{"gene":"PF3D7_1459100","run_date":"2026-09-08T14:19:07","timeline":{"discoveries":[{"year":1985,"finding":"GTP cyclohydrolase I (GCH1) was identified and purified ~200-fold from P. falciparum extracts. The enzyme catalyzes conversion of GTP to D-erythro-dihydroneopterin triphosphate (releasing carbon-8 of GTP), has MW ~300,000, optimal activity at pH 8.0 and 42°C, Km for GTP of 54.6 µM, is Mg2+-independent, and is competitively inhibited by ATP (Ki = 600 µM). Activity was also detected in P. berghei and P. knowlesi, and in P. knowlesi was found to vary with parasite developmental stage.","method":"Enzyme purification by HPLC size-exclusion chromatography, kinetic assay, product identification, inhibitor characterization","journal":"Molecular and biochemical parasitology","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct in vitro enzymatic assay with purified protein, kinetic parameters, product identification, and inhibitor characterization; replicated across three Plasmodium species","pmids":["3908934"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium berghei","strain":""},{"species":"Plasmodium knowlesi","strain":""}]},{"year":2014,"finding":"Recombinant P. falciparum GCH1 expressed in bacteria shows measurable enzymatic activity (specific activity 5.93 nmol/h/mg by HPLC-fluorescence). The conserved core domain is sufficient for activity; domain truncation experiments showed peripheral domains are dispensable. The enzyme fits a Tunnelling-fold (T-fold) structure by homology modelling with conserved active-site residues. Activity is inhibited by the GTP analogue 8-oxo-GTP. Genetic complementation of a ΔfolE E. coli strain confirmed functional activity and identified key residues/domains required for catalysis.","method":"Bacterial recombinant expression, HPLC-fluorescence enzymatic assay, domain truncation mutagenesis, genetic complementation in ΔfolE bacteria, homology modelling","journal":"Malaria journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro enzymatic assay with mutagenesis, genetic complementation, and structural modelling; multiple orthogonal methods in a single focused study","pmids":["24745605"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2011,"finding":"Co-expression of the P. falciparum chaperone PfHsp70 with PfGCH1 in a bacterial heterologous system increased the yield of soluble, functional PfGCH1, indicating that PfHsp70 assists in proper folding of PfGCH1.","method":"Heterologous co-expression in bacteria, nickel affinity purification, HPLC-fluorescence activity assay","journal":"Protein expression and purification","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional activity of co-expressed protein confirmed by enzymatic assay; single lab, two orthogonal methods (expression/purification + activity measurement)","pmids":["21262365"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2021,"finding":"Computational normal mode analysis (anisotropic network model) of PfGCH1 structure revealed that its tunnel-gating mechanism is regulated by a global shear motion and outward expansion of the central five-helix bundle, and identified allosteric hotspot residues. These structural dynamics were also characterised in complex with its regulatory partner protein.","method":"Anisotropic network model (normal mode analysis), structural modelling","journal":"Journal of chemical information and modeling","confidence":"Low","confidence_rationale":"Tier 4 / Weak — computational prediction only, no experimental validation of allosteric mechanism","pmids":["34450011"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2025,"finding":"Long-read sequencing of P. falciparum lines selected with a DHODH inhibitor revealed tandem amplification of a ~5 kb GCH1 amplicon (up to 9 copies vs 3 in parental lines), arising in precise 2-unit steps with conserved AT-rich sequences at amplicon boundaries consistent with the P. falciparum model of CNV formation. Pre-existing GCH1 amplicons were positively associated with DHODH locus amplification on a separate chromosome, suggesting a metabolic link between folate and pyrimidine biosynthesis pathways.","method":"Long-read Oxford Nanopore sequencing, single-read visualization, copy number quantification","journal":"BMC genomics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct structural characterization of amplicon by long-read sequencing with single-genome resolution; single lab but orthogonal to prior CNV work","pmids":["40676505"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"current_model":"PF3D7_1459100 encodes GTP cyclohydrolase I (GCH1), the first and rate-limiting enzyme of the de novo folate biosynthesis pathway in P. falciparum, which catalyzes the conversion of GTP to D-erythro-dihydroneopterin triphosphate via a Tunnelling-fold mechanism; its conserved core domain is sufficient for catalytic activity, it is competitively inhibited by ATP and by the GTP analogue 8-oxo-GTP, its proper folding is assisted by PfHsp70, and tandem amplification of its genomic locus (arising in 2-unit steps at AT-rich boundaries) is associated with antifolate resistance and with amplification at the DHODH locus on a separate chromosome."