Differences between murine arylamine N-acetyltransferase type 1 and human arylamine N-acetyltransferase type 2 defined by substrate specificity and inhibitor binding

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.

Treatment of Rats with Apocynin Has Considerable Inhibitory Effects on Arylamine N-Acetyltransferase Activity in the Liver

“…To date, only crystal structures of human NAT enzymes are available as mammalian species48; nevertheless, they have constituted highly reliable templates for inhibitor studies involving rodent NATs because of their high percentages of residue identity (>70%) (Table 1, Supplementary Figure S1)274950.…”

“…However, additional studies about functional selectivity of rat NATs can be useful to better establish structural analogies between rat and human NAT isoenzymes and build virtual models of rat NAT proteins based on human NAT structures27.…”

“…Thus, we initially investigated the impact of apocynin as un-metabolized natural biotic on liver NAT activity in rats fed normal diet or 50–100 mg apocynin/kg body for eight weeks. The activity of NAT enzymes in liver homogenates was measured using p -anisidine (pANS) which was previously shown to be a good arylamine substrate catalyzed by all active human and mouse NAT isoforms 27 .…”

“…4c ). Also, apocynin can be hypothesized to act as a non-covalent competitive inhibitor ligand of rat NAT enzymes for its similarity in steric size or chemical shape to known arylamine NAT substrates, such as 4-aminoveratrole 27 .…”

“…In particular, (RAT)NAT2 enzyme shows a high functional homology with (HUMAN)NAT1, whereas active (RAT)NAT1 and (RAT)NAT3 have a partial analogy in relation to (HUMAN)NAT2 and NATP respectively, with rodent NAT3 having a lower cytosolic production and very little catalytic activity ( Table 1 , Supplementary Figure S1 ) 22 23 . Moreover, the arrangement of the three rat NAT loci on chromosome 16 appeared to be highly similar to that of the three mouse NATs on chromosome 8 22 ; also, the mouse NAT enzymes (namely (MOUSE)NAT1, (MOUSE)NAT2 and (MOUSE)NAT3) were shown to N- acetylate similar probe substrates with an analogous catalytic efficiency as compared to (RAT)NAT1, (RAT)NAT2, and (RAT)NAT3 respectively ( Table 1 , Supplementary Figure S1 ) 22 24 25 26 27 .…”