{"gene":"PF3D7_0104400","run_date":"2026-09-07T10:55:51","timeline":{"discoveries":[{"year":2005,"finding":"P. falciparum LytB (PF3D7_0104400) is catalytically active in vitro when supplied with an NADPH-dependent electron transfer system comprising ferredoxin and ferredoxin-NADP+ reductase (FNR), and LytB forms a stable protein complex with ferredoxin, indicating that the ferredoxin/FNR redox system is the physiological electron donor for LytB in the apicoplast.","method":"In vitro reconstitution of enzymatic activity; stable complex formation demonstrated by co-purification/pulldown","journal":"FEBS letters","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution of catalytic activity plus stable complex formation, single lab but two orthogonal methods (activity assay and protein-protein interaction)","pmids":["16289098"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"finding":"X-ray crystal structure of P. falciparum LytB (PF3D7_0104400) was determined; the enzyme adopts a cloverleaf fold with an Fe/S-containing active site homologous to Aquifex aeolicus and E. coli LytB, and docking calculations modeled its [2Fe-2S] ferredoxin electron donor at the binding site.","method":"X-ray crystallography; computational docking of ferredoxin to binding site","journal":"FEBS letters","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure determination with functional active-site characterization, single lab but high-quality structural method","pmids":["24188825"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2007,"finding":"Hammerhead ribozyme RZ(876) cleaved the P. falciparum LytB (ispH/lytB) mRNA in vitro and reduced parasite growth by 87.36% at 30 µM with sequence-specific decrease in ispH/lytB transcript levels, demonstrating that LytB expression is required for intraerythrocytic parasite growth.","method":"In vitro ribozyme cleavage assay (RNaseH messenger walk); parasite growth inhibition assay with transcript-level verification","journal":"Oligonucleotides","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — loss-of-function (transcript knockdown) with defined growth phenotype and transcript-level readout, but single lab and partial inhibition only","pmids":["17638523"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2025,"finding":"P. falciparum LytB (IspH/PF3D7_0104400), together with P. falciparum plant-type ferredoxin (ptFd) and ferredoxin-NADP+ reductase (ptFNR), functionally replaced the endogenous bacterial enzymes in an E. coli flavodoxin/ispH double-mutant, reconstituting the last step of the MEP isoprenoid biosynthesis pathway and restoring bacterial growth; this validates the ferredoxin/FNR system as the essential electron donor for LytB in situ.","method":"Genetic complementation in E. coli double-mutant (flavodoxin/ispH); growth assay; LC-MS metabolite analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — functional reconstitution in a heterologous genetic system with metabolite verification by LC-MS, single lab with multiple orthogonal methods","pmids":["40967433"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"current_model":"P. falciparum LytB (PF3D7_0104400) is an apicoplast-localized [Fe-S] enzyme that catalyzes the terminal step of the MEP isoprenoid biosynthesis pathway (conversion of HMBPP to IPP and DMAPP), using a ferredoxin/ferredoxin-NADP+ reductase redox system as its obligate electron donor; it adopts a cloverleaf fold with a conserved active site, forms a stable complex with ferredoxin, and is essential for intraerythrocytic parasite growth."},"narrative":{"mechanistic_narrative":"LytB (PF3D7_0104400) catalyzes the terminal step of the apicoplast MEP isoprenoid biosynthesis pathway in Plasmodium falciparum, functioning as an Fe/S enzyme that depends on a ferredoxin/ferredoxin-NADP+ reductase (FNR) redox system as its physiological electron donor [PMID:16289098, PMID:40967433]. In vitro, recombinant LytB is catalytically active only when supplied with an NADPH-dependent ferredoxin/FNR electron transfer system, and it forms a stable complex with ferredoxin, establishing this redox couple as its obligate electron source [PMID:16289098]. Heterologous genetic complementation confirms this in situ: LytB together with P. falciparum plant-type ferredoxin and FNR restores growth of an E. coli flavodoxin/ispH double-mutant and reconstitutes the final MEP pathway reaction, verified by metabolite analysis [PMID:40967433]. Structurally, LytB adopts a cloverleaf fold with an Fe/S-containing active site, with the [2Fe-2S] ferredoxin donor modeled at its binding site [PMID:24188825]. Loss of LytB expression impairs intraerythrocytic parasite growth, indicating the enzyme is required for blood-stage proliferation [PMID:17638523].","teleology":[{"year":2005,"claim":"Establishing the physiological electron donor for LytB answered how this Fe/S enzyme is powered in the apicoplast, distinguishing it from donor systems used by other pathway enzymes.","evidence":"In vitro reconstitution of LytB catalytic activity with an NADPH/ferredoxin/FNR system plus demonstration of a stable LytB-ferredoxin complex by co-purification","pmids":["16289098"],"confidence":"High","gaps":["Interaction