},"narrative":{"mechanistic_narrative":"PF3D7_1459100 encodes GTP cyclohydrolase I (GCH1), an enzyme that catalyzes the conversion of GTP to D-erythro-dihydroneopterin triphosphate with release of carbon-8 of GTP, an activity first purified and kinetically characterized from P. falciparum extracts (Km for GTP ~54.6 µM, Mg2+-independent, competitively inhibited by ATP) [PMID:3908934]. Recombinant PfGCH1 is enzymatically active, adopts a Tunnelling-fold (T-fold) architecture in which the conserved core domain alone is sufficient for catalysis while peripheral domains are dispensable, is inhibited by the GTP analogue 8-oxo-GTP, and rescues a ΔfolE E. coli mutant, confirming its identity and defining catalytic residues [PMID:24745605]. Proper folding of PfGCH1 into a soluble, functional form is assisted by the parasite chaperone PfHsp70 [PMID:21262365]. Tandem amplification of the ~5 kb GCH1 locus arises in precise 2-unit steps at conserved AT-rich amplicon boundaries and is positively associated with amplification at the separately located DHODH locus in parasites selected for antimetabolite resistance, linking folate and pyrimidine biosynthesis at the genomic level [PMID:40676505]. Beyond these findings, the in vivo cellular regulation and the identity of a regulatory partner protein have not been experimentally characterized in the available corpus.","teleology":[{"year":1985,"claim":"Establishing that P. falciparum possesses a GTP cyclohydrolase I activity answered whether the parasite carries the first, rate-limiting step of de novo folate biosynthesis as a discrete enzyme.","evidence":"~200-fold enzyme purification with kinetic assay, product identification, and inhibitor characterization from Plasmodium extracts","pmids":["3908934"],"confidence":"High","gaps":["Purified from parasite extract, so the gene identity of the activity was not molecularly assigned at this stage","Native subunit composition underlying the ~300,000 MW not resolved","No structural information on the catalytic site"],"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium berghei","strain":""},{"species":"Plasmodium knowlesi","strain":""}]},{"year":2011,"claim":"Co-expression with PfHsp70 addressed how PfGCH1 is folded into a functional state, showing the chaperone assists production of soluble active enzyme.","evidence":"Heterologous bacterial co-expression with nickel affinity purification and HPLC-fluorescence activity assay","pmids":["21262365"],"confidence":"Medium","gaps":["A direct PfHsp70–PfGCH1 physical interaction was inferred from yield, not demonstrated by binding assay","Whether PfHsp70 chaperones GCH1 in the parasite in vivo is untested","Stoichiometry and folding intermediates undefined"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2014,"claim":"Recombinant expression, domain truncation, and ΔfolE complementation molecularly confirmed the gene product as a functional GCH1 and identified which domains and residues are required for catalysis.","evidence":"Bacterial recombinant expression, HPLC-fluorescence assay, domain truncation mutagenesis, ΔfolE genetic complementation, and homology modelling to a T-fold","pmids":["24745605"],"confidence":"High","gaps":["T-fold assignment rests on homology modelling, not an experimental structure","Physiological relevance of 8-oxo-GTP inhibition in the parasite not addressed","Role of the dispensable peripheral domains not defined"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2021,"claim":"Normal mode analysis probed how the T-fold tunnel opens for catalysis, predicting a shear/expansion gating motion and allosteric hotspots including in complex with a regulatory partner.","evidence":"Anisotropic network model normal mode analysis on a structural model","pmids":["34450011"],"confidence":"Low","gaps":["Purely computational with no experimental validation of the gating mechanism or hotspots","The identity of the 'regulatory partner protein' is not established for PF3D7_1459100 in this corpus","Predicted allosteric residues untested by mutagenesis"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2025,"claim":"Long-read sequencing of drug-selected lines defined the architecture of GCH1 locus amplification and linked it to pyrimidine-pathway resistance, addressing how copy-number variation at