Arylamine N-acetyltransferase 2 (NAT2) modifies drug efficacy/toxicity and cancer risk due to its role in bioactivation and detoxification of arylamine and hydrazine drugs and carcinogens. Human NAT2 alleles possess a combination of single nucleotide polymorphisms (SNPs) associated with slow acetylation phenotypes. Clinical and molecular epidemiology studies investigating associations of NAT2 genotype with drug efficacy/toxicity and/or cancer risk are compromised by incomplete and sometimes conflicting information regarding genotype/phenotype relationships. Studies in our laboratory and others have characterized the functional effects of SNPs alone, and in combinations present in alleles or haplotypes. We extrapolate this data generated following recombinant expression in yeast and COS-1 cells to assist in the interpretation of NAT2 structure. Whereas previous structural studies used homology models based on templates of N-acetyltransferase enzyme crystal structures from various prokaryotic species, alignment scores between bacterial and mammalian N-acetyltransferase protein sequences are low (~ 30%) with important differences between the bacterial and mammalian protein structures. Recently, the crystal structure of human NAT2 was released from the Protein Data Bank under accession number 2PFR. We utilized the NAT2 crystal structure to evaluate the functional effects of SNPs resulting in the protein substitutions R64Q (G191A), R64W (C190T), I114T (T341C), D122N (G364A), L137F (A411T), Q145P (A434C), E167K (G499A), R197Q (C590A), K268R (A803G), K282T (A845C), and G286E (G857A) of NAT2. This analysis advances understanding of NAT2 structure-function relationships, important for interpreting the role of NAT2 genetic polymorphisms in bioactivation and detoxification of arylamine and hydrazine drugs and carcinogens.

Arylamine N-acetyltransferases (NATs; EC 2.3.1.5) catalyze both the N-acetylation and O-acetylation of arylamines and Nhydroxyarylamines. Humans possess two functional N-acetyltransferase genes, NAT1 and NAT2, as well as a nonfunctional pseudogene, NATP. Previous studies have identified Nat1 and Nat2 genes in the rat. In this study, we identified and characterized a third rat N-acetyltransferase gene (Nat3) consisting of a single open reading frame of 870 base pairs encoding a 290-amino acid protein, analogous to the previously identified human and rat N-acetyltransferase genes. Rat Nat3 nucleotide sequence was 77.2 and 75.9% identical to human NAT1 and NAT2, respectively. Rat Nat3 amino acid sequence was 68.6 and 67.2% identical to human NAT1 and NAT2, respectively. Rat Nat1, Nat2, and Nat3 were each cloned and recombinantly expressed in Escherichia coli. Recombinant rat Nat3 exhibited thermostability intermediate between recombinant rat Nat1 and Nat2. Recombinant rat Nat3 was functional and catalyzed the N-acetylation of several arylamine substrates, including 3-ethylaniline, 3,5-dimethylaniline, 5-aminosalicylic acid, 4-aminobiphenyl, 4,4Ј-methylenedianiline, 4,4Ј-methylenebis(2-chloroaniline), and 2-aminofluorene, and the O-acetylation of N-hydroxy-4-aminobiphenyl. The relative affinities of arylamine carcinogens such as 4-aminobiphenyl, N-hydroxy-4-aminobiphenyl, and 2-aminofluorene for N-and O-acetylation via recombinant rat Nat3 were comparable with recombinant rat Nat1 and higher than for recombinant rat Nat2. This study is the first to report a third arylamine N-acetyltransferase isozyme with significant functional capacity.

Human N-acetyltransferase 1 (NAT1) alleles are characterized by one or more single nucleotide polymorphisms (SNPs) associated with rapid and slow acetylation phenotypes. NAT1 both activates and deactivates arylamine drugs and carcinogens, and NAT1 polymorphisms are associated with increased frequencies of many cancers and birth defects. The recently resolved human NAT1 crystal structure was used to evaluate SNPs resulting in the protein substitutions R64W, V149I, R187Q, M205V, S214A, D251V, E261K, and I263V. The analysis enhances knowledge of NAT1 structurefunction relationships, important for understanding associations of NAT1 SNPs with genetic predisposition to cancer, birth defects, and other diseases.

