Thermodynamic evaluation of ligand binding in the plant-like phosphoethanolamine methyltransferases of the parasitic nematode Haemonchus contortus

Abstract

Nematodes are a major cause of disease and the discovery of new pathways not found in hosts is critical for development of therapeutic targets. Previous studies suggest that Caenorhabditis elegans synthesizes phosphocholine via two S-adenosylmethionine (AdoMet)-dependent phosphoethanolamine methyltransferases (PMT). Here we examine two PMT from the parasitic nematode Haemonchus contortus. Sequence analysis suggests that HcPMT1 contains a methyltransferase domain in the N-terminal half of the protein and that HcPMT2 encodes a C-terminal methyltransferase domain, as in the C. elegans proteins. Kinetic analysis demonstrates that HcPMT1 catalyzes the conversion of phosphoethanolamine to phosphomonomethylethanolamine (pMME) and that HcPMT2 methylates pMME to phosphodimethylethanolamine (pDME) and pDME to phosphocholine. The IC(50) values for miltefosine, sinefungin, amodiaquine, diphenhydramine, and tacrine suggest differences in the active sites of these two enzymes. To examine the interaction of AdoMet and S-adenosylhomocysteine (AdoCys), isothermal titration calorimetry confirmed the presence of a single binding site in each enzyme. Binding of AdoMet and AdoCys is tight (K(d) ∼2-25 μm) over a range of temperatures (5-25 °C) and NaCl concentrations (0.05-0.5 m). Heat capacity changes for AdoMet and AdoCys binding suggests that each HcPMT differs in interaction surface area. Nonlinear van't Hoff plots also indicate a possible conformational change upon AdoMet/AdoCys binding. Functional analysis of the PMT from a parasitic nematode provides new insights on inhibitor and AdoMet/AdoCys binding to these enzymes.

FIGURE 1.

PMT overview. A, the three methylation reactions in the phosphobase methylation pathway found in plants, nematodes, and Plasmodium. B, methyltransferase domain organization in the type 1 (plant), type 2 (Plasmodium), and type 3 (nematode) PMT. MT-1 catalyzes conversion of pEA to pMME and MT-2 methylates pMME to pDME and pDME to pCho. SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine.

FIGURE 2.

ITC analysis of AdoCys binding to HcPMT1 and HcPMT2. A, titration of HcPMT1 with AdoCys. The top panel shows the ITC data plotted as heat signal (μcal s⁻¹) versus time (min). The experiment consisted of 30 injections of 10 μl of AdoCys (396 μm) into a solution containing protein (31.0 μm) at 20 °C. The botton panel shows the integrated heat response per injection from panel A plotted as normalized heat per mol of injectant. The solid line represents the fit to the data. B, titration of HcPMT2 (68.1 μm) with AdoCys (786 μm). Top and bottom panels are as described in A. SAH, S-adenosylhomocysteine.

FIGURE 3.

Temperature dependence of thermodynamic parameters for AdoCys/AdoMet binding. The temperature dependence of ΔG (circle), ΔH (open circle), and −TΔS (square) for binding of (A) AdoMet to HcPMT1, (B) AdoCys to HcPMT1, and (C) AdoCys to HcPMT2. The ΔH values were used to calculate the change in heat capacity (ΔC_p). D, van't Hoff plot of K_eq versus temperature. Data are shown for HcPMT1-AdoMet (circles), HcPMT1-AdoCys (open circles), and HcPMT2-AdoCys (squares).

FIGURE 4.

Ionic dependence of AdoCys/AdoMet binding. Effect of NaCl concentration on K_d for the binding of AdoMet to HcPMT1 (circles), AdoCys to HcPMT1 (open circles), and AdoCys to HcPMT2 (squares).