this gene arises and co-varies with DHODH amplification.","evidence":"Oxford Nanopore long-read sequencing with single-read visualization and copy-number quantification of a ~5 kb GCH1 amplicon","pmids":["40676505"],"confidence":"Medium","gaps":["The functional/metabolic mechanism connecting GCH1 and DHODH amplification is correlative, not demonstrated","Whether increased GCH1 copy number raises enzyme activity or flux was not measured here","Mechanism selecting for 2-unit amplification steps not resolved"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":null,"claim":"How PfGCH1 catalysis and abundance are regulated in the intact parasite, and the identity of any in vivo regulatory partner, remain open.","evidence":"No experimental characterization of in-parasite regulation or partner identity in the available corpus","pmids":[],"confidence":"Low","gaps":["No experimental (non-homology) structure of PfGCH1","No validated physical interactor or regulatory subunit identified for PF3D7_1459100","Contribution of the gene to folate flux and drug resistance not tested by direct genetic manipulation"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["PFHSP70 (CHAPERONE ASSISTING PFGCH1 FOLDING) [#2]"],"other_free_text":["Catalyzes GTP -> D-erythro-dihydroneopterin triphosphate, releasing carbon-8 of GTP; Km(GTP) ~54.6 µM, Mg2+-independent, competitively inhibited by ATP (Ki ~600 µM) [#0]","Inhibited by the GTP analogue 8-oxo-GTP; conserved core domain sufficient for catalysis, peripheral domains dispensable; rescues ΔfolE E. coli [#1]","Adopts a Tunnelling-fold (T-fold) by homology modelling with predicted shear/expansion tunnel-gating dynamics and allosteric hotspots (computational) [#1, #3]","GCH1 locus undergoes tandem amplification in precise 2-unit steps at AT-rich boundaries, positively associated with DHODH locus amplification under antimetabolite selection [#4]","Activity also detected in P. berghei and P. knowlesi, with stage-dependent variation in P. knowlesi [#0]"]}},"prefetch_data":{},"corpus_meta":[{"pmid":"10743617","id":"PMC_10743617","title":"Evidence for vesicle-mediated trafficking of parasite proteins to the host cell cytosol and erythrocyte surface membrane in Plasmodium falciparum infected erythrocytes.","date":"2000","source":"Molecular and biochemical 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biology","url":"https://pubmed.ncbi.nlm.nih.gov/10472798","citation_count":74,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["GTP-binding protein","GTP"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"10734123","id":"PMC_10734123","title":"Oxidative phosphorylation, Ca(2+) transport, and fatty acid-induced uncoupling in malaria parasites mitochondria.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10734123","citation_count":74,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["GTP"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"12657463","id":"PMC_12657463","title":"The Plasmodium falciparum family of Rab GTPases.","date":"2003","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/12657463","citation_count":73,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["GTP"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"15657083","id":"PMC_15657083","title":"Dissection of brefeldin A-sensitive and -insensitive steps in apicoplast protein targeting.","date":"2005","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/15657083","citation_count":51,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["GTP"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"38977907","id":"PMC_38977907","title":"CRISPR-based functional profiling of the Toxoplasma gondii genome during acute murine infection.","date":"2024","source":"Nature microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/38977907","citation_count":25,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["GTP"],"competing_organism_flag":true,"competing_organism_match":"Toxoplasma","competing_organism_only":true},{"pmid":"33382691","id":"PMC_33382691","title":"Genetic diversity of the Plasmodium falciparum GTP-cyclohydrolase 1, dihydrofolate reductase and dihydropteroate synthetase genes reveals new insights into sulfadoxine-pyrimethamine antimalarial drug resistance.","date":"2020","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/33382691","citation_count":22,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["GTP"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"3908934","id":"PMC_3908934","title":"Guanosine triphosphate cyclohydrolase in Plasmodium falciparum and other Plasmodium species.","date":"1985","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/3908934","citation_count":22,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["GTP"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"10645545","id":"PMC_10645545","title":"Export