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. Author manuscript; available in PMC: 2015 May 1.
Published in final edited form as: Trends Parasitol. 2014 Mar 28;30(5):241–250. doi: 10.1016/j.pt.2014.03.001

Nematode phospholipid metabolism: an example of closing the genome-structure-function circle

Soon Goo Lee 1, Joseph M Jez 1
PMCID: PMC4040950  NIHMSID: NIHMS581219  PMID: 24685202

Abstract

Parasitic nematodes that infect humans, animals, and plants cause health problems, livestock and agricultural losses, and economic damage worldwide and are important targets for drug development. The growing availability of nematode genomes supports the discovery of new pathways that differ from host organisms and are a starting point for structural and functional studies of novel antiparasitic targets. As an example of how genome data, structural biology, and biochemistry integrate into a research cycle targeting parasites, we summarize the discovery of the phosphobase methylation pathway for phospholipid synthesis in nematodes and compare the phosphoethanolamine methyltransferases from nematodes, plants, and Plasmodium. Crystallographic and biochemical studies of the phosphoethanolamine methyltransferases in this pathway provide a foundation that guides the next steps that close the genome-structure-function circle.

Keywords: Caenorhabditis elegans, nematode, phosphobase methylation, phospholipids, methyltransferase, phosphoethanolamine, phosphocholine, apicomplexa

Parasitic nematodes and current broad-spectrum drugs

Nematodes are extremely diverse creatures and are the most abundant animals [1]. Over 25,000 species of nematodes have been described, and millions of species are estimated to exist [2–3]. They are found worldwide in marine, aquatic, and terrestrial environments as either free-living or parasitic animals. Parasitic nematode infections of humans, animals, and plants cause significant medical health problems and agricultural damage, especially in tropical and impoverished regions. In humans, more than 3 billion people, mainly in the developing world, are infected by the most prevalent intestinal nematodes, including Ascaris lumbricoides, Trichuris trichiura, Necator americanus, and Ancylostoma duodenale [4–8]. The $3.5 billion global market for veterinary anthelmintics reflects the threat of parasitic nematodes, such as Haemonchus contortus and Dirofilaria immitis, to livestock, farm animals, and pets [9–10]. Likewise, plant parasitic nematodes, such as Heterodera glycines and Meloidogyne spp., cause major crop damage worldwide estimated at $100 billion annually [10–12].

Despite the worldwide impact of these organisms, there are few broad-spectrum compounds available that target intestinal and extra-intestinal nematode parasites [4, 13–16]. The most common antiparasitic compounds are benzimidazole derivatives, such as albendazole, menendazole, and thiabendazole [13]. These drugs target microtubules and the mitotic spindles of intestinal (i.e., Ascaris, Trichuris, N. americanus, A. duodenale, and Enterobius), extra-intestinal (i.e., Trichinella and Toxocara), and other tissue nematodes (i.e., Angiostrongylus, Baylisascaris, Gnathostoma, and Capillaria) [14]. The imidothiazole-tetrahydropyrimidine anthelmintics, including levamisole and tetramisole, are the second class of broad-spectrum compounds. Although the inhibition mechanism of imidothiazoles against parasites is not well understood, a study of Caenorhabditis elegans suggests that levamisole binds to a nicotinic acetylcholine receptor [17]. The avermectin-milbemycin macrolides, including ivermectin and moxidectin, are the third broad-spectrum and are some of the most effective therapeutics targeting nematodes available [18–19]. Ivermectin, which binds chloride channels of nerve and muscle cells in the target nematodes, is commonly used either as a primary treatment or a combination treatment with albendazole against many parasitic nematodes, especially Onchocerca, the causative agent of river blindness [18–22].

Even though the three major classes of nematicides remain effective controls of parasitic diseases, drug resistance in many parasites has been reported since the 1950s and multi-drug resistance has appeared globally [13, 21, 23–29]. To control drug-resistant nematodes, the development of new antiparasitic compounds and the identification of biochemical targets are inevitable. Because eukaryotes, including humans, protozoans, and nematodes, share many core metabolic pathways, discovering novel targets in these organisms and developing compounds that avoid non-specific, high toxicity present significant challenges and new opportunities.

