{"gene":"PF3D7_1302600","run_date":"2026-09-08T14:19:06","timeline":{"discoveries":[{"year":2009,"finding":"DOHH (PF3D7_1302600) from P. falciparum was cloned and expressed, and purified recombinant protein displayed hydroxylase activity in a novel in vitro DOHH assay, completing the modification of eIF-5A through hydroxylation of deoxyhypusine to hypusine. The protein is 412 amino acids (46.45 kDa, pI 4.96), contains E-Z-type HEAT-like repeats similar to phycocyanin lyase subunits, but phycocyanin lyase activity was absent. A signal peptide at the N-terminus may direct the protein to a different cellular compartment.","method":"Molecular cloning, recombinant expression, in vitro hydroxylase activity assay, bioinformatic analysis","journal":"The FEBS journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct in vitro enzymatic activity assay with purified recombinant protein, combined with functional characterization of substrate (eIF-5A) and negative result for alternative activity (phycocyanin lyase), single lab but multiple orthogonal methods","pmids":["19740108"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2009,"finding":"Plasmodium DOHH completes eIF-5A modification through hydroxylation of deoxyhypusine, and has only a FASTA score of 27 compared to its human ortholog. The HEAT-like repeats in the parasite DOHH differ in number and amino acid identity from the human ortholog. Plasmodium DOHH is proposed to have arisen from a cyanobacterial phycobilin lyase by loss of function.","method":"Sequence analysis, bioinformatic comparison, recombinant expression assessment","journal":"Amino acids","confidence":"Low","confidence_rationale":"Tier 4 / Weak — primarily computational/sequence-based analysis in a review/assessment paper, no new functional experiments reported","pmids":["19956997"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2011,"finding":"Purified recombinant DOHH from Plasmodium demonstrated a twofold higher specific activity than its human counterpart in an in vitro UPLC-based assay measuring conversion of deoxyhypusine to hypusine in eIF-5A. Iron-chelating compounds 2,2'-dipyridyl and mimosine abolished DOHH activity completely, consistent with a ferrous iron-dependent active site mechanism.","method":"In vitro enzymatic activity assay with purified recombinant DOHH, UPLC quantification of hypusine/deoxyhypusine, inhibitor testing","journal":"Amino acids","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — direct in vitro enzymatic assay with purified enzyme and defined inhibitors, single lab, single study","pmids":["21360085"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"finding":"DOHH from P. vivax was cloned and expressed as a histidine-tagged protein (39.01 kDa) in E. coli. P. vivax DOHH contains four E-Z-type HEAT-like repeats and shares significant amino acid identity with simian orthologs (P. knowlesi, P. yoelii). Phycocyanin lyase activity was absent. Recombinant P. vivax DOHH was specifically inhibited by zileuton (IC50 = 12.5 nmol) through complexation of ferrous iron in the active site, while the human ortholog was less affected (IC50 = 90 nmol), establishing a selective iron-chelating inhibition strategy.","method":"Molecular cloning, recombinant expression, in vitro DOHH activity assay (GC/MS-based quantification), inhibitor testing with zileuton","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct in vitro enzymatic assay with recombinant protein, inhibitor IC50 determination, comparative species selectivity established, single lab with multiple orthogonal methods","pmids":["23505486"],"is_preprint":false,"attribution":[{"species":"Plasmodium vivax","strain":""}]},{"year":2015,"finding":"P. falciparum DOHH is a dinuclear iron enzyme in which four conserved histidine and glutamate residues (predicted from structural modeling of the active site) coordinate two ferrous iron ions. Compounds from the Malaria Box with iron-chelating capacity (MMV666023, MMV007384, MMV665805) inhibited hypusine biosynthesis in a bioanalytical assay, with MMV665805 showing the strongest inhibitory effect consistent with the highest predicted iron-complexation capacity.","method":"In silico structural prediction, quantum-mechanical calculations, bioanalytical inhibition assay (GC-MS measurement of hypusine biosynthesis)","journal":"Amino acids","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — active site iron coordination established by structural prediction validated by inhibitor assay (GC-MS), single lab, computational plus bioanalytical","pmids":["25715757"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2003,"finding":"The second step of hypusine biosynthesis in Plasmodium is catalyzed by deoxyhypusine hydroxylase (DOHH/PF3D7_1302600), which hydroxylates the