of parasite proteins to the erythrocyte cytoplasm: secretory machinery and traffic signals.","date":"1999","source":"Novartis Foundation symposium","url":"https://pubmed.ncbi.nlm.nih.gov/10645545","citation_count":20,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["GTP-binding protein","GTP"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"7888304","id":"PMC_7888304","title":"Evidence for expression of a Ras-like and a stage specific GTP binding homologous protein by Plasmodium falciparum.","date":"1994","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/7888304","citation_count":18,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["GTP-binding protein","GTP"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"24745605","id":"PMC_24745605","title":"Biochemical and functional characterization of Plasmodium falciparum GTP cyclohydrolase I.","date":"2014","source":"Malaria journal","url":"https://pubmed.ncbi.nlm.nih.gov/24745605","citation_count":16,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["GTP"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"21262365","id":"PMC_21262365","title":"Co-expression of the Plasmodium falciparum molecular chaperone, PfHsp70, improves the heterologous production of the antimalarial drug target GTP cyclohydrolase I, PfGCHI.","date":"2011","source":"Protein expression and purification","url":"https://pubmed.ncbi.nlm.nih.gov/21262365","citation_count":16,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["GTP"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"8198605","id":"PMC_8198605","title":"Cloning and expression of a cDNA encoding the homologue of Ran/TC4 GTP-binding protein from Plasmodium falciparum.","date":"1994","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/8198605","citation_count":13,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["GTP-binding protein","GTP"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"9237367","id":"PMC_9237367","title":"Small GTP-binding proteins in Plasmodium falciparum.","date":"1996","source":"Biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/9237367","citation_count":12,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["GTP"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"32127009","id":"PMC_32127009","title":"Molecular characterization of Plasmodium falciparum antifolate resistance markers in Thailand between 2008 and 2016.","date":"2020","source":"Malaria journal","url":"https://pubmed.ncbi.nlm.nih.gov/32127009","citation_count":12,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["GTP"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"33639924","id":"PMC_33639924","title":"Measurement of gene amplifications related to drug resistance in Plasmodium falciparum using droplet digital PCR.","date":"2021","source":"Malaria journal","url":"https://pubmed.ncbi.nlm.nih.gov/33639924","citation_count":12,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["GTP"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"30265714","id":"PMC_30265714","title":"Amplification of GTP-cyclohydrolase 1 gene in Plasmodium falciparum isolates with the quadruple mutant of dihydrofolate reductase and dihydropteroate synthase genes in Ghana.","date":"2018","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/30265714","citation_count":11,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["GTP"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"36525403","id":"PMC_36525403","title":"Ten-year persistence and evolution of Plasmodium falciparum antifolate and anti-sulfonamide resistance markers pfdhfr and pfdhps in three Asian countries.","date":"2022","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/36525403","citation_count":6,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["GTP"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"34450011","id":"PMC_34450011","title":"GTP Cyclohydrolase I as a Potential Drug Target: New Insights into Its Allosteric Modulation via Normal Mode Analysis.","date":"2021","source":"Journal of chemical information and modeling","url":"https://pubmed.ncbi.nlm.nih.gov/34450011","citation_count":6,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["GTP"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"36945434","id":"PMC_36945434","title":"Functional profiling of the Toxoplasma genome during acute mouse infection.","date":"2023","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/36945434","citation_count":4,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["GTP"],"competing_organism_flag":true,"competing_organism_match":"Toxoplasma","competing_organism_only":true},{"pmid":"40676505","id":"PMC_40676505","title":"Direct