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

Background The 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. Methods To 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. Results Comparing (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. Conclusions Three 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 material The online version of this article (doi:10.1186/2050-6511-15-68) contains supplementary material, which is available to authorized users.


Background
Arylamine N-acetyltransferases (NATs, EC. 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 human genome contains two polymorphic NAT genes, (HUMAN)NAT1 and (HUMAN)NAT2 [3,4], which play important pharmacogenetic roles in cancer susceptibility and have the potential to contribute to personalised medicine [5,6]. The corresponding enzymes possess distinct substrate profiles: HUMAN(NAT1) preferentially N-acetylates the arylamines 4-aminobenzoic acid (4ABA), 4-aminobenzoyl glutamate (4ABglu) and 4-aminosalicylate (4AS), whereas (HUMAN)NAT2 has higher activity towards the hydrazines isoniazid (INH) and hydralazine (HDZ) and the arylamine sulfamethazine (SMZ) [7,8]. 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 also 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].
Human NATs are characterised by differing tissue distributions and patterns of gene expression during development [12][13][14]. In particular, while (HUMAN)NAT2 appears to be a well-established drug metabolising enzyme and is mainly expressed in the liver [15], the selective N-acetylation of the folate catabolites 4ABglu and 4ABA by (HUMAN)NAT1 suggests that this isoform may have an endogenous role in folate homeostasis [16,17]. Furthermore, recent studies indicate that (HUMAN)NAT1 has a novel catalytic function as a folate-dependent AcCoA hydrolase [18].
Rodents also have multiple NAT isoforms which have been investigated as animal models for the corresponding human enzymes. Mice, for example, have three NAT genes, (MOUSE)Nat1, (MOUSE)Nat2 and (MOUSE)Nat3. All of these are polymorphic; the (MOUSE)Nat3 gene exhibits the greatest extent of polymorphism and may effectively be considered a pseudogene like (HUMAN)NAT3 [19], although it does have enzymatic activity [20,21].
The (MOUSE)NAT1 enzyme is commonly assumed to be functionally equivalent to (HUMAN)NAT2 because both can metabolise isoniazid (INH) and sulfamethazine (SMZ) [29][30][31]. In order to determine whether these enzymes are true functional equivalents, we used a previously reported (MOUSE)Nat1*1 clone [32] to express and purify the (MOUSE)NAT1*1 enzyme and compared its substrate profile with those of other rodent and human NAT enzymes using a broad panel of aromatic amines and hydrazines. In addition, we used three site-directed mutants ((MOUSE) NAT2_F125S, (MOUSE)NAT2_R127G and (MOUSE) NAT2_R127L) to investigate the effects of key active site residues on the substrate specificity of (MOUSE)NAT2. In the present study we focused on residues 125 and 127; the role of the Y129 residue found in (HUMAN)NAT1 and (MOUSE)NAT2, at least with respect to inhibitor binding, has previously been investigated using (MESAU)NAT2, which has identical active site residues except for a leucine (L) at location 129 [33]. The results of this earlier study [26] suggest that Y129 is functionally important, at least in inhibitor recognition, and illustrate the value of (MESAU) NAT2 as a protein model for comparative studies with (HUMAN)NAT1 and (MOUSE)NAT2.
Finally, we modelled the binding of representative arylamine substrates within the active sites of reference and mutant mammalian NATs in order to elucidate key interactions within the NAT/substrate complex. The identification of (HUMAN)NAT1 as a potential therapeutic target in cancer means that understanding the molecular details of this series of enzymes in humans and potential animal models is important for their potential exploitation in both diagnostics [24] and therapy [34].

