Identification of inhibitors of Plasmodium falciparum phosphoethanolamine methyltransferase using an enzyme-coupled transmethylation assay
© Bobenchik et al; licensee BioMed Central Ltd. 2010
Received: 28 July 2009
Accepted: 19 January 2010
Published: 19 January 2010
The phosphoethanolamine methyltransferase, PfPMT, of the human malaria parasite Plasmodium falciparum, a member of a newly identified family of phosphoethanolamine methyltransferases (PMT) found solely in some protozoa, nematodes, frogs, and plants, is involved in the synthesis of the major membrane phospholipid, phosphatidylcholine. PMT enzymes catalyze a three-step S-adenosylmethionine-dependent methylation of the nitrogen atom of phosphoethanolamine to form phosphocholine. In P. falciparum, this activity is a limiting step in the pathway of synthesis of phosphatidylcholine from serine and plays an important role in the development, replication and survival of the parasite within human red blood cells.
We have employed an enzyme-coupled methylation assay to screen for potential inhibitors of PfPMT. In addition to hexadecyltrimethylammonium, previously known to inhibit PfPMT, two compounds dodecyltrimethylammonium and amodiaquine were also found to inhibit PfPMT activity in vitro. Interestingly, PfPMT activity was not inhibited by the amodiaquine analog, chloroquine, or other aminoquinolines, amino alcohols, or histamine methyltransferase inhibitors. Using yeast as a surrogate system we found that unlike wild-type cells, yeast mutants that rely on PfPMT for survival were sensitive to amodiaquine, and their phosphatidylcholine biosynthesis was inhibited by this compound. Furthermore NMR titration studies to characterize the interaction between amoidaquine and PfPMT demonstrated a specific and concentration dependent binding of the compound to the enzyme.
The identification of amodiaquine as an inhibitor of PfPMT in vitro and in yeast, and the biophysical evidence for the specific interaction of the compound with the enzyme will set the stage for the development of analogs of this drug that specifically inhibit this enzyme and possibly other PMTs.
Malaria is one of the most important parasitic diseases worldwide responsible for over 200 million clinical cases with an estimated 1 million deaths annually . Malaria is caused by intraerythrocytic protozoan parasites of the genus Plasmodium. Of the five species infective to humans, Plasmodium falciparum is responsible for the highest level of mortality and morbidity . The lack of an effective vaccine and the rapid rise of resistance to the most potent and affordable antimalarial drugs creates an urgent need for new therapies to prevent malaria pathogenesis and transmission. Novel strategies are now necessary to limit the appearance and spread of drug resistant malaria strains. One such a strategy involves targeting metabolic pathways proven to play an essential function in the parasite's infection and transmission. Recent studies indicate that the metabolic pathways for the synthesis of the major P. falciparum phospholipids are excellent targets for the development of lipid-based antimalarial therapies [2–4].
Studies in P. falciparum demonstrated that the synthesis of phosphatidylcholine during the intraerythrocytic life cycle of the parasite occurs via two pathways, the serine-decarboxylase phosphoethanolemine methyltransferase (SDPM) pathway and the CDP-choline pathway [2–4]. The SDPM pathway uses serine either transported from human serum or resulting from degradation of host hemoglobin as a starting precursor. Serine is first decarboxylated by a parasite serine decarboxylase to form ethanolamine. Ethanolamine is next phosphorylated by a parasite ethanolamine kinase to form phosphoethanolamine (P-EA). A parasite S-adenosylmethionine (SAM)-dependent methyltransferase, PfPMT, catalyzes a three-step methylation of P-EA to form phosphocholine [3, 5, 6], which is converted into phosphatidylcholine (PtdCho) via the activity of two parasite enzymes PfCCT and PfCEPT. The CDP-choline pathway uses choline transported from the host as a precursor. Choline is phosphorylated by a parasite choline kinase PfCK to phosphocholine, which is subsequently modified by PfCCT to CDP-choline and by PfCEPT to PtdCho.
