundant expression in liver, kidney, and jejunum, along with testis. Metabolic assays using recombinant proteins showed that cynomolgus NAT1 and NAT2 metabolized human NAT substrates, including p-aminobenzoic acid, sulfamethazine, isoniazid, and 2-aminofluorene. Interestingly, p-aminobenzoic acid and isoniazid were largely metabolized by NAT1 and NAT2, respectively, in cynomolgus macaques and humans; sulfamethazine, a human NAT2 substrate, was metabolized by both NAT enzymes in cynomolgus macaques. These results suggest molecular and enzymatic similarities of NAT1 and NAT2 between cynomolgus macaques and humans, despite some small differences in substrate specificity of the enzymes.

A web server for inferring the human N-acetyltransferase-2 (NAT2) enzymatic phenotype from NAT2 genotype.

UNLABELLED: N-acetyltransferase-2 (NAT2) is an important enzyme that catalyzes the acetylation of aromatic and heterocyclic amine carcinogens. Individuals in human populations are divided into three NAT2 acetylator phenotypes: slow, rapid and intermediate. NAT2PRED is a web server that implements a supervised pattern recognition method to infer NAT2 phenotype from SNPs found in NAT2 gene positions 282, 341, 481, 590, 803 and 857. The web server can be used for a fast determination of NAT2 phenotypes in genetic screens. AVAILABILITY: Freely available at http://nat2pred.rit.albany.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Arylamine N-acetyltransferases in prokaryotic and eukaryotic genomes: a survey of public databases.

Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes found in prokaryotes and eukaryotes. NATs have been characterized in bacteria (Bacilli, Mycobacteria, Salmonella etc.), laboratory animals (chicken, rabbit, rodents etc.) and humans, where the NAT loci occupy 230 kilobases on chromosome 8p22. Our previous comprehensive search for NAT genes involved 416 genomes (340 prokaryotic, 76 eukaryotic) and identified NAT homologues in several taxa, while also reporting on taxa that appeared to lack NAT genes [Boukouvala, S. and Fakis, G. (2005) Drug Metab. Rev. 37(3), 511-564]. Here, we present an update of this genomic search, covering 2138 genomes (1674 prokaryotic, 464 eukaryotic), of which 1167 (986 prokaryotic, 181 eukaryotic) were accessible using the advanced search algorithm tBLASTn. We have reconstructed the full-length open reading frames for putative proteins with sequence homology and features characteristic of NAT from 274 bacterial genomes (31 actinobacteria, 6 bacteroidetes/chlorobi, 2 cyanobacteria, 65 firmicutes and 170 proteobacteria) and 27 animals (1 sea-urchin, 5 fishes, 1 lizard, 1 bird and 19 mammals). Partial NAT sequences were recovered from several other organisms, including fungi, where NAT genes were found in 30 ascomycetes and 2 basidiomycetes. No NATs were found in arhaea, plants and lower invertebrates (insects and worms), while it is also uncertain whether NAT genes exist in protista. We present comparative genomic and phylogenetic analyses of the identified NAT homologues and announce a new database that will maintain information on non-human NATs and will provide recommendations for a standardized nomenclature, along the lines of the NAT Gene Nomenclature Committee.

Functional and structural characterization of the arylamine N-acetyltransferase from the opportunistic pathogen Nocardia farcinica.

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.

Heterologous co-expression of human cytochrome P450 1A2 and polymorphic forms of N-acetyltransferase 2 for studies on aromatic amines in V79 Chinese hamster cells.