Chemicals and reagents
All chemicals were purchased from Sigma-Aldrich unless otherwise stated. Molecular biology reagents were obtained from Promega.
Substrate and inhibitor selectivity and spectrometry of (MOUSE)NAT1*1 and three (MOUSE)NAT2 mutants Substrate specific N-acetylation profiles were determined according to published methods with minor modifications [8,36]. Recombinant proteins were used immediately after purification. The quantity of each protein used in assay mixes (100 μL) was: ( In each case the final concentration in the reaction mix was 500 μM. All measurements were performed in triplicate and are expressed as mean ± standard deviation. For each enzyme tested, average measures of specific activity were calculated relative to the substrate exhibiting the highest specific activity (100%) for that enzyme.
In order to highlight differences in substrate preference among murine NAT proteins, the substrate profiles of (MOUSE)NAT2*1, (MOUSE)NAT1*1, (MOUSE)NAT2_ F125S, (MOUSE)NAT2_R127G and (MOUSE)NAT2_ R127L were compared by ANOVA (confidence limit 95%) using the Shapiro-Wilk test to verify normal distribution and Cochran's test to check the equality of variances. Statistical significance was evaluated using Student's t-test with a Bonferroni adjustment if required.
Inhibition of NAT activity was determined as the ratio of specific activity in the presence and absence of the inhibitor naphthoquinone 1 using the preferred substrate for the enzyme under investigation. The final concentration of dimethyl sulphoxide (DMSO) in reaction mixes was 5% (v/v). IC 50 values were estimated using a doseresponse-based regression model in Kyplot® software. Curves were fitted by least squares analysis with confidence limits of 95%. Visible spectra (λ = 800 to 326 nm) of naphthoquinone 1 in the presence of pure recombinant NATs were recorded with a U-2001 spectrophotometer (Hitachi) using 50 μL UVettes® (Eppendorf ).

Results
Substrate selectivity of (MOUSE)NAT1*1 and (MOUSE)NAT2*1 We expressed (MOUSE)NAT1*1 in E. coli Rosetta (DE3) pLysS and used the resulting recombinant protein to determine the activity of (MOUSE)NAT1*1 towards a panel of chemicals commonly used as NAT substrates (Additional file 3: Table S1). The highest specific activities were with the hydrazines INH and HDZ and the arylamine SMZ ( Figure 1). A parallel screening experiment with (MOUSE)NAT2*1 yielded results corresponding to those published previously [22]. The differences between (MOUSE)NAT1*1 and (MOUSE)NAT2*1 for most substrates were statistically significant. (MOUSE) NAT1*1 did, however, have low but measurable activity towards certain arylamine substrates which are usually considered to be (MOUSE)NAT2-specific (4ABA and 4AS but not 4ABglu) and towards halogenated arylamines and alkyloxy-and aryloxy-substituted arylamines.

Site-directed mutagenesis of (MOUSE)NAT2
Three (MOUSE)NAT2 mutants ((MOUSE)NAT2_F125S, (MOUSE)NAT2_R127G and (MOUSE)NAT2_R127L) were used to explore the effects of mutating a single residue within the active site on the substrate specificity of (MOUSE)NAT2. Two of these, (MOUSE)NAT2_R127G and (MOUSE)NAT2_R127L, were reported previously [8,22]. The third, (MOUSE)NAT2_F125S was generated by site-directed mutagenesis, as described above and illustrated in Additional file 2: Figure S2.
The substrate panel listed in Additional file 3: Table S1 was used to characterise the activity profiles of the three mutants in comparison with that of (MOUSE)NAT2*1. The maximum specific activity of (MOUSE)NAT2_F125S (observed with EOA) was around 7.5 times lower than the maximal value of (MOUSE)NAT2*1 (observed with 4ABA), but the substrate preferences of (MOUSE)NAT2_ F125S and (MOUSE)NAT2*1 were similar. Some discrepancies were, however, observed: the main differences observed were marked reductions in the relative rate of N-acetylation of the arylamines 4ABglu and 4ABA and an augmentation of activities towards the hydrazines INH and HDZ.
Substituting R127 with glycine (R127G) or leucine (R127L) caused a dramatic loss of N-acetylation activity towards 4ABA and 4ABglu ( Figure 2). Aminosalicylic acids were differentially N-acetylated by (MOUSE)NAT2*1 and its R127 mutants: (MOUSE)NAT2_R127G and (MOUSE)NAT2_R127L had significantly lower N-acetylation activity towards the (MOUSE)NAT2-specific substrate 4AS, but higher catalytic activity with 5AS compared with (MOUSE)NAT2*1. These mutations had no marked effects on the N-acetylation of halogenated arylamines and alkyloxy-substituted arylamines with small alkyl chains (ANS, 4AV), but those with extended alkyloxy or bulkier aryloxy substituents (hexyloxy in HOA and phenoxy in POA) were strongly preferred by both R127 mutants compared with (MOUSE)NAT2*1. The mutants also had much higher N-acetylation activity towards hydrazines such as HDZ.

