Diadenosine tetraphosphate (Ap4A) turnover

A reusable metabolic module for the production and removal of diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A), a dinucleoside polyphosphate that accumulates under stress and has been proposed to act as an intracellular signal. Ap4A is made as a side reaction of aminoacyl-tRNA synthetases, either by transfer of the aminoacyl-adenylate AMP to ATP (as in lysyl-tRNA synthetase) or by direct condensation of two ATP molecules (as in glycyl-tRNA synthetase). It is removed by one of three unrelated enzyme chemistries: asymmetrical Nudix hydrolases that yield ATP + AMP (animals and other eukaryotes), symmetrical ApaH-type hydrolases that yield two ADP (proteobacteria), and Ap4A phosphorylases that yield ADP + ATP by phosphorolysis (fungi). Downstream Ap4A effectors (for example HINT1 or transcription factors) and the related Ap3A/FHIT system are outside the module boundary.

MODULE:diadenosine_tetraphosphate_turnoverDRAFTMetabolic Pathwaymodules/diadenosine_tetraphosphate_turnover.yaml
diadenosine polyphosphate metabolic processGO:0015959
GO:0015966
diadenosine tetraphosphate biosynthetic process
GO:0015966 describes the synthesis half of the module.
GO:0015967
diadenosine tetraphosphate catabolic process
GO:0015967 describes the removal half of the module.
PMID:19710017
Crystal structures and biochemical analyses suggest a unique mechanism and role for human glycyl-tRNA synthetase in Ap4A homeostasis.
Establishes that aminoacyl-tRNA synthetases divert the aminoacylation reaction to make Ap4A, either through the aminoacyl-adenylate or, for GlyRS, by direct condensation of two ATPs.
Crystal structures and biochemical analyses show that human glycyl-tRNA synthetase (GlyRS) produces Ap4A by direct condensation of two ATPs, independent of glycine concentration.
PMID:27144453
NUDT2 Disruption Elevates Diadenosine Tetraphosphate (Ap4A) and Down-Regulates Immune Response and Cancer Promotion Genes.
Loss of the asymmetrical hydrolase NUDT2 raises cellular Ap4A, showing that the hydrolase sets steady-state Ap4A levels in human cells.
PMID:2174863
Catabolism of bis(5'-nucleosidyl) tetraphosphates in Saccharomyces cerevisiae.
Yeast apa1 apa2 double mutants accumulate dinucleoside tetraphosphates, showing the phosphorylases act catabolically in vivo.
The concentrations of all bis(5'-nucleosidyl) tetraphosphates are increased in an apa1 apa2 double mutant, as compared with the parental wild-type strain.
file:modules/diadenosine_tetraphosphate_turnover-deep-research-openscientist.md
OpenScientist research for Ap4A turnover
Supports the synthesis-plus-removal boundary, the exclusion of HINT1, Fhit and extracellular ApnA signalling, and identified the HD-domain YqeK family as an additional removal implementation. It also highlights that Np4A can be incorporated as 5' RNA caps and that ApaH, YqeK and NUDT2 also act as decapping enzymes; that RNA-cap biology is recorded in the notes but kept outside the module boundary.
file:interpro/panther/PTHR21340/PTHR21340-review.yaml
PTHR21340 family review
Family-level assessment of the asymmetrical Ap4A hydrolase family, including the PAINT node PTN000482012 that carries GO:0004081.
file:worm/ndx-4/ndx-4-ai-review.yaml
C. elegans ndx-4 gene review
Q9U2M7 is a biochemically and structurally characterised asymmetrical Ap4A hydrolase.
file:human/NUDT2/NUDT2-ai-review.yaml
NUDT2 (human) gene review
P50583 is the human asymmetrical Ap4A hydrolase.
file:mouse/Nudt2/Nudt2-ai-review.yaml
Nudt2 (mouse) gene review
P56380 is the mouse asymmetrical Ap4A hydrolase.
file:DROME/Datp/Datp-ai-review.yaml
Drosophila Datp gene review
Q4V6M1 is the Drosophila asymmetrical Ap4A hydrolase (formerly Apf).
9Nodes
2Parts
2Variant Sets
6Variants
6Annotons
1Connections

Derived QC

Recommended-field compliance

100.0% recommended fields populated

All recommended fields populated.

