Denis M. Grant

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.

Characterization of the microsomal epoxide hydrolase gene in patients with anticonvulsant adverse drug reactions.

Therapy with the aromatic anticonvulsants phenytoin, phenobarbital and carbamazepine has been associated with the occurrence of rare idiosyncratic hypersensitivity reactions. These drugs are thought to be activated to potentially reactive arene oxide (epoxide) metabolites by cytochrome P450-dependent monooxygenation, while liver microsomal epoxide hydrolase (mEH) plays a detoxifying role by converting such reactive intermediates to non-toxic dihydrodiols. Evidence from in vitro lymphocyte toxicity tests and enzyme inhibitor studies has suggested that an inherited defect in mEH function may be responsible for the enhanced drug toxicity observed in affected individuals. To test this hypothesis we designed methods to directly compare mEH gene structure in patients presenting with anticonvulsant adverse reactions and in control subjects in which no in vivo or in vitro toxicity to anticonvulsants could be demonstrated. Southern analysis of peripheral lymphocyte DNA using a full-length mEH cDNA as hybridization probe revealed no gross differences in mEH gene structure in selected patients when compared with DNA samples from unaffected control subjects. The human mEH gene was then cloned and characterized from a control individual. Nine exons were identified within a 22 kb region and sequences of selected regions, including all exons, were determined. Single strand conformation polymorphism (SSCP) analysis was performed on all exonic regions in genomic DNA from each of 26 subjects, including six unrelated patients with previous toxicity to anticonvulsant therapy and seven siblings (three of whom had displayed toxicity). Several distinct SSCP patterns could be observed among the subjects tested, each corresponding to a specific point mutation within one of the amplified fragments of the mEH gene. However, none of the SSCP patterns reflecting point mutations was correlated with the occurrence of anticonvulsant toxicity. From these observations we conclude that a genetic defect altering the structure and function of the mEH protein is unlikely to be responsible for predisposing patients to anticonvulsant adverse reactions.

Structural Basis of Substrate-binding Specificity of Human Arylamine N-Acetyltransferases

The human arylamine N-acetyltransferases NAT1 and NAT2 play an important role in the biotransformation of a plethora of aromatic amine and hydrazine drugs. They are also able to participate in the bioactivation of several known carcinogens. Each of these enzymes is genetically variable in human populations, and polymorphisms in NAT genes have been associated with various cancers. Here we have solved the high resolution crystal structures of human NAT1 and NAT2, including NAT1 in complex with the irreversible inhibitor 2-bromoacetanilide, a NAT1 active site mutant, and NAT2 in complex with CoA, and have refined them to 1.7-, 1.8-, and 1.9-Å resolution, respectively. The crystal structures reveal novel structural features unique to human NATs and provide insights into the structural basis of the substrate specificity and genetic polymorphism of these enzymes.

Variants of N-acetyltransferase NAT1 and a case-control study of colorectal adenomas.

N-acetyltransferase NAT1, together with enzymes CYP1A2 and NAT2, helps convert heterocyclic amines to mutagens. Epidemiologic studies of the association of variants of these enzymes with colorectal cancer may provide indirect support for a heterocyclic amine mechanism. We used single strand conformation polymorphism and heteroduplex analysis to screen fro mutations in the NAT1 coding region in a case-control study (n = 932) of colorectal adenomas, which are precursors to cancer. Thirteen different single-base mutations were found: C97T, C190T, T402C, G445A-G459A-T640G ( a combination of three mutations), C559T, G560A, A613G, A752T, T777C, G781A, and A787G. Function of novel mutations was tested by bacterial production of enzymes and measurements of Km, Vmax, and stability. However, on 24-control individuals and 18 cases carried an inactivating NAT1 mutation. When combined with our data on the NAT2 acetylation polymorphism, we saw no evidence for an association between N-acetyltransferases and prevalence of adenomas. Larger sample sizes are required for further evaluation.

Genotyping of the polymorphic N-acetyltransferase (NAT2*) gene locus in two native African populations.

The hepatic N-acetyltransferase enzyme encoded by the NAT2* gene locus is responsible for the human polymorphic acetylation of numerous arylamine or hydrazine-containing drugs and xenobiotics including AIDS-related therapeutic agents such as isoniazid and sulphonamides. The genetic basis underlying the human acetylation polymorphism has been extensively studied in several populations but native African populations were poorly documented. In the present study, 117 unrelated black Africans, namely Dogons from Mali and Gabonese, were investigated for NAT2* allelic variability and genotype distribution. Thirteen NAT2* alleles were unambiguously identified by combined use of allele-specific reamplifications and restriction endonuclease digestions. Our results confirm the African origin of G191->A substitution in the NAT2* coding region which was previously associated with slow acetylation in African-Americans. The finding of high allelic diversity in the studied populations is consistent with the hypothesis of a single African origin for NAT2*-associated polymorphism. Finally, no excess of the slow acetylator phenotype is predicted in these populations, implying no need for fitting NAT2* polymorphism-sensitive therapies to black Africans, compared to Caucasians.

Identification of amino acids imparting acceptor substrate selectivity to human arylamine acetyltransferases NAT1 and NAT2.