Combining genomics, structural biology, biochemistry, and parasitology

A hallmark feature in modern drug discovery and development is a research cycle that combines genomic information, structural biology, and functional analysis of potential macromolecular targets. With regard to parasites and the development of compounds targeting these organisms, C. elegans has long been a tractable system for exploring nematode biology. Completion of the C. elegans genome in 1998 combined with the development of RNA interference (RNAi) in 2000 led to this model system becoming a powerful tool for biological discovery and for the characterization of new drug targets and molecules [30–34]. For example, the automation of RNAi and high-throughput assays make C. elegans a first step in many drug development efforts [35–38]. Since then, next-generation sequencing technologies have lowered the barriers for genome and transcriptome sequencing to enable comparative studies of nematodes and other organisms [39–40]. The low cost of sequencing and improvements in computational methods have expanded the scope of nematode genomes currently available and driven the development of multiple open-access resources that provide information on nematode parasites and tools for browsing genes of interest (Table 1) [41–43]. For nematode parasitology and pharmacology, the enormous amount of genomic information is a gateway for the development of new antiparasitic compounds, for the identification of novel targets, and for the discovery of biochemical pathways that differ from host organisms.

Table 1.

Genomic and structural resources for nematode studies

Resources Description
Base (http://www.wormbase.org/)

  • Genetics, genomics, and biology of C. elegans and related nematodes

  • BLAST/BLAT SEARCH: Genome database for 9 free-living, 3 human-parasitic, 4 animal-parasitic, 2 plant-parasitic, and 1 entomopathogenic nematodes
Nematode.net (http://nematode.net/)

  • Resource for parasitic nematodes at the Genome Institute; Washington University in St. Louis

  • NemaBLAST; Genome database for 3 free-living, 4 human-parasitic, 9 animal-parasitic, 14 plant-parasitic nematodes
Nematodes.org (http://www.nematodes.org/)

  • Resource for nematode genomics at the University of Edinburgh

  • NEMBASE4: Available transcriptome database for 63 nematode species

  • 959 Nematode Genomes: Provide genome database URL for 40 nematode strains
RCSB Protein Data Bank

  • Biological macromolecular structures

  • Available three-dimensional structures of nematode proteins : 276 X-ray crystallography and 39 solution NMR
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To complement and extend genomic approaches, molecular studies aimed at elucidating protein function and structural biology methods, such as X-ray crystallography, are critical for target characterization and provide an experimental foundation for computational chemistry, molecular docking, and virtual screening during the drug discovery and development process [44–47]. Protein crystallography was traditionally regarded as a complex and laborious technique, but recent advances in experimental methods (e.g., protein purification, crystallization, and X-ray data collection), software (e.g., data processing, structure determination, structure fitting, and structure refinement software), and X-ray light sources have led to it becoming an integral and accessible tool for understanding biological and chemical functions of macromolecules [45]. Indeed, drug development projects blending genomics, structural biology, and parasitology are underway at multiple structural genomics centers, such as Medical Structural Genomics of Pathogenic Protozoa group (http://www.msgpp.org) and the Structural Parasitology at the Structural Genomics Consortium (http://www.thesgc.org/science/parasitology). Although these projects are focused mainly on diseases caused by pathogenic protozoa, such as Plasmodium and Toxoplasma, as the genome information on parasitic nematodes grows, the number of crystal structures of proteins from C. elegans and other nematodes has also increased (Figure 1). Since 2010, the total number of three-dimensional structures of proteins from nematodes, and in particular parasitic nematodes, has rapidly increased. Although proteins from C. elegans and A. suum account for most of the nematode proteins in the Protein Data Bank (PDB), a growing number of structures from a variety of parasitic nematodes are being deposited. This trend promises new insights on important proteins of interest to parasitologists focused on nematodes.

Figure 1. Structural biology of nematode proteins.

Figure 1

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The yearly total of X-ray crystal structures from nematodes deposited in Protein Data Bank (PDB; (http://www.pdb.org/) from 1995 to 2013 are shown as blue bars. The numbers of X-ray crystal structures from parasitic nematodes are shown as a red line. The inset pie chart shows the distribution of X-ray crystal structures by nematode species. Genus and species for organisms indicated are as follows: Caenorhabditis elegans, Ascaris suum, Brugia malayi, Ancylostoma ceylanicum, Necator americanus, Ancylostoma caninum, Trichinella spiralis, Haemonchus contortus, Onchocerca volvulus, Wuchereria bancrofti, Strongyloides stercoralis, Ostertagia osetertagi, Toxascaris leonina, and Heligmosomoides polygyrus.

