Diphthamide biosynthesis on translation elongation factor 2

Diphthamide is a post-translationally modified histidine residue found only on archaeal and eukaryotic translation elongation factor 2 (EF2), at the tip of domain IV. It is the target of diphtheria toxin and Pseudomonas exotoxin A, which ADP-ribosylate it and so inactivate EF2. The modification is built on the folded EF2 protein in the cytosol in four enzymatic steps. First, a [4Fe-4S]-cluster enzyme cleaves S-adenosyl-L-methionine at the methionine C(gamma)-S bond, unlike canonical radical-SAM enzymes, and transfers the 3-amino-3-carboxypropyl (ACP) group to C2 of the histidine imidazole ring. In archaea this enzyme is a Dph2 homodimer; in eukaryotes it is a Dph1-Dph2 heterodimer that also needs the CSL-zinc-finger protein Dph3, which reduces its iron-sulfur clusters, and the J-domain protein Dph4. Second, the SAM-dependent methyltransferase Dph5 trimethylates the amino group and methylates the carboxylate, giving diphthine methyl ester. Third, the WD40-repeat methylesterase Dph7 hydrolyses the ester to diphthine. Fourth, the ATP-binding diphthine-ammonia ligase Dph6 amidates the carboxylate with ammonium to form diphthamide. Diphthamide is not essential in yeast, but its loss reduces translational fidelity (more -1 ribosomal frameshifting) and confers resistance to diphtheria toxin and the antifungal sordarin.

MODULE:diphthamide_biosynthesisDRAFTABSTRACTBiological Processmodules/diphthamide_biosynthesis.yaml
protein histidyl modification to diphthamideGO:0017183
YeastPathways:PWY-6482-1
diphthamide biosynthesis (S. cerevisiae, SGD YeastPathways)
YeastCyc lists RXN-11371 (DPH1, DPH2, KTI11/DPH3), three single-methyl DPH5 reactions (RXN-11370, RXN-11374, RXN-11373) ending at diphthine, and DIPHTINE--AMMONIA-LIGASE-RXN catalysed by RRT2/YBR246W. Four corrections were made. (1) The amidation enzyme is DPH6/YLR143W (PMID:23169644), not RRT2. (2) RRT2 is DPH7, the diphthine methylesterase acting between DPH5 and DPH6 (PMID:24739148). (3) The DPH5 product is diphthine methyl ester, not diphthine (PMID:24739148). (4) DPH4/JJJ3, which the first step requires (PMID:15485916), was added.
GO:0017183
protein histidyl modification to diphthamide
The module is grounded in the GO biological-process term for diphthamide formation on EF2.
PMID:15485916
Identification of the proteins required for biosynthesis of diphthamide, the target of bacterial ADP-ribosylating toxins on translation elongation factor 2.
Identifies yeast Dph1, Dph3 and Dph4 alongside Dph2 and Dph5, names the human homologs, and shows that DPH4 encodes a CSL-zinc-finger DnaJ-like protein and that Dph1-Dph3 act as a complex in vivo.
PMID:20559380
Diphthamide biosynthesis requires an organic radical generated by an iron-sulphur enzyme.
Pyrococcus horikoshii Dph2 is a [4Fe-4S]-cluster homodimer that cleaves SAM to a 3-amino-3-carboxypropyl radical for the first step, defining the archaeal single-protein architecture.
PMID:24422557
Dph3 is an electron donor for Dph1-Dph2 in the first step of eukaryotic diphthamide biosynthesis.
Reconstitution shows that yeast Dph1-Dph2 is the eukaryotic equivalent of the PhDph2 homodimer and that reduced Dph3 (KTI11) donates electrons to the Dph1-Dph2 Fe-S clusters.
PMID:22367199
Structure and mechanistic insights into novel iron-mediated moonlighting functions of human J-protein cochaperone, Dph4.
Human Dph4 (DNAJC24) and yeast Jjj3 bind iron through the CSL domain; iron-bound Dph4 has redox and electron-carrier properties. How this contributes to step 1 remains unresolved.
PMID:1508200
DPH5, a methyltransferase gene required for diphthamide biosynthesis in Saccharomyces cerevisiae.
DPH5 encodes the AdoMet-dependent EF2 methyltransferase. It is related to uroporphyrinogen III methyltransferases, and recombinant Dph5 methylates EF2 from a dph5 mutant.
PMID:22188241
YBR246W is required for the third step of diphthamide biosynthesis.
Deleting YBR246W causes a diphthine-like intermediate to accumulate. YeastCyc cites this paper for its RRT2 amidation assignment, but PMID:24739148 later superseded that interpretation.
PMID:24739148
Dph7 catalyzes a previously unknown demethylation step in diphthamide biosynthesis.
Dph5 produces methylated diphthine (diphthine methyl ester). Dph7 (YBR246W/WDR85) is the methylesterase that converts it to diphthine, which Dph6 then amidates.
PMID:23169644
Chemogenomic approach identified yeast YLR143W as diphthamide synthetase.
YLR143W/DPH6 is the ATP- and ammonium-dependent diphthamide synthetase. Human ATPBD4/DPH6 fully rescues the yeast deletion.
PMID:23468660
The amidation step of diphthamide biosynthesis in yeast requires DPH6, a gene identified through mining the DPH1-DPH5 interaction network.
dph6 and dph7 mutants accumulate diphthine-modified eEF2. Dph6 carries an essential adenine-nucleotide hydrolase domain and binds eEF2. The dph mutants share increased -1 frameshifting.
7Nodes
4Parts
1Variant Sets
2Variants
7Annotons
3Connections