The 266 amino acid PfPMT is a member of a new class of SAM-dependent methyltransferases that acts on P-EA . Homologs of this enzyme are found in plants, nematodes, frogs, fish and other protozoa but not in mammals [7–11]. While phosphoethanolamine methyltransferases (PEAMT) share significant homology in their primary structure, the organization of their catalytic domains differ. The plant PEAMTs have two tandem catalytic domains, with the N-terminal domain catalyzing the methylation of P-EA to monomethylphosphoethanolamine and the C-terminal domain acting in the last two methylation reactions to form phosphocholine [7, 9, 10]. C. elegans expresses two PEAMT enzymes each containing only one methyltransferase domain located at either the N-terminus of the protein, in the case of Pmt1, or at the C-terminus of the protein in Pmt2 [8, 11]. Pmt1 catalyzes only the first methylation reaction, whereas Pmt2 catalyzes the last two methylation reactions. The Plasmodium PfPMT is only half the size of the plant and nematode proteins, possesses a single catalytic domain, and catalyzes all three methylation steps . No crystal or solution structures of these enzymes are available.
Confocal and immunoelectron microscopy studies have shown that PfPMT is expressed in the Golgi apparatus of the parasite . Biochemical and genetic analyses using yeast as a surrogate system allowed identification of residues in this enzyme that play a critical role in PfPMT catalysis and substrate binding . The finding that PfPMT has no homologs in mammalian databases suggested that this protein could be an ideal target for the development of novel antimalarial inhibitors targeting lipid metabolism. Interestingly, biochemical studies revealed that PfPMT activity was inhibited by S-adenosyl homocysteine (SAH) as well as by phosphocholine and its analog and anticancer drug, hexadecylphosphocholine (Miltefosine) . When tested against P. falciparum, hexadecylphosphocholine was found to inhibit the growth of the parasite within human erythrocytes with 50% inhibitory concentrations in the low micromolar range . Genetic studies in P. falciparum demonstrated that PfPMT plays an important role during the intraerythrocytic life cycle of the parasite . Parasites lacking PfPMT display severe alterations in development, replication and survival within human red blood cells . These defects were only partially complemented by choline supplementation.
Here we provide biochemical and biophysical data indicating that PfPMT activity is inhibited by amodiaquine (AQ). Using yeast as a surrogate system we show that AQ inhibits PfPMT activity and blocks PtdCho biosynthesis.
S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), Phosphoethanolamine (P-Etn), Cholorquine (CQ), Amodiaquine (AQ), Hexadecyltrimethylammonium bromide (HDTA), Dodecyltrimethylammonium bromide (DDTA), 2,3-Dichloro- a- methylbenzylamine (DCMB), Chlorpromazine, and Diphenhydramine were purchased from Sigma. Miltefosine (HePC) was purchased from Cayman chemicals. SKF91488 and Tacrine were purchased from Tocris Bioscience. Recombinant SAH nucleosidase (SAHN) was purified from an E. coli strain generously given by K. Cornell (VAMC, Portland, OR, USA), as previously described .
Expression and purification of recombinant PfPMT and BsAda
P. falciparum PfPMT cDNA cloned in the expression vector pET-15b and expressed in E. coli BL21-CodonPlus strain was purified as described earlier [3, 6]. Expression strains were grown at 37°C in LB medium containing 100 μg/ml ampicillin and 35 μg/ml chloramphenicol. A 1 L culture of E. coli was grown until an A600 ~ 0.6, and PfPMT expression was induced by addition of 1 mM isopropyl-β-D-thiogalactopyranosidase (IPTG). The cells were harvested 3 h after induction by centrifugation, and resuspended in 25 ml lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) containing a protease inhibitor cocktail (complete Mini, EDTA-free, Roche Diagnostics). The cells were lysed by sonication using 10 s discontinuous cycles for three minutes on ice, and the cell debris and unbroken cells were removed by centrifugation at 10,000 rpm at 4°C for 30 min. The supernatant was directly applied to a 5 ml Ni-NTA (Qiagen) column pre-equilibrated in lysis buffer. The column was subsequently washed with 40 ml of buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0) and the protein was eluted from the column in a single 15 ml fraction of buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). The protein was incubated at 4°C for 1 h in the presence of 2 mM Na2EDTA, followed by dialysis and concentration against HEPES (N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid) assay buffer (100 mM HEPES-KOH, pH 7.5) using Amicon Ultra centrifugal filter devices. The purity of the recombinant enzyme was examined by SDS-PAGE, and the protein concentration was measured by the method of Bradford using bovine serum albumin as a standard.