Structural parasitology targeting nematodes of medical, veterinary, and agricultural interest adds another strategy for developing mechanistic insight on target protein function, for defining relevant functional features, and as a tool for understanding protein-drug interactions. Not only does this approach aid in drug discovery, but also the detailed information obtained about the target protein can be used in conjunction with genomic data to develop new questions about a particular pathway or a protein target across a variety of parasites. The recent identification of the phosphobase methylation pathway for phospholipid metabolism in nematodes and Plasmodium provides an example of how to close the circle of genomics, structural biology, and functional analysis to open new avenues in parasite research [48–51].

Phosphatidylcholine synthesis and the phosphobase methylation pathway

Phospholipids are major structural components of cellular membranes, but can also play important roles in signal transduction pathways, as membrane anchors for proteins and glycans, or as modulators of protein activity [52]. In eukaryotes, phosphatidylcholine (PtdCho) is the predominant membrane phospholipid, accounting for 40–60% of membrane content [52]. Physically, the composition of polar head groups in phospholipids influences cellular structure with phosphatidylethanolamine affecting membrane curvature and vesicle formation and PtdCho driving formation of lipid bilayers [53]. In addition to defining membrane architecture, PtdCho also serves as a structural component of serum lipoproteins and as a precursor for lipid second messengers [54]. In nematodes, changes in membrane composition occur at different developmental stages and as an adaptation to environmental conditions with phosphorylcholine-containing glycolipids also as elements in the extracellular matrix [55]. As described for apicomplexan parasites [56–57], nematode parasites likely use host lipids as source material for tailoring phospholipid composition. As the most abundant membrane phospholipid, parasitic organisms need PtdCho for adaptation to the host and for increased membrane biogenesis to support growth and proliferation.

The biosynthesis of PtdCho occurs primarily through three possible metabolic routes (Figure 2A). In mammals, PtdCho is mainly synthesized through the de novo choline (or Kennedy pathway) (Figure 2A, pink) [57]. The de novo choline pathway consists of three enzymes, choline kinase, CTP:phophosphocholine cytidyltransferase, and diacylglycerol choline phosphotransferase [53, 58]. Yeast and mammalian liver cells primarily use an endogenous route - the Bremer-Greenberg pathway (Figure 2A, gold) [59]. The Bremer-Greenberg pathway consists of two sequential methylation reactions that convert phosphatidylethanolamine (PtdEA) to PtdCho. The S-adenosylmethionine (SAM)-dependent methylation reactions are catalyzed by phosphatidylethanolamine N-methyltransferases known as CHO2 (or PEMT1) and OPI3 (or PEMT2) in yeast [60–61]. Plants produce phosphocholine (pCho) by the phosphobase methylation pathway (Figure 2A, blue). This third route accounts for nearly all the metabolic flux into the de novo choline pathway in plants [62]. It also provides an alternate pathway for exogenous or recycled choline to be used as a precursor for PtdCho synthesis in the Kennedy pathway. The phosphobase methylation pathway consists of successive methylation reactions from phosphoethanolamine (pEA) to pCho, mediated by the SAM-dependent enzyme phosphoethanolamine methyltransferase (PMT) [62–64].

Figure 2. Phosphatidylcholine biosynthesis and the PMT.