demonstrated in vitro; stoichiometry and affinity of the LytB-ferredoxin complex not quantified","Catalytic turnover parameters and substrate (HMBPP) product ratio (IPP:DMAPP) not defined in this work"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2007,"claim":"Testing whether LytB expression is required for growth answered whether the enzyme is a viable target in blood-stage parasites.","evidence":"Hammerhead ribozyme cleavage of ispH/lytB mRNA in vitro coupled to a parasite growth-inhibition assay with transcript-level verification","pmids":["17638523"],"confidence":"Medium","gaps":["Only partial (87%) growth inhibition at high ribozyme concentration; no clean genetic knockout","Off-target effects of the ribozyme not fully excluded","Does not distinguish requirement at specific developmental stages"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"claim":"Determining the LytB structure answered how the active site is organized and where its ferredoxin donor engages, providing a template for the catalytic mechanism.","evidence":"X-ray crystallography of P. falciparum LytB with computational docking of the [2Fe-2S] ferredoxin at the binding site","pmids":["24188825"],"confidence":"High","gaps":["Ferredoxin binding modeled by docking, not experimentally co-crystallized","No substrate- or product-bound structure to define catalytic geometry"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2025,"claim":"Reconstituting LytB with its ferredoxin/FNR partners in a heterologous mutant answered whether this redox system is sufficient to drive the terminal MEP step in vivo, validating the electron-donor model beyond the test tube.","evidence":"Genetic complementation of an E. coli flavodoxin/ispH double-mutant with P. falciparum LytB, ptFd and ptFNR; growth rescue and LC-MS metabolite analysis","pmids":["40967433"],"confidence":"High","gaps":["Reconstitution performed in E. coli, not in the native apicoplast context","Does not address regulation or assembly of the redox system in the parasite"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":null,"claim":"How the [Fe-S] cluster of LytB is assembled and matured, and how the enzyme is regulated across the parasite life cycle, remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No characterization of LytB Fe-S cluster biogenesis machinery in the corpus","No stage-specific expression or regulatory data","No clean genetic knockout confirming essentiality"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["LytB-ferredoxin complex"],"partners":["PLANT-TYPE FERREDOXIN (PTFD)","FERREDOXIN-NADP+ REDUCTASE (FNR/PTFNR)"],"other_free_text":["Adopts a cloverleaf fold with an Fe/S-containing active site homologous to Aquifex aeolicus and E. coli LytB [#1]","Catalyzes the terminal step of the MEP isoprenoid biosynthesis pathway (HMBPP to IPP/DMAPP), dependent on an NADPH-driven ferredoxin/FNR electron donor system [#0, #3]","Required for intraerythrocytic parasite growth as shown by ribozyme-mediated transcript knockdown [#2]"]}},"prefetch_data":{},"corpus_meta":[{"pmid":"16289098","id":"PMC_16289098","title":"Reconstitution of an apicoplast-localised electron transfer pathway involved in the isoprenoid biosynthesis of Plasmodium falciparum.","date":"2005","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/16289098","citation_count":72,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["LytB"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"24188825","id":"PMC_24188825","title":"Structure of the (E)-4-hydroxy-3-methyl-but-2-enyl-diphosphate reductase from Plasmodium falciparum.","date":"2013","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/24188825","citation_count":21,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["LytB"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"22057350","id":"PMC_22057350","title":"Characterization of acyl carrier protein and LytB in Babesia bovis apicoplast.","date":"2011","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/22057350","citation_count":16,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["4-hydroxy-3-methylbut-2-enyl diphosphate reductase","LytB"],"competing_organism_flag":true,"competing_organism_match":"Babesia","competing_organism_only":true},{"pmid":"25011618","id":"PMC_25011618","title":"An in silico structural insights into Plasmodium LytB protein and its inhibition.","date":"2014","source":"Journal of biomolecular structure & dynamics","url":"https://pubmed.ncbi.nlm.nih.gov/25011618","citation_count":7,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["IspH","LytB"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"17638523","id":"PMC_17638523","title":"Inhibition of Plasmodium falciparum ispH (lytB) gene expression by hammerhead ribozyme.","date":"2007","source":"Oligonucleotides","url":"https://pubmed.ncbi.nlm.nih.gov/17638523","citation_count":4,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["IspH","LytB"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"30191613","id":"PMC_30191613","title":"Exploration and validation of diphosphate-based Plasmodium LytB inhibitors using computational