The gene encoding Bacillus subtilis adenine deaminase (BsAda) was amplified from genomic DNA by PCR and ligated into a pET15b plasmid vector between XhoI and BamHI sites to yield an N-terminal 6His-epitope-tagged encoding construct. The resulting plasmid was then transformed into E. coli BL21-CodonPlus strain for enzyme expression. The purification protocol for BsAda is similar to PfPMT purification except that the 1 hr incubation step with 2 mM Na2EDTA was omitted.
PfPMT enzyme-coupled spectrophotometric assay
Assays were performed in 96-well UV-transparent plates (acrylic, non-sterile, Costar) at 37°C. Manganese sulfate (MnSO4) was added to a final concentration of 1 mM . The assay mixture in 1X HEPES assay buffer (100 mM HEPES-KOH, pH 7.5) contained 200 μM SAM, 200 μM P-EA, 1 mM MnSO4, 0.5 μM BsAda, 4.72 μM SAHN and 2.5 μM PfPMT in a total volume of 200 μl. A mixture without PfPMT was pre-incubated at 37°C for 10 min and the reaction was initiated with the addition of PfPMT. Absorbance at 265 nm was continuously recorded with a UV, visible plate-reader (Synergy HT Multi-Mode Microplate Reader, Biotek) under kinetic mode. A negative control containing all the reaction components except for PfPMT was conducted on the same plate as the experimental reactions. The absorbance value of the control reaction was subtracted from the experimental absorbance values to eliminate the background signal.
All inhibitor containing stock solutions were prepared in H2O, except for HDTA, which was prepared in DMSO. The final concentration of DMSO in the assays did not exceed 10%. Control experiments containing reaction components and inhibitors were performed in the absence of PfPMT to quantify the intrinsic absorbance of inhibitors at 265 nm. These inhibitors were added before the pre-incubation step. The absorbance at 265 nm for a PfPMT assay without inhibitor was assigned as 100% PfPMT activity. The absorbance values obtained for the various inhibitor concentrations were then converted into a PfPMT activity percentage and plotted against inhibitor concentration. Control experiments were performed to ensure that the inhibitors did not affect the coupling enzymes. To measure coupling enzyme sensitivity to inhibitors, 100 μM SAH was used in place of SAM and P-EA in a 200 μl reaction in the presence of the inhibitor at the highest concentration used in PfPMT assay. For Ki determination e nzymatic assays were performed with increasing concentrations of P-EA (50, 75, 100, 125, 150, 175, and 200 μM), 200 μM SAM, 1 mM MnSO4, 0.5 μM BsAda, 4.72 μM SAHN and 2.5 μM PfPMT in a HEPES assay buffer, along with the inhibitor AQ (0, 2, 4, 10 μM). A reciprocal plot was generated from the absorbance readings at 265 nm versus the P-EA concentrations for each concentration of AQ. The fitted line was extrapolated through the X axis to determine the estimated Ki.
Yeast growth assays
BY4741-pYes2.1 (wild-type) and mutant pem1Δpem2Δ- pYes2.1-PfPMT yeast strains were pre-grown overnight in uracil dropout synthetic galactose (4%) (SG-ura) medium supplemented with 10 μM ethanolamine. The next day, cells were harvested by centrifugation, washed twice by resuspension in water and diluted to an A600 = 0.005 in fresh SG-ura medium supplemented with 100 μM ethanolamine, in the absence or presence of 10, 50, or 100 μM AQ. In a second set of experiments BY4741-pYes2.1, pem1Δpem2Δ-pYes2.1 and pem1Δpem2Δ-pYes2.1-PfPMT yeast strains were pre-grown overnight in SG-ura medium supplemented with 2 mM ethanolamine. The next day, cells were harvested by centrifugation, washed by resuspension in water and diluted to an A600 = 0.005 in fresh SG-ura medium lacking or supplemented with 1 mM choline in the presence of 0 or 200 μM AQ. In both sets of experiments, cells were grown at 30°C and monitored by measuring the A600.