V79 Chinese hamster cells were genetically engineered for the stable co-expression of human cytochrome P450 1A2 and the polymorphic N-acetyltransferase 2 alleles *4, *5B, *6A and *13, in order to generate an in vitro tool for studying the metabolism-dependent toxicity of aromatic amines. N-acetyltransferase 2*4-encoding cDNA was generated by the polymerase chain reaction (PCR) with defined primers from the genomic DNA of a human liver donor homozygous for *4, and served as a template to generate the *5B, *6A and *13 isoforms by site-directed mutagenesis. Human cytochrome P450 (CYP) 1A2-encoding cDNA was generated by the PCR from genomic DNA of the recombinant V79MZh1A2 cell line. All the cDNAs were inserted into a CMV promoter-containing plasmid in conjunction with the selectable marker genes, neomycin and hydromycin. The recombinant expression plasmids were transfected for stable integration into the genomic DNA of the V79 cells. Several cellular clones were obtained and checked for the genomic integration of intact cDNAs with the PCR on the genomic DNA of the recombinant cells. Stable expression was confirmed by the reverse transcriptase PCR (RT-PCR) on RNA preparations. Metabolic function was tested with ethoxyresorufin as a marker substrate for CYP1A2, and 2-aminofluorene and N-sulphametazine for N-acetyltransferase activity, and compared to data obtained from biological samples. 7-Ethoxyresorufin-O-deethylase activities ranged from 0.2 to 4 pmol resorufin/min/mg total protein. The N-acetylation of sulphametazine ranged from 0.07 to 1.7 nmol N-acetyl-sulphametazine/mg total protein/min. Selected clones showing activities in the range of physiological activities were submitted to metabolism dependent mutagenicity studies. In particular, the polymorphism-dependent N-acetylation of 2-aminofluorene and the role of CYP1A2 and N-acetyltransferase in the mutagenicity of 2-aminofluorene, were investigated. Surprisingly, the mutagenicity of 2-aminofluorene is dramatically reduced in V79 cells co-expressing CYP1A2 and N-acetyltransferase, compared to V79 cells expressing CYP1A2 only, pointing to a significant species-dependent difference in the metabolic activation of aromatic amines between rats and humans.

Identification and functional characterization of arylamine N-acetyltransferases in eubacteria: evidence for highly selective acetylation of 5-aminosalicylic acid.

Arylamine N-acetyltransferase activity has been described in various bacterial species. Bacterial N-acetyltransferases, including those from bacteria of the gut flora, may be involved in the metabolism of xenobiotics, thereby exerting physiopathological effects. We characterized these enzymes further by steady-state kinetics, time-dependent inhibition, and DNA hybridization in 40 species, mostly from the human intestinal microflora. We report for the first time N-acetyltransferase activity in 11 species of Proteobacteriaceae from seven genera: Citrobacter amalonaticus, Citrobacter farmeri, Citrobacter freundii, Klebsiella ozaenae, Klebsiella oxytoca, Klebsiella rhinoscleromatis, Morganella morganii, Serratia marcescens, Shigella flexneri, Plesiomonas shigelloides, and Vibrio cholerae. We estimated apparent kinetic parameters and found that 5-aminosalicylic acid, a compound efficient in the treatment of inflammatory bowel diseases, was acetylated with a catalytic efficiency 27 to 645 times higher than that for its isomer, 4-aminosalicylic acid. In contrast, para-aminobenzoic acid, a folate precursor in bacteria, was poorly acetylated. Of the wild-type strains studied, Pseudomonas aeruginosa was the best acetylator in terms of both substrate spectrum and catalytic efficiency. DNA hybridization with a Salmonella enterica serovar Typhimurium-derived probe suggested the presence of this enzyme in eight proteobacterial and four gram-positive species. Molecular aspects together with the kinetic data suggest distinct functional features for this class of microbial enzymes.

N-acetyltransferase polymorphisms and colorectal cancer: a HuGE review.

The two expressed genes coding for N-acetyltransferase (NAT) activity, NAT1 and NAT2, are located on chromosome 8 at 8p21.3-23.1 and are polymorphic. Both enzymes are capable of N-acetylation, O-acetylation, and N,O-acetylation and are implicated in the activation and detoxification of known carcinogens. Single base-pair substitutions in NAT2 tend to occur in combination with other substitutions within the gene. As yet, less work has been done to characterize NAT1 allelic variants. Various methods for the detection of the reported polymorphisms exist. It is important to select a method that is appropriate to the population being studied. The functional significance of many NAT allelic variants has not been determined. Geographic and ethnic variation in the frequency of NAT2 genotypes associated with fast or intermediate acetylation has been observed. Insufficient data for NAT1 genotypes are available to reveal a clear geographic pattern. No consistent association has been found between acetylator phenotype or genotype and colorectal cancer. The lack of consistency can in part be accounted for by methodological factors, including limited statistical power. Possible interactions between the NAT genes and either environmental exposures or other polymorphic genes encoding xenobiotic metabolizing enzymes have been investigated in only a minority of these studies, and these studies have lacked statistical power to detect interactions.