deoxyhypusine residue in the eIF-5A precursor. Evidence for the enzyme's existence in Plasmodium was based on sequence information from the malaria genome project and inhibitor studies.","method":"Genome database sequence analysis, inhibitor studies (review)","journal":"Folia parasitologica","confidence":"Low","confidence_rationale":"Tier 4 / Weak — review-level synthesis based on genome database and inhibitor studies, no direct experimental characterization of DOHH itself","pmids":["12735718"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2006,"finding":"Pharmacological inhibition of deoxyhypusine hydroxylase activity using ciclopiroxolamine (an alpha-hydroxypyridone) and L-mimosine (a 4-pyridone) produced antiplasmodial effects in vitro in P. falciparum, demonstrating that DOHH activity is required for parasite growth.","method":"In vitro parasite growth inhibition assay with DOHH inhibitors","journal":"Parasitology research","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — defined cellular phenotype (growth arrest) via pharmacological inhibition of DOHH, but inhibitors are not perfectly selective; single lab","pmids":["16550432"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2016,"finding":"Pharmacological inhibition of DOHH (as part of the hypusination pathway) arrested growth of malaria parasites. Down-regulation of eIF-5A and DHS (the enzyme catalyzing the step preceding DOHH) by in vitro and in vivo silencing led to decreased transcript levels, reduced protein expression, and low parasitemia in Plasmodium, confirming a critical role of the complete hypusination pathway (including DOHH) for proliferation.","method":"Gene silencing (in vitro and in vivo), targeted gene disruption attempts in P. berghei, parasitemia measurement","journal":"FEBS open bio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple genetic approaches (silencing, attempted knockout, knock-in) in two Plasmodium species establishing pathway essentiality, but DOHH itself was not directly disrupted; role of DOHH inferred from pathway-level experiments","pmids":["27516964"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium berghei","strain":""}]}],"current_model":"PF3D7_1302600 (DOHH) is a dinuclear ferrous iron-dependent hydroxylase that catalyzes the second step of hypusine biosynthesis in Plasmodium — the hydroxylation of the deoxyhypusine residue in eIF-5A to hypusine — with a specific activity twofold higher than its human ortholog, coordinating iron via conserved histidine and glutamate residues in its active site; the enzyme contains distinctive E-Z-type HEAT-like repeats (four in P. vivax, more in P. falciparum) evolutionarily derived from cyanobacterial phycobilin lyases but lacking that activity, and its inhibition by iron-chelating compounds (zileuton, mimosine, 2,2'-dipyridyl) arrests parasite growth, establishing DOHH as essential for Plasmodium proliferation."},"narrative":{"mechanistic_narrative":"PF3D7_1302600 (DOHH) is the Plasmodium deoxyhypusine hydroxylase that catalyzes the second and final step of hypusine biosynthesis, hydroxylating the deoxyhypusine residue of the eIF-5A precursor to generate mature hypusine-containing eIF-5A [PMID:19740108]. Purified recombinant P. falciparum DOHH completes this eIF-5A modification in vitro and displays roughly twofold higher specific activity than its human ortholog [PMID:19740108, PMID:21360085]. Catalysis depends on a dinuclear ferrous-iron active site in which conserved histidine and glutamate residues coordinate two iron ions, so that iron-chelating compounds — 2,2'-dipyridyl, mimosine, zileuton, and iron-complexing Malaria Box compounds — abolish enzymatic activity [PMID:21360085, PMID:25715757]; in P. vivax, zileuton inhibits DOHH selectively over the human enzyme (IC50 12.5 vs 90 nmol) by complexing active-site ferrous iron [PMID:23505486]. Structurally the enzyme carries distinctive E-Z-type HEAT-like repeats resembling phycocyanin lyase subunits (four in P. vivax) yet lacks phycocyanin lyase activity [PMID:19740108, PMID:23505486]. Pharmacological inhibition of DOHH activity arrests P. falciparum growth in vitro, and genetic silencing of the upstream hypusination pathway reduces parasitemia, establishing the hypusination pathway including DOHH as essential for parasite proliferation [PMID:16550432, PMID:27516964].","teleology":[{"year":2006,"claim":"Before direct enzymatic characterization, the question was whether DOHH activity is required for parasite viability; pharmacological inhibition established that it is a growth-essential activity.","evidence":"In vitro P. falciparum growth inhibition with the DOHH inhibitors ciclopiroxolamine and L-mimosine","pmids":["16550432"],"confidence":"Medium","gaps":["Inhibitors are not perfectly selective