Figure 1.
Potential role of (HUMAN)NAT1 and (MOUSE)NAT2 in folate degradation.
One endogenous role of (HUMAN)NAT1 and (MOUSE)NAT2 appears to be related to the breakdown of folate through the acetylation of the folate catabolites pABAglu and pABA. Small molecule inhibitors such as naphthoquinone 1 (shown in red) are valuable tools which can be used to enhance our understanding of this proposed role.
Figure 2.
(HUMAN)NAT1 hydrolyses AcCoA in the presence of folate.
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.
Figure 3.
AcCoA hydrolysis by (MOUSE)NAT2 in the presence of folate.
(A) Specific pABA (150 µM) acetylation activity by (MOUSE)NAT2 (5 ng) was followed in the absence (grey columns) or in the presence (white columns) of an equimolar concentration of folate. The concentration of AcCoA was 400 µM in all assays. Specific AcCoA (400 µM) hydrolysis activity by (MOUSE)NAT2 (5 ng) was also followed in the presence of folate (150 µM), but in the absence of an arylamine substrate (black columns). For each set of reagents, both primary amine acetylation and AcCoA hydrolysis methods were used in parallel, yielding equivalent results. No acetylation of folate was detected in a corresponding acetylation assay. (B) Time course of assay containing AcCoA (400 µM) and folate (150 µM), with 5 ng (MOUSE)NAT2 (squares); negative control: assay containing AcCoA (400 µM) and folate (150 µM) only, without (MOUSE)NAT2 (diamonds). (C) Activity of mammalian and bacterial NATs as AcCoA hydrolases in the presence of folate. Different NAT enzymes were tested in AcCoA hydrolysis assays in the presence of either the arylamine substrate 5AS (150 µM, grey columns), both 5AS and folate (each at 150 µM, white columns) or folate alone (150 µM, black columns). (D) Initial velocities of AcCoA hydrolysis by (HUMAN)NAT1 (triangles) and (MOUSE)NAT2 (circles) in the presence of different concentrations of folate. A Michaelis-Menten curve was fitted using the formula: y = (Vmax×[folate])/(Km+[folate]).
Figure 4.
Two hypotheses for the mechanism of the AcCoA hydrolysis reaction catalysed by (MOUSE)NAT2 in the presence of folate.
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 N-acetylfolate intermediate which immediately decomposes, releasing free AcOH.
Figure 5.
Real-time 1H-NMR analysis of (MOUSE)NAT2 activity as a folate-dependent AcCoA hydrolase performed in PBS-D2O.
(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 versus d6-DMSO as an internal standard with appropriate correction. Diamonds: AcCoA; Squares: CoA; Triangles: AcOH.
Figure 6.
Inhibition of (MOUSE)NAT2-dependent AcCoA hydrolysis by the small molecule inhibitor naphthoquinone 1.
(A) Structure of naphthoquinone 1, a selective inhibitor of (HUMAN)NAT1. (B) Screening of different enzymatic assays with (MOUSE)NAT2 in the presence of naphthoquinone 1. AcCoA hydrolysis assays were carried out in order to monitor the inhibitory potency of naphthoquinone 1 at 10 µM. AcCoA was present at 400 µM in all assays. Specific pABA (150 µM) acetylation activities by (MOUSE)NAT2 (5 ng) were performed in the absence (grey columns) or presence (white columns) of an equimolar concentration of folate. Specific AcCoA hydrolysis activity was also detected in the presence of folate (150 µM) in a DTNB assay without an arylamine substrate (black columns). The percentage inhibition was calculated in terms of the reduction in (MOUSE)NAT2 specific activity measured against a control in which DMSO (5% (v/v)) alone was added to the assay. (C) Naphthoquinone 1 as an inhibitor of the folate-dependent AcCoA hydrolytic activity of (MOUSE)NAT2. AcCoA hydrolysis assays were performed using (MOUSE)NAT2 (510 ng) and AcCoA (400 µM) in the presence of folate (150 µM) and serial dilutions of naphthoquinone 1. Data points represent the mean ± standard deviation of triplicate assays. Percentage inhibition was determined as the ratio of specific activity with the inhibitor related to specific activity without inhibitor (100%); 100% activity was 198 µM.sec−1.mg−1. The regression equation used was: y = 100/[1+10∧(x-log(IC50)].
Figure 7.
In silico docking of folate into the active site of (HUMAN)NAT1.
(A) Folic acid was docked into the active site of (HUMAN)NAT1 using the program GOLD [62]; the highest scoring solution is shown. The structure of (HUMAN)NAT1 (pdb code: 2PQT) [6] 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 C-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.
Figure 8.
Structural similarity between folate and AcCoA.
(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.