Polymorphic NATs and cancer predisposition.

The acetylation polymorphism, discovered 40 years ago, holds a special place as one of the first described examples of a pharmacogenetic defect affecting xenobiotic biotransformation capacity in human populations. The genetically determined N-acetyltransferase activity is involved in activation/inactivation reactions of numerous xenobiotics. Therefore, it has been suggested that slow acetylator status may modify the individual responses to various chemicals. In humans, two genes, NAT1 and NAT2, are responsible for N-acetyltransferase activity. To date several allelic variants of both NAT1 and NAT2 have been detected, and it has been suggested that some of them modify individual susceptibility to cancer. Slow NAT2 acetylation capacity has been suggested as conferring increased risk of bladder, breast, liver and lung cancers, and decreased risk of colon cancer, whereas a prominent change in the NAT1 gene, putatively associated with increased NAT1 activity, has been suggested as increasing the risk of bladder and colon cancer and decreasing that of lung cancer. While three of the NAT2 variants have been shown to account for most of the slow NAT2 acetylator genotypes in Caucasians, less complete data are available on how the NAT1 variants modify NAT1 activity in vivo. This review discusses present knowledge on NAT polymorphisms, particularly in relation to individual cancer predisposition.

1998 International Meeting on the Arylamine N-Acetyltransferases: synopsis of the workshop on nomenclature, biochemistry, molecular biology, interspecies comparisons, and role in human disease risk.

On October 22-24, 1998, a workshop was held at Kuranda, Queensland, Australia. The purpose of the meeting was to provide a forum for discussion of a number of diverse research areas of the biochemistry and molecular biology of arylamine N-acetyltransferases and to foster collaboration among several major groups of investigators around the world. In addition, participants were asked to consider how the nomenclature system for arylamine N-acetyltransferases could be strengthened to cope with the burgeoning number of new alleles discovered in the last 3 years. The full text of all meeting abstracts can be viewed at.

Nomenclature for N-acetyltransferases.

A consolidated classification system is described for prokaryotic and eukaryotic N-acetyltransferases in accordance with the international rules for gene nomenclature. The root symbol (NAT) specifically identifies the genes that code for the N-acetyltransferases, and NAT* loci encoding proteins with similar function are distinguished by Arabic numerals. Allele characters, denoted by Arabic numbers or by a combination of Arabic numbers and uppercase Latin letters, are separated from gene loci by an asterisk, and the entire gene-allele symbols are italicized. Alleles at the different NAT* loci have been numbered chronologically irrespective of the species of origin. For designation of genotypes at a single NAT* locus, a slash serves to separate the alleles; in phenotype designations, which are not italicized, alleles are separated by a comma.

N-hydroxyarylamine O-acetyltransferase in hamster liver: identity with arylhydroxamic acid N,O-acetyltransferase and arylamine N-acetyltransferase.

N-Hydroxyarylamine O-acetyltransferase, arylhydroxamic acid N,O-acetyltransferase, and arylamine N-acetyltransferase in hamster liver cytosol were co-purified almost to electrophoretical homogeneity by ion exchange chromatography on DEAE-cellulose, gel filtration on Cellulofine GCL-2000-sf and high-performance KB-hydroxyapatite chromatography. The molecular weight of the acetyltransferase was estimated to be 33,000 by gel filtration and SDS-polyacrylamide gel electrophoresis. The three acetyltransferase activities were inhibited by iodoacetamide, pentachlorophenol, and 1-nitro-2-naphthol. Furthermore, 2-aminofluorene, a substrate for arylamine N-acetyltransferase, inhibited the reactions of N-hydroxyarylamine O-acetyl transfer and arylhydroxamic acid N,O-acetyl transfer. These results suggest that the same enzyme catalyzes the three types of acetyl transfer reactions. The acetyltransferase could activate N-hydroxyarylamines, such as 2-hydroxyamino-6-methyldipyrido[1,2-alpha:3',2'-d]imidazole, 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole, and N-hydroxy-2-aminofluorene, to the corresponding N-acetoxyarylamines, which are capable of binding to nucleic acid. Polyguanylic acid was most efficiently modified by the N-acetoxyarylamines formed by the acetyltransferase.