Abstract
BackgroundThe mouse has three arylamine N-acetyltransferase genes, (MOUSE)Nat1, (MOUSE)Nat2 and (MOUSE)Nat3. These are believed to correspond to (HUMAN)NAT1, (HUMAN)NAT2 and NATP in humans. (MOUSE)Nat3 encodes an enzyme with poor activity and human NATP is a pseudogene. (MOUSE)Nat2 is orthologous to (HUMAN)NAT1 and their corresponding proteins are functionally similar, but the relationship between (MOUSE)Nat1 and (HUMAN)NAT2 is less clear-cut.MethodsTo determine whether the (MOUSE)NAT1 and (HUMAN)NAT2 enzymes are functionally equivalent, we expressed and purified (MOUSE)NAT1*1 and analysed its substrate specificity using a panel of arylamines and hydrazines. To understand how specific residues contribute to substrate selectivity, three site-directed mutants of (MOUSE)NAT2*1 were prepared: these were (MOUSE)NAT2_F125S, (MOUSE)NAT2_R127G and (MOUSE)NAT2_R127L. All three exhibited diminished activity towards “(MOUSE)NAT2-specific” arylamines but were more active against hydrazines than (MOUSE)NAT1*1. The inhibitory and colorimetric properties of a selective naphthoquinone inhibitor of (HUMAN)NAT1 and (MOUSE)NAT2 were investigated.ResultsComparing (MOUSE)NAT1*1 with other mammalian NAT enzymes demonstrated that the substrate profiles of (MOUSE)NAT1 and (HUMAN)NAT2 are less similar than previously believed. Three key residues (F125, R127 and Y129) in (HUMAN)NAT1*4 and (MOUSE)NAT2*1 were required for enzyme inhibition and the associated colour change on naphthoquinone binding. In silico modelling of selective ligands into the appropriate NAT active sites further implicated these residues in substrate and inhibitor specificity in mouse and human NAT isoenzymes.ConclusionsThree non-catalytic residues within (HUMAN)NAT1*4 (F125, R127 and Y129) contribute both to substrate recognition and inhibitor binding by participating in distinctive intermolecular interactions and maintaining the steric conformation of the catalytic pocket. These active site residues contribute to the definition of substrate and inhibitor selectivity, an understanding of which is essential for facilitating the design of second generation (HUMAN)NAT1-selective inhibitors for diagnostic, prognostic and therapeutic purposes. In particular, since the expression of (HUMAN)NAT1 is related to the development and progression of oestrogen-receptor-positive breast cancer, these structure-based tools will facilitate the ongoing design of candidate compounds for use in (HUMAN)NAT1-positive breast tumours.Electronic supplementary materialThe online version of this article (doi:10.1186/2050-6511-15-68) contains supplementary material, which is available to authorized users.
Highlights
The mouse has three arylamine N-acetyltransferase genes, (MOUSE)Nat1, (MOUSE)Nat2 and (MOUSE) Nat3
When the arylamine POA was docked into the active sites of (MOUSE)NAT2_F125S, (MOUSE)NAT2_R127G and (MOUSE)NAT2_R127L, the results suggested that accommodation of this substrate via stacking interactions is facilitated by the larger cavity created by these single substitutions
The virtual models of mammalian Arylamine N-acetyltransferase (NAT) generated in this study, used in conjunction with X-ray structures of human NATs, constitute a rich resource for investigating the roles of particular residues within the NAT active site in relation to both NAT activity and inhibitor selectivity
Summary
The mouse has three arylamine N-acetyltransferase genes, (MOUSE)Nat, (MOUSE)Nat and (MOUSE) Nat. 2.3.1.5) [1] are drug metabolising enzymes which catalyse the conjugation of an acetyl group from acetyl Coenzyme A (AcCoA) to arylamines, hydrazines and N-hydroxyarylamines They participate in the detoxification and metabolic activation of xenobiotics and are found in a wide variety of prokaryotic and eukaryotic species [2]. The crystal structures of human NATs [9] resemble the three-domain conformation observed in prokaryotic NATs [10] Like their prokaryotic counterparts, each has a catalytic triad comprising cysteine (C), histidine (H) and aspartic acid (D) at the active site; they contain an additional loop of 17 residues which many prokaryotic enzymes lack. These observations helped to lay the foundations for studies addressing the structural determinants of their catalytic selectivity [11]
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