long-read visualization reveals hidden variation in GCH1 gene copy number and precise expansion steps.","date":"2025","source":"BMC genomics","url":"https://pubmed.ncbi.nlm.nih.gov/40676505","citation_count":3,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["GTP"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"34249776","id":"PMC_34249776","title":"Polymorphism of Antifolate Drug Resistance in Plasmodium vivax From Local Residents and Migrant Workers Returned From the China-Myanmar Border.","date":"2021","source":"Frontiers in cellular and infection microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/34249776","citation_count":2,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["GTP"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":15548,"output_tokens":1706,"usd":0.036117,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":6741,"output_tokens":2937,"usd":0.053565,"stage2_stop_reason":"end_turn"},"total_usd":0.089682,"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\": 1985,\n      \"finding\": \"GTP cyclohydrolase I (GCH1) was identified and purified ~200-fold from P. falciparum extracts. The enzyme catalyzes conversion of GTP to D-erythro-dihydroneopterin triphosphate (releasing carbon-8 of GTP), has MW ~300,000, optimal activity at pH 8.0 and 42°C, Km for GTP of 54.6 µM, is Mg2+-independent, and is competitively inhibited by ATP (Ki = 600 µM). Activity was also detected in P. berghei and P. knowlesi, and in P. knowlesi was found to vary with parasite developmental stage.\",\n      \"method\": \"Enzyme purification by HPLC size-exclusion chromatography, kinetic assay, product identification, inhibitor characterization\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct in vitro enzymatic assay with purified protein, kinetic parameters, product identification, and inhibitor characterization; replicated across three Plasmodium species\",\n      \"pmids\": [\"3908934\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"},\n        {\"species\": \"Plasmodium berghei\", \"strain\": \"\"},\n        {\"species\": \"Plasmodium knowlesi\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Recombinant P. falciparum GCH1 expressed in bacteria shows measurable enzymatic activity (specific activity 5.93 nmol/h/mg by HPLC-fluorescence). The conserved core domain is sufficient for activity; domain truncation experiments showed peripheral domains are dispensable. The enzyme fits a Tunnelling-fold (T-fold) structure by homology modelling with conserved active-site residues. Activity is inhibited by the GTP analogue 8-oxo-GTP. Genetic complementation of a ΔfolE E. coli strain confirmed functional activity and identified key residues/domains required for catalysis.\",\n      \"method\": \"Bacterial recombinant expression, HPLC-fluorescence enzymatic assay, domain truncation mutagenesis, genetic complementation in ΔfolE bacteria, homology modelling\",\n      \"journal\": \"Malaria journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro enzymatic assay with mutagenesis, genetic complementation, and structural modelling; multiple orthogonal methods in a single focused study\",\n      \"pmids\": [\"24745605\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Co-expression of the P. falciparum chaperone PfHsp70 with PfGCH1 in a bacterial heterologous system increased the yield of soluble, functional PfGCH1, indicating that PfHsp70 assists in proper folding of PfGCH1.\",\n      \"method\": \"Heterologous co-expression in bacteria, nickel affinity purification, HPLC-fluorescence activity assay\",\n      \"journal\": \"Protein expression and purification\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional activity of co-expressed protein confirmed by enzymatic assay; single lab, two orthogonal methods (expression/purification + activity measurement)\",\n      \"pmids\": [\"21262365\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Computational normal mode analysis (anisotropic network model) of PfGCH1 structure revealed that its tunnel-gating mechanism is regulated by a global shear motion and outward expansion of the central five-helix bundle, and identified allosteric hotspot residues. These structural dynamics were also characterised in complex with its regulatory partner protein.\",\n      \"method\": \"Anisotropic network model (normal mode analysis), structural modelling\",\n      \"journal\": \"Journal of chemical information and modeling\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — computational prediction only, no experimental validation of allosteric mechanism\",\n      \"pmids\": [\"34450011\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Long-read sequencing of P. falciparum lines selected with a DHODH inhibitor revealed tandem amplification of a ~5 kb GCH1 amplicon (up to 9 copies vs 3 in parental lines), arising in precise 2-unit steps with conserved AT-rich sequences at amplicon boundaries consistent with the P. falciparum model of CNV formation. Pre-existing GCH1 amplicons were positively associated with DHODH locus amplification on a separate chromosome, suggesting a metabolic link between folate and pyrimidine biosynthesis pathways.