Affinage

DPH2

2-(3-amino-3-carboxypropyl)histidine synthase subunit 2 · UniProt Q9BQC3

Length
489 aa
Mass
52.1 kDa
Annotated
2026-04-28
12 papers in source corpus 11 papers cited in narrative 11 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

DPH2 is an essential subunit of the Dph1–Dph2 heterodimeric radical SAM enzyme that catalyzes the first committed step of diphthamide biosynthesis—the transfer of a 3-amino-3-carboxypropyl (ACP) radical from SAM to a conserved histidine on elongation factor 2 (EF2) (PMID:24422557, PMID:20931132). Within the heterodimer, DPH2 harbors a [4Fe-4S] cluster coordinated by a non-canonical tandem cysteine motif; this cluster does not directly cleave SAM but instead serves as an electron-transfer conduit, accepting electrons from the Dph3/Cbr1/NADH reducing system and relaying them to the catalytic [4Fe-4S] cluster on Dph1, which performs reductive SAM cleavage (PMID:31463593, PMID:34154323). The DPH2 cluster is oxygen-sensitive and degrades to an inactive [3Fe-4S] form, which Dph3 repairs by donating a single iron atom, thereby enabling aerobic diphthamide production (PMID:34154323). Loss-of-function DPH2 variants (e.g., H105P, C341Y) impair diphthamide synthesis in yeast and mammalian cells, establishing DPH2 as a diphthamide-deficiency susceptibility gene (PMID:37675463).

Mechanistic history

Synthesis pass · year-by-year structured walk · 8 steps
  1. 1993 Medium

    Identification of DPH2 as a non-essential yeast gene required for diphthamide biosynthesis established it as a dedicated pathway component rather than a general translation factor.

    Evidence Gene cloning by complementation and disruption in S. cerevisiae

    PMID:8406038

    Open questions at the time
    • Biochemical activity of Dph2 protein unknown
    • Human ortholog not functionally validated
    • Relationship to other dph genes not characterized
  2. 2010 High

    Determination that archaeal Dph2 is a radical SAM enzyme using a [4Fe-4S] cluster to cleave SAM into an ACP radical (rather than the canonical 5′-deoxyadenosyl radical) revealed an unprecedented SAM fragmentation mechanism for the first step of diphthamide synthesis.

    Evidence Crystal structure, EPR spectroscopy, and in vitro reconstitution of Pyrococcus horikoshii Dph2

    PMID:20931132

    Open questions at the time
    • Eukaryotic Dph1-Dph2 heterodimer not yet reconstituted
    • Reducing system for cluster activation unknown
    • Substrate-enzyme complex structure not solved
  3. 2014 High

    Reconstitution of the yeast Dph1–Dph2 heterodimer showed it is the functional equivalent of the archaeal homodimer and identified Dph3 as the physiological electron donor, linking the reducing system to ACP transfer activity.

    Evidence In vitro reconstitution with EPR and iron-binding assays in yeast system

    PMID:24422557

    Open questions at the time
    • Individual roles of Dph1 vs. Dph2 clusters not resolved
    • Mechanism of Dph3 electron donation not defined at atomic level
    • Eukaryotic crystal structure of the heterodimer lacking
  4. 2019 High

    Mutagenesis of cluster-binding cysteines in each subunit demonstrated that Dph1 and Dph2 have asymmetric roles: Dph1 is catalytic while Dph2 functions as an electron relay from the Dph3/Cbr1/NADH system, resolving the division of labor within the heterodimer.

    Evidence Site-directed mutagenesis combined with in vivo diphthamide assay, in vitro reconstitution, and EPR

    PMID:31463593

    Open questions at the time
    • Structural basis for inter-subunit electron transfer unknown
    • Whether human DPH2 has identical relay function not directly tested
    • Kinetic parameters for electron transfer not measured
  5. 2019 High

    The crystal structure of the archaeal Dph2–EF-2 complex revealed how EF-2 domain IV docks into the active site and identified a conserved arginine critical for SAM carboxylate binding and catalysis, providing the first structural view of substrate recognition.

    Evidence X-ray crystallography at 3.5 Å of MsDph2–MsEF-2 complex with mutagenesis validation

    PMID:31566354

    Open questions at the time
    • Eukaryotic heterodimer–EF2 complex structure not available
    • Transition-state structure not captured
    • Role of corresponding residues in human DPH2 not validated
  6. 2021 High

    Discovery that the [4Fe-4S] cluster degrades to [3Fe-4S] under oxygen and that Dph3 repairs it by donating iron explained how diphthamide biosynthesis proceeds aerobically despite the oxygen sensitivity of radical SAM enzymes.