for DOHH","Growth arrest not tied to direct on-target inhibition of DOHH protein"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2009,"claim":"The unknown was whether the genome-predicted DOHH is a bona fide functional hydroxylase; cloning and in vitro assay of recombinant P. falciparum protein demonstrated it completes eIF-5A modification by hydroxylating deoxyhypusine to hypusine.","evidence":"Molecular cloning, recombinant expression, and a novel in vitro hydroxylase assay on eIF-5A substrate; bioinformatic identification of E-Z-type HEAT-like repeats with absent phycocyanin lyase activity","pmids":["19740108"],"confidence":"High","gaps":["Cellular localization only inferred from a predicted N-terminal signal peptide","Native complex partners not identified"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2011,"claim":"It was unclear whether the parasite enzyme differs kinetically or mechanistically from the human ortholog; quantitative assays showed higher specific activity and confirmed ferrous-iron dependence.","evidence":"In vitro UPLC-based conversion assay with purified recombinant DOHH plus iron-chelator inhibition (2,2'-dipyridyl, mimosine)","pmids":["21360085"],"confidence":"Medium","gaps":["No crystal structure defining the active site","Single lab, single study"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"claim":"Whether iron chelation could achieve parasite-versus-host selectivity was open; the P. vivax enzyme showed selective inhibition by zileuton over the human ortholog.","evidence":"Cloning and recombinant expression of P. vivax DOHH, GC/MS-based activity assay, and zileuton IC50 comparison (12.5 vs 90 nmol)","pmids":["23505486"],"confidence":"High","gaps":["Structural basis of selectivity not resolved","Selectivity shown in vitro, not in parasite culture"],"attribution":[{"species":"Plasmodium vivax","strain":""}]},{"year":2015,"claim":"The active-site architecture underlying iron dependence was undefined; modeling and inhibitor assays established a dinuclear iron site coordinated by conserved His/Glu residues.","evidence":"In silico structural prediction and quantum-mechanical calculations with GC-MS bioanalytical inhibition using iron-chelating Malaria Box compounds (MMV666023, MMV007384, MMV665805)","pmids":["25715757"],"confidence":"Medium","gaps":["Iron coordination predicted rather than crystallographically resolved","Residue assignments not tested by mutagenesis"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2016,"claim":"Whether the hypusination pathway including DOHH is genetically essential (not only pharmacologically vulnerable) was addressed via pathway-level silencing.","evidence":"In vitro and in vivo gene silencing and targeted disruption attempts of pathway components in P. falciparum and P. berghei with parasitemia measurement","pmids":["27516964"],"confidence":"Medium","gaps":["DOHH itself was not directly disrupted; essentiality inferred from upstream eIF-5A/DHS and pharmacology","No conditional knockout of DOHH"],"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium berghei","strain":""}]},{"year":null,"claim":"Direct genetic proof of DOHH essentiality and an experimentally solved structure defining iron coordination and inhibitor binding remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No conditional knockout/knockdown of DOHH itself","No experimental crystal or cryo-EM structure","Subcellular localization not experimentally confirmed"],"attribution":[]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["EIF-5A (SUBSTRATE, HYDROXYLATION OF ITS DEOXYHYPUSINE RESIDUE)"],"other_free_text":["Contains E-Z-type HEAT-like repeats resembling cyanobacterial phycocyanin lyase subunits but lacks phycocyanin lyase activity [#0, #3].","Twofold higher specific activity than the human ortholog [#2].","Inhibited by iron-chelating compounds: 2,2'-dipyridyl, mimosine, zileuton, ciclopiroxolamine, and Malaria Box compounds; zileuton is selective over the human enzyme in P. vivax [#2, #3, #4, #6].","Dinuclear ferrous-iron active site coordinated by conserved histidine and glutamate residues (structural prediction) [#4].","Pharmacological inhibition arrests P. falciparum growth; hypusination pathway including DOHH is essential for proliferation [#6, #7]."]