Polymorphism p.Val231Ile alters substrate selectivity of drug-metabolizing arylamine N-acetyltransferase 2 (NAT2) isoenzyme of rhesus macaque and human

Abstract

Arylamine N-acetyltransferases (NATs) are polymorphic enzymes mediating the biotransformation of arylamine/arylhydrazine xenobiotics, including pharmaceuticals and environmental carcinogens. The NAT1 and NAT2 genes, and their many polymorphic variants, have been thoroughly studied in humans by pharmacogeneticists and cancer epidemiologists. However, little is known about the function of NAT homologues in other primate species, including disease models. Here, we perform a comparative functional investigation of the NAT2 homologues of the rhesus macaque and human. We further dissect the functional impact of a previously described rhesus NAT2 gene polymorphism, causing substitution of valine by isoleucine at amino acid position 231. Gene constructs of rhesus and human NAT2, bearing or lacking non-synonymous polymorphism c.691G>A (p.Val231Ile), were expressed in Escherichia coli for comparative enzymatic analysis against various NAT1- and NAT2-selective substrates. The results suggest that the p.Val231Ile polymorphism does not compromise the stability or overall enzymatic activity of NAT2. However, substitution of Val231 by the bulkier isoleucine appears to alter enzyme substrate selectivity by decreasing the affinity towards NAT2 substrates and increasing the affinity towards NAT1 substrates. The experimental observations are supported by in silico modelling localizing polymorphic residue 231 close to amino acid loop 125-129, which forms part of the substrate binding pocket wall and determines the substrate binding preferences of the NAT isoenzymes. The p.Val231Ile polymorphism is the first natural polymorphism demonstrated to affect NAT substrate selectivity via this particular mechanism. The study is also the first to thoroughly characterize the properties of a polymorphic NAT isoenzyme in a non-human primate model.

Keywords: 5,5′-dithiobis-(2-nitrobenzoic acid); 5-AS; 5-aminosalicylate; Acetyl-CoA; Animal model; CoA; DMAB; DSF; DTNB; Drug metabolism; INH; NAT; NAT2 gene; PA; PABA; PANS; PAS; Polymorphic gene variant; Primate; SMZ; SNP; Xenobiotic metabolizing enzyme; acetyl-coenzyme A; arylamine N-acetyltransferase; coenzyme A; differential scanning fluorimetry; dimethylaminobenzaldehyde; isoniazid; p-aminobenzoate; p-aminosalicylate; p-anisidine; procainamide; single nucleotide polymorphism; sulphamethazine.