Module deep research

✓ present

  • diadenosine_tetraphosphate_turnover-deep-research-openscientist.md (openscientist)

Leaf nodes lacking representative members

✓ every leaf node grounds to a representative protein.

Template conformance

✓ every declared conforms_to bundle matches its template motif.

Function consistency and core coverage

4 gene-function check(s) reflected in core functions · 0 conflict(s) · 7 evidence or coverage gap(s) (13 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
lysrs_ap4a_synthesis KARS1 (human)
UniProtKB:Q15046
Family Representative 		ATP:ATP adenylyltransferase activity
GO:0141192 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
glyrs_ap4a_synthesis GARS1 (human)
UniProtKB:P41250
Family Representative 		ATP:ATP adenylyltransferase activity
GO:0141192 		Non Core Supported
The review explicitly retains this function as non-core; promotion into core_functions is not implied.
nudt2_asymmetrical_hydrolysis NUDT2 (human)
UniProtKB:P50583
Family Representative 		bis(5'-nucleosyl)-tetraphosphatase (asymmetrical) activity
GO:0004081 		Core Supported
The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
nudt2_asymmetrical_hydrolysis NUDT2 (pig)
UniProtKB:P50584
Family Representative 		bis(5'-nucleosyl)-tetraphosphatase (asymmetrical) activity
GO:0004081 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
nudt2_asymmetrical_hydrolysis Nudt2 (mouse)
UniProtKB:P56380
Family Representative 		bis(5'-nucleosyl)-tetraphosphatase (asymmetrical) activity
GO:0004081 		Core Supported
The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
nudt2_asymmetrical_hydrolysis ndx-4 (Caenorhabditis elegans)
UniProtKB:Q9U2M7
Family Representative 		bis(5'-nucleosyl)-tetraphosphatase (asymmetrical) activity
GO:0004081 		Core Supported
The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
nudt2_asymmetrical_hydrolysis Datp / Apf (Drosophila melanogaster)
UniProtKB:Q4V6M1
Family Representative 		bis(5'-nucleosyl)-tetraphosphatase (asymmetrical) activity
GO:0004081 		Core Supported
The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
apah_symmetrical_hydrolysis apaH (Escherichia coli K-12)
UniProtKB:P05637
Gene 		bis(5'-nucleosyl)-tetraphosphatase (symmetrical) activity
GO:0008803 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
yqek_symmetrical_hydrolysis yqeK (Staphylococcus aureus NCTC 8325)
UniProtKB:Q2G297
Gene 		bis(5'-nucleosyl)-tetraphosphatase (symmetrical) activity
GO:0008803 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
apa_phosphorolysis APA1 (Saccharomyces cerevisiae)
UniProtKB:P16550
Gene 		ATP:ADP adenylyltransferase activity
GO:0003877 		Review Missing
No ai-review exists for this protein; its function and core-function coverage cannot yet be checked.
lysrs_ap4a_synthesis lysyl-tRNA synthetases (class II)
PANTHER:PTHR42918:SF9
Family 		ATP:ATP adenylyltransferase activity
GO:0141192 		Family Function Unreviewed
The family review has no applicable adjudication supporting or excluding GO:0141192.
glyrs_ap4a_synthesis glycyl-tRNA synthetases (class II, eukaryotic type)
PANTHER:PTHR10745:SF0
Family 		ATP:ATP adenylyltransferase activity
GO:0141192 		Family Review Missing
No supported structured family review is available for PANTHER:PTHR10745:SF0.
nudt2_asymmetrical_hydrolysis asymmetrical Ap4A hydrolases (Nudix)
PANTHER:PTHR21340:SF0
Family 		bis(5'-nucleosyl)-tetraphosphatase (asymmetrical) activity
GO:0004081 		Family Supported
Family review supports GO:0004081 for PANTHER:PTHR21340:SF0. Safe for SF0, the subfamily of every characterized asymmetrical Ap4A hydrolase (NUDT2 orthologs, ndx-4, Datp/Apf, Plasmodium) and of Thermus Ndx1, which also releases ATP and AMP from Ap4A. The term is ancestral to the family (see the PTN000482012 node assessment). SF7 is left out of the applicable set because none of its members has been assayed. That is a lack of evidence, not evidence of loss. Inside SF0, the large DNA virus enzymes are the weakest cases. ASFV g5R does hydrolyse diadenosine and diguanosine polyphosphates, but its preferred substrates are diphosphoinositol polyphosphates and RNA-linked mRNA caps, so whether its Ap4A cleavage is asymmetrical, and whether it matters, is not established. Members whose catalytic Nudix glutamates are changed should be checked against site nudix_catalytic_glutamates.

