¹H, ¹³C, and ¹⁵N chemical shift assignments for PfPMT, a phosphoethanolamine methyltransferase from Plasmodium falciparum

Abstract

Phosphoethanolamine methyltransferases (PMTs also known as PEAMTs) catalyze the three-step s-adenosylmethionione-dependent methylation of phosphoethanolamine to form phosphocholine. These enzymes play an important function in the synthesis of phosphatidylcholine, the major phospholipid in the membranes of lower and higher eukaryotes, as well as in the production of the compatible solute and osmoprotectant glycine betaine in plants. Genetic studies in plants, Caenhorhabditis elegans and Plasmodium falciparum have demonstrated that disruption of PMT activity results in severe defects in important cellular processes such as development, replication, survival and sexual maturation and differentiation. Here we report chemical shift assignments for PfPMT, the PMT from Plasmodium falciparum. X-ray crystal structures have been recently reported for complexes of PfPMT, but the structure of the apoenzyme remains unknown. The solution structure of the apoenzyme will help to elucidate important details of the mechanism of substrate binding by PfPMT, as residues comprising the substrate binding site are inaccessible to solvent in the conformation evident in the available crystal structures. In addition to enabling determination of the solution structure of the apoenzyme, the assignments will facilitate additional investigations into the interaction of PfPMT with its substrates and inhibitors.

Biological context

The PfPMT enzyme of Plasmodium falciparum, the agent of severe human malaria, is a defining member of a large family of phosphoethanolamine methyltransferases (PMTs) recently identified in plants, worms, frogs, and some proteobacteria (Bobenchik et al. 2011). This family is divided into four classes depending on the size of the enzyme members and whether they contain one or two catalytic domains (Bobenchik et al. 2011). Functional studies revealed that PfPMT plays a critical role in the synthesis of phosphatidylcholine (PtdCho) via a plant-like pathway involving serine decarboxylation and phosphoethanolamine methylation (Pessi et al. 2004). Genetic studies in P. falciparum have shown that this enzyme is critical for parasite development and survival within human erythrocytes (Witola et al. 2008). PtdCho constitutes half of the phospholipid content of the parasite membranes. Biochemical studies demonstrated that PtdCho synthesis occurs via two metabolic routes (Pessi et al. 2004; Bobenchik et al. 2011). The first route is the CDP-choline pathway, which uses host choline as a precursor. The second route is the serine decarboxylation-phosphoethanolamine methylation pathway, which uses serine, either transported from the host or generated by degradation of host proteins as a phospholipid precursor. The serine is first decarboxylated to produce ethanolamine, by an unknown serine decarboxylase. The ethanolamine is next phosphorylated by a parasite-specific ethanolamine kinase. A SAM-dependent triple methylation of the resulting phosphoethanolamine (P-Etn) by PfPMT results in the synthesis of phosphocholine (P-Cho). P-Cho then integrates into the CDP-choline pathway for the synthesis of PtdCho.

The 266 amino acid PfPMT enzyme is half the size that of plant PMT enzymes and contains a single SAM-dependent catalytic domain. Its specificity for its substrates and co-substrates was demonstrated using both biochemical and genetic studies (Reynolds et al. 2008). Interestingly, no PfPMT homologs are found in mammalian databases, suggesting that PfPMT could be an ideal target for development of novel inhibitors. Despite recent crystal structures solved for complexes of PfPMT, important questions about substrate recognition remain unresolved (Lee et al. 2012). Residues involved in substrate recognition by PfPMT are inaccessible to solvent in the crystal structures of the complexes, suggesting that conformation dynamics plays an important role in recognition. The detailed nature of the conformational changes remains the subject of speculation until the structure of the apoenzyme is determined.

Relatively little is known about the evolution of substrate specificity in PMTs. PfPMT has a single catalytic domain that catalyzes the three successive methyl transfer reactions in the formation of P-Cho from P-Etn (Pessi et al. 2004), in contrast to plant and worm PMTs that have catalytic domains with distinct specificities for different substrates (reviewed in Bobenchik et al. 2011). The solution structure of apo-PfPMT, which will be enabled by the chemical shift assignments, should yield significant new insights into the mechanism of substrate recognition, and as an exemplar of the simplest class of PMTs, further advance understanding of the evolution of specificity in substrate recognition by PMTs in other organisms.

Methods and experiments

Protein expression and purification

Full length PfPMT was expressed in the Escherichia coli BL21-CodonPlus strain (Stratagene) as a His-tag fusion protein. Isotope labeling was performed using M9 media, supplemented using a micronutrient mixture similar to one previously described (Weber et al. 1992), containing uniformly ¹⁵N-labeled ammonium chloride and uniformly ¹³C-labeled glucose. His-tagged PfPMT was purified using Ni–NTA Agarose (Qiagen) using a standard protocol of imidazole elution. The eluate was further purified by Superdex75 gel filtration chromatography using an AKTApurifier (GE) system. The final NMR sample contained 0.4 mM uniformly ¹³C/¹⁵N-labelled PfPMT in 50 mM HEPES, pH 6.9, 50 mM NaCl, 5 mM DTT, 1 mM EDTA and 10% (v/v) D₂O.

NMR spectroscopy

Most NMR experiments were recorded at 25°C on Agilent VNMRS spectrometers operating at 500, 600, and 800 MHz, all equipped with cryogenically-cooled triple-resonance pulse field gradient probes. Supplemental data were collected using Bruker Avance spectrometers operating at 800 MHz (Landsman Research Facility, Brandeis) and 900 MHz (MIT), both equipped with TXI cryoprobes. The backbone and side-chain resonances were assigned using ¹⁵N-HSQC, HNCACB, CBCA(CO)NH, HBHA(CO)NH, 13C-HSQC, (H)CCH-TOCSY and H(C)CH-TOCSY experiments (Kay 1995). NMR data sets were processed using NMRPipe (Shen et al. 2009). Nonuniformly sampled data were processed with the Rowland NMR Toolkit (http://rnmrtk.uchc.edu). Spectra were analyzed using Sparky (Goddard, T. D. and Kneller, D. G., SPARKY-NMR, 2003, University of California San Francisco). The backbone Φ and ψ torsion angles were derived from chemical shifts of backbone atoms using TALOS+ (Shen et al. 2009).

Assignments and data deposition

The ¹H-¹⁵N HSQC spectrum of the full length, 266 amino acid residue long, PfPMT is shown in Figure 1a. The assigned backbone N and HN resonances are annotated in the supplemental material, Fig. 1S. The resonance assignments were made for 95% of backbone residues and 75% of side chain residues. The assignment of remaining resonances was not possible due to spectral overlap and ambiguity. The regular secondary structure elements of the PfPMT were predicted based on ¹Hα, ¹³Cα, ¹³Cβ and ¹³C′ secondary chemical shifts (Wishart and Sykes 1994) and are shown in Fig. 1b.

Fig. 1.

¹⁵N-¹H HSQC spectrum of uniformly ¹⁵N-labeled PfPMT. Assigned backbone resonances are indicated by black dots in the center of a peak. The annotated spectrum is available in “Supplementary materials”

The ¹H, ¹³C and ¹⁵N chemical shifts of PfPMT have been deposited into the BioMagResBank (http://www.bmrb.wisc.edu; accession no. 18303) (Fig. 2).

Fig. 2.

Elements of PfPMT secondary structure. Secondary structure propensities calculated based on ¹Hα, ¹³Cα, ¹³Cβ and C′ secondary chemical shifts (Wishart and Sykes 1994) are shown as red and blue bars for α-helix and β-sheet, respectively