Yeast phospholipid analysis
pem1Δpem2Δ-pYes2.1 and pem1Δpem2Δ- pYes2.1-PfPMT yeast strains were pre-grown in SG-ura medium supplemented with 2 mM ethanolamine and 2 mM choline for 24 hours. The cells were harvested by centrifugation, washed twice by resuspension in water and diluted to an A600 = 0.03 in the SG-ura medium supplemented with 2 mM ethanolamine and grown overnight to an A600 ~ 1.5. The cells were next harvested and the lipids extracted for two dimensional thin layer chromatography using previously described methods [5, 6, 16]. Lipids were stained with iodine vapor and excised from the plate for quantification by measuring phosphorus . The results are shown as the percentage of total phosphorus in each phospholipid fraction.
For NMR analysis, E. coli cells expressing 6his-epitope-tagged PfPMT were grown in minimal medium (M9) containing 15 N-ammonium chloride. The purification of the recombinant enzyme followed the same procedure described above except for the addition of a second step of purification through a Superdex G75 gel filtration column in a buffer containing 50 mM HEPES, 50 mM NaCl, 5 mM DTT pH 6.9. The sample was concentrated and D2O was added to a final concentration of 7% (v/v). Amodiaquine stock solution was prepared in ddH2O due to its low solubility. Serial dilutions were prepared and 5 μl of the diluted samples were added to the NMR sample containing 310 μM of purified PfPMT. The AQ concentration was determined spectrophotometrically using extinction coefficients ε238 = 27 × 103M-1cm-1, ε251 = 23 × 103M-1cm-1 and ε341 = 19 × 103M-1cm-1 . The PfPMT concentration was determined using the Edelhoch method [19–21]. Standard 1H-15N HSQC experiments were performed on 15N-labeled PfPMT using a Varian Inova spectrometer operating at a 1H frequency of 600 MHz. Data were processed using nmrPipe  and analyzed using Sparky  or the Rowland NMR Toolkit http://rnmrtk.uchc.edu.
Coupling PfPMT activity to that of SAH nucleosidase and adenine deaminase
Application of the PfPMT spectrophotometric assay for screening inhibitors of the enzyme
Inhibition of PfPMT activity by amodiaquine in vitro and in yeast
Structural analysis of the interaction between PfPMT and amodiaquine
We also performed NMR titrations using CQ (Fig. 9B). While the effects of CQ are similarly restricted to about 20 residues, indicating site-specific binding, the affinity of CQ for PfPMT is much lower than that of AQ. Complete characterization of CQ binding by NMR is hampered by nonspecific effects that begin to dominate at concentrations above 3 mM (molar ratio of 10 in Fig. 9). Detailed comparison of the concentration dependence of the chemical shifts for two residues, tentatively assigned to Gly32 and Gly68, revealed the difference in the affinities of AQ and CQ for PfPMT. For AQ, the chemical shift changes approached an asymptotic value approximately exponentially, consistent with all binding sites occupied around a 3:1 molar ratio (Figs. 9C and 9D). In contrast, the chemical shifts of Gly32 and Gly68 changed only linearly up to a molar ratio of 10:1. The fact that the same residues are perturbed in response to both AQ and CQ suggest that they interact with the same site on PfPMT, but the different concentration dependence suggests that the affinity of CQ for PfPMT is at least an order of magnitude lower than that of AQ.