Gene-review completeness (5/10 grounded genes reviewed)

4 complete review(s) · 4 with deep research · 5 missing review · 1 reviewed but lacking deep research

Gene Review Complete Deep research
Datp Q4V6M1 ✓ ✓ ✓
GARS1 P41250 ✓ 50/51 ✗
ndx-4 Q9U2M7 ✓ ✓ ✓
NUDT2 P50583 ✓ ✓ ✓
Nudt2 P56380 ✓ ✓ ✓
apaH (Escherichia coli K-12) P05637 ✗ — —
APA1 (Saccharomyces cerevisiae) P16550 ✗ — —
NUDT2 (pig) P50584 ✗ — —
KARS1 (human) Q15046 ✗ — —
yqeK (Staphylococcus aureus NCTC 8325) Q2G297 ✗ — —

Details

Diadenosine tetraphosphate (Ap4A) turnoverMetabolic Pathwaydiadenosine_tetraphosphate_turnover
diadenosine polyphosphate metabolic processGO:0015959

The module boundary is synthesis plus removal of Ap4A. Effectors that bind Ap4A (HINT1, MITF/USF2 signalling, Ap4A-binding proteins in bacteria) and the Ap3A-specific FHIT branch are downstream or parallel biology and are left out. Np4A/Ap4A can also be incorporated as non-canonical 5' RNA caps, and the ApaH, YqeK and NUDT2 removal enzymes also decap such RNAs; that epitranscriptomic role is a candidate for a separate, connected module. Whether Ap4A is a regulated signal or a constitutively cleared by-product appears to differ between organisms and is not resolved by this module. The asymmetrical Nudix variant is grounded on four completed gene reviews and the PTHR21340 family review; the ApaH, APA1 and synthetase exemplars (including YqeK) have no gene review in this repository yet. The KARS1 and GARS1 subfamily assignments were confirmed in the PANTHER 19.0 human classification file, which lists UniProtKB=Q15046 under PTHR42918:SF9 and UniProtKB=P41250 under PTHR10745:SF0. PANTHER families are not asserted for ApaH or APA1/APA2 because their PANTHER placements (ApaH in PTHR40942:SF4, APA1 and APA2 in different families) were not checked against the family trees. PTN000482012 is the PTHR21340 root node in the local PAINT data and also carries AMP and ATP biosynthetic process IBDs that the reviews judge to be over-annotations; only its GO:0004081 assertion is used here.

Connections

ap4a_synthesis_step -> ap4a_removal_step Provides Input For
Aminoacyl-tRNA synthetases supply the Ap4A that the removal enzymes degrade.
Part 1: Ap4A synthesis
Ap4A synthesis by aminoacyl-tRNA synthetasesReactionap4a_synthesis_step
diadenosine tetraphosphate biosynthetic processGO:0015966
Variant set: Ap4A synthesis mechanism by enzyme mechanism (One Or More)

Both routes have the same net stoichiometry (2 ATP = Ap4A + diphosphate), which is what GO:0141192 describes. Several class I and class II synthetases can make Ap4A in vitro; lysyl- and glycyl-tRNA synthetases are used as exemplars because their Ap4A production is best characterised in cells.