Comparison of substrate selectivity among mammalian NATs
The assays described here were performed under the conditions described in our previous publications [8,22]. We were therefore able to compare previously published substrate selectivity profiles of (HUMAN)NAT1*4, (HUMAN) NAT2*4 and (MESAU)NAT2*1 with those of (MOUSE) NAT1*1, (MOUSE)NAT2*1 and the three engineered (MOUSE)NAT2 mutants reported here (Table 1). In Table 1, the NAT enzymes are arranged according to divergence of the active site sequence from that of (HUMAN)NAT1*4 ( Figure 3) [9] and plotted against arylamine and hydrazine substrates organised according to their chemical functionalities and physicochemical properties. Overall, as the active site diverged further from that of (HUMAN)NAT1*4, substrate preference moved from arylamines with negatively charged para-substituents towards aromatic amines and hydrazines without ionised, polar or para-substituents around the aromatic core. In particular, 4ABglu, 4ABA and 4AS have an acetyl acceptor amine with pK aH <3 (Additional file 3: Table S1) and are subject to N-acetylation by (HUMAN)NAT1*4 and its homologues (MOUSE)NAT2*1 and (MESAU)NAT2*1. The other substrates have an acceptor amine with pK aH ≥3. These are better substrates for (HUMAN) NAT2*4, (MOUSE)NAT1*1 and the two R127-mutated (MOUSE)NAT2 enzymes. In general, the results obtained corresponded with previous reports of functional similarities between (HUMAN) NAT1*4, (MOUSE)NAT2*1 and (MESAU)NAT2*1 [8,22]; however, the substrate profile of (MOUSE)NAT1*1 did not, as expected, correspond to that of (HUMAN)NAT2*4 [29,31]. Indeed, (MOUSE)NAT1*1 was able to N-acetylate      Percentage specific activity values of native and engineered mammalian NAT proteins are shown. A value of 100% is attributed to the substrate towards which the isoform in question has the highest specific activity. All results shown are the mean of 3 measurements ± standard deviation. Percentages ≥90% for each isoenzyme were compared by ANOVA using a Student's t-test and statistically similar values are emboldened. Isoforms are ordered according to increasing number of residue differences within the active site in relation to (HUMAN)NAT1*4. Residues identical to those in (HUMAN)NAT1*4 are labelled with a hyphen; residues different from the corresponding residue of (HUMAN)NAT1*4 are specified. Substrates are ordered according to the number of negative charges at pH 8.0, then to electronrichness and increasing size of aromatic substituents. The chemical structures and physicochemical properties of the chemicals used as substrates for NAT in this study are shown in Additional file 3: Table S1.  Figure S3, Table 2).

In silico analysis of substrate selectivity in mammalian NATs
The structural features underlying substrate selectivity were investigated by in silico analysis of interactions between the NAT proteins and their arylamine substrates. Structural models of (MOUSE)NAT2*1 and (MESAU) NAT2*1 were generated using the crystal structure of (HUMAN)NAT1*4 [PDB:2PQT] [9] because these three isoforms have >80% amino acid identity (Additional file 5: Table S2). Swiss-Model simulations generated models of (MOUSE)NAT2*1 and (MESAU)NAT2*1 which were of high quality according to their scoring functions and background noise, thus confirming the reliability of the template used. The mutations (MOUSE)NAT2_F125S, (MOUSE)NAT2_R127G and (MOUSE)NAT2_R127L did not abolish the catalytic reactivity compared with the reference enzyme, so each individual residue modification was considered unlikely to have altered overall protein folding. The same modelling procedure was therefore used to create structural models of (MOUSE)NAT2_F125S, (MOUSE)NAT2_R127G and (MOUSE)NAT2_R127L. However, attempts to model (MOUSE)NAT1*1 (64% identity; Additional file 5: Table S2) based on the structure of (HUMAN)NAT2*4 [PDB:2PFR] [9] did not yield reliable results.
Substrates were docked into the catalytic site of each enzyme as shown in the selectivity profile summarised in Table 1 Figure S4).
We also attempted to model the selective hydrazine substrates INH and HDZ within the active site of (HUMAN)NAT2*4 and the (MOUSE)NAT2 mutants. No proximity between the hydrazine functionality and C68 thiolate compatible with the NAT catalytic mechanism [44] was observed, possibly because of the smaller steric size of the substrate docked. However, when a bulkier selective arylamine substrate such as POA was docked into (HUMAN)NAT2*4 and the three (MOUSE) NAT2 mutants, the results indicated proximity between the primary amine of the substrate and the key active site residues C68 and H106 (<4 Å) ( Figure 5). These simulations indicated that the 4-aminoarene of POA could be sandwiched between the benzyl ring of F217 and the apolar side chain of F93 in (HUMAN)NAT2*4 and F125 in (MOUSE)NAT2_R127G and (MOUSE)NAT2_ R127L via hydrophobic interactions. In the (MOUSE) NAT2 mutants, the phenoxy group of POA was predicted to make further π-stacking interactions with the side Active site residues 125 chain of Y129 (~3.9 Å), whereas the equivalent residue in (HUMAN)NAT2*4, S129, is incapable of this interaction.

