{"gene":"PF3D7_1343000","run_date":"2026-09-08T14:19:06","timeline":{"discoveries":[{"year":2004,"finding":"PMT (PF3D7_1343000) encodes a phosphoethanolamine N-methyltransferase that catalyzes a three-step S-adenosyl-L-methionine-dependent methylation of phosphoethanolamine to phosphocholine, functioning as a monopartite enzyme with a single catalytic domain responsible for all three methylation steps. PMT activity is inhibited by its product phosphocholine and by the phosphocholine analog miltefosine.","method":"Biochemical characterization of recombinant enzyme; in vitro enzymatic assay; inhibitor studies","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct enzymatic characterization of the recombinant protein, in vitro activity assays, product inhibition demonstrated, replicated across multiple subsequent studies","pmids":["15073329"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2005,"finding":"PMT (PF3D7_1343000) acts specifically on phosphoethanolamine (not phosphatidylethanolamine) as its in vivo substrate, demonstrated by complementation of a yeast pem1Δpem2Δ mutant. PMT activity is directly coupled to the Kennedy (CDP-choline) pathway for phosphatidylcholine synthesis, as mutation in yeast choline-phosphate cytidylyltransferase abrogates complementation by PMT.","method":"Yeast genetic complementation assay; lipid analysis; epistasis via double mutant","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with multiple mutant backgrounds, lipid metabolic profiling, replicated with biochemical analysis","pmids":["15664981"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2006,"finding":"PMT (PF3D7_1343000) localizes to the Golgi apparatus of P. falciparum, as demonstrated by complete co-localization with the Golgi marker Rab6 in transgenic parasites expressing GFP targeted to different compartments, and by immunoelectron microscopy. PMT protein levels are regulated in a stage-specific manner, with highest expression during the trophozoite stage.","method":"Confocal microscopy; immunoelectron microscopy; GFP co-localization with organelle markers (Rab6 for Golgi, BiP for ER); immunoblotting across life stages","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct localization by multiple orthogonal methods (IEM, confocal with multiple markers, transgenic GFP), functional stage-specific regulation shown","pmids":["16704982"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2007,"finding":"PMT (PF3D7_1343000) is regulated by choline at two levels: choline induces transcriptional repression of the PfPMT gene in a dose-dependent manner, and also targets the PMT protein for proteasomal degradation. The proteasome inhibitor bortezomib blocks choline-mediated PMT protein degradation. This regulation occurs in both wild-type and transgenic parasites constitutively expressing PMT.","method":"Transcript analysis; immunoblotting; pulse-chase experiments; immunoprecipitation; proteasome inhibitor (bortezomib) treatment","journal":"Eukaryotic cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (transcript analysis, pulse-chase, immunoprecipitation, pharmacological inhibition) in single study establishing mechanism of regulation","pmids":["17644653"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2008,"finding":"Mutational analysis of PMT (PF3D7_1343000) identified residues Asp-61, Gly-83, and Asp-128 as critical for enzymatic activity and substrate/SAM binding, demonstrated by loss-of-function in a yeast complementation system.","method":"Site-directed mutagenesis of 24 residues; yeast complementation assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — mutagenesis with functional readout in yeast system, single study, no structural validation at this stage","pmids":["18178564"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2008,"finding":"Genetic knockout of PMT (PF3D7_1343000) in P. falciparum completely abrogates phosphatidylcholine biosynthesis via the SDPM pathway. Unlike in mammalian and yeast cells, methylation of phosphatidylethanolamine to phosphatidylcholine does not occur in P. falciparum, making the SDPM and CDP-choline pathways the only two routes for phosphatidylcholine biosynthesis. Loss of PMT causes significant defects in parasite growth, multiplication, and viability.","method":"Genetic knockout; lipid analysis in knockout parasites; parasite growth and viability assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic knockout with biochemical (lipid) and phenotypic (growth/viability) readouts, defines pathway architecture","pmids":["18694927"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2010,"finding":"PMT (PF3D7_1343000) activity is inhibited in vitro by hexadecyltrimethylammonium, dodecyltrimethylammonium, and amodiaquine, but not by chloroquine or other aminoquinolines tested. NMR titration studies demonstrated specific, concentration-dependent binding of amodiaquine to the PMT protein. In a yeast surrogate system, yeast mutants relying on PMT for survival were sensitive to amodiaquine and showed inhibited phosphatidylcholine biosynthesis.","method":"Enzyme-coupled methylation assay; yeast genetic surrogate system; NMR titration","journal":"BMC biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro enzyme assay, NMR binding, and yeast functional system, single lab with multiple orthogonal approaches","pmids":["20085640"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2010,"finding":"PMT (phosphoethanolamine N-methyltransferase) activity is absent in rodent Plasmodium species (P. berghei, P. yoelii, P. chabaudi, P. vinckei), as no PMT gene ortholog was detected in silico and biochemical experiments showed that ethanolamine and serine are not incorporated into phosphatidylcholine in rodent parasites, unlike in P. falciparum.","method":"In silico genome analysis; biochemical radiolabeling experiments with labeled choline, ethanolamine, and serine","journal":"Journal of lipid research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical radiolabeling with multiple precursors across multiple species, consistent with genomic analysis","pmids":["19561325"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium berghei","strain":""},{"species":"Plasmodium yoelii","strain":""},{"species":"Plasmodium chabaudi","strain":""},{"species":"Plasmodium vinckei","strain":""}]},{"year":2011,"finding":"The 1.55 Å resolution crystal structure of PMT (PF3D7_1343000) in complex with AdoMet was solved by single-wavelength anomalous dispersion phasing, and additional structures at 1.19–1.52 Å resolution were determined with phosphoethanolamine (substrate), phosphocholine (product), sinefungin (inhibitor), and both phosphoethanolamine and S-adenosylhomocysteine. Domain rearrangements occur upon ligand binding. Functional characterization of 27 site-directed mutants identified Tyr-19 and His-132 as forming a catalytic dyad; kinetic analysis, isothermal titration calorimetry, and structural analysis of Y19F and H132A mutants showed these residues are required for phosphobase methylation and form a 'catalytic latch' that locks ligands in the active site.","method":"X-ray crystallography (multiple structures); site-directed mutagenesis of 27 residues; kinetic analysis; isothermal titration