Figure 2

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(A) Routes for phosphocholine (pCho) synthesis are shown, as follows: the de novo choline (or Kennedy) pathway (red), the Bremer-Greenberg (blue), and the phosphobase methylation pathway (gold). Names of metabolites consist of a prefix (p, phospho; CDP-, cytidine 5’-diphosphate; or Ptd, phosphatidyl) and a core name (EA, ethanolamine; MME, monomethylethanolamine; DME, dimethylethanolamine; Cho, choline). Enzymes corresponding to each metabolic step are shown in italics. (B-E) Three-dimensional structures and methyltransferase domain organization in the PMT. Panel B shows a homology model of the type I PMT from Arabidopsis thaliana (At3g18000). The structure was modeled using Protein Homology/analogY Recognition Engine V 2.0 [86]. Plant PMT contain two methyltransferase domains (MT) in tandem. MT1 (yellow) catalyzes the conversion of pEA to pMME. MT2 (blue) catalyzes the methylation of pMME to pDME and pDME to pCho. Panel C shows the protein crystal structure of the P. falciparum PMT [PDB: 3UJ6; 76], which contains a single bifunctional domain that catalyzes all three methylation reactions. Panels D and E show the crystal structures of H. contortus PMT1 (PDB: 4KRG) and PMT2 (PDB: 4KRH), respectively [79]. The monofunctional type III PMT contain either an MT1 (gold) or MT2 (blue) domain and vestigial MT domains (gray). Abbreviations: PMT, phosphoethalamine methyltransferases; CK, choline kinase; CCT, choline cytidylyltransferase; CPT, cholinephosphotransferase; EK, ethanolamine kinase; ECT, phosphoethanolamine cytidylyltransferase; EPT, ethanolaminephosphotransferase; PEMT, Phosphatidylethanolamine methyltransferase; PMT, Phosphoethanolamine methyltransferase.

The PtdCho biosynthesis pathways in nematodes are not well understood at the molecular level. Genome data indicates the presence of the genes for both the de novo choline and Bremer-Greenberg pathways in C. elegans [65]. High-throughput RNAi screens have examined the de novo choline and Bremer-Greenberg pathways to varying degrees. Generally, RNAi screens show the importance of the later enzymes in the de novo choline pathway and a lesser role of the Bremer-Greenberg pathway for worm growth and development. RNAi of choline kinase yields mixed results with a majority describing no phenotypic effects [32–33, 66–68], although some studies report abnormal embryo and post-embryo developmental phenotypes [31, 34, 69]. Targeting of CTP:phophosphocholine cytidyltransferase and diacylglycerol choline phosphotransferase results in phenotypes ranging from the arrest of larva growth to embryo lethality [31–32, 66, 68]. In comparison, RNAi of the PEMT of the Bremer-Greenberg pathway produces no observable phenotypes [34, 66]. These studies suggested the presence of an alternate route to provide metabolites for the synthesis of PtdCho in C. elegans.

Recent studies show that C. elegans, as well as the protozoan parasite Plasmodium falciparum, synthesize pCho through the plant-like phosphobase pathway [48–51]. Because the plant-like phosphobase pathway is not found in mammals, this raises the possibility of targeting the PMT in nematode and protozoan parasites for inhibitor development.

PMT from plants, Plasmodium, and nematodes

The phosphobase methylation pathway in plants was originally discovered in the mid-1980s, but the cloning and characterization of the PMT from spinach suggested a link to nematode and protozoan phospholipid metabolism [70–72]. Sequence searches using the plant PMT identified two hypothetical genes in C. elegans (ZK622.3a/b; pmt1 and F54D11.1; pmt2) sharing ∼20% identity with the PMT from plants. Interestingly, each of the C. elegans genes were more closely related to the plant PMT than to each other, as they share only 12% sequence identity. This similarity led to targeted gene silencing of pmt1 and pmt2 by RNAi, which resulted in pronounced growth and developmental phenotypes [49–50]. Expression of each gene is required for normal growth and development of C. elegans at the L1 (early larva), L4 (late larva), and dauer (stasis) stages [49–50]. Subsequent studies revealed that loss of pmt1 results in the accumulation of large lipid droplets in the intestines of C. elegans, indicating that pmt1 is also involved in the homeostasis of cholesterol, triacylglycerol, and PtdCho [73–74]. Biochemical analyses of the C. elegans PMT confirmed the enzymatic activity of the enzymes but also revealed significant differences from the plant enzymes [49–50].

Functional studies of the PMT from plants (type I), P. falciparum (type II), and nematodes (type III) confirm the essential roles in the phosphobase methylation pathway in these organisms [48–51, 70–72]. Like many SAM-dependent methyltransferases, the amino acid sequences of the PMT catalytic domains share four consensus sequence motifs (I-IV) that define canonical SAM-binding sites [75]. Interestingly, each type of PMT differs in overall domain architecture. Recently solved X-ray crystal structures reveal that the physical organization of active sites that catalyze distinct reaction chemistry in methyltransferase domains (MT1 and MT2) of the PMT also dramatically varies (Figure 2B – E).