    Evidence EPR, Mössbauer spectroscopy, and in vitro iron reconstitution/activity assays

    PMID:34154323

    Open questions at the time
    • Whether Dph2's cluster or Dph1's cluster is preferentially repaired not distinguished
    • In vivo rates of cluster damage and repair unknown
    • Whether additional cellular iron chaperones participate is untested
  7. 2023 Medium

    Functional assessment of human DPH2 missense variants (H105P, C341Y) in yeast and mammalian cells demonstrated that specific substitutions impair diphthamide synthesis, linking DPH2 variation to diphthamide deficiency.

    Evidence Yeast complementation and mammalian cell functional assays with patient-derived variants

    PMID:37675463

    Open questions at the time
    • No Mendelian disease formally established with segregation data in the timeline
    • Structural impact of each variant not experimentally determined
    • Genotype-phenotype correlation across broader variant spectrum unknown
  8. 2024 Medium

    Characterization of the non-canonical tandem cysteine motif (TCM) in DPH2 showed that the fourth cysteine is required for both [4Fe-4S] cluster coordination and protein stability, establishing this atypical ligand as essential for DPH2 function.

    Evidence Site-directed mutagenesis with in vivo diphthamide assay and cycloheximide chase stability analysis

    PMID:38672486

    Open questions at the time
    • Structural model of eukaryotic TCM not experimentally validated
    • Whether TCM mutations affect inter-subunit electron transfer directly not tested
    • No high-resolution structure of the eukaryotic Dph1–Dph2 heterodimer available

Open questions

Synthesis pass · forward-looking unresolved questions
  • A high-resolution structure of the eukaryotic Dph1–Dph2 heterodimer—ideally in complex with EF2 and SAM—is needed to define the inter-subunit electron-transfer pathway and the structural basis for human disease-associated variants.
  • No eukaryotic Dph1–Dph2 crystal or cryo-EM structure exists
  • Kinetic mechanism of inter-cluster electron transfer not resolved
  • Clinical significance of DPH2 variants in human disease not fully established

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0016740 transferase activity 3
Localization
GO:0005829 cytosol 1
Pathway
R-HSA-392499 Metabolism of proteins 4
Partners
DPH1DPH3EEF2
Complex memberships
Dph1-Dph2 heterodimer