calorimetry","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution crystal structures with multiple ligands, extensive mutagenesis, kinetics, and ITC in single study with multiple orthogonal methods","pmids":["22117061"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2012,"finding":"The 2.0 Å resolution X-ray crystal structure of PMT (PF3D7_1343000) in complex with amodiaquine was determined, revealing the structural basis for inhibitor binding at the active site.","method":"X-ray crystallography; IC50 determination by radiochemical assay for a series of aminoquinolines","journal":"Bioorganic & medicinal chemistry letters","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with inhibitor, functional IC50 measurements, single study","pmids":["22771008"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"finding":"PMT (PF3D7_1343000) is essential for P. falciparum gametocytogenesis and malaria transmission. Parasites lacking PMT are severely altered in gametocyte development, unable to produce mature-stage gametocytes, and cannot be transmitted to mosquitoes. Chemical inhibitor NSC-158011 inhibits PMT activity in vitro, blocks intraerythrocytic asexual replication and gametocyte differentiation, and inhibits phosphatidylcholine biosynthesis from ethanolamine in a yeast functional complementation system.","method":"Genetic knockout; gametocyte development assays; mosquito transmission assay; chemical inhibitor screening; kinetic studies in vitro; yeast functional complementation; lipid metabolic analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout with defined transmission phenotype, pharmacological corroboration, in vitro kinetics, and metabolic analysis using multiple orthogonal approaches","pmids":["24145416"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2014,"finding":"QM/MM computational analysis and mutagenesis of PMT (PF3D7_1343000) revealed that the reaction mechanism involves Asp-128 (not the previously proposed Tyr-19–His-132 dyad) in the initial methylation step: a proton is transferred to Asp-128 via a bridging water molecule, followed by SN2-type methyl transfer from SAM to phosphoethanolamine. D128A, D128E, D128Q, and D128N mutants lose activity with phosphoethanolamine as substrate. Crystal structures of D128A mutant in complex with S-adenosylhomocysteine and either phosphoethanolamine or phosphocholine show disruption of the hydrogen bond network in the active site.","method":"QM/MM calculations; site-directed mutagenesis (D128A/E/Q/N); enzymatic activity assays; X-ray crystallography of mutant complexes","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — combined QM/MM, mutagenesis, enzymatic assays, and crystal structures provide orthogonal mechanistic evidence in a single study","pmids":["25288796"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2015,"finding":"PMT orthologs from Plasmodium vivax (PvPMT) and Plasmodium knowlesi (PkPMT) were characterized by X-ray crystallography and biochemical assays; both are inhibited by amodiaquine and NSC158011. Metabolic studies in yeast mutants relying on PkPMT or PvPMT showed that these compounds inhibit phosphatidylcholine biosynthesis from ethanolamine.","method":"X-ray crystallography; biochemical inhibitor assays; yeast functional complementation; metabolic labeling","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structures, biochemical assays, and yeast metabolic studies provide orthogonal mechanistic evidence for PMT orthologs in two Plasmodium species","pmids":["25761669"],"is_preprint":false,"attribution":[{"species":"Plasmodium vivax","strain":""},{"species":"Plasmodium knowlesi","strain":""}]},{"year":2021,"finding":"PMT (PF3D7_1343000) is phosphorylated by PfCDPK7 in P. falciparum; in the absence of PfCDPK7, PMT is hypophosphorylated and undergoes degradation. PfCDPK7 regulates phosphatidylcholine synthesis via PMT (and ethanolamine kinase), placing PMT downstream of the PfPI4K/4'-phosphoinositide/PfCDPK7 signaling axis.","method":"Quantitative phosphoproteomics; phospholipid analysis; conditional knockdown of PfCDPK7; PI4K inhibitor (MMV39048) treatment","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phosphoproteomic identification of PMT as hypophosphorylated substrate of PfCDPK7 combined with lipidomics and pharmacological perturbation, single lab","pmids":["34866326"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"current_model":"PMT (PF3D7_1343000) is a Golgi-localized, monopartite phosphoethanolamine N-methyltransferase that catalyzes all three SAM-dependent methylation steps converting phosphoethanolamine to phosphocholine via a mechanism requiring Asp-128 (proton transfer via bridging water) and with structural contributions from Tyr-19 and His-132; its product phosphocholine feeds directly into the Kennedy pathway for phosphatidylcholine synthesis, it is essential for intraerythrocytic growth, gametocytogenesis, and mosquito transmission, its expression is controlled by choline-mediated transcriptional repression and proteasomal degradation, and its activity is further regulated by phosphorylation downstream of the PfPI4K/PfCDPK7 signaling axis."},"narrative":{"mechanistic_narrative":"PMT (PF3D7_1343000) is a phosphoethanolamine N-methyltransferase that supplies phosphocholine for phosphatidylcholine biosynthesis in Plasmodium falciparum, and it is essential for both intraerythrocytic asexual growth and transmission to mosquitoes [PMID:15073329, PMID:18694927, PMID:24145416]. As a monopartite single-domain enzyme, PMT catalyzes all three sequential SAM-dependent methylation steps that convert phosphoethanolamine to phosphocholine, acting specifically on the free phosphobase rather than on phosphatidylethanolamine, and its product feeds directly into the Kennedy (CDP-choline) pathway [PMID:15073329, PMID:15664981]. Because P. falciparum lacks phosphatidylethanolamine-to-phosphatidylcholine methylation, genetic ablation of PMT abolishes phosphatidylcholine synthesis via the serine-decarboxylase-phosphoethanolamine-methyltransferase route and impairs parasite growth and viability, while PMT-null parasites fail to produce mature gametocytes and cannot be transmitted to mosquitoes [PMID:18694927, PMID:24145416]. High-resolution crystal structures with substrate, product, cofactor, and inhibitors, combined with extensive mutagenesis and QM/MM analysis, define an active site in which Asp-128 mediates proton transfer through a bridging water to enable SN2 methyl transfer from SAM, with Tyr-19 and His-132 contributing to ligand positioning [PMID:22117061, PMID:25288796]. Enzyme abundance and activity are tightly controlled: choline both represses PfPMT transcription and drives proteasomal degradation of the protein [PMID:17644653], and PMT is phosphorylated by PfCDPK7 downstream of the PfPI4K/PfCDPK7 signaling axis, with loss of PfCDPK7 leaving PMT hypophosphorylated and degraded [PMID:34866326]. The enzyme localizes to the Golgi apparatus [PMID:16704982] and is inhibited by product phosphocholine, miltefosine, amodiaquine, and other compounds, making it a validated antimalarial target [PMID:15073329, PMID:20085640, PMID:24145416].","teleology":[{"year":2004,"claim":"Establishing the core biochemical activity answered what PMT does: it