Methyltransferases represent a large group of enzymes divided into 160 classes (EC 18.104.22.168 to EC22.214.171.124). These enzymes catalyze the methylation of a large number of different substrates including DNA, RNAs, phospholipids, fatty acids, inositol, phosphoethanolamine, phenylethanolamine, tocopherol, catechol and histamine. Although some methyl donor molecules such as S-methylmethionine, betaine and folate can be used, the majority of biologically active methyltransferase enzymes utilize SAM as a methyl donor . SAM-methyltransferases are recognized by the primary structure and topology of the core fold containing the SAM binding domain . These enzymes play important functions in various biological systems and have been linked to different human diseases such as Alzheimer's disease, attention deficit disorder and pre-eclampsia [33–35]. Phosphoethanolamine methyltransferases (PMTs) represent a recently identified class of enzymes found mostly in plants, nematodes, frogs, fish and some species of protozoa. Their absence in humans and the finding that these enzymes play important physiological functions make them good targets for the development of novel inhibitors to treat worm and protozoan parasitic diseases [3, 8, 11]. The use of radiolabeled SAM to assay PMTs in vitro makes it difficult to perform large-scale screenings of available chemical libraries to search for inhibitors of these enzymes. Here we have modified a continuous coupled assay previously described by Dorgan et al  for the rat arginine N-methyltransferase 1 (PRMT1) to measure PfPMT activity in vitro and demonstrated its feasibility for drug inhibition assays using HePC, which was previously shown to inhibit PfPMT with a radioactivity-based assay . In this report, PfPMT activity was inhibited ~60% at a concentration of 100 μM of HePC and 150 μM of this compound resulted in a complete loss of activity. The enzyme-coupled assay was also useful in identifying HDTA as an inhibitor of PfPMT activity. HDTA was found to have an inhibitory activity similar to that of HePC with ~50% inhibition of PfPMT activity observed at 100 μM of the compound. HDTA inhibits the growth of P. falciparum in culture with an IC50 of 2.1 μM . Using this assay we have also identified AQ as an inhibitor of PfPMT. In addition to its antimalarial activity, AQ is also known as a potent inhibitor of the human histamine N-methyltransferase (HNMT) . Other more potent inhibitors of HNMT such as SKF91488, tacrine, diphenhydramine and chlorpromazine [37–39] had no effect on PfPMT, suggesting that the interaction between PfPMT and AQ is unique.
Amodiaquine is a structural analog of CQ. Analysis of uncomplicated falciparum malaria cases in Africa suggested that AQ is more effective in clearing parasites, including CQ resistant strains, and resulted in better clinical recovery compared to CQ [40–42]. Amodiaquine is used mostly in combination therapy with artesunate and sulfadoxine-pyrimethamine to treat malaria infections, especially those caused by CQ resistant strains . Following oral administration, AQ is detected in the plasma between 30 min and 8 h, but is rapidly converted to desethylamodiaquine (AQm) with a peak plasma concentration of 181 ± 26 ng ml-1 . In the liver AQ is converted to AQm by the polymorphic P450 isoform CYP2C8 . Up to 96 hours after administration, AQm could be detected in the plasma, a property that made AQ an ideal compound in combination therapy . In vitro both AQ and AQm inhibit parasite intraerythrocytic growth with IC50 values of 18.2 and 67.5 nM, respectively . The modes of action of AQ and AQm are unknown. Field studies suggest that low levels of resistance to AQ associate with mutations in the PfCRT and PfMDR1 genes, whereas high AQ resistance involves unknown mechanisms . These findings suggest that AQ might have CQ like properties in the food vacuole as well as novel activities that remain to be elucidated. Interestingly, whereas AQ inhibited PfPMT activity, CQ did not. PfPMT activity was also not affected by the antimalarials quinine, quinidine and quinacrine at concentrations as high as 200 μM. The specificity of inhibition by AQ was demonstrated in vivo by using yeast as a surrogate system. In yeast, the growth of mutants that rely on PfPMT for survival was dramatically altered in the presence of this compound. Consistent with this phenotype, the synthesis of PtdCho in these complemented cells was strongly inhibited by AQ. Surprisingly, the amplitude of inhibition of pem1Δpem2Δ+PfPMT cells by AQ was dependent on the concentration of ethanolamine in the culture medium. This is likely due to the fact that these complemented cells grow faster on medium containing high concentrations of ethanolamine as a result of increased substrate availability for PfPMT.