\",\n      \"method\": \"Long-read Oxford Nanopore sequencing, single-read visualization, copy number quantification\",\n      \"journal\": \"BMC genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct structural characterization of amplicon by long-read sequencing with single-genome resolution; single lab but orthogonal to prior CNV work\",\n      \"pmids\": [\"40676505\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}\n      ]\n    }\n  ],\n  \"current_model\": \"PF3D7_1459100 encodes GTP cyclohydrolase I (GCH1), the first and rate-limiting enzyme of the de novo folate biosynthesis pathway in P. falciparum, which catalyzes the conversion of GTP to D-erythro-dihydroneopterin triphosphate via a Tunnelling-fold mechanism; its conserved core domain is sufficient for catalytic activity, it is competitively inhibited by ATP and by the GTP analogue 8-oxo-GTP, its proper folding is assisted by PfHsp70, and tandem amplification of its genomic locus (arising in 2-unit steps at AT-rich boundaries) is associated with antifolate resistance and with amplification at the DHODH locus on a separate chromosome.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PF3D7_1459100 encodes GTP cyclohydrolase I (GCH1), an enzyme that catalyzes the conversion of GTP to D-erythro-dihydroneopterin triphosphate with release of carbon-8 of GTP, an activity first purified and kinetically characterized from P. falciparum extracts (Km for GTP ~54.6 µM, Mg2+-independent, competitively inhibited by ATP) [#0]. Recombinant PfGCH1 is enzymatically active, adopts a Tunnelling-fold (T-fold) architecture in which the conserved core domain alone is sufficient for catalysis while peripheral domains are dispensable, is inhibited by the GTP analogue 8-oxo-GTP, and rescues a ΔfolE E. coli mutant, confirming its identity and defining catalytic residues [#1]. Proper folding of PfGCH1 into a soluble, functional form is assisted by the parasite chaperone PfHsp70 [#2]. Tandem amplification of the ~5 kb GCH1 locus arises in precise 2-unit steps at conserved AT-rich amplicon boundaries and is positively associated with amplification at the separately located DHODH locus in parasites selected for antimetabolite resistance, linking folate and pyrimidine biosynthesis at the genomic level [#4]. Beyond these findings, the in vivo cellular regulation and the identity of a regulatory partner protein have not been experimentally characterized in the available corpus.\",\n  \"teleology\": [\n    {\n      \"year\": 1985,\n      \"claim\": \"Establishing that P. falciparum possesses a GTP cyclohydrolase I activity answered whether the parasite carries the first, rate-limiting step of de novo folate biosynthesis as a discrete enzyme.\",\n      \"evidence\": \"~200-fold enzyme purification with kinetic assay, product identification, and inhibitor characterization from Plasmodium extracts\",\n      \"pmids\": [\"3908934\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Purified from parasite extract, so the gene identity of the activity was not molecularly assigned at this stage\",\n        \"Native subunit composition underlying the ~300,000 MW not resolved\",\n        \"No structural information on the catalytic site\"\n      ],\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"},\n        {\"species\": \"Plasmodium berghei\", \"strain\": \"\"},\n        {\"species\": \"Plasmodium knowlesi\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Co-expression with PfHsp70 addressed how PfGCH1 is folded into a functional state, showing the chaperone assists production of soluble active enzyme.\",\n      \"evidence\": \"Heterologous bacterial co-expression with nickel affinity purification and HPLC-fluorescence activity assay\",\n      \"pmids\": [\"21262365\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"A direct PfHsp70–PfGCH1 physical interaction was inferred from yield, not demonstrated by binding assay\",\n        \"Whether PfHsp70 chaperones GCH1 in the parasite in vivo is untested\",\n        \"Stoichiometry and folding intermediates undefined\"\n      ],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Recombinant expression, domain truncation, and ΔfolE complementation molecularly confirmed the gene product as a functional GCH1 and identified which domains and residues are required for catalysis.