defined the enzyme as a single-domain catalyst performing all three methylations converting phosphoethanolamine to phosphocholine.","evidence":"Biochemical characterization of recombinant enzyme with in vitro methylation assays and product/analog inhibition","pmids":["15073329"],"confidence":"High","gaps":["Did not establish the in vivo substrate specificity","No structural basis for catalysis at this stage"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2005,"claim":"Yeast complementation resolved whether PMT acts on the free phosphobase or the lipid and how its product connects to lipid synthesis, showing phosphoethanolamine is the in vivo substrate coupled to the Kennedy pathway.","evidence":"Complementation of yeast pem1Δpem2Δ mutant with epistasis via choline-phosphate cytidylyltransferase mutation and lipid analysis","pmids":["15664981"],"confidence":"High","gaps":["Yeast surrogate may not fully recapitulate parasite context","Did not address subcellular localization in the parasite"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2006,"claim":"Localization studies answered where PMT operates in the parasite, placing the enzyme in the Golgi apparatus and revealing stage-dependent abundance.","evidence":"Confocal and immunoelectron microscopy with GFP organelle markers plus stage-resolved immunoblotting","pmids":["16704982"],"confidence":"High","gaps":["Mechanism of Golgi targeting not defined","Functional significance of Golgi localization for phosphocholine supply unclear"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2007,"claim":"Regulation studies answered how PMT levels respond to nutrient availability, revealing dual control by choline at the transcriptional level and via proteasomal degradation.","evidence":"Transcript analysis, pulse-chase, immunoprecipitation, and bortezomib proteasome inhibition in wild-type and transgenic parasites","pmids":["17644653"],"confidence":"High","gaps":["Degradation signal/E3 ligase not identified","Transcriptional repressor mediating choline response unknown"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2008,"claim":"Mutagenesis began mapping catalytically critical residues, identifying Asp-61, Gly-83, and Asp-128 as required for activity and cofactor/substrate binding.","evidence":"Site-directed mutagenesis of 24 residues scored by yeast complementation","pmids":["18178564"],"confidence":"Medium","gaps":["No structural validation at this stage","Functional readout limited to yeast survival, not direct kinetics"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2008,"claim":"Genetic knockout answered whether PMT is dispensable, showing it is required for phosphatidylcholine synthesis via the SDPM pathway and for asexual growth, and defining the pathway architecture in P. falciparum.","evidence":"Genetic knockout with lipid analysis and growth/viability assays","pmids":["18694927"],"confidence":"High","gaps":["Did not address sexual-stage requirement","Residual growth via CDP-choline pathway not fully quantified"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2010,"claim":"Species comparison addressed conservation of the pathway, showing PMT activity and gene are absent in rodent Plasmodium species, distinguishing P. falciparum phospholipid metabolism.","evidence":"In silico genome analysis and radiolabeling with choline, ethanolamine, and serine across P. falciparum and four rodent species","pmids":["19561325"],"confidence":"Medium","gaps":["Does not clarify how rodent parasites compensate for the missing route","Limits use of rodent models to study PMT function in vivo"],"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium berghei","strain":""},{"species":"Plasmodium yoelii","strain":""},{"species":"Plasmodium chabaudi","strain":""},{"species":"Plasmodium vinckei","strain":""}]},{"year":2010,"claim":"Inhibitor profiling identified small molecules that block PMT and demonstrated direct binding, advancing PMT as a druggable target.","evidence":"Enzyme-coupled methylation assay, NMR titration binding, and yeast surrogate sensitivity with amodiaquine and quaternary ammonium compounds","pmids":["20085640"],"confidence":"Medium","gaps":["No structural basis for inhibitor binding yet","Selectivity over human methyltransferases untested"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2011,"claim":"High-resolution crystallography with substrate, product, cofactor, and inhibitor resolved the structural mechanism, defining ligand-induced domain rearrangements and implicating a Tyr-19/His-132 catalytic dyad acting as a latch.","evidence":"Multiple X-ray structures (1.19–1.55 Å), mutagenesis of 27 residues, kinetics, and ITC","pmids":["22117061"],"confidence":"High","gaps":["Assignment of the catalytic base was later revised","Order of the three methylation steps not structurally resolved"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2012,"claim":"A PMT-amodiaquine complex structure provided the structural rationale for inhibitor binding at the active site, guiding structure-based inhibitor design.","evidence":"2.0 Å X-ray structure with amodiaquine and IC50 determination across a series of aminoquinolines","pmids":["22771008"],"confidence":"High","gaps":["Cellular potency and selectivity of aminoquinoline series not established here"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"claim":"Transmission-stage genetics answered whether PMT matters beyond asexual growth, showing it is essential for gametocyte maturation and mosquito transmission, with a chemical inhibitor phenocopying the loss.","evidence":"Genetic knockout with gametocyte and mosquito transmission assays plus NSC-158011 inhibitor characterization and yeast complementation","pmids":["24145416"],"confidence":"High","gaps":["Mechanistic basis of the gametocyte defect not resolved","Inhibitor specificity in vivo not fully defined"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2014,"claim":"Combined QM/MM, mutagenesis, and mutant crystal structures revised the catalytic mechanism, establishing Asp-128 as the proton acceptor via a bridging water preceding SN2 methyl transfer.","evidence":"QM/MM calculations, D128A/E/Q/N mutagenesis with activity assays, and crystal structures of the D128A mutant with SAH and phosphobase ligands","pmids":["25288796"],"confidence":"High","gaps":["Whether later methylation steps use the same residue is not fully resolved","Roles of Tyr-19/His-132 reconciled but hierarchy of contributions still incomplete"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2015,"claim":"Structural and biochemical characterization of P. vivax and P. knowlesi orthologs tested conservation of the drug target, showing both are inhibited by amodiaquine and NSC158011.","evidence":"X-ray crystallography, inhibitor assays, and yeast metabolic labeling for PvPMT and PkPMT","pmids":["25761669"],"confidence":"High","gaps":["Findings are on orthologs, not PF3D7_1343000 directly","Cross-species inhibitor potency in parasites not compared"],"attribution":[{"species":"Plasmodium vivax","strain":""},{"species":"Plasmodium knowlesi","strain":""}]},{"year":2021,"claim":"Signaling studies answered how PMT is regulated post-translationally, placing it downstream of the PfPI4K/PfCDPK7 axis where PfCDPK7 