From arylamine N-acetyltransferase to folate-dependent acetyl CoA hydrolase : impact of folic acid on the activity of (HUMAN)NAT1 and its homologue (MOUSE)NAT2

Acetyl Coenzyme A-dependent N-, O- and N,O-acetylation of aromatic amines and hydrazines by arylamine N-acetyltransferases is well characterised. Here, we describe experiments demonstrating that human arylamine N-acetyltransferase Type 1 and its murine homologue (Type 2) can also catalyse the direct hydrolysis of acetyl Coenzyme A in the presence of folate. This folate-dependent activity is exclusive to these two isoforms; no acetyl Coenzyme A hydrolysis was found when murine arylamine N-acetyltransferase Type 1 or recombinant bacterial arylamine N-acetyltransferases were incubated with folate. Proton nuclear magnetic resonance spectroscopy allowed chemical modifications occurring during the catalytic reaction to be analysed in real time, revealing that the disappearance of acetyl CH3 from acetyl Coenzyme A occurred concomitantly with the appearance of a CH3 peak corresponding to that of free acetate and suggesting that folate is not acetylated during the reaction. We propose that folate is a cofactor for this reaction and suggest it as an endogenous function of this widespread enzyme. Furthermore, in silico docking of folate within the active site of human arylamine N-acetyltransferase Type 1 suggests that folate may bind at the enzyme's active site, and facilitate acetyl Coenzyme A hydrolysis. The evidence presented in this paper adds to our growing understanding of the endogenous roles of human arylamine N-acetyltransferase Type 1 and its mouse homologue and expands the catalytic repertoire of these enzymes, demonstrating that they are by no means just xenobiotic metabolising enzymes but probably also play an important role in cellular metabolism. These data, together with the characterisation of a naphthoquinone inhibitor of folate-dependent acetyl Coenzyme A hydrolysis by human arylamine N-acetyltransferase Type 1/murine arylamine N-acetyltransferase Type 2, open up a range of future avenues of exploration, both for elucidating the developmental role of these enzymes and for improving chemotherapeutic approaches to pathological conditions including estrogen receptor-positive breast cancer

Editorial : Competence in scientific agriculture

<div><p>Human arylamine <i>N</i>-acetyltransferase 1 (hNAT1) has become an attractive potential biomarker for estrogen-receptor-positive breast cancers. We describe here the mechanism of action of a selective non-covalent colorimetric biosensor for the recognition of hNAT1 and its murine homologue, mNat2, over their respective isoenzymes, leading to new opportunities in diagnosis. On interaction with the enzyme, the naphthoquinone probe undergoes an instantaneous and striking visible color change from red to blue. Spectroscopic, chemical, molecular modelling and biochemical studies reported here show that the color change is mediated by selective recognition between the conjugate base of the sulfonamide group within the probe and the conjugate acid of the arginine residue within the active site of both hNAT1 and mNat2. This represents a new mechanism for selective biomarker sensing and may be exploited as a general approach to the specific detection of biomarkers in disease.</p></div

Real-time <i>¹H</i>-NMR analysis of (MOUSE)NAT2 activity as a folate-dependent AcCoA hydrolase performed in PBS-D₂O.

<p>(A) Purified recombinant (MOUSE)NAT2 (25 µg) was mixed with folate (150 µM) and AcCoA (400 µM) in buffer PBS-D₂O (pD 7.4) and incubated at 37°C. NMR data were collected before adding the enzyme (t = 0 min), and subsequently 5 min after enzyme addition. A selected region of the spectra (3.2 to 1.6 ppm) is shown, the signals being labelled as follows: folate (f), AcCoA (A), CoA (C), AcOH (a). (B) Enzymatic reaction progress curves of folate-dependent AcCoA hydrolytic activity by (MOUSE)NAT2. Folate-dependent AcCoA hydrolysis by (MOUSE)NAT2 was monitored by following the characteristic peaks of both reagent and products <i>versus d₆</i>-DMSO as an internal standard with appropriate correction. Diamonds: AcCoA; Squares: CoA; Triangles: AcOH.</p

Two hypotheses for the mechanism of the AcCoA hydrolysis reaction catalysed by (MOUSE)NAT2 in the presence of folate.

<p>Pathway (a) corresponds to hydrolysis of AcCoA with direct release of AcOH into the bulk solvent; Pathway (b) corresponds to the formation of an unstable <i>N-</i>acetylfolate intermediate which immediately decomposes, releasing free AcOH.</p

<i>In silico</i> docking of folate into the active site of (HUMAN)NAT1.