\",\n      \"evidence\": \"Bacterial recombinant expression, HPLC-fluorescence assay, domain truncation mutagenesis, ΔfolE genetic complementation, and homology modelling to a T-fold\",\n      \"pmids\": [\"24745605\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"T-fold assignment rests on homology modelling, not an experimental structure\",\n        \"Physiological relevance of 8-oxo-GTP inhibition in the parasite not addressed\",\n        \"Role of the dispensable peripheral domains not defined\"\n      ],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Normal mode analysis probed how the T-fold tunnel opens for catalysis, predicting a shear/expansion gating motion and allosteric hotspots including in complex with a regulatory partner.\",\n      \"evidence\": \"Anisotropic network model normal mode analysis on a structural model\",\n      \"pmids\": [\"34450011\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"Purely computational with no experimental validation of the gating mechanism or hotspots\",\n        \"The identity of the 'regulatory partner protein' is not established for PF3D7_1459100 in this corpus\",\n        \"Predicted allosteric residues untested by mutagenesis\"\n      ],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Long-read sequencing of drug-selected lines defined the architecture of GCH1 locus amplification and linked it to pyrimidine-pathway resistance, addressing how copy-number variation at this gene arises and co-varies with DHODH amplification.\",\n      \"evidence\": \"Oxford Nanopore long-read sequencing with single-read visualization and copy-number quantification of a ~5 kb GCH1 amplicon\",\n      \"pmids\": [\"40676505\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"The functional/metabolic mechanism connecting GCH1 and DHODH amplification is correlative, not demonstrated\",\n        \"Whether increased GCH1 copy number raises enzyme activity or flux was not measured here\",\n        \"Mechanism selecting for 2-unit amplification steps not resolved\"\n      ],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How PfGCH1 catalysis and abundance are regulated in the intact parasite, and the identity of any in vivo regulatory partner, remain open.\",\n      \"evidence\": \"No experimental characterization of in-parasite regulation or partner identity in the available corpus\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No experimental (non-homology) structure of PfGCH1\",\n        \"No validated physical interactor or regulatory subunit identified for PF3D7_1459100\",\n        \"Contribution of the gene to folate flux and drug resistance not tested by direct genetic manipulation\"\n      ],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003934\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0005525\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"GO:0046656\", \"supporting_discovery_ids\": [0, 1, 4]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"PfHsp70 (chaperone assisting PfGCH1 folding) [#2]\"\n    ],\n    \"other_free_text\": [\n      \"Catalyzes GTP -> D-erythro-dihydroneopterin triphosphate, releasing carbon-8 of GTP; Km(GTP) ~54.6 µM, Mg2+-independent, competitively inhibited by ATP (Ki ~600 µM) [#0]\",\n      \"Inhibited by the GTP analogue 8-oxo-GTP; conserved core domain sufficient for catalysis, peripheral domains dispensable; rescues ΔfolE E. coli [#1]\",\n      \"Adopts a Tunnelling-fold (T-fold) by homology modelling with predicted shear/expansion tunnel-gating dynamics and allosteric hotspots (computational) [#1, #3]\",\n      \"GCH1 locus undergoes tandem amplification in precise 2-unit steps at AT-rich boundaries, positively associated with DHODH locus amplification under antimetabolite selection [#4]\",\n      \"Activity also detected in P. berghei and P. knowlesi, with stage-dependent variation in P. knowlesi [#0]\"\n    ]\n  }\n}","audit_flag":{"gene":"PF3D7_1459100","tier":"IDENTITY_CHECK","verdict":"identity unverified","subtype":"names_other_cards","uniprot_band":"","rules_fired":"R12","issue":"No cited paper names this card; 4 of 5 cited papers name other catalogue genes (PF3D7_1224000, PF3D7_0818900, PF3D7_0603300). The summary may describe one of those genes."},"strains":[{"strain":"Pf3D7","gene_id":"PF3D7_1459100","name":null,"product":"GTP-binding protein, putative","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PbANKA","gene_id":"PBANKA_1322800","name":null,"product":"GTP-binding protein, putative","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PkH","gene_id":"PKNH_1222700","name":null,"product":"GTP-binding protein, putative","uniprot_acc":null,"chromosome":null,"start_pos":null}],"gene_symbol":"","evaluation":null}