Affinage · Plasmodium

DOHH

PF3D7_1302600 · deoxyhypusine hydroxylase

Annotated
2026-09-08
11 papers in source corpus 8 papers cited in narrative 8 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

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).

Mechanistic history

Synthesis pass · year-by-year structured walk · 6 steps
  1. 2006 Medium

    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

    PMID:16550432

    Open questions at the time
    • Inhibitors are not perfectly selective for DOHH
    • Growth arrest not tied to direct on-target inhibition of DOHH protein
  2. 2009 High

    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

    PMID:19740108

    Open questions at the time
    • Cellular localization only inferred from a predicted N-terminal signal peptide
    • Native complex partners not identified
  3. 2011 Medium

    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)

    PMID:21360085

    Open questions at the time
    • No crystal structure defining the active site
    • Single lab, single study
  4. 2013 High

    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)

    PMID:23505486

    Open questions at the time
    • Structural basis of selectivity not resolved
    • Selectivity shown in vitro, not in parasite culture
  5. 2015 Medium

    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)

    PMID:25715757

    Open questions at the time
    • Iron coordination predicted rather than crystallographically resolved
    • Residue assignments not tested by mutagenesis
  6. 2016 Medium

    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

    PMID:27516964

    Open questions at the time
    • DOHH itself was not directly disrupted; essentiality inferred from upstream eIF-5A/DHS and pharmacology
    • No conditional knockout of DOHH

Open questions

Synthesis pass · forward-looking unresolved questions
  • Direct genetic proof of DOHH essentiality and an experimentally solved structure defining iron coordination and inhibitor binding remain unresolved.
  • No conditional knockout/knockdown of DOHH itself
  • No experimental crystal or cryo-EM structure
  • Subcellular localization not experimentally confirmed

Mechanism profile

Synthesis pass · controlled-vocabulary classification
Partners
EIF-5A (SUBSTRATE, HYDROXYLATION OF ITS DEOXYHYPUSINE RESIDUE)

Evidence

Reading pass · 8 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2009 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. Molecular cloning, recombinant expression, in vitro hydroxylase activity assay, bioinformatic analysis The FEBS journal High 19740108
2009 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. Sequence analysis, bioinformatic comparison, recombinant expression assessment Amino acids Low 19956997
2011 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. In vitro enzymatic activity assay with purified recombinant DOHH, UPLC quantification of hypusine/deoxyhypusine, inhibitor testing Amino acids Medium 21360085
2013 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. Molecular cloning, recombinant expression, in vitro DOHH activity assay (GC/MS-based quantification), inhibitor testing with zileuton PloS one High 23505486
2015 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. In silico structural prediction, quantum-mechanical calculations, bioanalytical inhibition assay (GC-MS measurement of hypusine biosynthesis) Amino acids Medium 25715757
2003 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. Genome database sequence analysis, inhibitor studies (review) Folia parasitologica Low 12735718
2006 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. In vitro parasite growth inhibition assay with DOHH inhibitors Parasitology research Medium 16550432
2016 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. Gene silencing (in vitro and in vivo), targeted gene disruption attempts in P. berghei, parasitemia measurement FEBS open bio Medium 27516964

Source papers

Stage 0 corpus · 11 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2007 In search of novel agents for therapy of tropical diseases and human immunodeficiency virus. Journal of medicinal chemistry 16 18159921
2013 Deoxyhypusine hydroxylase from Plasmodium vivax, the neglected human malaria parasite: molecular cloning, expression and specific inhibition by the 5-LOX inhibitor zileuton. PloS one 14 23505486
2009 Completing the hypusine pathway in Plasmodium. The FEBS journal 13 19740108
2009 Assessment of deoxyhypusine hydroxylase as a putative, novel drug target. Amino acids 13 19956997
2015 New insights into novel inhibitors against deoxyhypusine hydroxylase from plasmodium falciparum: compounds with an iron chelating potential. Amino acids 12 25715757
2006 Piperidones with activity against Plasmodium falciparum. Parasitology research 12 16550432
2020 Inhibition of EIF-5A prevents apoptosis in human cardiomyocytes after malaria infection. Amino acids 9 32367435
2016 A suggested vital function for eIF-5A and dhs genes during murine malaria blood-stage infection. FEBS open bio 9 27516964
2013 Chemical profiling of deoxyhypusine hydroxylase inhibitors for antimalarial therapy. Amino acids 8 23943044
2003 Spermidine metabolism in parasitic protozoa--a comparison to the situation in prokaryotes, viruses, plants and fungi. Folia parasitologica 7 12735718
2011 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. Amino acids 6 21360085