<p>(A) Folic acid was docked into the active site of (HUMAN)NAT1 using the program GOLD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096370#pone.0096370-Verdonk1" target="_blank">[62]</a>; the highest scoring solution is shown. The structure of (HUMAN)NAT1 (pdb code: 2PQT) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096370#pone.0096370-Wu1" target="_blank">[6]</a> is shown in surface format with the three domains labelled and coloured in different shades of blue. The domains are numbered from the amino terminus with Domain 3 corresponding to the <i>C-</i>terminus of the protein. Folic acid is labelled with carbon atoms in orange, nitrogen in blue, oxygen in red, and hydrogen in white. (B) Maximised view of the (HUMAN)NAT1 active site with folic acid docked. The structure of (HUMAN)NAT1 is shown in cartoon format with the three domains coloured as above. The side chains of the key residues involved in predicted folic acid binding within the catalytic pocket are labelled with nitrogen in blue, oxygen in red, and sulfur in yellow. Folic acid is labelled with carbon atoms in orange, nitrogen in blue, oxygen in red, and hydrogen in white.</p

Structural similarity between folate and AcCoA.

<p>(A) Predicted alignment between folic acid and CoA in free space using the software program FORGE, based on the conformation of CoA as it is bound in the co-crystal structure with (HUMAN)NAT2 (pdb code: 2PFR). Folic acid is shown in thick sticks, with carbons in grey, and CoA is shown in thin sticks, with carbon atoms in green. Nitrogen atoms are shown in blue, oxygens in red, and hydrogens in white. Regions of negative charge are shown in cyan, regions of positive charge are shown in dark red, and lipophilic regions are shown in yellow. Icosahedra represent the electronic fields of CoA and spheres represent those of folic acid. (B) Predicted alignment between folic acid and CoA using (HUMAN)NAT2 as an excluded volume. Folic acid, CoA and their respective electron density distributions are represented as above. The (HUMAN)NAT2 structure is represented in thin sticks with the carbon atoms in lilac.</p

(HUMAN)NAT1 hydrolyses AcCoA in the presence of folate.

<p>The time course of AcCoA hydrolysis by (HUMAN)NAT1 in the presence of folate was monitored. The reaction mixture was prepared and incubated as described. Aliquots were taken every 2 minutes over a 30 minute time course and analysed by HPLC.</p

Structure-activity relationships and colorimetric properties of specific probes for the putative cancer biomarker human arylamine [Nu]-acetyltransferase 1

A naphthoquinone inhibitor of human arylamine N-acetyltransferase 1 (hNAT1), a potential cancer biomarker and therapeutic target, has been reported which undergoes a distinctive concomitant color change from red to blue upon binding to the enzyme. Here we describe the use of in silico modeling alongside structure-activity relationship studies to advance the hit compound towards a potential probe to quantify hNAT1 levels in tissues. Derivatives with both a fifty-fold higher potency against hNAT1 and a two-fold greater absorption coefficient compared to the initial hit have been synthesized; these compounds retain specificity for hNAT1 and its murine homologue mNat2 over the isoenzyme hNAT2. A relationship between pKa, inhibitor potency and colorimetric properties has also been uncovered. The high potency of representative examples against hNAT1 in ZR-75-1 cell extracts also paves the way for the development of inhibitors with improved intrinsic sensitivity which could enable detection of hNAT1 in tissue samples and potentially act as tools for elucidating the unknown role hNAT1 plays in ER+ breast cancer; this could in turn lead to a therapeutic use for such inhibitors

Inhibitor binding pocket of hNAT1 and mNat2.

<p>(<b>a</b>) The active site of hNAT1 crystal structure (PDB:2PQT) in surface representation with <b>1</b> docked in stick representation. The hNAT1 residues involved in inhibitor binding and selectivity are shown in stick representation and labeled with carbon in green, nitrogen in blue, oxygen in red, and sulfur in yellow. <b>1</b> is labeled with carbon atoms in light orange, nitrogen in blue, oxygen in red, and sulfur in yellow. (<b>b</b>) The active site of mNat2 structural model with docked compound <b>1</b> is shown using the same representation as in (a).</p

Competitive inhibition of 1 towards mNat2 and active site differences in mammalian NATs

<p>(<b>a</b>) <b>Left panel</b>: Structure of compound <b>1</b>; <b>Right panel</b>: Dixon plot shows competitive inhibition of mNat2 (9 ng) by <b>1</b> at different pABA concentrations (25 µM (circles), 50 µM (triangles), 100 µM (diamonds), and 250 µM (squares)). Initial rates of the mNat2 catalysed reaction were determined by monitoring the rate of hydrolysis of AcCoA (400 μM) (<b>b</b>) Summary table of active site differences of human and murine NATs and the effects of their interaction with <b>1</b>. Blue and red columns indicate the color of <b>1</b> on interaction with the protein.</p