}},"prefetch_data":{},"corpus_meta":[{"pmid":"18159921","id":"PMC_18159921","title":"In search of novel agents for therapy of tropical diseases and human immunodeficiency virus.","date":"2007","source":"Journal of medicinal chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/18159921","citation_count":16,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["deoxyhypusine hydroxylase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"23505486","id":"PMC_23505486","title":"Deoxyhypusine hydroxylase from Plasmodium vivax, the neglected human malaria parasite: molecular cloning, expression and specific inhibition by the 5-LOX inhibitor zileuton.","date":"2013","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/23505486","citation_count":14,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["DOHH","deoxyhypusine hydroxylase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"19740108","id":"PMC_19740108","title":"Completing the hypusine pathway in Plasmodium.","date":"2009","source":"The FEBS journal","url":"https://pubmed.ncbi.nlm.nih.gov/19740108","citation_count":13,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["DOHH","deoxyhypusine hydroxylase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"19956997","id":"PMC_19956997","title":"Assessment of deoxyhypusine hydroxylase as a putative, novel drug target.","date":"2009","source":"Amino acids","url":"https://pubmed.ncbi.nlm.nih.gov/19956997","citation_count":13,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["DOHH","deoxyhypusine hydroxylase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"25715757","id":"PMC_25715757","title":"New insights into novel inhibitors against deoxyhypusine hydroxylase from plasmodium falciparum: compounds with an iron chelating potential.","date":"2015","source":"Amino acids","url":"https://pubmed.ncbi.nlm.nih.gov/25715757","citation_count":12,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["DOHH","deoxyhypusine hydroxylase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"16550432","id":"PMC_16550432","title":"Piperidones with activity against Plasmodium falciparum.","date":"2006","source":"Parasitology research","url":"https://pubmed.ncbi.nlm.nih.gov/16550432","citation_count":12,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["deoxyhypusine hydroxylase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"27516964","id":"PMC_27516964","title":"A suggested vital function for eIF-5A and dhs genes during murine malaria blood-stage infection.","date":"2016","source":"FEBS open bio","url":"https://pubmed.ncbi.nlm.nih.gov/27516964","citation_count":9,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["DOHH","deoxyhypusine hydroxylase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"32367435","id":"PMC_32367435","title":"Inhibition of EIF-5A prevents apoptosis in human cardiomyocytes after malaria infection.","date":"2020","source":"Amino acids","url":"https://pubmed.ncbi.nlm.nih.gov/32367435","citation_count":9,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["deoxyhypusine hydroxylase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"23943044","id":"PMC_23943044","title":"Chemical profiling of deoxyhypusine hydroxylase inhibitors for antimalarial therapy.","date":"2013","source":"Amino acids","url":"https://pubmed.ncbi.nlm.nih.gov/23943044","citation_count":8,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["DOHH","deoxyhypusine hydroxylase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"12735718","id":"PMC_12735718","title":"Spermidine metabolism in parasitic protozoa--a comparison to the situation in prokaryotes, viruses, plants and fungi.","date":"2003","source":"Folia parasitologica","url":"https://pubmed.ncbi.nlm.nih.gov/12735718","citation_count":7,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["deoxyhypusine hydroxylase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"21360085","id":"PMC_21360085","title":"A rapid and robust assay for the determination of the amino acid hypusine as a possible biomarker for a high-throughput screening of antimalarials and for the diagnosis and therapy of different diseases.","date":"2011","source":"Amino acids","url":"https://pubmed.ncbi.nlm.nih.gov/21360085","citation_count":6,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["DOHH","deoxyhypusine hydroxylase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9323,"output_tokens":2566,"usd":0.03323,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":8046,"output_tokens":2847,"usd":0.055703,"stage2_stop_reason":"end_turn"},"total_usd":0.088933,"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\": 2009,\n      \"finding\": \"DOHH (PF3D7_1302600) from P. falciparum was cloned and expressed, and purified recombinant protein displayed hydroxylase activity in a novel in vitro DOHH assay, completing the modification of eIF-5A through hydroxylation of deoxyhypusine to hypusine. The protein is 412 amino acids (46.45 kDa, pI 4.96), contains E-Z-type HEAT-like repeats similar to phycocyanin lyase subunits, but phycocyanin lyase activity was absent. A signal peptide at the N-terminus may direct the protein to a different cellular compartment.