In the case of the type I PMT from plants, a single 470–500 amino acid polypeptide contains two catalytic methyltransferase domains - one at the N-terminus (MT1) and another at the C-terminus (MT2) (Figure 2B). In this di-domain enzyme, the N-terminal MT1 active site only converts pEA to phosphomonomethyl-ethanolamine (pMME) [71]. The C-terminal MT2 active site catalyzes the last two methylation reactions from pMME to phosphodimethyl-ethanolamine (pDME) and from pDME to pCho [71–72].

Characterization of the PMT from the malaria parasite P. falciparum revealed that this enzyme is also multifunctional, but of smaller size (length of ∼260 – 280 amino acids) [48]. Moreover, the type II PMT contains a single methyltransferase domain that catalyzes all three methylation reactions in the phosphobase pathway. This enzyme is required for normal growth and transmission of the parasite [48, 51]. X-ray crystal structures of the P. falciparum PMT (Figure 2C) provided the first molecular insight on how the PMT catalyze phosphobase methylation. The Plasmodium enzyme shares the same overall three-dimensional fold as the C-terminal domain of the nematode PMT2 protein [76–77]. Structures of P. falciparum PMT complexed with pEA, pCho, sinefungin (a methyltransferase inhibitor), and both pEA and S-adenosylhomocysteine (SAH) suggest that conformational rearrangements occur upon ligand binding to cover the active site [76]. These structures and extensive site-directed mutagenesis of key residues identified a catalytic dyad consisting of a tyrosine and histidine, which also form a 'catalytic latch' that locks ligands in the active site for catalysis [76].

The type III PMT found in nematodes are structurally distinct and reveal another variation of active site organization in the phosphobase methylation pathway. Each nematode PMT contains a methyltransferase domain in either half of the molecule [49–50, 78]. The original sequence analysis suggested that PMT from C. elegans lacked the dual methylation domain architecture. In contrast to plants and Plasmodium, the nematode phosphobase methylation pathway requires two separate PMT proteins, which are PMT1 and PMT2 (Figure 2D – E).

Steady-state kinetic analysis of PMT1 from C. elegans and H. contortus showed that these proteins contain the N-terminal MT1 domain that only performs the first methylation reaction of the pathway (i.e., conversion of pEA to pMME) using a random Bi Bi kinetic mechanism [50, 78–79]. Similarly, biochemical studies of the PMT2 from these nematodes show that the C-terminal methyltransferase domain uses a random Bi Bi kinetic mechanism to catalyze the methylation of pMME to pDME and pDME to pCho [49, 78–79]. Thermodynamic analysis of SAM and SAH binding to PMT1 and PMT2 from H. contortus demonstrates the presence of a single ligand-binding site in each enzyme and highlights structural differences in their active sites [78]. Collectively, the chemistry of the phosphobase pathway in nematodes is split between two proteins with each enzyme having distinct reaction specificity.

Crystal structures of H. contortus PMT1 and PMT2 reveal the structural evolution that led to specialization of each protein (Figure 2D–E) [79]. The structure of HcPMT1 complexed as a dead-end ternary complex with pEA and SAH and that of HcPMT2 with SAH and pMME bound clearly show different di-domain structures with a functional methyltransferase domain in either the N- (PMT1) or C-terminal (PMT2). Interestingly, the C-terminal region of PMT1 and the N-terminal region of PMT2 contain vestigial methyltransferase folds that lack key features required for enzymatic activity. It appears that in nematodes, gene duplication and divergence of function into enzymes that perform distinct methylation reactions has occurred.

Although the PMT from nematodes, plants, and Plasmodium catalyze a common overall chemical reaction, that is, transfer of a methyl group to an amine, each domain adopts a distinct active site structure and uses a different reaction mechanism for catalysis (Figure 3) [79]. In the PMT1 active site (Figure 3A), Tyr127 hydrogen bonds to the substrate amine group, and Thr178 is part of an interaction network that links a water molecule and the substrate phosphate group. The positioning of the substrate phosphate group relative to the water molecule bound by Tyr127 and Thr178 suggests a possible proton relay that facilitates substrate-assisted catalysis. In the proposed reaction mechanism for PMT1, the hydroxyl proton of Tyr127 is relayed via the water molecule to the phosphate group of pEA, which activates the hydroxyl group of the tyrosine to deprotonate the amine for transfer of the methyl group from SAM. The proposed catalytic residues and amino acids forming the phosphobase-binding site of PMT1 are highly conserved (Figure 3B).