Aminoacyl-adenylate route (LysRS type)Reactionaminoacyl_adenylate_route

The aminoacyl-AMP intermediate of tRNA aminoacylation is attacked by ATP instead of tRNA, releasing the amino acid and Ap4A. Ap4A output therefore depends on the cognate amino acid. In stimulated mammalian mast cells, phosphorylated LysRS leaves the multi-tRNA synthetase complex and produces Ap4A in the nucleus.

Annotons

LysRS Ap4A synthesis
lysrs_ap4a_synthesis
Participant: Family: lysyl-tRNA synthetases (class II)
Family:
lysyl-tRNA synthetases (class II)PANTHER:PTHR42918:SF9
Representative Members: KARS1 (human)UniProtKB:Q15046

Function

ATP:ATP adenylyltransferase activityGO:0141192
Substrates: ATP
Products: P1,P4-bis(5'-adenosyl) tetraphosphate diphosphate

Makes Ap4A from the lysyl-adenylate intermediate and ATP; the canonical lysine-tRNA ligase activity is the primary function of the enzyme and is not part of this module.

PMID:23159739
Structural switch of lysyl-tRNA synthetase between translation and transcription.
Phosphorylation releases LysRS from the multi-tRNA synthetase complex and triggers LysRS-directed Ap4A production.
triggers LysRS-directed production of the second messenger Ap(4)A that activates MITF
Direct ATP condensation route (GlyRS type)Reactiondirect_atp_condensation_route

An insertion domain in glycyl-tRNA synthetase forms a second ATP pocket, allowing direct condensation of two ATP molecules independently of glycine.

Annotons

GlyRS Ap4A synthesis
glyrs_ap4a_synthesis
Participant: Family: glycyl-tRNA synthetases (class II, eukaryotic type)
Family:
glycyl-tRNA synthetases (class II, eukaryotic type)PANTHER:PTHR10745:SF0
Representative Members: GARS1 (human)UniProtKB:P41250

Function

ATP:ATP adenylyltransferase activityGO:0141192
Substrates: ATP
Products: P1,P4-bis(5'-adenosyl) tetraphosphate diphosphate

Condenses two ATP molecules directly into Ap4A.

PMID:19710017
Crystal structures and biochemical analyses suggest a unique mechanism and role for human glycyl-tRNA synthetase in Ap4A homeostasis.
GlyRS forms Ap4A from two ATPs without glycine.
the second ATP pocket is formed by an insertion domain that is unique to GlyRS, suggesting that GlyRS is the only tRNA synthetase catalyzing direct Ap4A synthesis
Part 2: Ap4A removal
Ap4A removalReactionap4a_removal_step
diadenosine tetraphosphate catabolic processGO:0015967
Variant set: Ap4A removal chemistry by enzyme chemistry and lineage (One Or More)

The four implementations are evolutionarily unrelated and use three chemistries with different products. A given organism usually relies on one of them, but some carry more than one Ap4A-degrading activity, so the selection is ONE_OR_MORE.

Asymmetrical Nudix hydrolase (NUDT2 type)Reactionasymmetrical_nudix_hydrolase

A small Nudix-fold hydrolase cleaves Ap4A between the gamma and delta phosphates to give ATP + AMP. This is the animal implementation; the same subfamily occurs in other eukaryotes, archaea and many bacteria.

Annotons

NUDT2-type asymmetrical Ap4A hydrolysis
nudt2_asymmetrical_hydrolysis
Participant: Family: asymmetrical Ap4A hydrolases (Nudix)
Family:
asymmetrical Ap4A hydrolases (Nudix)PANTHER:PTHR21340:SF0
Representative Members: NUDT2 (human)UniProtKB:P50583 NUDT2 (pig)UniProtKB:P50584 Nudt2 (mouse)UniProtKB:P56380 ndx-4 (Caenorhabditis elegans)UniProtKB:Q9U2M7 Datp / Apf (Drosophila melanogaster)UniProtKB:Q4V6M1

Function

bis(5'-nucleosyl)-tetraphosphatase (asymmetrical) activityGO:0004081
Substrates: P1,P4-bis(5'-adenosyl) tetraphosphate water
Products: ATP AMP

Hydrolyses Ap4A asymmetrically to ATP + AMP.