\",\n      \"method\": \"Molecular cloning, recombinant expression, in vitro hydroxylase activity assay, bioinformatic analysis\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct in vitro enzymatic activity assay with purified recombinant protein, combined with functional characterization of substrate (eIF-5A) and negative result for alternative activity (phycocyanin lyase), single lab but multiple orthogonal methods\",\n      \"pmids\": [\"19740108\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Plasmodium DOHH completes eIF-5A modification through hydroxylation of deoxyhypusine, and has only a FASTA score of 27 compared to its human ortholog. The HEAT-like repeats in the parasite DOHH differ in number and amino acid identity from the human ortholog. Plasmodium DOHH is proposed to have arisen from a cyanobacterial phycobilin lyase by loss of function.\",\n      \"method\": \"Sequence analysis, bioinformatic comparison, recombinant expression assessment\",\n      \"journal\": \"Amino acids\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — primarily computational/sequence-based analysis in a review/assessment paper, no new functional experiments reported\",\n      \"pmids\": [\"19956997\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Purified recombinant DOHH from Plasmodium demonstrated a twofold higher specific activity than its human counterpart in an in vitro UPLC-based assay measuring conversion of deoxyhypusine to hypusine in eIF-5A. Iron-chelating compounds 2,2'-dipyridyl and mimosine abolished DOHH activity completely, consistent with a ferrous iron-dependent active site mechanism.\",\n      \"method\": \"In vitro enzymatic activity assay with purified recombinant DOHH, UPLC quantification of hypusine/deoxyhypusine, inhibitor testing\",\n      \"journal\": \"Amino acids\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — direct in vitro enzymatic assay with purified enzyme and defined inhibitors, single lab, single study\",\n      \"pmids\": [\"21360085\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"DOHH from P. vivax was cloned and expressed as a histidine-tagged protein (39.01 kDa) in E. coli. P. vivax DOHH contains four E-Z-type HEAT-like repeats and shares significant amino acid identity with simian orthologs (P. knowlesi, P. yoelii). Phycocyanin lyase activity was absent. Recombinant P. vivax DOHH was specifically inhibited by zileuton (IC50 = 12.5 nmol) through complexation of ferrous iron in the active site, while the human ortholog was less affected (IC50 = 90 nmol), establishing a selective iron-chelating inhibition strategy.\",\n      \"method\": \"Molecular cloning, recombinant expression, in vitro DOHH activity assay (GC/MS-based quantification), inhibitor testing with zileuton\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct in vitro enzymatic assay with recombinant protein, inhibitor IC50 determination, comparative species selectivity established, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"23505486\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium vivax\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"P. falciparum DOHH is a dinuclear iron enzyme in which four conserved histidine and glutamate residues (predicted from structural modeling of the active site) coordinate two ferrous iron ions. Compounds from the Malaria Box with iron-chelating capacity (MMV666023, MMV007384, MMV665805) inhibited hypusine biosynthesis in a bioanalytical assay, with MMV665805 showing the strongest inhibitory effect consistent with the highest predicted iron-complexation capacity.\",\n      \"method\": \"In silico structural prediction, quantum-mechanical calculations, bioanalytical inhibition assay (GC-MS measurement of hypusine biosynthesis)\",\n      \"journal\": \"Amino acids\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — active site iron coordination established by structural prediction validated by inhibitor assay (GC-MS), single lab, computational plus bioanalytical\",\n      \"pmids\": [\"25715757\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The second step of hypusine biosynthesis in Plasmodium is catalyzed by deoxyhypusine hydroxylase (DOHH/PF3D7_1302600), which hydroxylates the deoxyhypusine residue in the eIF-5A precursor. Evidence for the enzyme's existence in Plasmodium was based on sequence information from the malaria genome project and inhibitor studies.\",\n      \"method\": \"Genome database sequence analysis, inhibitor studies (review)\",\n      \"journal\": \"Folia parasitologica\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — review-level synthesis based on genome database and inhibitor studies, no direct experimental characterization of DOHH itself\",\n      \"pmids\": [\"12735718\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Pharmacological inhibition of deoxyhypusine hydroxylase activity using ciclopiroxolamine (an alpha-hydroxypyridone) and L-mimosine (a 4-pyridone) produced antiplasmodial effects in vitro in P. falciparum, demonstrating that DOHH activity is required for parasite growth.