Evidence

Reading pass · 11 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1993 DPH2 encodes a 534-amino acid protein required for diphthamide biosynthesis in S. cerevisiae; gene disruption shows it is not essential for viability but is required for diphthamide synthesis. Gene cloning by complementation, Northern blot, gene disruption Gene Medium 8406038
1998 A human paralog DPH2L2 (489 aa, chromosome 1p34) is more closely related to yeast Dph2 than OVCA1, identifying it as a candidate human diphthamide biosynthesis protein distinct from the previously proposed human homolog OVCA1. RACE cloning, sequence analysis, chromosomal mapping Genomics Low 9782084
2010 Archaeal Dph2 (PhDph2) is a homodimeric [4Fe-4S] radical SAM enzyme that cleaves SAM to generate a 3-amino-3-carboxypropyl (ACP) radical, which is added to the imidazole ring of EF2 histidine rather than undergoing hydrogen abstraction; only one [4Fe-4S] cluster per dimer is required for in vitro activity. Crystal structure, EPR spectroscopy, in vitro biochemical assay, mutagenesis Molecular bioSystems High 20931132
2014 Yeast Dph1 and Dph2 form a heterodimeric complex (Dph1-Dph2) equivalent to the archaeal PhDph2 homodimer; this complex is sufficient to catalyze the first step of diphthamide biosynthesis (ACP transfer to EF2 histidine) in vitro; yeast Dph3 (KTI11), a CSL-type zinc finger protein that binds iron, acts as a physiological electron donor to reduce the Fe-S clusters in Dph1-Dph2. In vitro reconstitution, EPR spectroscopy, iron-binding assay Journal of the American Chemical Society High 24422557
2019 In the eukaryotic Dph1-Dph2 heterodimer, the [4Fe-4S] cluster-binding cysteine residues in both subunits are required for diphthamide biosynthesis in vivo; the Dph1 cluster plays a catalytic role while the Dph2 cluster facilitates reduction of the Dph1 cluster by the Dph3/Cbr1/NADH reducing system, demonstrating asymmetric functional roles of the two subunits. Site-directed mutagenesis, in vivo diphthamide assay, in vitro reconstitution with mutants, EPR Journal of biological inorganic chemistry High 31463593
2019 Crystal structure of archaeal MsDph2 homodimer bound to MsEF-2 at 3.5 Å reveals that EF2 domain IV is inserted into the Dph2 active site with the target histidine positioned for modification; a conserved arginine (pre-oriented by conserved Phe and Asp) binds the SAM carboxylate and is functionally important for catalysis. X-ray crystallography, site-directed mutagenesis, in vitro activity assay Biochemistry High 31566354
2021 The [4Fe-4S] cluster in Dph1-Dph2 is oxygen-sensitive and degrades to a [3Fe-4S] cluster; Dph3 donates one iron atom to convert the inactive [3Fe-4S] cluster back to a functional [4Fe-4S] cluster, enabling aerobic diphthamide biosynthesis. EPR spectroscopy, Mössbauer spectroscopy, in vitro iron reconstitution assay, activity assay Journal of the American Chemical Society High 34154323
2021 QM/MM calculations show that reductive cleavage of SAM's S-Cγ bond by the Dph2 [4Fe-4S] cluster is spin-state dependent; the net spin on the Fe4 atom (ligated to SAM) dictates electron transfer and exchange-enhanced reactivity, providing a mechanistic basis for ACP radical generation. MD simulations, QM/MM calculations Angewandte Chemie Low 34302311
2023 The SAM-binding pocket in the Dph1•Dph2 heterodimer is located near the FeS cluster domain and is conserved in Dph1 but not Dph2; site-directed mutagenesis of predicted SAM-pocket residues in Dph1 abolishes diphthamide formation in vivo, identifying residues critical for SAM cleavage and ACP radical formation. Site-directed mutagenesis, in vivo diphthamide assay, structural modeling Biomolecules Medium 38002337
2023 Functional assessment of human DPH2 missense variants (H105P, C341Y) in yeast and mammalian cell models showed reduced activity, identifying these as diphthamide deficiency susceptibility alleles; variants near the active site are proposed to affect catalysis. Yeast complementation assay, mammalian cell functional assay, missense variant analysis Disease models & mechanisms Medium 37675463
2024 Each subunit of the Dph1•Dph2 heterodimer contains a non-canonical tandem cysteine motif (TCM) for Fe-S cluster binding; mutagenesis of the second (ill-defined) cysteine in the Dph2 TCM causes mild single-substitution defects and near-complete inactivation when combined with the first cysteine substitution; TCM mutations also destabilize the protein, indicating this fourth cysteine in Dph2 is required for structural integrity and catalytic function. Site-directed mutagenesis, in vivo diphthamide assay, cycloheximide chase (protein stability), structural modeling Biomolecules Medium 38672486

Source papers

Stage 0 corpus · 12 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2014 Dph3 is an electron donor for Dph1-Dph2 in the first step of eukaryotic diphthamide biosynthesis. Journal of the American Chemical Society 58 24422557
1993 Diphthamide synthesis in Saccharomyces cerevisiae: structure of the DPH2 gene. Gene 35 8406038
2010 Mechanistic understanding of Pyrococcus horikoshii Dph2, a [4Fe-4S] enzyme required for diphthamide biosynthesis. Molecular bioSystems 34 20931132
1998 Cloning and localization of a human diphthamide biosynthesis-like protein-2 gene, DPH2L2. Genomics 16 9782084
2019 The asymmetric function of Dph1-Dph2 heterodimer in diphthamide biosynthesis. Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry 14 31463593
2019 The Crystal Structure of Dph2 in Complex with Elongation Factor 2 Reveals the Structural Basis for the First Step of Diphthamide Biosynthesis. Biochemistry 14 31566354
2021 Dph3 Enables Aerobic Diphthamide Biosynthesis by Donating One Iron Atom to Transform a [3Fe-4S] to a [4Fe-4S] Cluster in Dph1-Dph2. Journal of the American Chemical Society 11 34154323
2021 Spin-Regulated Electron Transfer and Exchange-Enhanced Reactivity in Fe4 S4 -Mediated Redox Reaction of the Dph2 Enzyme During the Biosynthesis of Diphthamide. Angewandte Chemie (International ed. in English) 10 34302311
2023 DPH1 and DPH2 variants that confer susceptibility to diphthamide deficiency syndrome in human cells and yeast models. Disease models & mechanisms 7 37675463
2023 DPH1 Gene Mutations Identify a Candidate SAM Pocket in Radical Enzyme Dph1•Dph2 for Diphthamide Synthesis on EF2. Biomolecules 4 38002337
2025 Diphthamide formation in Arabidopsis requires DPH1-interacting DPH2 for light and oxidative stress resistance. Plant physiology 1 40200557
2024 Functional Integrity of Radical SAM Enzyme Dph1•Dph2 Requires Non-Canonical Cofactor Motifs with Tandem Cysteines. Biomolecules 0 38672486