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.Arylamine N-acetyltransferases (NATs, EC 2.3.1.5) 2 catalyze the acetyl-CoA-dependent N-acetylation of primary aromatic amines and hydrazines, as well as the O-acetylation of their N-hydroxylated metabolites, thereby influencing the biological activity and toxicity of this class of chemicals (1-3). NAT enzyme activity therefore plays an important role in determining the duration of action and pharmacokinetics of aromatic amine-containing drugs used in clinical therapy, as well as in influencing the balance between detoxification and metabolic activation of aromatic amine procarcinogens (4). In the latter regard, N-acetylation reactions are considered to be protective, because the resulting arylacetamide derivatives are chemically stable, whereas O-acetylation of hydroxylamines produces acetoxy esters that can spontaneously decompose to electrophilic, DNA-binding nitrenium ions (5). Thus a better understanding of the structural features of NATs that contribute to their relative abilities to catalyze such reactions for various amine substrates may be of considerable predictive value in optimizing drug development and biomedical toxicology.Two NATs, NAT1 and NAT2, have been annotated in the human genome. Each of these enzymes is genetically variable in human populations, with variable activity of NAT2 being responsible for the classic acetylation polymorphism that was discovered over half a century ago with the advent of isoniazid therapy for tuberculosis (6 -8). In addition to isoniazid, the disposition of several other therapeutically useful drugs is affected by defective NAT2 function, including hydralazine, phenelzine, procainamide, and some of the sulfonamide antibacterials. NAT1 is highly homologous to NAT2 yet kinetically distinct, such that certain aromatic amines (such as isoniazid and sulfamethazine) are preferentially acetylated by NAT2, whereas others (such as p-aminosalicylic acid and p-aminobenzoic acid) are selective substrates for NAT1. NAT1 is also genetically variable in human populations. Numerous epidemiological studies have reported associations between both NAT1 and NAT2 variation and the occurrence of cancers related to exposure to aromatic...

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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 Cys68 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 Arg127 had the greatest effect on specificity for PAS, whereas changing Phe125 had the greatest effect on specificity for SMZ. Selected NAT mutants exhibited Km 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 Cys68 and function as important determinants of NAT substrate selectivity.

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.

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