Derived QC

Recommended-field compliance

40.0% recommended fields populated
  • knowledge_gaps[0] · boundary (0/1)
  • knowledge_gaps[0] · status (0/1)
  • knowledge_gaps[0] · provenance (0/1)

Module deep research

✗ none found

No MODULE:diphthamide_biosynthesis deep-research report alongside the module YAML.

Leaf nodes lacking representative members

✓ representative grounding skipped for abstract module.

Template conformance

✓ every declared conforms_to bundle matches its template motif.

Reaction chaining (advisory)

✓ every PRECEDES step chains, or its break is acknowledged via chaining_status.

  • acp_transfer_step → dph5_step [NOT_CHECKED]
    Intermediates are protein-bound EF2 modifications, not CoA carriers.
  • dph5_step → dph7_step [NOT_CHECKED]
    Intermediates are protein-bound EF2 modifications, not CoA carriers.
  • dph7_step → dph6_step [NOT_CHECKED]
    Intermediates are protein-bound EF2 modifications, not CoA carriers.

Function consistency and core coverage

4 gene-function check(s) reflected in core functions · 0 conflict(s) · 7 evidence or coverage gap(s) (16 comparison(s))

Retained noncore functions are reported separately; they do not require promotion to core functions. Support from representative genes does not establish conservation across the whole family. Missing reviews or identifiers leave a claim unverified.