phosphorylation stabilizes the enzyme.","evidence":"Quantitative phosphoproteomics, lipidomics, conditional PfCDPK7 knockdown, and PI4K inhibitor treatment","pmids":["34866326"],"confidence":"Medium","gaps":["Direct kinase-substrate relationship not reconstituted","Phosphosites on PMT and their functional consequences not mapped"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":null,"claim":"How Golgi localization, choline-driven turnover, and PfCDPK7 phosphorylation are integrated to control PMT-dependent phosphocholine flux across asexual and sexual stages remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No integrated model linking regulatory inputs to phospholipid flux","E3 ligase and phosphosites governing PMT stability unidentified","Mechanistic cause of the gametocyte maturation defect unknown"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["PFCDPK7 (PHOSPHORYLATES PF3D7_1343000; LOSS LEADS TO HYPOPHOSPHORYLATION AND DEGRADATION) [#13]"],"other_free_text":["Monopartite single-domain enzyme catalyzing all three SAM-dependent methylation steps from phosphoethanolamine to phosphocholine [#0]","Acts on the free phosphobase phosphoethanolamine, not phosphatidylethanolamine; couples to the Kennedy/CDP-choline pathway [#1]","Essential for intraerythrocytic asexual growth and viability [#5]","Essential for gametocyte maturation and mosquito transmission [#10]","Catalytic mechanism: Asp-128 accepts a proton via a bridging water preceding SN2 methyl transfer; Tyr-19 and His-132 position ligands [#8, #11]","Product-inhibited by phosphocholine and inhibited by miltefosine, amodiaquine, NSC-158011, and quaternary ammonium compounds [#0, #6, #10]","Regulated by choline via transcriptional repression and proteasomal degradation [#3]","Post-translationally stabilized by phosphorylation downstream of the PfPI4K/PfCDPK7 axis [#13]","Absent in rodent Plasmodium species (P. berghei, P. yoelii, P. chabaudi, P. vinckei) [#7]","Orthologs in P. vivax and P. knowlesi share amodiaquine/NSC158011 inhibition [#12]"]}},"prefetch_data":{},"corpus_meta":[{"pmid":"15073329","id":"PMC_15073329","title":"A pathway for phosphatidylcholine biosynthesis in Plasmodium falciparum involving phosphoethanolamine methylation.","date":"2004","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/15073329","citation_count":131,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"15130134","id":"PMC_15130134","title":"Quantitative proteomics of the human malaria parasite Plasmodium falciparum and its application to studies of development and inhibition.","date":"2004","source":"Molecular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/15130134","citation_count":88,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["phosphoethanolamine N-methyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"18694927","id":"PMC_18694927","title":"Disruption of the Plasmodium falciparum PfPMT gene results in a complete loss of phosphatidylcholine biosynthesis via the serine-decarboxylase-phosphoethanolamine-methyltransferase pathway and severe growth and survival defects.","date":"2008","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/18694927","citation_count":68,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"15664981","id":"PMC_15664981","title":"In vivo evidence for the specificity of Plasmodium falciparum phosphoethanolamine methyltransferase and its coupling to the Kennedy pathway.","date":"2005","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15664981","citation_count":66,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"24145416","id":"PMC_24145416","title":"Plasmodium falciparum phosphoethanolamine methyltransferase is essential for malaria transmission.","date":"2013","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/24145416","citation_count":53,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["phosphoethanolamine N-methyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"22117061","id":"PMC_22117061","title":"Structure and reaction mechanism of phosphoethanolamine methyltransferase from the malaria parasite Plasmodium falciparum: an antiparasitic drug target.","date":"2011","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/22117061","citation_count":47,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PMT"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"19561325","id":"PMC_19561325","title":"Rodent and nonrodent malaria parasites differ in their phospholipid metabolic pathways.","date":"2010","source":"Journal of lipid research","url":"https://pubmed.ncbi.nlm.nih.gov/19561325","citation_count":45,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["phosphoethanolamine N-methyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"16704982","id":"PMC_16704982","title":"Localization of the phosphoethanolamine methyltransferase of the human malaria parasite Plasmodium falciparum to the Golgi apparatus.","date":"2006","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/16704982","citation_count":31,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"20085640","id":"PMC_20085640","title":"Identification of inhibitors of Plasmodium falciparum phosphoethanolamine methyltransferase using an enzyme-coupled transmethylation assay.","date":"2010","source":"BMC biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/20085640","citation_count":24,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PMT"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"17644653","id":"PMC_17644653","title":"Choline induces transcriptional repression and proteasomal degradation of the malarial phosphoethanolamine methyltransferase.","date":"2007","source":"Eukaryotic cell","url":"https://pubmed.ncbi.nlm.nih.gov/17644653","citation_count":23,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"18178564","id":"PMC_18178564","title":"Biochemical and genetic analysis of the phosphoethanolamine methyltransferase of the human malaria parasite Plasmodium falciparum.","date":"2008","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/18178564","citation_count":20,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PMT"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"22771008","id":"PMC_22771008","title":"Crystal structure of phosphoethanolamine methyltransferase from Plasmodium falciparum in complex with amodiaquine.","date":"2012","source":"Bioorganic & medicinal chemistry letters","url":"https://pubmed.ncbi.nlm.nih.gov/22771008","citation_count":18,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PMT","phosphoethanolamine N-methyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"25761669","id":"PMC_25761669","title":"Structure, function and inhibition of the phosphoethanolamine methyltransferases of the human malaria parasites Plasmodium vivax and Plasmodium knowlesi.","date":"2015","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/25761669","citation_count":17,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PMT"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"34866326","id":"PMC_34866326","title":"PI4-kinase and PfCDPK7 signaling regulate phospholipid biosynthesis in Plasmodium falciparum.","date":"2021","source":"EMBO reports","url":"https://pubmed.ncbi.nlm.nih.gov/34866326","citation_count":13,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PMT","phosphoethanolamine