| Pathway step | Enzyme(s) / gene(s) | Core reaction chemistry | Key cofactors / partners | Evidence type | Selected supporting citation(s) |
|---|---|---|---|---|---|
| 1. ACP transfer to eEF2 histidine (first committed step) | DPH1-DPH4; catalytic core is DPH1•DPH2 | Transfer of the 3-amino-3-carboxypropyl (ACP) group from SAM/AdoMet to the imidazole C2 of the target histidine on eEF2 (His715 in human; His699 in yeast), initiating diphthamide biosynthesis (pqac-00000007, pqac-00000003, pqac-00000011) | DPH1•DPH2 heterodimer; DPH3 electron donation; DPH4 required in eukaryotes (pqac-00000003, pqac-00000008, pqac-00000011) | Biochemical, review, genetics, cell biology | Liu et al. 2004, Mol Cell Biol, 2004-11, https://doi.org/10.1128/mcb.24.21.9487-9497.2004; Su et al. 2013, Crit Rev Biochem Mol Biol, 2013-11, https://doi.org/10.3109/10409238.2013.831023; Ütkür et al. 2023, Dis Model Mech, 2023-09, https://doi.org/10.1242/dmm.050207 (pqac-00000007, pqac-00000003, pqac-00000011) |
| 1a. Mechanistic detail of the DPH2-containing step | DPH2 (archaeal homodimeric Dph2; eukaryotic DPH1•DPH2 analog) | Non-canonical radical-SAM-like chemistry: cleavage of the Cγ,Met-S bond of SAM to generate MTA plus an ACP radical, rather than the classical 5'-deoxyadenosyl radical; the ACP radical then attacks eEF2 histidine (pqac-00000001, pqac-00000003, pqac-00000006) | One Fe-S cluster per subunit; atypical cysteine motifs instead of canonical CX3CX2C radical-SAM motif (pqac-00000002, pqac-00000003, pqac-00000006) | Biochemical, review | Su et al. 2013, Crit Rev Biochem Mol Biol, 2013-11, https://doi.org/10.3109/10409238.2013.831023 (pqac-00000003); X. Su thesis summary 2013 (pqac-00000001, pqac-00000006) |
| 1b. 2024 motif-level advance for DPH2 function | DPH1•DPH2 | Conserved tandem cysteine motifs adjacent to known Fe-S/SAM-binding cysteines are required for full activity and structural integrity; combined DPH2 cysteine substitutions nearly abolish activity and accelerate subunit decay (pqac-00000013, pqac-00000014) | Non-canonical Fe-S/cofactor motifs in DPH1 and DPH2; dimer integrity depends on these residues (pqac-00000002, pqac-00000013, pqac-00000014) | Biochemical, genetics | Ütkür et al. 2024, Biomolecules, 2024-04, https://doi.org/10.3390/biom14040470 (pqac-00000002, pqac-00000013, pqac-00000014) |
| 2. Trimethylation of ACP intermediate to diphthine | DPH5 | Methylation of the ACP-modified eEF2 intermediate to form diphthine (pqac-00000007, pqac-00000008, pqac-00000011) | SAM/AdoMet methyl donor (pqac-00000007, pqac-00000008) | Review, genetics, cell biology | Liu et al. 2004, Mol Cell Biol, 2004-11, https://doi.org/10.1128/mcb.24.21.9487-9497.2004; Ütkür et al. 2023, Dis Model Mech, 2023-09, https://doi.org/10.1242/dmm.050207; Zhao et al. 2024, ACS Cent Sci, 2024-09, https://doi.org/10.1021/acscentsci.4c00967 (pqac-00000007, pqac-00000011, pqac-00000008) |
| 3. Demethylation / preparation for amidation | DPH7 | Converts diphthine to the intermediate used for final amidation; described as a demethylation/preparatory step in recent pathway summaries (pqac-00000008, pqac-00000011) | Functions in downstream maturation of modified eEF2 (pqac-00000008, pqac-00000011) | Genetics, cell biology | Ütkür et al. 2023, Dis Model Mech, 2023-09, https://doi.org/10.1242/dmm.050207; Zhao et al. 2024, ACS Cent Sci, 2024-09, https://doi.org/10.1021/acscentsci.4c00967 (pqac-00000011, pqac-00000008) |
| 4. Final amidation to diphthamide | DPH6 | ATP-dependent amidation of the carboxyl group to generate mature diphthamide on eEF2 (pqac-00000007, pqac-00000008, pqac-00000011) | ATP-dependent amidation machinery (pqac-00000007, pqac-00000008) | Biochemical, review, genetics | Liu et al. 2004, Mol Cell Biol, 2004-11, https://doi.org/10.1128/mcb.24.21.9487-9497.2004; Ütkür et al. 2023, Dis Model Mech, 2023-09, https://doi.org/10.1242/dmm.050207; Zhao et al. 2024, ACS Cent Sci, 2024-09, https://doi.org/10.1021/acscentsci.4c00967 (pqac-00000007, pqac-00000011, pqac-00000008) |
| Biological consequence of complete pathway | DPH1-DPH7 pathway acting on eEF2 | Mature diphthamide on eEF2 supports translational fidelity and suppresses spurious -1 frameshifting; deficiency perturbs translation of proteins such as RRM1 and elevates DNA replication stress (pqac-00000008, pqac-00000009) | eEF2 as modified substrate; pathway defects linked to developmental disease and toxin resistance (pqac-00000009, pqac-00000013) | Cell biology, genetics | Zhao et al. 2024, ACS Cent Sci, 2024-09, https://doi.org/10.1021/acscentsci.4c00967; Ütkür et al. 2023, Dis Model Mech, 2023-09, https://doi.org/10.1242/dmm.050207; Ütkür et al. 2024, Biomolecules, 2024-04, https://doi.org/10.3390/biom14040470 (pqac-00000008, pqac-00000009, pqac-00000013) |


*Table: This table summarizes the diphthamide biosynthesis pathway with emphasis on the DPH2-containing first step, the enzymes involved across DPH1-7, the reaction chemistry and cofactors, and key recent and foundational citations. It is useful as a compact reference for functional annotation of DPH2 and its pathway context.*