Discussion
The present study has extended our understanding of (MOUSE)NAT1 by demonstrating that it is functionally different from (HUMAN)NAT2. In addition to its known activity in N-acetylating the hydrazine INH and the arylamine SMZ [29,30], (MOUSE)NAT1*1 was shown to have significant activity towards the hydrazine HDZ and two arylamine substrates which were previously considered to be (HUMAN)NAT1 and (MOUSE)NAT2-specific (4ABA and 4AS). The substrate-binding interactions involved in Nacetylation were previously characterised using nuclear magnetic resonance [33,45], demonstrating that an arylamine or hydrazine could bind to a non-acetylated NAT active site. In the present study, arylamine substrates were docked within the catalytic pockets of different non-acetylated NATs in silico in order to identify possible selective interactions underlying the formation of the enzyme-substrate complex.
In (HUMAN)NAT1*4, the positively charged guanidinium (at pH 8.0) of R127 plays an essential role in recognising the negatively charged carboxylate of 4ABA, as illustrated by modelling 4ABA (Additional file 6: Figure S4) or 4AS [9] within the active site. The significant N-acetylation activity of (MOUSE)NAT1*1 towards 4ABA, 4AS and ANS in the present study was difficult to reconcile with the presence of an apolar G127 instead of an R127. However, it is possible that the presence of a sterically smaller residue (G) at this position in (MOUSE)NAT1*1 permits bulky or highly polar substrates to enter and allows their charged para-substituents to interact with other polar side chains in the active site (e.g. Y125 and Y129). Similarly, essential π-π stacking interactions observed between the aromatic core of 4ABA and F125, as seen in (HUMAN)NAT1*4, may be permitted by the side chain arene of Y125 within the active site of (MOUSE)NAT1*1. The lack of a structural model suitable for docking studies and the restricted commonality of substrate selectivity between (MOUSE) NAT1*1 and either of the two human NATs precluded the construction of a structural model for (MOUSE) NAT1*1, so this hypothesis could not be tested.
When 4ABglu was docked into the structure of (HUMAN)NAT1*4 and models of (MOUSE)NAT2*1 and (MESAU)NAT2*1, the essential role of R127 in forming ionic interactions with the electron-negative para-substituent of the substrate was evident; this may explain why a bulky charged arylamine such as 4ABglu can enter a small, very hydrophobic microenvironment such as the active site crevice of (HUMAN)NAT1*4 [9]. The preference of (HUMAN)NAT1*4 and its homologues (MOUSE) NAT2*1 and (MESAU)NAT2*1 for arylamine substrates with a negatively charged para-substituent (e.g. 4ABA and 4AS) may therefore be due to the positively charged guanidinium moiety of R127.
Previous studies have shown that mutation of F125 to S125 modifies the catalytic preference of (HUMAN) NAT1*4 from the conventional probe substrate 4AS to SMZ [27]. When we examined the effect of the equivalent mutation on (MOUSE)NAT2, no such shift in substrate preference was observed; neither did the general substrate preferences of (MOUSE)NAT2 change to resemble those of (MOUSE)NAT1*1. The higher reactivity of (MOUSE)NAT2_F125S with conformationally flexible hydrazines than with planar arylamines could be associated with increased active site space in (MOUSE) NAT2*1 after the substitution of the bulky benzyl group of F with the less hindering hydroxymethyl of S.
Site-directed mutagenesis of R127 to G127 or L127 within (MOUSE)NAT2 markedly altered the enzyme's substrate selectivity. Overall, the non-polar side chains of G and L appeared to have similar effects in terms of modifying the substrate preferences of (MOUSE)NAT2: the metabolism of 4ABglu and 4ABA was dramatically decreased whereas N-acetylation of the hydrazines INH and HDZ, which are commonly used as probe substrates  [9] and (MOUSE)NAT2 mutants in light blue. The side chains of the key residues involved in substrate binding within the active site are drawn in stick representation and labelled with carbon atoms in the corresponding colour of the enzyme, nitrogen in blue, oxygen in red and sulphur in yellow. The arylamine substrate POA is labelled with carbon atoms in orange, nitrogen in blue, oxygen in red and polar hydrogen in gray. The figures were generated using PyMOL [41].