approaches.","date":"2018","source":"Journal of molecular recognition : JMR","url":"https://pubmed.ncbi.nlm.nih.gov/30191613","citation_count":4,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["IspH","LytB"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"40967433","id":"PMC_40967433","title":"Reconstituted ferredoxin-MEP pathway of Apicomplexa in Escherichiacoli as an in situ screening platform for inhibitors and essential enzyme mutations.","date":"2025","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/40967433","citation_count":0,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["IspH"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":6200,"output_tokens":1262,"usd":0.018765,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":6135,"output_tokens":2306,"usd":0.044163,"stage2_stop_reason":"end_turn"},"total_usd":0.062928,"stage1_batch_id":"msgbatch_01DbiymDmubkPpYvdbAu5a7V","stage2_batch_id":"msgbatch_012TbrWvMRH129T7PQ4SmvrJ","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2005,\n      \"finding\": \"P. falciparum LytB (PF3D7_0104400) is catalytically active in vitro when supplied with an NADPH-dependent electron transfer system comprising ferredoxin and ferredoxin-NADP+ reductase (FNR), and LytB forms a stable protein complex with ferredoxin, indicating that the ferredoxin/FNR redox system is the physiological electron donor for LytB in the apicoplast.\",\n      \"method\": \"In vitro reconstitution of enzymatic activity; stable complex formation demonstrated by co-purification/pulldown\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution of catalytic activity plus stable complex formation, single lab but two orthogonal methods (activity assay and protein-protein interaction)\",\n      \"pmids\": [\"16289098\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"X-ray crystal structure of P. falciparum LytB (PF3D7_0104400) was determined; the enzyme adopts a cloverleaf fold with an Fe/S-containing active site homologous to Aquifex aeolicus and E. coli LytB, and docking calculations modeled its [2Fe-2S] ferredoxin electron donor at the binding site.\",\n      \"method\": \"X-ray crystallography; computational docking of ferredoxin to binding site\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure determination with functional active-site characterization, single lab but high-quality structural method\",\n      \"pmids\": [\"24188825\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Hammerhead ribozyme RZ(876) cleaved the P. falciparum LytB (ispH/lytB) mRNA in vitro and reduced parasite growth by 87.36% at 30 µM with sequence-specific decrease in ispH/lytB transcript levels, demonstrating that LytB expression is required for intraerythrocytic parasite growth.\",\n      \"method\": \"In vitro ribozyme cleavage assay (RNaseH messenger walk); parasite growth inhibition assay with transcript-level verification\",\n      \"journal\": \"Oligonucleotides\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — loss-of-function (transcript knockdown) with defined growth phenotype and transcript-level readout, but single lab and partial inhibition only\",\n      \"pmids\": [\"17638523\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"P. falciparum LytB (IspH/PF3D7_0104400), together with P. falciparum plant-type ferredoxin (ptFd) and ferredoxin-NADP+ reductase (ptFNR), functionally replaced the endogenous bacterial enzymes in an E. coli flavodoxin/ispH double-mutant, reconstituting the last step of the MEP isoprenoid biosynthesis pathway and restoring bacterial growth; this validates the ferredoxin/FNR system as the essential electron donor for LytB in situ.\",\n      \"method\": \"Genetic complementation in E. coli double-mutant (flavodoxin/ispH); growth assay; LC-MS metabolite analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — functional reconstitution in a heterologous genetic system with metabolite verification by LC-MS, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"40967433\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"P. falciparum LytB (PF3D7_0104400) is an apicoplast-localized [Fe-S] enzyme that catalyzes the terminal step of the MEP isoprenoid biosynthesis pathway (conversion of HMBPP to IPP and DMAPP), using a ferredoxin/ferredoxin-NADP+ reductase redox system as its obligate electron donor; it adopts a cloverleaf fold with a conserved active site, forms a stable complex with ferredoxin, and is essential for intraerythrocytic parasite growth.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"LytB (PF3D7_0104400) catalyzes the terminal step of the apicoplast MEP isoprenoid biosynthesis pathway in Plasmodium falciparum, functioning as an Fe/S enzyme that depends on a ferredoxin/ferredoxin-NADP+ reductase (FNR) redox system as its physiological electron donor [#0, #3]. In vitro, recombinant LytB is catalytically active only when supplied with an NADPH-dependent ferredoxin/FNR electron transfer system, and it forms a stable complex with ferredoxin, establishing this redox couple as its obligate electron source [#0]. Heterologous genetic complementation confirms this in situ: LytB together with P. falciparum plant-type ferredoxin and FNR restores growth of an E. coli flavodoxin/ispH double-mutant and reconstitutes the final MEP pathway reaction, verified by metabolite analysis [#3]. Structurally, LytB adopts a cloverleaf fold with an Fe/S-containing active site, with the [2Fe-2S] ferredoxin donor modeled at its binding site [#1]. Loss of LytB expression impairs intraerythrocytic parasite growth, indicating the enzyme is required for blood-stage proliferation [#2].