AnnotonParticipantFunctionAssessment
dph1_dph2_acp_transferase PROTEIN_COMPLEX
Gene 		2-(3-amino-3-carboxypropyl)histidine synthase activity
GO:0090560 		Participant Unchecked
No direct UniProt participant to compare. Abstract selectors and complex-wide activities cannot be inferred from donor or subunit gene reviews.
dph3_electron_donor KTI11/DPH3 (Saccharomyces cerevisiae)
UniProtKB:Q3E840
Family Representative 		electron transfer activity
GO:0009055 		Core Supported
The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
dph3_electron_donor DPH3 (human)
UniProtKB:Q96FX2
Family Representative 		electron transfer activity
GO:0009055 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
archaeal_dph2_acp_transferase dph2 (Pyrococcus horikoshii OT3)
UniProtKB:O58832
Family Representative 		2-(3-amino-3-carboxypropyl)histidine synthase activity
GO:0090560 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
dph5_methyltransferase DPH5 (Saccharomyces cerevisiae)
UniProtKB:P32469
Family Representative 		diphthine methyl ester synthase activity
GO:0141133 		Core Supported
The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
dph5_methyltransferase DPH5 (human)
UniProtKB:Q9H2P9
Family Representative 		diphthine methyl ester synthase activity
GO:0141133 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
dph7_methylesterase RRT2/DPH7 (Saccharomyces cerevisiae)
UniProtKB:P38332
Family Representative 		diphthine methylesterase activity
GO:0061685 		Core Supported
The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
dph7_methylesterase DPH7/WDR85 (human)
UniProtKB:Q9BTV6
Family Representative 		diphthine methylesterase activity
GO:0061685 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
dph6_ligase DPH6/YLR143W (Saccharomyces cerevisiae)
UniProtKB:Q12429
Family Representative 		diphthine-ammonia ligase activity
GO:0017178 		Core Supported
The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
dph6_ligase DPH6/ATPBD4 (human)
UniProtKB:Q7L8W6
Family Representative 		diphthine-ammonia ligase activity
GO:0017178 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
dph3_electron_donor Dph3 CSL-type zinc-finger family
PANTHER:PTHR21454:SF31
Family 		electron transfer activity
GO:0009055 		Family Supported
Family review supports GO:0009055 for PANTHER:PTHR21454:SF31. Dph3 orthologs only. This is the function of the module's dph3_electron_donor annoton, which selects the base family PTHR21454. Because the family also contains Dph4 (SF46, SF49), plant EXPP1 and DnaJ-CSL proteins, the base-family selector over-includes. Dph4 was tested and judged unlikely to be the Dph1-Dph2 electron donor. One study did report electron carrier activity for human iron-bound Dph4 (PMID:22367199), so the Dph4 exclusion reflects the direct reconstitution result rather than a settled negative.
dph3_electron_donor Dph3 CSL-type zinc-finger family
PANTHER:PTHR21454:SF24
Family 		electron transfer activity
GO:0009055 		Family Supported
Family review supports GO:0009055 for PANTHER:PTHR21454:SF24. Dph3 orthologs only. This is the function of the module's dph3_electron_donor annoton, which selects the base family PTHR21454. Because the family also contains Dph4 (SF46, SF49), plant EXPP1 and DnaJ-CSL proteins, the base-family selector over-includes. Dph4 was tested and judged unlikely to be the Dph1-Dph2 electron donor. One study did report electron carrier activity for human iron-bound Dph4 (PMID:22367199), so the Dph4 exclusion reflects the direct reconstitution result rather than a settled negative.
archaeal_dph2_acp_transferase archaeal Dph2 family
Family 		2-(3-amino-3-carboxypropyl)histidine synthase activity
GO:0090560 		Family Id Missing
The family has no identifier linking it to a curated family review.
dph5_methyltransferase Dph5 diphthine synthase family
PANTHER:PTHR10882
Family 		diphthine methyl ester synthase activity
GO:0141133 		Family Function Unreviewed
Family review leaves GO:0141133 UNRESOLVED. This is the term the diphthamide module asserts for the Dph5 role, and it is correct for the eukaryotic members: yeast Dph5 makes the methyl ester (IDA/IMP on DPH5, accepted in the gene review). It is not correct for the archaeal members, whose Dph5 does not esterify the carboxylate, and GO itself restricts the term to Eukaryota. The boundary is taxonomic (eukaryotic versus archaeal Dph5), and PANTHER puts both in SF0, so neither family-wide nor subfamily scope is right. No PAINT node exists in this family that could carry the eukaryote-only assertion. Apply only to eukaryotic members.
dph7_methylesterase Dph7 WD40-repeat diphthine methylesterase family
PANTHER:PTHR46042
Family 		diphthine methylesterase activity
GO:0061685 		Family Supported
Family review supports GO:0061685 FAMILY_WIDE. This is the term the diphthamide module asserts for the Dph7 role. It is shown biochemically for yeast Dph7 (IDA/IMP, accepted in the RRT2 review), and the family is a single eukaryote-only orthologous group that tracks the eukaryote-specific methyl-ester step. No PAINT node currently asserts it; the MF has to be added to members individually. It could be placed at PTN000522690, where the process term already sits.
dph6_ligase Dph6 (ATPBD4, DUF71) diphthine-ammonia ligase family
PANTHER:PTHR12196:SF2
Family 		diphthine-ammonia ligase activity
GO:0017178 		Family Supported
Family review supports GO:0017178 for PANTHER:PTHR12196:SF2. This is the term the diphthamide module asserts for the Dph6 role. It is safe for the eukaryotic and archaeal Dph6 group under PTN000257633. SF2 is listed only because it is PANTHER's single subfamily. Its bacterial DUF71 members, which sit outside PTN000257633, must not inherit the term, because bacteria have no diphthamide pathway. Apply by the PAINT node, not by the subfamily label.