\",\n      \"method\": \"In vitro parasite growth inhibition assay with DOHH inhibitors\",\n      \"journal\": \"Parasitology research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — defined cellular phenotype (growth arrest) via pharmacological inhibition of DOHH, but inhibitors are not perfectly selective; single lab\",\n      \"pmids\": [\"16550432\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Pharmacological inhibition of DOHH (as part of the hypusination pathway) arrested growth of malaria parasites. Down-regulation of eIF-5A and DHS (the enzyme catalyzing the step preceding DOHH) by in vitro and in vivo silencing led to decreased transcript levels, reduced protein expression, and low parasitemia in Plasmodium, confirming a critical role of the complete hypusination pathway (including DOHH) for proliferation.\",\n      \"method\": \"Gene silencing (in vitro and in vivo), targeted gene disruption attempts in P. berghei, parasitemia measurement\",\n      \"journal\": \"FEBS open bio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple genetic approaches (silencing, attempted knockout, knock-in) in two Plasmodium species establishing pathway essentiality, but DOHH itself was not directly disrupted; role of DOHH inferred from pathway-level experiments\",\n      \"pmids\": [\"27516964\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}, {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"PF3D7_1302600 (DOHH) is a dinuclear ferrous iron-dependent hydroxylase that catalyzes the second step of hypusine biosynthesis in Plasmodium — the hydroxylation of the deoxyhypusine residue in eIF-5A to hypusine — with a specific activity twofold higher than its human ortholog, coordinating iron via conserved histidine and glutamate residues in its active site; the enzyme contains distinctive E-Z-type HEAT-like repeats (four in P. vivax, more in P. falciparum) evolutionarily derived from cyanobacterial phycobilin lyases but lacking that activity, and its inhibition by iron-chelating compounds (zileuton, mimosine, 2,2'-dipyridyl) arrests parasite growth, establishing DOHH as essential for Plasmodium proliferation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PF3D7_1302600 (DOHH) is the Plasmodium deoxyhypusine hydroxylase that catalyzes the second and final step of hypusine biosynthesis, hydroxylating the deoxyhypusine residue of the eIF-5A precursor to generate mature hypusine-containing eIF-5A [#0]. Purified recombinant P. falciparum DOHH completes this eIF-5A modification in vitro and displays roughly twofold higher specific activity than its human ortholog [#0, #2]. Catalysis depends on a dinuclear ferrous-iron active site in which conserved histidine and glutamate residues coordinate two iron ions, so that iron-chelating compounds — 2,2'-dipyridyl, mimosine, zileuton, and iron-complexing Malaria Box compounds — abolish enzymatic activity [#2, #4]; in P. vivax, zileuton inhibits DOHH selectively over the human enzyme (IC50 12.5 vs 90 nmol) by complexing active-site ferrous iron [#3]. Structurally the enzyme carries distinctive E-Z-type HEAT-like repeats resembling phycocyanin lyase subunits (four in P. vivax) yet lacks phycocyanin lyase activity [#0, #3]. Pharmacological inhibition of DOHH activity arrests P. falciparum growth in vitro, and genetic silencing of the upstream hypusination pathway reduces parasitemia, establishing the hypusination pathway including DOHH as essential for parasite proliferation [#6, #7].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Before direct enzymatic characterization, the question was whether DOHH activity is required for parasite viability; pharmacological inhibition established that it is a growth-essential activity.\",\n      \"evidence\": \"In vitro P. falciparum growth inhibition with the DOHH inhibitors ciclopiroxolamine and L-mimosine\",\n      \"pmids\": [\"16550432\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Inhibitors are not perfectly selective for DOHH\", \"Growth arrest not tied to direct on-target inhibition of DOHH protein\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"The unknown was whether the genome-predicted DOHH is a bona fide functional hydroxylase; cloning and in vitro assay of recombinant P. falciparum protein demonstrated it completes eIF-5A modification by hydroxylating deoxyhypusine to hypusine.