N-methyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"29505599","id":"PMC_29505599","title":"Phosphoethanolamine-N-methyltransferase is a potential biomarker for the diagnosis of P. knowlesi and P. falciparum malaria.","date":"2018","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/29505599","citation_count":13,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PMT","phosphoethanolamine N-methyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"24209716","id":"PMC_24209716","title":"Kinetic modelling of phospholipid synthesis in Plasmodium knowlesi unravels crucial steps and relative importance of multiple pathways.","date":"2013","source":"BMC systems biology","url":"https://pubmed.ncbi.nlm.nih.gov/24209716","citation_count":11,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PMT","phosphoethanolamine N-methyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"25288796","id":"PMC_25288796","title":"An alternative mechanism for the methylation of phosphoethanolamine catalyzed by Plasmodium falciparum phosphoethanolamine methyltransferase.","date":"2014","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/25288796","citation_count":11,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PMT"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"36710008","id":"PMC_36710008","title":"Functional analysis of phosphoethanolamine N-methyltransferase in plants and parasites: Essential S-adenosylmethionine-dependent methyltransferase in choline and phospholipid metabolism.","date":"2022","source":"Methods in enzymology","url":"https://pubmed.ncbi.nlm.nih.gov/36710008","citation_count":4,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PMT","phosphoethanolamine N-methyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"22392340","id":"PMC_22392340","title":"(1)H, (13)C, and (15)N chemical shift assignments for PfPMT, a phosphoethanolamine methyltransferase from Plasmodium falciparum.","date":"2012","source":"Biomolecular NMR assignments","url":"https://pubmed.ncbi.nlm.nih.gov/22392340","citation_count":3,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PMT"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"19523215","id":"PMC_19523215","title":"The effect of mimicking febrile temperature and drug stress on malarial development.","date":"2009","source":"Annals of clinical microbiology and antimicrobials","url":"https://pubmed.ncbi.nlm.nih.gov/19523215","citation_count":3,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["phosphoethanolamine N-methyltransferase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"37665563","id":"PMC_37665563","title":"Discovery of dual-target natural antimalarial agents against DHODH and PMT of Plasmodium falciparum: pharmacophore modelling, molecular docking, quantum mechanics, and molecular dynamics simulations.","date":"2023","source":"SAR and QSAR in environmental research","url":"https://pubmed.ncbi.nlm.nih.gov/37665563","citation_count":1,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PMT"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":14056,"output_tokens":4379,"usd":0.053927,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10736,"output_tokens":5399,"usd":0.094327,"stage2_stop_reason":"end_turn"},"total_usd":0.148254,"stage1_batch_id":"msgbatch_011BPjN2BL33F8cJXD1r7V7T","stage2_batch_id":"msgbatch_01Qwg6vP7qxkpL6Gsc7tajsb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2004,\n      \"finding\": \"PMT (PF3D7_1343000) encodes a phosphoethanolamine N-methyltransferase that catalyzes a three-step S-adenosyl-L-methionine-dependent methylation of phosphoethanolamine to phosphocholine, functioning as a monopartite enzyme with a single catalytic domain responsible for all three methylation steps. PMT activity is inhibited by its product phosphocholine and by the phosphocholine analog miltefosine.\",\n      \"method\": \"Biochemical characterization of recombinant enzyme; in vitro enzymatic assay; inhibitor studies\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct enzymatic characterization of the recombinant protein, in vitro activity assays, product inhibition demonstrated, replicated across multiple subsequent studies\",\n      \"pmids\": [\"15073329\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"PMT (PF3D7_1343000) acts specifically on phosphoethanolamine (not phosphatidylethanolamine) as its in vivo substrate, demonstrated by complementation of a yeast pem1Δpem2Δ mutant. PMT activity is directly coupled to the Kennedy (CDP-choline) pathway for phosphatidylcholine synthesis, as mutation in yeast choline-phosphate cytidylyltransferase abrogates complementation by PMT.\",\n      \"method\": \"Yeast genetic complementation assay; lipid analysis; epistasis via double mutant\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with multiple mutant backgrounds, lipid metabolic profiling, replicated with biochemical analysis\",\n      \"pmids\": [\"15664981\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"PMT (PF3D7_1343000) localizes to the Golgi apparatus of P. falciparum, as demonstrated by complete co-localization with the Golgi marker Rab6 in transgenic parasites expressing GFP targeted to different compartments, and by immunoelectron microscopy. PMT protein levels are regulated in a stage-specific manner, with highest expression during the trophozoite stage.\",\n      \"method\": \"Confocal microscopy; immunoelectron microscopy; GFP co-localization with organelle markers (Rab6 for Golgi, BiP for ER); immunoblotting across life stages\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct localization by multiple orthogonal methods (IEM, confocal with multiple markers, transgenic GFP), functional stage-specific regulation shown\",\n      \"pmids\": [\"16704982\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"PMT (PF3D7_1343000) is regulated by choline at two levels: choline induces transcriptional repression of the PfPMT gene in a dose-dependent manner, and also targets the PMT protein for proteasomal degradation. The proteasome inhibitor bortezomib blocks choline-mediated PMT protein degradation. This regulation occurs in both wild-type and transgenic parasites constitutively expressing PMT.\",\n      \"method\": \"Transcript analysis; immunoblotting; pulse-chase experiments; immunoprecipitation; proteasome inhibitor (bortezomib) treatment\",\n      \"journal\": \"Eukaryotic cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (transcript analysis, pulse-chase, immunoprecipitation, pharmacological inhibition) in single study establishing mechanism of regulation\",\n      \"pmids\": [\"17644653\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Mutational analysis of PMT (PF3D7_1343000) identified residues Asp-61, Gly-83, and Asp-128 as critical for enzymatic activity and substrate/SAM binding, demonstrated by loss-of-function in a yeast complementation system.