Figure 3. Active site motifs in the type III PMT from nematodes.

Figure 3

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(A) Active site of H. contortus PMT1 (PDB: 4KRG). Amino acids involved in catalysis (red) and phosphobase binding (gold) are shown. (B) Sequence alignment of PMT1 from nematodes. Residues in the canonical SAM-binding motifs are shown in gray. Active site residues are colored as in panel A. Invariant residues are in white text. (C) Active site of H. contortus PMT2 (PDB:4KRI). Amino acids involved in catalysis (green) and phosphobase binding (blue) are shown. (D) Sequence alignment of PMT2 from nematodes. Residues in the canonical SAM-binding motifs are shown in gray. Active site residues are colored as in panel C. Invariant residues are in white text.

In contrast to the proximity and desolvation reaction of PMT1, the PMT2 active site employs acid/base catalysis for methylation [79]. The active site residues of PMT2 are nearly identical to those found in the Plasmodium PMT [76]. In the active sites of the nematode PMT2 and the Plasmodium PMT, an invariant histidine serves as a general base to abstract a proton from the tyrosine hydroxyl group to activate it for catalysis (Figure 3C). The phosphobase binding site uses hydrogen bonds from the hydroxyl groups of four tyrosine residues (Tyr191, Tyr325, Tyr339, and Tyr345 in HcPMT2) and the charge-charge interactions with two basic residues, that is, Arg373 and Lys411 in HcPMT2, to lock substrates in place for catalysis. Among the nematode PMT, the sequences of the consensus methyltransferase motifs (I-IV), the catalytic dyad, and residues in the phosphobase binding are almost invariant (Figure 3D). The differences between the MT1 and MT2 active sites provide distinct molecular targets for development of small molecule inhibitors targeting these proteins and identify key features for the identification of PMT in other organisms.

PMT as potential broad-spectrum antiparasitic targets

The three-dimensional structures of the PMT provide views of the structural and functional evolution of an essential biochemical pathway in these organisms. Our understanding of the phosphobase methylation pathway began with genome analysis, which led to extensive structural and in vitro and in vivo functional studies that are a foundation for the development of inhibitors targeting parasites of mammals that rely on the phosphobase methylation pathway for membrane biogenesis. Importantly, the molecular details obtained from crystallographic studies can help guide subsequent genome analysis and inform the development of new research in different organisms, thus, closing the genome-structure-function circle.

In multiple early studies of the PMT from plants, nematodes, and Plasmodium, database searches using BLAST identified multiple putative PMT homologs in other species [48–51, 65, 70–72, 81]; however, the conserved SAM-binding motifs in these sequences tended to drive the overall sequence alignment, which raises the possibility of 'false-positive' identification. Now, with detailed knowledge of the relevant structural features in the active sites of the PMT, that is, catalytic residues, SAM binding residues, and phosphobase recognition residues (Figure 3), additional criteria can be used to evaluate sequences to increase the likelihood of successful identification of authentic PMT homologs. Sequence searches using the type III PMT1 and PMT2 from C. elegans and H. contortus reveal that the PMT genes are widely distributed across the nematode species (Table 2). The proteins encoded by these genes contain the SAM-binding consensus motifs, the key catalytic residues, and conserved amino acids forming the phosphobase binding site of each methyltransferase domain. In the open-access genomic resources, full-length PMT1 and PMT2 homologs are found in the three major clades of nematodes (clade III, clade IV, and clade V), including the human, animal, and plant parasitic nematodes [80]. These organisms all encode highly homologous (∼35–90% amino acid sequence identity) type III PMT with shared methyltransferase domain architectures (Figure 4) [48–50].

Table 2.