\",\n      \"evidence\": \"Molecular cloning, recombinant expression, and a novel in vitro hydroxylase assay on eIF-5A substrate; bioinformatic identification of E-Z-type HEAT-like repeats with absent phycocyanin lyase activity\",\n      \"pmids\": [\"19740108\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cellular localization only inferred from a predicted N-terminal signal peptide\", \"Native complex partners not identified\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"It was unclear whether the parasite enzyme differs kinetically or mechanistically from the human ortholog; quantitative assays showed higher specific activity and confirmed ferrous-iron dependence.\",\n      \"evidence\": \"In vitro UPLC-based conversion assay with purified recombinant DOHH plus iron-chelator inhibition (2,2'-dipyridyl, mimosine)\",\n      \"pmids\": [\"21360085\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No crystal structure defining the active site\", \"Single lab, single study\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Whether iron chelation could achieve parasite-versus-host selectivity was open; the P. vivax enzyme showed selective inhibition by zileuton over the human ortholog.\",\n      \"evidence\": \"Cloning and recombinant expression of P. vivax DOHH, GC/MS-based activity assay, and zileuton IC50 comparison (12.5 vs 90 nmol)\",\n      \"pmids\": [\"23505486\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of selectivity not resolved\", \"Selectivity shown in vitro, not in parasite culture\"],\n      \"attribution\": [{\"species\": \"Plasmodium vivax\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"The active-site architecture underlying iron dependence was undefined; modeling and inhibitor assays established a dinuclear iron site coordinated by conserved His/Glu residues.\",\n      \"evidence\": \"In silico structural prediction and quantum-mechanical calculations with GC-MS bioanalytical inhibition using iron-chelating Malaria Box compounds (MMV666023, MMV007384, MMV665805)\",\n      \"pmids\": [\"25715757\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Iron coordination predicted rather than crystallographically resolved\", \"Residue assignments not tested by mutagenesis\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Whether the hypusination pathway including DOHH is genetically essential (not only pharmacologically vulnerable) was addressed via pathway-level silencing.\",\n      \"evidence\": \"In vitro and in vivo gene silencing and targeted disruption attempts of pathway components in P. falciparum and P. berghei with parasitemia measurement\",\n      \"pmids\": [\"27516964\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"DOHH itself was not directly disrupted; essentiality inferred from upstream eIF-5A/DHS and pharmacology\", \"No conditional knockout of DOHH\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}, {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Direct genetic proof of DOHH essentiality and an experimentally solved structure defining iron coordination and inhibitor binding remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No conditional knockout/knockdown of DOHH itself\", \"No experimental crystal or cryo-EM structure\", \"Subcellular localization not experimentally confirmed\"],\n      \"attribution\": []\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0019135\", \"supporting_discovery_ids\": [0, 2, 3]},\n      {\"term_id\": \"GO:0005506\", \"supporting_discovery_ids\": [2, 4]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"GO:0008612\", \"supporting_discovery_ids\": [0, 5, 7]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"eIF-5A (substrate, hydroxylation of its deoxyhypusine residue)\"],\n    \"other_free_text\": [\n      \"Contains E-Z-type HEAT-like repeats resembling cyanobacterial phycocyanin lyase subunits but lacks phycocyanin lyase activity [#0, #3].\",\n      \"Twofold higher specific activity than the human ortholog [#2].\",\n      \"Inhibited by iron-chelating compounds: 2,2'-dipyridyl, mimosine, zileuton, ciclopiroxolamine, and Malaria Box compounds; zileuton is selective over the human enzyme in P. vivax [#2, #3, #4, #6].\",\n      \"Dinuclear ferrous-iron active site coordinated by conserved histidine and glutamate residues (structural prediction) [#4].\",\n      \"Pharmacological inhibition arrests P. falciparum growth; hypusination pathway including DOHH is essential for proliferation [#6, #7].\"\n    ]\n  }\n}","audit_flag":null,"strains":[{"strain":"Pf3D7","gene_id":"PF3D7_1302600","name":null,"product":"deoxyhypusine hydroxylase","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PbANKA","gene_id":"PBANKA_1401100","name":null,"product":"deoxyhypusine hydroxylase, putative","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PkH","gene_id":"PKNH_1402800","name":null,"product":"deoxyhypusine hydroxylase, putative","uniprot_acc":null,"chromosome":null,"start_pos":null}],"gene_symbol":"DOHH","evaluation":null}