Symmetrical ApaH-type hydrolaseReactionsymmetrical_apah_hydrolase

A metallophosphoesterase-fold hydrolase cleaves Ap4A symmetrically between the beta and gamma phosphates to give two ADP. This is the gamma-proteobacterial implementation.

Annotons

ApaH symmetrical Ap4A hydrolysis
apah_symmetrical_hydrolysis
Participant: Gene Product: apaH (Escherichia coli K-12)
Gene Product:
apaH (Escherichia coli K-12)UniProtKB:P05637

Function

bis(5'-nucleosyl)-tetraphosphatase (symmetrical) activityGO:0008803
Substrates: P1,P4-bis(5'-adenosyl) tetraphosphate water
Products: ADP

Hydrolyses Ap4A symmetrically to two ADP.

PMID:2986688
Catabolism of bis(5'-nucleosidyl) oligophosphates in Escherichia coli: metal requirements and substrate specificity of homogeneous diadenosine-5',5'''-P1,P4-tetraphosphate pyrophosphohydrolase.
The purified E. coli enzyme always releases a nucleoside diphosphate from dinucleoside polyphosphates.
Among the products, a nucleoside diphosphate is always formed.
Symmetrical YqeK-type hydrolase (HD domain)Reactionsymmetrical_yqek_hydrolase

An HD-domain diiron hydrolase of the YqeK family (COG1713) cleaves Ap4A symmetrically to two ADP. It is structurally unrelated to ApaH and replaces it in a group of Gram-positive bacteria that lack ApaH.

Annotons

YqeK symmetrical Ap4A hydrolysis
yqek_symmetrical_hydrolysis
Participant: Gene Product: yqeK (Staphylococcus aureus NCTC 8325)
Gene Product:
yqeK (Staphylococcus aureus NCTC 8325)UniProtKB:Q2G297

Function

bis(5'-nucleosyl)-tetraphosphatase (symmetrical) activityGO:0008803
Substrates: P1,P4-bis(5'-adenosyl) tetraphosphate water
Products: ADP

Hydrolyses Ap4A symmetrically to two ADP in ApaH-lacking Gram-positive bacteria.

PMID:32152217
Functional Characterization of COG1713 (YqeK) as a Novel Diadenosine Tetraphosphate Hydrolase Family.
YqeK is a symmetrically cleaving Ap4A hydrolase with an HD-domain fold, found in Gram-positive bacteria lacking ApaH, and it affects Ap4A levels in vivo in B. subtilis.
we identified the YqeK family of proteins (COG1713) as an unprecedented class of symmetrically cleaving Ap4A hydrolases
Ap4A phosphorylase (fungal APA1/APA2 type)Reactionfungal_ap4a_phosphorylase

A HIT-superfamily phosphorylase cleaves Ap4A by phosphorolysis to ADP + ATP. The reaction is reversible in vitro, but yeast genetics show that it acts catabolically in vivo.

Annotons

APA1/APA2 Ap4A phosphorolysis
apa_phosphorolysis
Participant: Gene Product: APA1 (Saccharomyces cerevisiae)
Gene Product:
APA1 (Saccharomyces cerevisiae)UniProtKB:P16550

Function

ATP:ADP adenylyltransferase activityGO:0003877
Substrates: P1,P4-bis(5'-adenosyl) tetraphosphate phosphate
Products: ADP ATP

Phosphorolyses Ap4A to ADP + ATP. GO:0003877 is written in the synthetic direction; the module uses it for the catabolic direction shown by apa1 apa2 mutants. The paralogue APA2 (UniProtKB:P22108) has the same activity.

PMID:2556364
Isolation, characterization, and inactivation of the APA1 gene encoding yeast diadenosine 5',5'''-P1,P4-tetraphosphate phosphorylase.
APA1 disruption shows the phosphorylase degrades Ap4N in vivo.
in vivo, Ap4A phosphorylase catabolizes the Ap4N nucleotides