PMT orthologs in nematode species

PMT1
pecies Accession No. Source Lengthd % Identitya
CePMT1 HcPMT1
Ascaris suum ERG79882.1 GenBank 460 aa 52.3 53.5
Meloidogyne incognita US 2007/0009981 A1 ID NO: 9 US Patent 457 aa 39.7 40.8
Meloidogyne hapla MhA1_Contig212.frz3.gene4 WormBase 412 aa 34.4 34.8
Strongyloides stercoralis US 2007/0009981 A1 ID NO: 10 US Patent 469 aa 36.1 37.4
Bursaphelenchus xylophilus BUX.s01513.225 WormBase 454 aa 41.1 44.5
Pristionchus pacificus PPA22786 WormBase 470 aa 53.6 53.7
Haemonchus contortus CDJ81011.1 GenBank 460 aa 62.2 100.0
Necator americanus ETN83758.1 GenBank 431 aa 64.2 80.5
Caenorhabditis japonica CJA07932 WormBase 460 aa 79.5 60.4
Caenorhabditis elegans NP_494990.2 GenBank 475 aa 100.0 62.2
Caenorhabditis sp.11 Csp11.Scaffold630.g21423.t1 WormBase 477 aa 88.8 62.5
Caenorhabditis brenneri CBN10892 WormBase 478 aa 89.9 62.5
Caenorhabditis remanei CRE11930 WormBase 463 aa 88.0 62.4
Caenorhabditis sp.5 Csp5_scaffold_01891.g23737.t1 WormBase 480 aa 85.2 62.2
Caenorhabditis briggsae CBG02363 WormBase 482 aa 84.2 61.4
PMT2
Species Accession No. Source Length % Identitya
CePMT2 HcPMT2
Ascaris suum GS_23928 WormBase 440 aa 47.7 46.2
Meloidogyne hapla MhA1_Contig1 162.frz3.gene5 WormBase 472 aa 42.7 45.2
Meloidogyne javanica US 2007/0009981 A1 ID NO: 12 US Patent 472 aa 42.0 45.7
Bursaphelenchus xylophilus BUX.s01143.358 WormBase 416 aa 33.1 36.5
Pristionchus pacificus PPA16775 WormBase 434 aa 50.1 49.7
Haemonchus contortus CDJ96940.1 GenBank 431 aa 53.8 100.0
Necator americanus ETN82894.1 GenBank 431 aa 54.5 67.1
Heterorhabditis bacteriophora Hba_11105 WormBase 448 aa 54.1 55.3
Caenorhabditis japonica CJA04052 WormBase 473 aa 83.8 53.8
Caenorhabditis elegans NP504248.1 GenBank 437 aa 100.0 53.8
Caenorhabditis brenneri CB_N22056 WormBase 437 aa 89.2 54.0
Caenorhabditis remanei CRE18566 WormBase 436 aa 89.9 55.0
Caenorhabditis briggsae CBG08775 WormBase 437 aa 86.7 54.1
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Abbreviations: PMT, phosphoethalamine methyltransferase; CePMT1, C. elegans PMT1; CePMT2, C. elegans PMT2; HcPMT1, H. contortus PMT1; HcPMT2, H. contortus PMT2.

a

BLAST searches to identify PMT homologs in nemaodes used CePMT1, CePMT2, HcPMT1, and HcPMT2 as queries. Percent amino acid sequence identity of various PMT with these queries are shown for comparison across the full-length gene products.

Figure 4. Phylogeny of PMT enzymes and domain organizations.

Figure 4

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Selected PMT amino acid sequences were aligned at the MultAlin server [87]. The phylogenetic tree was generated in MEGA5 software [88] using the Neighbor-Joining method [89] and the bootstrap test (1000 replicates) [90]. Species are shown in green, red, gray, and yellow, indicating plants, animals, microorganisms, and apicomplexa, respectively. The scale bar under the phylogenetic tree represents the number of amino acid differences per site. The MT domains of PMT enzymes are shown in gold (MT1), blue (MT2), or green (MT1/2, Plasmodium-like). All of the nematode PMT1 and PMT2 contain the corresponding MT2 and MT1 vestigial domains, respectively, although these are not shown in the cartoons. The scale bar under the domain illustration represents the amino acid length of each enzyme.