Recent genetic studies have shown that PfPMT plays an important function during P. falciparum intraerythrocytic development, multiplication and survival , and an essential role in sexual differentiation (Bobenchik et al., unpublished data). Interestingly, silencing of the A. thaliana PEAMT results in multiple morphological phenotypes, including pale-green leaves, early senescence, temperature-sensitive male sterility and increased sensitivity to salts . Furthermore, characterization of an A. thaliana mutant with the T-DNA inserted at the At3g18000 locus (XIPOTL1), which encodes PEAMT revealed severe alterations in root developmental and induces cell death in root epidermal cells . In C. elegans, studies aimed at silencing the PEAMT genes PMT-1 and PMT-2 demonstrated an essential function of these genes in worm, growth, development and survival [8, 11]. Altogether, these genetic studies validate PEAMT enzymes as possible targets for the development of inhibitors that could be used for the treatment of specific human and veterinary protozoa and nematode related infections as well as in agriculture.
P. falciparum parasites lacking PfPMT were, however, equally sensitive to AQ as wild-type parasites in medium containing choline. This result suggests that PfPMT is not the sole target of AQ in choline-containing medium. Our finding that AQ inhibits PfPMT activity, and the fact that this compound also inhibits other eukaryotic methyltransferases, suggest that it may exert its antimalarial activity by targeting several parasite methyltransferases. Several studies have suggested that aminoquinolines may exert their antimalarial activity by interfering with biological functions other than heme detoxification in the digestive vacuole. Famin and Ginsburg reported inhibition of parasite's 6-phosphogluconate dehydrogenase by CQ . Sharma and Mishra reported inhibition of parasite's tyrosine kinase by this compound . Recently, CQ was reported to inhibit vesicular trafficking within the parasite , and to induce proteome oxidative damage . More recent studies aimed at investigating the stage specificity of CQ and comparing the effect of continuous vs bolus dosing on P. falciparum strains revealed that rings (prior to the formation of the digestive vacuole) and schizont stage parasites (after hemoglobin degradation and heme detoxification activities have plateaued) were equally sensitive to this compound . The bolus dosing concentrations of CQ used by Gligorijevic and colleagues are in the range of the compound's peak plasma concentration (low micromolar range) . Together these studies suggest that heme detoxification, although important, is unlikely to be the only target of CQ. Although, the stage specificity and the effect of bolus dosing of AQ on P. falciparum have not been investigated, our results indicate that at its peak plasma concentration, AQ is likely to inhibit PfPMT and possibly other parasite methyltransferases. Future studies will aim to identify Plasmodium methyltransferases inhibited by AQ and to characterize the mechanism of sensitivity and resistance to AQ. From a chemistry standpoint, AQ represents an excellent lead compound for the rational design of better inhibitors of PfPMT and other parasite methyltransferases as well as plant and nematode PMTs.
Horton and coworkers have previously shown that AQ acts both as a competitive and a noncompetitive inhibitor of human HNMT . The crystal structure of the HNMT-AQ complex  revealed that one AQ molecule binds to the histamine-binding site (Site 1), while the second one tucks into an adjacent pocket on the outer surface of the protein (Site 2) (Fig. 8A). Both quinoline rings fit into a sandwich-like structure formed by the aromatic side chains of Tyr15 and Phe19, and Phe190 and Tyr198, respectively (Fig. 8B). The side chains of Trp183 and Phe243 also contribute to the stabilization of the branched alkylamino tail of the AQ in the histamine-binding site . It should be noted that the branch structure of the second AQ is disordered in the outer-surface pocket, suggesting a weaker binding interaction. The strong hydrophobic interactions between AQ and the aromatic side chains of the enzyme have been proposed to account for the affinity and specificity of the inhibitor for HNMT . Sequence alignment analysis of HNMT and PfPMT shows that Tyr15, Trp183, and Phe243 are conserved in PfPMT, while Phe190 is substituted by tyrosine (Fig. 8B and Additional file 