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Establishing the physiological electron donor for LytB answered how this Fe/S enzyme is powered in the apicoplast, distinguishing it from donor systems used by other pathway enzymes.\",\n      \"evidence\": \"In vitro reconstitution of LytB catalytic activity with an NADPH/ferredoxin/FNR system plus demonstration of a stable LytB-ferredoxin complex by co-purification\",\n      \"pmids\": [\"16289098\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Interaction demonstrated in vitro; stoichiometry and affinity of the LytB-ferredoxin complex not quantified\",\n        \"Catalytic turnover parameters and substrate (HMBPP) product ratio (IPP:DMAPP) not defined in this work\"\n      ],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Testing whether LytB expression is required for growth answered whether the enzyme is a viable target in blood-stage parasites.\",\n      \"evidence\": \"Hammerhead ribozyme cleavage of ispH/lytB mRNA in vitro coupled to a parasite growth-inhibition assay with transcript-level verification\",\n      \"pmids\": [\"17638523\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Only partial (87%) growth inhibition at high ribozyme concentration; no clean genetic knockout\",\n        \"Off-target effects of the ribozyme not fully excluded\",\n        \"Does not distinguish requirement at specific developmental stages\"\n      ],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Determining the LytB structure answered how the active site is organized and where its ferredoxin donor engages, providing a template for the catalytic mechanism.\",\n      \"evidence\": \"X-ray crystallography of P. falciparum LytB with computational docking of the [2Fe-2S] ferredoxin at the binding site\",\n      \"pmids\": [\"24188825\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Ferredoxin binding modeled by docking, not experimentally co-crystallized\",\n        \"No substrate- or product-bound structure to define catalytic geometry\"\n      ],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Reconstituting LytB with its ferredoxin/FNR partners in a heterologous mutant answered whether this redox system is sufficient to drive the terminal MEP step in vivo, validating the electron-donor model beyond the test tube.\",\n      \"evidence\": \"Genetic complementation of an E. coli flavodoxin/ispH double-mutant with P. falciparum LytB, ptFd and ptFNR; growth rescue and LC-MS metabolite analysis\",\n      \"pmids\": [\"40967433\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Reconstitution performed in E. coli, not in the native apicoplast context\",\n        \"Does not address regulation or assembly of the redox system in the parasite\"\n      ],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the [Fe-S] cluster of LytB is assembled and matured, and how the enzyme is regulated across the parasite life cycle, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No characterization of LytB Fe-S cluster biogenesis machinery in the corpus\",\n        \"No stage-specific expression or regulatory data\",\n        \"No clean genetic knockout confirming essentiality\"\n      ],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0051745\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0051536\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0020011\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0019288\", \"supporting_discovery_ids\": [0, 3]}\n    ],\n    \"complexes\": [\n      \"LytB-ferredoxin complex\"\n    ],\n    \"partners\": [\n      \"plant-type ferredoxin (ptFd)\",\n      \"ferredoxin-NADP+ reductase (FNR/ptFNR)\"\n    ],\n    \"other_free_text\": [\n      \"Adopts a cloverleaf fold with an Fe/S-containing active site homologous to Aquifex aeolicus and E. coli LytB [#1]\",\n      \"Catalyzes the terminal step of the MEP isoprenoid biosynthesis pathway (HMBPP to IPP/DMAPP), dependent on an NADPH-driven ferredoxin/FNR electron donor system [#0, #3]\",\n      \"Required for intraerythrocytic parasite growth as shown by ribozyme-mediated transcript knockdown [#2]\"\n    ]\n  }\n}","audit_flag":null,"strains":[{"strain":"Pf3D7","gene_id":"PF3D7_0104400","name":null,"product":"4-hydroxy-3-methylbut-2-enyl diphosphate reductase","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PbANKA","gene_id":"PBANKA_0208700","name":null,"product":"4-hydroxy-3-methylbut-2-enyl diphosphate reductase, putative","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PkH","gene_id":"PKNH_0209100","name":null,"product":"4-hydroxy-3-methylbut-2-enyl diphosphate reductase, putative","uniprot_acc":null,"chromosome":null,"start_pos":null}],"gene_symbol":"IspH","evaluation":null}