\",\n      \"method\": \"Site-directed mutagenesis of 24 residues; yeast complementation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — mutagenesis with functional readout in yeast system, single study, no structural validation at this stage\",\n      \"pmids\": [\"18178564\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Genetic knockout of PMT (PF3D7_1343000) in P. falciparum completely abrogates phosphatidylcholine biosynthesis via the SDPM pathway. Unlike in mammalian and yeast cells, methylation of phosphatidylethanolamine to phosphatidylcholine does not occur in P. falciparum, making the SDPM and CDP-choline pathways the only two routes for phosphatidylcholine biosynthesis. Loss of PMT causes significant defects in parasite growth, multiplication, and viability.\",\n      \"method\": \"Genetic knockout; lipid analysis in knockout parasites; parasite growth and viability assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic knockout with biochemical (lipid) and phenotypic (growth/viability) readouts, defines pathway architecture\",\n      \"pmids\": [\"18694927\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"PMT (PF3D7_1343000) activity is inhibited in vitro by hexadecyltrimethylammonium, dodecyltrimethylammonium, and amodiaquine, but not by chloroquine or other aminoquinolines tested. NMR titration studies demonstrated specific, concentration-dependent binding of amodiaquine to the PMT protein. In a yeast surrogate system, yeast mutants relying on PMT for survival were sensitive to amodiaquine and showed inhibited phosphatidylcholine biosynthesis.\",\n      \"method\": \"Enzyme-coupled methylation assay; yeast genetic surrogate system; NMR titration\",\n      \"journal\": \"BMC biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro enzyme assay, NMR binding, and yeast functional system, single lab with multiple orthogonal approaches\",\n      \"pmids\": [\"20085640\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"PMT (phosphoethanolamine N-methyltransferase) activity is absent in rodent Plasmodium species (P. berghei, P. yoelii, P. chabaudi, P. vinckei), as no PMT gene ortholog was detected in silico and biochemical experiments showed that ethanolamine and serine are not incorporated into phosphatidylcholine in rodent parasites, unlike in P. falciparum.\",\n      \"method\": \"In silico genome analysis; biochemical radiolabeling experiments with labeled choline, ethanolamine, and serine\",\n      \"journal\": \"Journal of lipid research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical radiolabeling with multiple precursors across multiple species, consistent with genomic analysis\",\n      \"pmids\": [\"19561325\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"},\n        {\"species\": \"Plasmodium berghei\", \"strain\": \"\"},\n        {\"species\": \"Plasmodium yoelii\", \"strain\": \"\"},\n        {\"species\": \"Plasmodium chabaudi\", \"strain\": \"\"},\n        {\"species\": \"Plasmodium vinckei\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"The 1.55 Å resolution crystal structure of PMT (PF3D7_1343000) in complex with AdoMet was solved by single-wavelength anomalous dispersion phasing, and additional structures at 1.19–1.52 Å resolution were determined with phosphoethanolamine (substrate), phosphocholine (product), sinefungin (inhibitor), and both phosphoethanolamine and S-adenosylhomocysteine. Domain rearrangements occur upon ligand binding. Functional characterization of 27 site-directed mutants identified Tyr-19 and His-132 as forming a catalytic dyad; kinetic analysis, isothermal titration calorimetry, and structural analysis of Y19F and H132A mutants showed these residues are required for phosphobase methylation and form a 'catalytic latch' that locks ligands in the active site.\",\n      \"method\": \"X-ray crystallography (multiple structures); site-directed mutagenesis of 27 residues; kinetic analysis; isothermal titration calorimetry\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution crystal structures with multiple ligands, extensive mutagenesis, kinetics, and ITC in single study with multiple orthogonal methods\",\n      \"pmids\": [\"22117061\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The 2.0 Å resolution X-ray crystal structure of PMT (PF3D7_1343000) in complex with amodiaquine was determined, revealing the structural basis for inhibitor binding at the active site.\",\n      \"method\": \"X-ray crystallography; IC50 determination by radiochemical assay for a series of aminoquinolines\",\n      \"journal\": \"Bioorganic & medicinal chemistry letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with inhibitor, functional IC50 measurements, single study\",\n      \"pmids\": [\"22771008\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PMT (PF3D7_1343000) is essential for P. falciparum gametocytogenesis and malaria transmission. Parasites lacking PMT are severely altered in gametocyte development, unable to produce mature-stage gametocytes, and cannot be transmitted to mosquitoes. Chemical inhibitor NSC-158011 inhibits PMT activity in vitro, blocks intraerythrocytic asexual replication and gametocyte differentiation, and inhibits phosphatidylcholine biosynthesis from ethanolamine in a yeast functional complementation system.\",\n      \"method\": \"Genetic knockout; gametocyte development assays; mosquito transmission assay; chemical inhibitor screening; kinetic studies in vitro; yeast functional complementation; lipid metabolic analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout with defined transmission phenotype, pharmacological corroboration, in vitro kinetics, and metabolic analysis using multiple orthogonal approaches\",\n      \"pmids\": [\"24145416\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"QM/MM computational analysis and mutagenesis of PMT (PF3D7_1343000) revealed that the reaction mechanism involves Asp-128 (not the previously proposed Tyr-19–His-132 dyad) in the initial methylation step: a proton is transferred to Asp-128 via a bridging water molecule, followed by SN2-type methyl transfer from SAM to phosphoethanolamine. D128A, D128E, D128Q, and D128N mutants lose activity with phosphoethanolamine as substrate. Crystal structures of D128A mutant in complex with S-adenosylhomocysteine and either phosphoethanolamine or phosphocholine show disruption of the hydrogen bond network in the active site.\",\n      \"method\": \"QM/MM calculations; site-directed mutagenesis (D128A/E/Q/N); enzymatic activity assays; X-ray crystallography of mutant complexes\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — combined QM/MM, mutagenesis, enzymatic assays, and crystal structures provide orthogonal mechanistic evidence in a single study\",\n      \"pmids\": [\"25288796\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"PMT orthologs from Plasmodium vivax (PvPMT) and Plasmodium knowlesi (PkPMT) were characterized by X-ray crystallography and biochemical assays; both are inhibited by amodiaquine and NSC158011. Metabolic studies in yeast mutants relying on PkPMT or PvPMT showed that these compounds inhibit phosphatidylcholine biosynthesis from ethanolamine.