ABSTRACT:Arylamine N-acetyltransferases (NATs) play an important role in the metabolism of arylamine and hydrazine drugs and many arylamine procarcinogens. The two human N-acetyltransferases, NAT1 and NAT2, are widely distributed in human tissues and are highly polymorphic. Although many xenobiotic procarcinogens and drugs are known mammalian NAT substrates, it is unclear what physiological roles these enzymes might play, what endogenous substrates they primarily act upon, or the mechanisms underlying the functional effects of specific NAT gene coding region single-nucleotide polymorphisms. Analyses of mammalian NAT protein structures can greatly help to answer these questions. Homology modeling techniques can be used to approximate mammalian NAT structures using known bacterial NAT crystal structures as templates. In comparison to the bacterial template NATs used for homology modeling, mammalian NATs have a 17-residue insert of unknown structure and function. Homology modeling analyses yielded two different alignments (Modeler 8v1 or 3DCof-fee algorithms) that placed this insert in two likely alternative locations. Secondary structure prediction techniques and experimental analyses of a series of human NAT2 mutants with artificial deletions/replacements of the insert region distinguished one of these alternatives as the most likely insert location and provided a better understanding of its structure and function. This study demonstrates both the utility and limitations of computational structural modeling with proteins that differ as much as the mammalian and bacterial NATs.Arylamine N-acetyltransferases (NATs; EC 2.3.1.5) catalyze the N-acetylation of arylamines and hydrazines and O-acetylation of N-hydroxy-arylamines and heterocyclic amines. These reactions are important for the activation and deactivation of exocyclic aminecontaining procarcinogens, and for the metabolism of some pharmaceutical drugs (Weber and Hein, 1985). In a ping-pong bi-bi reaction mechanism, the enzyme first acetylates the active site cysteine using acetyl-coenzyme A, and then transfers the acetyl group to the substrate's exocyclic nitrogen (N-acetylation) or the oxygen of its oxidized exocyclic nitrogen (O-acetylation) (Hein, 1988). Whereas Nacetylation is typically considered a deactivation step, O-acetylation is an activation step, resulting in the formation of reactive arylnitrenium species that can react with DNA to form adducts (Hanna, 1996). Because human NAT genes are highly polymorphic, it is important to understand how different NAT genotypes alter N-acetylation phenotypes, thereby influencing cancer susceptibilities and pharmaceutical drug toxicities (Hein et al., 2000).Expression of human NAT1 and NAT2 is widely distributed throughout body tissues (Barker et al., 2006;Husain et al., 2007). Although many xenobiotic NAT substrates have been discovered, only one potential endogenous substrate, p-aminobenzoylglutamate, has been discovered (Minchin, 1995). Therefore, it is highly probable that other, yet unknown endogenous NAT s...

ABSTRACT:Human N-acetyltransferase 1 (NAT1) and 2 (NAT2) are important phase II enzymes involved in the biotransformation of xenobiotics. In toxicity and carcinogenicity studies, functional polymorphism of rat N-acetyltransferase is considered a model for similar human variability. To accurately quantitate expression of the three rat N-acetyltransferases, we developed sensitive, specific assays for Nat1, Nat2, and Nat3 mRNAs. In male F344 rats, tissue-specific expression varied over a limited range for both Nat1 (ϳ19-fold) and Nat2 (ϳ30-fold), with the highest expression of both genes in colon. Expression of Nat3 mRNA was at least 2 to 3 orders of magnitude less than that of Nat1 or Nat2. Comparison of Nat1 and Nat2 mRNA expression in bladder, colon, liver, and lung of male and female F344 rats detected no significant gender-specific difference. In Sprague-Dawley and F344 rats ranging in age from neonate to mature adult, colon showed a >10-fold increase in Nat2 during the first postnatal month that did not correlate with changes in Nat1. In contrast, Nat2 showed no developmental change in Sprague-Dawley or F344 liver as Nat1 increased modestly. These measures of rat Nat expression confirm that Nat3 expression is negligible and that Nat1 and Nat2 are the primary determinants of arylamine acetylation activity in all tissues tested. The findings demonstrate differential tissue-specific and developmental regulation of the rat Nat1 and Nat2 genes and contribute to more complete understanding of tissue-, gender-, and developmentspecific expression patterns of the cognate N-acetyltransferase genes of humans and other species.Human N-acetyltransferase genes NAT1 and NAT2 exhibit genetic polymorphisms that modify pharmaceutical drug toxicities and influence individual susceptibility to cancers caused by exposure to environmental arylamine and heterocyclic amine procarcinogens (Hein, 2000;Hein et al., 2000). The N-acetyltransferase enzymes participate in the metabolism of these amines by transferring an acetyl group from acetyl-coenzyme A to an exocyclic amine (N-acetylation) or to an oxygen (O-acetylation) after the exocyclic amine has been oxidized by a cytochrome P450 (Hein, 1988). N-Acetylation is typically a metabolic detoxification step, whereas O-acetylation leads to formation of reactive nitrenium intermediates that can form mutagenic DNA adducts (Hanna, 1994).The rat is often used as a model for studies of carcinogenicity and toxicity of arylamines, and functional polymorphism of rat Nat2 is often considered to be a model for similar polymorphism in humans. Understanding the expression and activity of the rat N-acetyltransferases is thus of great value for making relevant extrapolations to humans. The substrate specificity and kinetic properties of the rat and human enzymes and the effects of biological differences and environmental exposures on transcription, translation, and in vivo enzyme activity are all of potential importance. Previous studies reported similarities of rat Nat1 and mouse Nat1 with human NAT2...