Genomic data screening using the same structural criteria suggest that potential PMT homologs are also present in some species of bacteria: (i) actinobacteria, β-proteobacteria, δ-proteobacteria, γ-proteobacteria, cyanobacteria, and planctomycetes; (ii) apicomplexan parasites, including four species of Plasmodium and five species of Eimeria, the coccidiosis causing parasites; (iii) oomycetes–the water molds; (iv) plants–from mosses to eudicots; and (v) some animals, including sea anemones to lower vertebrates, such as frogs and fishes (Figure 4). Given the structural and sequence homology of the PMT found in plants and plant parasitic nematodes, the likelihood to targeting these enzymes for the development of inhibitors of agricultural utility is low. However, the genome data analyses indicate that the PMT are not found in humans and other mammals. Although the biochemical and physiological roles of the putative PMT in these phylogenetically diverse organisms remain to be examined, the wide distribution in parasitic nematodes and protozoans suggests these enzymes as candidate targets for new broad-spectrum antiparasitic drug discovery efforts.

Currently, the development of small molecule inhibitors targeting PMT is proceeding, but is still at an early stage. Prior to the first crystal structure of PMT from Plasmodium, human histamine N-methyltransferase (HNMT) was regarded as a homologous model for the type II PMT and utilized to screen the potential inhibitors [81–83]. Multiple inhibitors of HNMT, such as amodiaquine, SKF91488, tacrine, chlorpromazine, and diphenhydramine, were examined on the type II P. falciparum PMT and the type III PMT from C. elegans and H. contortus [77–78, 81]; however, the tested HNMT inhibitors showed either weak inhibition (mM) or no inhibition effects. Instead, biochemical studies identified the SAM analog sinefungin, a nucleoside antibiotic from fungi, and the pCho analogs hexadecylphosphocholine (miltefosine) and hexadecyltrimethylammonium bromide as µM inhibitors of both Plasmodium and nematode PMT enzymes [48, 77–78, 81]. A recent report describes chemical screening that identified 11 potential inhibitors of the Plasmodium PMT from 3161 compounds from the National Cancer Institute Open Chemical Repository [51]. Of these, the NSC-158011 compound was further characterized (Ki = 10.9 ± 4.1 µM) [51]. These compounds remain to be counter-screened against other types of PMT.

Combined with high-throughput screening methods, the X-ray crystal structures of type II and type III PMT can accelerate the drug discovery and development by providing molecular insight on how these enzymes function and on structural features that could be used to improve inhibitor efficacy. Crystallographic information can be used for virtual screening of compound libraries prior to high-throughput assays of potential inhibitors [84]. Moreover, the crystal structures of the nematode and Plasmodium PMT have been determined at high (1.19 Å to 1.99 Å) resolution and are publicly available from the PDB [76–77, 79]. In the case of the type II PMT from Plasmodium, the eight structures of different ligand-bound forms–substrates, products, and inhibitors–provide various protein conformations and reduce the potential bias of virtual screening results toward specific classes of ligand [85]. As a first step toward virtual screening, two of the type II PMT structures in complex with the inhibitors sinefungin and amodiaquine were applied to docking methods, which suggest possible target binding sites for small molecules, such as a quinolones [77]. In the type III PMT of nematodes, the structurally different active sites provide distinct molecular targets for small molecule inhibitors, which may increase the odds of identifying novel compounds that target the phosphobase methylation pathway.

Concluding remarks

Genome science has transformed parasitology as sequence information on free-living and parasitic nematodes, including C. elegans, Brugia malayi, O. volvulus, N. americanus, A. lumbricoides, Trichinella spiralis, and protozoans (Babesia, Eimeria, Giardia, Leishmania, Plasmodium, Toxoplasma, Trichomonas, and Trypanosoma) allows for testing of the hypothesis developed by comparative bioinformatics [35–37, 46–47]. Now, structural biology is handed the baton. Combined with in vitro and in vivo biochemical studies, protein structure can provide significant insight on parasite biology to open new opportunities for the identification of essential metabolic differences from hosts and the elucidation of molecular features for drug discovery targeted at neglected diseases endemic in the developing regions of Africa, Asia, and the Americas.

HIGHLIGHTS.

  • Integrating genomic, structural, and biochemical studies provides new insights.

  • Nematodes use a plant-like phosphobase methylation pathway in phospholipid synthesis.

  • The phosphobase methylation pathway is a potential anti-parasitic target.

  • Protein crystal structures and functional studies can guide genome data analysis.

Acknowledgements

The authors acknowledge support from the National Institutes of Health (AI-097119).

Footnotes

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