Gene-review completeness (9/15 grounded genes reviewed)

9 complete review(s) · 7 with deep research · 6 missing review · 2 reviewed but lacking deep research

Gene Review Complete Deep research
DNAJC24 Q6P3W2 ✓ ✓ ✗
DPH1 P40487 ✓ ✓ ✓
DPH1 Q9BZG8 ✓ ✓ ✗
DPH2 P32461 ✓ ✓ ✓
DPH5 P32469 ✓ ✓ ✓
DPH6 Q12429 ✓ ✓ ✓
JJJ3 P47138 ✓ ✓ ✓
KTI11 Q3E840 ✓ ✓ ✓
dph2 (Pyrococcus horikoshii OT3) O58832 ✗ — —
DPH6/ATPBD4 (human) Q7L8W6 ✗ — —
DPH3 (human) Q96FX2 ✗ — —
DPH2 (human) Q9BQC3 ✗ — —
DPH7/WDR85 (human) Q9BTV6 ✗ — —
DPH5 (human) Q9H2P9 ✗ — —
RRT2 P38332 ✓ ✓ ✓

Details

Context
EukaryotaNCBITaxon:2759 ArchaeaNCBITaxon:2157
cytosolGO:0005829
Diphthamide biosynthesisBiological Processdiphthamide_biosynthesis

Four ordered enzymatic modifications of a single histidine residue of EF2. Each step uses the product of the previous step, which stays attached to EF2 throughout, as its substrate.

protein histidyl modification to diphthamideGO:0017183
Context
EukaryotaNCBITaxon:2759 ArchaeaNCBITaxon:2157
cytosolGO:0005829

Boundary: the module begins with the first ACP transfer onto EF2 and ends with diphthamide. Synthesis of SAM, ATP and the Fe-S clusters is outside it, and so are the toxin ADP-ribosylation of diphthamide and any role of diphthamide in translation. The EF2 substrate (yeast EFT1/EFT2) is the acceptor of every step and performs none of them, so it is not a module participant. KTI11/DPH3 also has a separate role with the Elongator tRNA modification complex, which is out of scope. The Dph7 methylesterase step is established for yeast and human. Archaeal EF2 also carries diphthamide, but the cached literature does not establish whether archaea use a Dph7-like esterase or a different chemistry, so this step is modelled from eukaryotic evidence. The S. pombe orthologs (dph1-dph5 are named in UniProt) are not listed because this group has no PomBase GO-CAM.

Connections

The ACP-modified histidine on EF2 is the Dph5 substrate.
dph5_step -> dph7_step Precedes
Diphthine methyl ester made by Dph5 is hydrolysed by Dph7.
dph7_step -> dph6_step Precedes
Diphthine produced by Dph7 is amidated by Dph6.
Part 1: first step (3-amino-3-carboxypropyl transfer from SAM to EF2 histidine)
EF2 histidine to 2-(3-amino-3-carboxypropyl)histidineReactionacp_transfer_step

A non-canonical radical-SAM reaction. A [4Fe-4S] cluster cleaves SAM at the methionine C(gamma)-S bond, releasing S-methyl-5'-thioadenosine, and the 3-amino-3-carboxypropyl radical is added to C2 of the target histidine's imidazole ring.