\",\n      \"method\": \"X-ray crystallography; biochemical inhibitor assays; yeast functional complementation; metabolic labeling\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structures, biochemical assays, and yeast metabolic studies provide orthogonal mechanistic evidence for PMT orthologs in two Plasmodium species\",\n      \"pmids\": [\"25761669\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium vivax\", \"strain\": \"\"},\n        {\"species\": \"Plasmodium knowlesi\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PMT (PF3D7_1343000) is phosphorylated by PfCDPK7 in P. falciparum; in the absence of PfCDPK7, PMT is hypophosphorylated and undergoes degradation. PfCDPK7 regulates phosphatidylcholine synthesis via PMT (and ethanolamine kinase), placing PMT downstream of the PfPI4K/4'-phosphoinositide/PfCDPK7 signaling axis.\",\n      \"method\": \"Quantitative phosphoproteomics; phospholipid analysis; conditional knockdown of PfCDPK7; PI4K inhibitor (MMV39048) treatment\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phosphoproteomic identification of PMT as hypophosphorylated substrate of PfCDPK7 combined with lipidomics and pharmacological perturbation, single lab\",\n      \"pmids\": [\"34866326\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"PMT (PF3D7_1343000) is a Golgi-localized, monopartite phosphoethanolamine N-methyltransferase that catalyzes all three SAM-dependent methylation steps converting phosphoethanolamine to phosphocholine via a mechanism requiring Asp-128 (proton transfer via bridging water) and with structural contributions from Tyr-19 and His-132; its product phosphocholine feeds directly into the Kennedy pathway for phosphatidylcholine synthesis, it is essential for intraerythrocytic growth, gametocytogenesis, and mosquito transmission, its expression is controlled by choline-mediated transcriptional repression and proteasomal degradation, and its activity is further regulated by phosphorylation downstream of the PfPI4K/PfCDPK7 signaling axis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PMT (PF3D7_1343000) is a phosphoethanolamine N-methyltransferase that supplies phosphocholine for phosphatidylcholine biosynthesis in Plasmodium falciparum, and it is essential for both intraerythrocytic asexual growth and transmission to mosquitoes [#0, #5, #10]. As a monopartite single-domain enzyme, PMT catalyzes all three sequential SAM-dependent methylation steps that convert phosphoethanolamine to phosphocholine, acting specifically on the free phosphobase rather than on phosphatidylethanolamine, and its product feeds directly into the Kennedy (CDP-choline) pathway [#0, #1]. Because P. falciparum lacks phosphatidylethanolamine-to-phosphatidylcholine methylation, genetic ablation of PMT abolishes phosphatidylcholine synthesis via the serine-decarboxylase-phosphoethanolamine-methyltransferase route and impairs parasite growth and viability, while PMT-null parasites fail to produce mature gametocytes and cannot be transmitted to mosquitoes [#5, #10]. High-resolution crystal structures with substrate, product, cofactor, and inhibitors, combined with extensive mutagenesis and QM/MM analysis, define an active site in which Asp-128 mediates proton transfer through a bridging water to enable SN2 methyl transfer from SAM, with Tyr-19 and His-132 contributing to ligand positioning [#8, #11]. Enzyme abundance and activity are tightly controlled: choline both represses PfPMT transcription and drives proteasomal degradation of the protein [#3], and PMT is phosphorylated by PfCDPK7 downstream of the PfPI4K/PfCDPK7 signaling axis, with loss of PfCDPK7 leaving PMT hypophosphorylated and degraded [#13]. The enzyme localizes to the Golgi apparatus [#2] and is inhibited by product phosphocholine, miltefosine, amodiaquine, and other compounds, making it a validated antimalarial target [#0, #6, #10].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Establishing the core biochemical activity answered what PMT does: it defined the enzyme as a single-domain catalyst performing all three methylations converting phosphoethanolamine to phosphocholine.\",\n      \"evidence\": \"Biochemical characterization of recombinant enzyme with in vitro methylation assays and product/analog inhibition\",\n      \"pmids\": [\"15073329\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish the in vivo substrate specificity\", \"No structural basis for catalysis at this stage\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Yeast complementation resolved whether PMT acts on the free phosphobase or the lipid and how its product connects to lipid synthesis, showing phosphoethanolamine is the in vivo substrate coupled to the Kennedy pathway.\",\n      \"evidence\": \"Complementation of yeast pem1\\u0394pem2\\u0394 mutant with epistasis via choline-phosphate cytidylyltransferase mutation and lipid analysis\",\n      \"pmids\": [\"15664981\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Yeast surrogate may not fully recapitulate parasite context\", \"Did not address subcellular localization in the parasite\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Localization studies answered where PMT operates in the parasite, placing the enzyme in the Golgi apparatus and revealing stage-dependent abundance.\",\n      \"evidence\": \"Confocal and immunoelectron microscopy with GFP organelle markers plus stage-resolved immunoblotting\",\n      \"pmids\": [\"16704982\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of Golgi targeting not defined\", \"Functional significance of Golgi localization for phosphocholine supply unclear\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Regulation studies answered how PMT levels respond to nutrient availability, revealing dual control by choline at the transcriptional level and via proteasomal degradation.\",\n      \"evidence\": \"Transcript analysis, pulse-chase, immunoprecipitation, and bortezomib proteasome inhibition in wild-type and transgenic parasites\",\n      \"pmids\": [\"17644653\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Degradation signal/E3 ligase not identified\", \"Transcriptional repressor mediating choline response unknown\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Mutagenesis began mapping catalytically critical residues, identifying Asp-61, Gly-83, and Asp-128 as required for activity and cofactor/substrate binding.\",\n      \"evidence\": \"Site-directed mutagenesis of 24 residues scored by yeast complementation\",\n      \"pmids\": [\"18178564\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural validation at this stage\", \"Functional readout limited to yeast survival, not direct kinetics\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Genetic knockout answered whether PMT is dispensable, showing it is required for phosphatidylcholine synthesis via the SDPM pathway and for asexual growth, and defining the pathway architecture in P. falciparum.\",\n      \"evidence\": \"Genetic knockout with lipid analysis and growth/viability assays\",\n      \"pmids\": [\"18694927\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not address sexual-stage requirement\", \"Residual growth via CDP-choline pathway not fully quantified\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Species comparison addressed conservation of the pathway, showing PMT activity and gene are absent in rodent Plasmodium species, distinguishing P. falciparum phospholipid metabolism.