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

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.     

Crystal Structures of Wild-type and Mutant Methicillin-resistant Staphylococcus aureus Dihydrofolate Reductase Reveal an Alternate Conformation of NADPH That May Be Linked to Trimethoprim Resistance

Both hospital- and community-acquired Staphylococcus aureus infections have become major health concerns in terms of morbidity, suffering and cost. Trimethoprim-sulfamethoxazole (TMP-SMZ) is an alternative treatment for methicillin-resistant S. aureus (MRSA) infections. However, TMP-resistant strains have arisen with point mutations in dihydrofolate reductase (DHFR), the target for TMP. A single point mutation, F98Y, has been shown biochemically to confer the majority of this resistance to TMP. Using a structure-based approach, we have designed a series of novel propargyl-linked DHFR inhibitors that are active against several trimethoprim-resistant enzymes. We screened this series against wild-type and mutant (F98Y) S. aureus DHFR and found that several are active against both enzymes and specifically that the meta-biphenyl class of these inhibitors is the most potent. In order to understand the structural basis of this potency, we determined eight high-resolution crystal structures: four each of the wild-type and mutant DHFR enzymes bound to various propargyl-linked DHFR inhibitors. In addition to explaining the structure-activity relationships, several of the structures reveal a novel conformation for the cofactor, NADPH. In this new conformation that is predominantly associated with the mutant enzyme, the nicotinamide ring is displaced from its conserved location and three water molecules complete a network of hydrogen bonds between the nicotinamide ring and the protein. In this new position, NADPH has reduced interactions with the inhibitor. An equilibrium between the two conformations of NADPH, implied by their occupancies in the eight crystal structures, is influenced both by the ligand and the F98Y mutation. The mutation induced equilibrium between two NADPH-binding conformations may contribute to decrease TMP binding and thus may be responsible for TMP resistance.