Variant set: ACP-transferase enzyme architecture by taxon / enzyme architecture (Exactly One)
Eukaryotic Dph1-Dph2 heterodimer with Dph3 and Dph4Protein Complexeukaryotic_dph1_dph2_variant

Two paralogous catalytic subunits, each binding a [4Fe-4S] cluster, form the ACP transferase. The reduced CSL zinc-finger protein Dph3 supplies electrons to those clusters, and the CSL-domain J-protein Dph4 is also required for this step.

Annotons

Dph1-Dph2 2-(3-amino-3-carboxypropyl)histidine synthase
dph1_dph2_acp_transferase
Participant: Protein Complex: 2-(3-amino-3-carboxypropyl)histidine synthase complex
Protein Complex:
2-(3-amino-3-carboxypropyl)histidine synthase complexGO:0120513
Active units:
Dph1 catalytic subunit
Participant: Family: Dph1/Dph2 diphthamide-synthesis family (Dph1 subfamily)
Family:
Dph1/Dph2 diphthamide-synthesis family (Dph1 subfamily)PANTHER:PTHR10762
Representative Members: DPH1 (Saccharomyces cerevisiae)UniProtKB:P40487 DPH1 (human)UniProtKB:Q9BZG8
Role: One of the two paralogous [4Fe-4S]-cluster subunits of the heterodimeric ACP transferase.
Dph2 catalytic subunit
Participant: Family: Dph1/Dph2 diphthamide-synthesis family (Dph2 subfamily)
Family:
Dph1/Dph2 diphthamide-synthesis family (Dph2 subfamily)PANTHER:PTHR10762
Representative Members: DPH2 (Saccharomyces cerevisiae)UniProtKB:P32461 DPH2 (human)UniProtKB:Q9BQC3
Role: The second paralogous [4Fe-4S]-cluster subunit of the heterodimeric ACP transferase.

Function

2-(3-amino-3-carboxypropyl)histidine synthase activityGO:0090560
Substrates: L-histidyl-[translation elongation factor 2] S-adenosyl-L-methionine
Products: 2-[(3S)-3-amino-3-carboxypropyl]-L-histidyl-[translation elongation factor 2] S-methyl-5'-thioadenosine

Locations

cytosolGO:0005829

Catalytic heterodimer of the first step. Both subunits are needed, and neither substitutes for the other.

Dph3 (KTI11) electron donor to the Dph1-Dph2 Fe-S clusters
dph3_electron_donor
Participant: Family: Dph3 CSL-type zinc-finger family
Family:
Dph3 CSL-type zinc-finger family
Representative Members: KTI11/DPH3 (Saccharomyces cerevisiae)UniProtKB:Q3E840 DPH3 (human)UniProtKB:Q96FX2

Function

electron transfer activityGO:0009055 Reduced, iron-bound Dph3 transfers electrons to the [4Fe-4S] clusters of Dph1-Dph2.

Processes

protein histidyl modification to diphthamideGO:0017183

Reductant for the first step. In yeast KTI11 also acts as a subunit-like partner of the Elongator complex, but that role lies outside this module.

Dph4 (JJJ3/DNAJC24) CSL-domain J-protein required for step 1
dph4_j_protein
Participant: Family: Dph4 CSL-zinc-finger type III J-protein
Family:
Dph4 CSL-zinc-finger type III J-protein PANTHER does not place yeast JJJ3 and human DNAJC24 in the same family, so no family id is asserted.
Representative Members: JJJ3/DPH4 (Saccharomyces cerevisiae)UniProtKB:P47138 DNAJC24/DPH4 (human)UniProtKB:Q6P3W2

Processes

protein histidyl modification to diphthamideGO:0017183

Required for the first step (PMID:15485916). Its CSL domain binds iron, and the iron-bound protein has redox properties (PMID:22367199). Its exact molecular contribution to ACP transfer is not established, so no molecular function is asserted.