The human arylamine N-acetyltransferases NAT1 and NAT2 catalyse the acetyl-CoA-dependent N- and O-acetylation of primary arylamine and hydrazine xenobiotics and their N-hydroxylated metabolites. We previously used a panel of recombinant NAT1/NAT2 chimaeric proteins to identify linear amino acid segments that have roles in imparting the distinct catalytic specificities to these proteins [Dupret, Goodfellow, Janezic and Grant (1994) J. Biol. Chem. 269, 26830-26835]. These studies indicated that a conserved central region (residues 112-210) distinct from that containing the active-site cysteine residue Cys(68) was important in determining NAT substrate selectivity. In the present study we have refined our analysis through further chimaera generation of this conserved region and by subsequent site-directed mutagenesis of individual amino acids. Enzyme-kinetic analysis of these mutant proteins with the NAT1-selective and NAT2-selective substrates p-aminosalicylic acid (PAS) and sulphamethazine (SMZ) respectively suggests that residues 125, 127 and 129 are important determinants of NAT1-type and NAT2-type substrate selectivity. Modification of Arg(127) had the greatest effect on specificity for PAS, whereas changing Phe(125) had the greatest effect on specificity for SMZ. Selected NAT mutants exhibited K(m) values for acetyl-CoA that were comparable with those of the wild-type NATs, implying that the mutations affected acceptor substrate specificity rather than cofactor binding affinity. Taken together with previous observations, these results suggest that residues 125, 127 and 129 might contribute to the formation of the active-site pocket surrounding Cys(68) and function as important determinants of NAT substrate selectivity.

Pharmacogenetics of the Human Arylamine N-Acetyltransferases

This review briefly describes current understanding of one of the earliest discovered pharmacogenetic polymorphisms of drug biotransformation affecting acetylation of certain homo- and heterocyclic aromatic amines and hydrazines. This so-called acetylation polymorphism arises from allelic variation in one of the two known human arylamine N-acetyltransferase genes, namely NAT2, which results in production of NAT2 proteins with variable enzyme activity or stability. The NAT1 gene locus encodes a structurally related enzyme, NAT1, with catalytic specificity for arylamine acceptor substrates distinct from that exhibited by NAT2. NAT1 function is also genetically variable in human populations. Clinical and toxicological consequences of genetic variation in NAT1 and NAT2 activity are discussed.

Evidence for two closely related isozymes of arylamine N-acetyltransferase in human liver.

Acetyl CoA‐dependent arylamine N‐acetyltransferase (EC 2.3.1.5) is the target of a genetic polymorphism in the metabolism of drugs and carcinogens. N‐Acetyltransferase was purified 1000‐fold from cytosol of human liver and its identity was verified by amino acid sequence homology of two of its tryptic peptides with published rabbit and chicken N‐acetyltransferase sequences. Enzyme activity correlated with the presence of two proteins, NAT‐1 and NAT‐2, with indistinguishable molecular masses (31 kDa). NAT‐1 and NAT‐2 could be separated by anion‐exchange chromatography and were functionally distinguished by their different apparent affinities for the acceptor amine sulfamethazine (SMZ). Antibodies raised against NAT‐1 were able to recognize both isozymes on Western blots.

Homology modelling and structural analysis of human arylamine N-acetyltransferase NAT1: evidence for the conservation of a cysteine protease catalytic domain and an active-site loop.

Arylamine N-acetyltransferases (EC 2.3.1.5) (NATs) catalyse the biotransformation of many primary arylamines, hydrazines and their N-hydroxylated metabolites, thereby playing an important role in both the detoxification and metabolic activation of numerous xenobiotics. The recently published crystal structure of the Salmonella typhimurium NAT (StNAT) revealed the existence of a cysteine protease-like (Cys-His-Asp) catalytic triad. In the present study, a three-dimensional homology model of human NAT1, based upon the crystal structure of StNAT [Sinclair, Sandy, Delgoda, Sim and Noble (2000) Nat. Struct. Biol. 7, 560-564], is demonstrated. Alignment of StNAT and NAT1, together with secondary structure predictions, have defined a consensus region (residues 29-131) in which 37% of the residues are conserved. Homology modelling provided a good quality model of the corresponding region in human NAT1. The location of the catalytic triad was found to be identical in StNAT and NAT1. Comparison of active-site structural elements revealed that a similar length loop is conserved in both species (residues 122-131 in NAT1 model and residues 122-133 in StNAT). This observation may explain the involvement of residues 125, 127 and 129 in human NAT substrate selectivity. Our model, and the fact that cysteine protease inhibitors do not affect the activity of NAT1, suggests that human NATs may have adapted a common catalytic mechanism from cysteine proteases to accommodate it for acetyl-transfer reactions.

Changes in consensus arylamine n- acetyltransferase gene nomenclature

Changes in consensus arylamine N-acetyltransferase (NAT) gene nomenclature determined at the 2007 International NAT Workshop include: (1) Alleles in all species except mouse and rat are all uppercase. For mouse and rat, the first letter is upper case followed by lower case. (2) The nomenclature system is now species-specific. Thus, NAT2(*)1 (chicken), NAT2(*)2 and NAT2(*)3 (rabbit), Nat2(*)8 Nat2(*)9, Nat2(*)22 and Nat2(*)23 (mouse), NAT2(*)15, NAT2(*)16A and NAT2(*)16B (Syrian hamster), and NAT2(*)20, NAT2(*)21A and NAT2(*)21B (rat) are retired and renumbered within a species. A species modifier incorporated into the allele designation is written in upper case Roman font, e.g., (MOUSE)Nat1(*)1 is now the reference Nat1 allele in mouse; and (3) The NAT website also can now be accessed at a webalias address: http://N-acetyltransferasenomenclature.louisville.edu. New NAT alleles should continue to be submitted to the NAT Nomenclature Committee for inclusion on the website to ensure proper categorization and to continue consistency in nomenclature.