2, Fig. S2). This structural conservation is significant, as the two proteins have an overall 14% pairwise sequence identity. The majority of the other invariant residues are within the classic SAM-dependent methyltransferase fold in HNMT (Fig. 8B and Additional file 2, Fig. S2). Our inhibition studies revealed that whereas AQ inhibited PfPMT activity, CQ did not. PfPMT activity was also not affected by the aminoquinolines, quinine, quinidine and quinacrine at concentrations as high as 200 μM, or by the histamine methyltransferase inhibitors SKF91488, diphenhydramine and tacrine at concentrations as high as 100 μM. Unlike AQ, diphenhydramine and tacrine have been shown to interact only with Site 1 of HNMT. If this property is also valid in the case of PfPMT, it may account for the difference in inhibition of PfPMT activity between AQ and other aminoquinolines. The NMR studies reported here confirmed the site-specific binding of AQ to PfPMT, as less than 10% of residues in PfPMT showed a significant change in chemical shift in the presence of the compound. Nine of these residues have thus far been assigned. Six of those (corresponding to Glu28, Ser26, Ala63, Glu65, Ile66 and Met36 in HNMT) are proximal to the histamine-binding site (Site 1), while the 7th (Leu261 in HNMT) is proximal to Site 2. The 8th (Cys196 in HNMT) is located between the two sites. The last residue (Leu108 in HNMT) is further from Site 1, but is proximal to residues in contact with this site, and could be a relayed effect. The three residues (Gly68, Gly32 and Gly40 corresponding to Glu65, Glu28 and Met36 in HNMT) with the largest shift changes are closest to site 1. The binding of AQ to the free enzyme suggests that the inhibition is not uncompetitive, i.e. via binding to the enzyme-substrate complex, and therefore either competitive or noncompetitive. The NMR experiments at this stage do not definitively reveal whether AQ binds competitively to the substrate binding site. NMR studies to determine the structure of PfPMT alone and in combination with AQ are underway. They will address the question of noncompetitive vs. competitive inhibition, and should shed new light on the interaction between the enzyme and the inhibitor. However, based on our preliminary assignment of two of the nine assigned residues, Gly32 and Gly68, and the known structure of HNMT, we can postulate a structural hypothesis consistent with the NMR results for Gly32 and Gly68. Gly32 and Gly68 of PfPMT correspond to Glu28 and Glu65 of HNMT (Fig. 9). Glu28 lies at the bottom of the cleft comprising site 1 of HNMT. Glu65 also lines the surface of the cleft, closer to the enzyme surface. The similarity of the chemical shift perturbation curves (to within the experimental uncertainty of ~ 0.006 ppm) for Gly32 and Gly68 is thus consistent with binding at site 1. Despite the low sequence homology between PfPMT and HNMT, these data suggest that PfPMT and HNMT nevertheless exhibit structural homology, and are consistent with two AQ binding sites. The proximity of the two binding sites suggests that titration data might reflect coupling between sites. Indeed, we do not observe partitioning of the titration data into two distinct classes, as would be expected for independent binding sites.
The enzyme-coupled assay adapted for PfPMT is a simple and reliable method for measuring PfPMT activity and screening for specific inhibitors of this enzyme. The identification of AQ as an inhibitor of PfPMT may help in the future design of compounds that specifically inhibit this enzyme and possibly other PMTs.
amodiaquine AQm: desethylamodiaquine CQ: chloroquine PtdCho: phosphatidylcholine
serine decarboxylase phosphoethanolamine methyltransferase
thin layer chromatography
nuclear magnetic resonance
We thank Dr. Peter Setlow for providing Bacillus subtilis DNA. We are grateful to Dr. Ken Cornell, Boise State University, for providing the SAH nucleosidase expression vector, and to Dr. Zhaohui Sunny Zhou, Washington State University, for helpful discussions about the assay. We thank Oksana Gorbatyuk, Li Luo and William Witola for help with the expression and purification of PfPMT. This research was supported by NIH and DOD grants [AI51507], [PR033005] and BWF award  to CBM. CBM is a recipient of the Burroughs Wellcome Award, Investigators of Pathogenesis of Infectious Disease. JCH acknowledges support from NIH [RR020125].
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