Archaeal Dph2 homodimerReactionarchaeal_dph2_homodimer_variant

Annotons

Archaeal Dph2 homodimeric ACP transferase
archaeal_dph2_acp_transferase
Participant: Family: archaeal Dph2 family
Family:
archaeal Dph2 family Archaeal homolog of the eukaryotic Dph1/Dph2 subunits. PANTHER places the archaeal Dph2 proteins (O58832, Q57907) in PTHR10762:SF1 together with eukaryotic Dph1 (see the PTHR10762 family review), so no subfamily separates the archaeal homodimer; no family id is asserted for this variant.
Representative Members: dph2 (Pyrococcus horikoshii OT3)UniProtKB:O58832

Function

2-(3-amino-3-carboxypropyl)histidine synthase activityGO:0090560

A single-gene homodimer that does the work of the eukaryotic Dph1-Dph2 heterodimer.

Part 2: trimethylation and methyl esterification (Dph5)
ACP-histidine to diphthine methyl esterReactiondph5_step

Annotons

Dph5 diphthine methyl ester synthase
dph5_methyltransferase
Participant: Family: Dph5 diphthine synthase family
Family:
Dph5 diphthine synthase familyPANTHER:PTHR10882
Representative Members: DPH5 (Saccharomyces cerevisiae)UniProtKB:P32469 DPH5 (human)UniProtKB:Q9H2P9

Function

diphthine methyl ester synthase activityGO:0141133
Substrates: 2-[(3S)-3-amino-3-carboxypropyl]-L-histidyl-[translation elongation factor 2] S-adenosyl-L-methionine (4 equivalents)
Products: diphthine methyl ester-[translation elongation factor 2] S-adenosyl-L-homocysteine

Locations

cytosolGO:0005829

One SAM-dependent enzyme of the uroporphyrinogen-methyltransferase fold carries out all four methyl transfers.

Part 3: methyl ester hydrolysis (Dph7)
diphthine methyl ester to diphthineReactiondph7_step

Annotons

Dph7 diphthine methylesterase
dph7_methylesterase
Participant: Family: Dph7 WD40-repeat diphthine methylesterase family
Family:
Dph7 WD40-repeat diphthine methylesterase familyPANTHER:PTHR46042
Representative Members: RRT2/DPH7 (Saccharomyces cerevisiae)UniProtKB:P38332 DPH7/WDR85 (human)UniProtKB:Q9BTV6

Function

diphthine methylesterase activityGO:0061685
Substrates: diphthine methyl ester-[translation elongation factor 2] water
Products: diphthine-[translation elongation factor 2] methanol

Locations

cytosolGO:0005829

Removes the methyl ester that Dph5 installs, producing the free carboxylate that Dph6 amidates. YeastCyc wrongly assigns RRT2 to the amidation reaction.

Part 4: ATP-dependent amidation (Dph6)
diphthine to diphthamideReactiondph6_step

Annotons

Dph6 diphthine-ammonia ligase
dph6_ligase
Participant: Family: Dph6 (ATPBD4, DUF71) diphthine-ammonia ligase family
Family:
Dph6 (ATPBD4, DUF71) diphthine-ammonia ligase family
Representative Members: DPH6/YLR143W (Saccharomyces cerevisiae)UniProtKB:Q12429 DPH6/ATPBD4 (human)UniProtKB:Q7L8W6

Function

diphthine-ammonia ligase activityGO:0017178
Substrates: diphthine-[translation elongation factor 2] ammonium ATP
Products: diphthamide-[translation elongation factor 2] AMP diphosphate

Locations

cytosolGO:0005829

Final step. The ATP-binding domain is essential, and Dph6 binds eEF2 (PMID:23468660).