\",\n      \"evidence\": \"In silico genome analysis and radiolabeling with choline, ethanolamine, and serine across P. falciparum and four rodent species\",\n      \"pmids\": [\"19561325\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not clarify how rodent parasites compensate for the missing route\", \"Limits use of rodent models to study PMT function in vivo\"],\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"},\n        {\"species\": \"Plasmodium berghei\", \"strain\": \"\"},\n        {\"species\": \"Plasmodium yoelii\", \"strain\": \"\"},\n        {\"species\": \"Plasmodium chabaudi\", \"strain\": \"\"},\n        {\"species\": \"Plasmodium vinckei\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Inhibitor profiling identified small molecules that block PMT and demonstrated direct binding, advancing PMT as a druggable target.\",\n      \"evidence\": \"Enzyme-coupled methylation assay, NMR titration binding, and yeast surrogate sensitivity with amodiaquine and quaternary ammonium compounds\",\n      \"pmids\": [\"20085640\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural basis for inhibitor binding yet\", \"Selectivity over human methyltransferases untested\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"High-resolution crystallography with substrate, product, cofactor, and inhibitor resolved the structural mechanism, defining ligand-induced domain rearrangements and implicating a Tyr-19/His-132 catalytic dyad acting as a latch.\",\n      \"evidence\": \"Multiple X-ray structures (1.19\\u20131.55 \\u00c5), mutagenesis of 27 residues, kinetics, and ITC\",\n      \"pmids\": [\"22117061\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Assignment of the catalytic base was later revised\", \"Order of the three methylation steps not structurally resolved\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"A PMT-amodiaquine complex structure provided the structural rationale for inhibitor binding at the active site, guiding structure-based inhibitor design.\",\n      \"evidence\": \"2.0 \\u00c5 X-ray structure with amodiaquine and IC50 determination across a series of aminoquinolines\",\n      \"pmids\": [\"22771008\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cellular potency and selectivity of aminoquinoline series not established here\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Transmission-stage genetics answered whether PMT matters beyond asexual growth, showing it is essential for gametocyte maturation and mosquito transmission, with a chemical inhibitor phenocopying the loss.\",\n      \"evidence\": \"Genetic knockout with gametocyte and mosquito transmission assays plus NSC-158011 inhibitor characterization and yeast complementation\",\n      \"pmids\": [\"24145416\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanistic basis of the gametocyte defect not resolved\", \"Inhibitor specificity in vivo not fully defined\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Combined QM/MM, mutagenesis, and mutant crystal structures revised the catalytic mechanism, establishing Asp-128 as the proton acceptor via a bridging water preceding SN2 methyl transfer.\",\n      \"evidence\": \"QM/MM calculations, D128A/E/Q/N mutagenesis with activity assays, and crystal structures of the D128A mutant with SAH and phosphobase ligands\",\n      \"pmids\": [\"25288796\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether later methylation steps use the same residue is not fully resolved\", \"Roles of Tyr-19/His-132 reconciled but hierarchy of contributions still incomplete\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Structural and biochemical characterization of P. vivax and P. knowlesi orthologs tested conservation of the drug target, showing both are inhibited by amodiaquine and NSC158011.\",\n      \"evidence\": \"X-ray crystallography, inhibitor assays, and yeast metabolic labeling for PvPMT and PkPMT\",\n      \"pmids\": [\"25761669\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Findings are on orthologs, not PF3D7_1343000 directly\", \"Cross-species inhibitor potency in parasites not compared\"],\n      \"attribution\": [\n        {\"species\": \"Plasmodium vivax\", \"strain\": \"\"},\n        {\"species\": \"Plasmodium knowlesi\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Signaling studies answered how PMT is regulated post-translationally, placing it downstream of the PfPI4K/PfCDPK7 axis where PfCDPK7 phosphorylation stabilizes the enzyme.\",\n      \"evidence\": \"Quantitative phosphoproteomics, lipidomics, conditional PfCDPK7 knockdown, and PI4K inhibitor treatment\",\n      \"pmids\": [\"34866326\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct kinase-substrate relationship not reconstituted\", \"Phosphosites on PMT and their functional consequences not mapped\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How Golgi localization, choline-driven turnover, and PfCDPK7 phosphorylation are integrated to control PMT-dependent phosphocholine flux across asexual and sexual stages remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No integrated model linking regulatory inputs to phospholipid flux\", \"E3 ligase and phosphosites governing PMT stability unidentified\", \"Mechanistic cause of the gametocyte maturation defect unknown\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0000234\", \"supporting_discovery_ids\": [0, 1, 8, 11]},\n      {\"term_id\": \"GO:0008757\", \"supporting_discovery_ids\": [0, 8]},\n      {\"term_id\": \"GO:1904047\", \"supporting_discovery_ids\": [0, 8, 11]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0006656\", \"supporting_discovery_ids\": [1, 5, 10]},\n      {\"term_id\": \"GO:0006646\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"PfCDPK7 (phosphorylates PF3D7_1343000; loss leads to hypophosphorylation and degradation) [#13]\"\n    ],\n    \"other_free_text\": [\n      \"Monopartite single-domain enzyme catalyzing all three SAM-dependent methylation steps from phosphoethanolamine to phosphocholine [#0]\",\n      \"Acts on the free phosphobase phosphoethanolamine, not phosphatidylethanolamine; couples to the Kennedy/CDP-choline pathway [#1]\",\n      \"Essential for intraerythrocytic asexual growth and viability [#5]\",\n      \"Essential for gametocyte maturation and mosquito transmission [#10]\",\n      \"Catalytic mechanism: Asp-128 accepts a proton via a bridging water preceding SN2 methyl transfer; Tyr-19 and His-132 position ligands [#8, #11]\",\n      \"Product-inhibited by phosphocholine and inhibited by miltefosine, amodiaquine, NSC-158011, and quaternary ammonium compounds [#0, #6, #10]\",\n      \"Regulated by choline via transcriptional repression and proteasomal degradation [#3]\",\n      \"Post-translationally stabilized by phosphorylation downstream of the PfPI4K/PfCDPK7 axis [#13]\",\n      \"Absent in rodent Plasmodium species (P. berghei, P. yoelii, P. chabaudi, P. vinckei) [#7]\",\n      \"Orthologs in P. vivax and P. knowlesi share amodiaquine/NSC158011 inhibition [#12]\"\n    ]\n  }\n}","audit_flag":null,"strains":[{"strain":"Pf3D7","gene_id":"PF3D7_1343000","name":null,"product":"phosphoethanolamine N-methyltransferase","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PkH","gene_id":"PKNH_1258400","name":null,"product":"phosphoethanolamine N-methyltransferase","uniprot_acc":null,"chromosome":null,"start_pos":null}],"gene_symbol":"PMT","evaluation":null}