for (MOUSE)NAT1 and (HUMAN)NAT2, was augmented. R127 seemed to play a crucial role in discriminating between arylamines with highly hydrophilic substituents and hydrazines with less polar aromatic functional groups. Moreover, the (MOUSE)NAT2_R127G mutation improved the overall catalytic activity of the enzyme, possibly due to the larger size of the active site cavity generated after substitution. Similarly, 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. When POA was docked into the active pocket of (HUMAN)NAT2*4, the results of substrate fitting were very similar to previous modelling results obtained with SMZ [9], consistent with the preference of (HUMAN)NAT2*4 for apolar and flexible substrates [11].
The individual (MOUSE)NAT2 mutants tested did not affect the specificity of (MOUSE)NAT2 for SMZ, which is a very poor substrate for this isoform. This may be related to the need for a larger active site to accommodate SMZ, which is a bulky arylamine; a single amino acid change may not be sufficient to permit access to the active site. Further 3D structural data and thermal stability studies on the mutants would help to ascertain the extent of the folding perturbations produced by each mutation. However, it is also intriguing that small hydrazines such as INH and phenylhydrazine are poor substrates for (HUMAN)NAT1 and its rodent homologues [8,22], whereas arylamines of similar steric size such as 4ABA, 4AS and ANS are good substrates.
While the amine nitrogen and the aromatic carbons of an arylamine substrate are on the same plane, crystallographic studies of INH, HDZ and phenylhydrazine as single molecules [46][47][48][49][50] and protein co-crystallised ligands [51,52], showed that the hydrazine bond could also be off the plane defined by the remaining aryl and acyl carbon atoms, thereby giving the hydrazine substrate a non-planar conformation. Our docking simulations using the crystal structure of (HUMAN)NAT1*4 and the model of (MOUSE)NAT2*1 show that the bulky side chain of R127, the benzyl ring of F125 and the 4hydroxybenzyl of Y129 create a characteristic constrained microenvironment which appears to allow preferable accommodation of planar arylamines rather than conformationally flexible hydrazines in the active site of (HUMAN) NAT1*4. This is consistent with the volumes of the active sites in human NAT structures: the (HUMAN)NAT1*4 cavity has a volume of 162 Å 3 , whereas (HUMAN)NAT2*4 (with S at positions 125, 127 and 129) has a larger active pocket (257 Å 3 ). In this context it is also noteworthy that the crystal structures of prokaryotic NATs from Mycobacterium tuberculosis, M. marinum and M. smegmatis, which preferentially N-acetylate conformationally flexible hydrazine substrates, have much larger active sites (>200 Å 3 ) than (HUMAN)NAT1*4 [53][54][55].
Recent studies have suggested that F125, R127 and Y129 in the active sites of (HUMAN)NAT1*4 and (MOUSE)NAT2*1 are essential for selective recognition and binding of the potent inhibitor naphthoquinone 1 [24][25][26]. In particular, ionic recognition between the conjugate base of naphthoquinone 1 and the guanidinium moiety of R127 is known to be essential for inhibitor binding. The results shown here confirm that these key residues are required for the interaction with naphthoquinone 1, since the absence of any of them results in a protein which is resistant to inhibition and colour shifting in response to naphthoquinone 1 (Table 2).
Overall, our results suggest that the substrate selectivity of the mammalian NATs investigated in this study is influenced by two major factors; firstly, the planarity of the acceptor arylamine and the polarity of its para substituent (which appear to affect both the type of the intermolecular interactions within the enzyme and the pK aH strength and nucleophilicity of the acceptor amine); and secondly, the size of the catalytic cavity and its overall polarity, as determined by the residues at positions 125, 127 and 129. In particular, F125 in (HUMAN)NAT1*4 and (MOUSE)NAT2*1 appears to play a key role in discriminating between planar arylamines and conformationally flexible hydrazines, while the ionised (at physiological pH) side chain of R127 perturbs the characteristic hydrophobicity of the NAT active site and, in cooperation with the 4-hydroxybenzyl side chain of Y129, contributes to the narrowness of the (HUMAN)NAT1*4 active site.

Conclusions
In conclusion, this evaluation of the substrate profiles of various native and engineered mammalian NATs in the context of their structures has highlighted the features which influence NAT substrate selectivity in mammals. The virtual models of mammalian NATs 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. Three 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-receptorpositive breast cancer, these structure-based tools will facilitate the ongoing design of candidate compounds for use in (HUMAN)NAT1-positive breast tumours.