ADO (2-aminoethanethiol / cysteamine dioxygenase) — curation notes

UniProt: Q96SZ5 (AEDO_HUMAN). Gene ADO (synonym C10orf22). Human, 270 aa, cytosolic.
EC 1.13.11.19. HGNC:23506. Chromosome 10.

Overview / core biology

ADO is a cytosolic non-heme mononuclear Fe(II) thiol dioxygenase of the cupin
superfamily. It is one of only two mammalian thiol dioxygenases (the other is
cysteine dioxygenase, CDO1), and is unusual in having two well-established
activities acting on chemically distinct substrate classes:

Cysteamine (2-aminoethanethiol) dioxygenase — oxidizes the sulfur of the
small molecule cysteamine to hypotaurine (a sulfinic acid), the second route of
hypotaurine/taurine biosynthesis complementing the CDO1/CSAD cysteinesulfinate
route. This was the founding assigned activity.
[file:human/ADO/ADO-uniprot.txt "Catalyzes the oxidation of cysteamine (2-aminoethanethiol) to hypotaurine"]
PMID:17581819
N-terminal cysteine dioxygenase of the Cys/N-degron (oxygen-sensing) pathway
— oxidizes the thiol of N-terminal cysteine residues of substrate proteins to
Cys-sulfinic acid, priming them for arginylation (by ATE1) and downstream
ubiquitin-mediated degradation. This makes ADO an enzymatic cellular O2 sensor.
Verified substrates: RGS4, RGS5, IL-32 (and, in reporter assays, the plant
ERF-VII RAP2.12 N-terminus).
PMID:31273118
PMID:31273118

Both activities use O2 as a co-substrate and incorporate both oxygen atoms
(true dioxygenase). ADO has a high KmO2 (>500 uM for peptide substrates),
significantly above the physiological range, which underpins its oxygen-sensing role.
PMID:31273118
PMID:31273118

Cofactor / metal center

Non-heme mononuclear iron center, 3-His facial triad (His112, His114, His193).
[file:human/ADO/ADO-uniprot.txt "Name=Fe cation"] (COFACTOR block, ECO:0000269|PubMed:34508780)
PMID:34508780
First crystal structure (1.78 Å, Ni-substituted C18S/C239S mutant, PDB 7REI).
Monomer. Cupin jelly-roll β-barrel with unusually extensive flexible loops that
accommodate both small-molecule and protein substrates; active-site architecture
resembles plant cysteine oxidases (PCOs) rather than CDO.
PMID:34508780
PMID:34508780
Autocatalytically forms a Cys220–Tyr222 thioether cross-linked cofactor that acts
as a "catalytic amplifier" (boosts efficiency, not strictly required).
PMID:29752763
Substrates bind the ferrous iron through their free thiol in a monodentate mode
(distinct from CDO's bidentate binding).
PMID:32601061

Subcellular location

Cytosol is the authoritative location.
[file:human/ADO/ADO-uniprot.txt "GO:0005829; C:cytosol; TAS:Reactome."]
Reactome R-HSA-6814153 places the cysteamine→hypotaurine reaction in the cytosol.
Mitochondrion appears in GOA via IBA (GO_REF:0000033), Ensembl-Compara IEA
(GO_REF:0000107), and a large-scale HTP mito-proteome dataset (PMID:34800366).
These are inconsistent with the curated cytosolic localization in UniProt (which
gives no mitochondrial location) and with the enzyme's known cytosolic substrates
(cytosolic RGS4/5, IL-32; cytosolic N-degron pathway). The HTP mitochondrial
detection is likely co-purification/contaminant signal; ADO is not mentioned by
name in the cached full text of PMID:34800366 (its listing is in supplementary
tables). Treated as over-annotation / non-core, not the primary function.

Existing-annotation decisions (summary)

GO:0047800 cysteamine dioxygenase activity — CORE MF. IMP/IDA (PMID:17581819,
29752763, 31273118, 32601061) ACCEPT; the IEA (GO_REF:0000120) is a redundant but
correct electronic call → ACCEPT.
GO:0005506 iron ion binding — CORE cofactor MF. IDA (PMID:34508780) ACCEPT;
ISS/IEA redundant-correct → ACCEPT.
GO:0071456 cellular response to hypoxia — CORE BP (oxygen sensing). IDA
(PMID:31273118) ACCEPT.
GO:0042412 taurine biosynthetic process — CORE BP (via hypotaurine). IEA ACCEPT.
GO:0005829 cytosol — CORE CC. TAS (Reactome) ACCEPT; the IEA cytosol is redundant.
GO:0016702 (oxidoreductase, single donors, 2 O atoms incorporated) — correct
parent MF but generic; MODIFY → GO:0047800.
GO:0005739 mitochondrion (IBA, IEA, HTP) — likely mislocalization / contaminant;
MARK_AS_OVER_ANNOTATED (IBA/HTP cannot be REMOVEd per policy; IEA-Compara → REMOVE
is permissible but conservatively MARK_AS_OVER_ANNOTATED given the two experimental
siblings). See below.
GO:0005515 protein binding (IPI, x3 rows) — high-throughput interactome hits
(FAM76B, CAP2, DISC1); uninformative bare protein binding →
MARK_AS_OVER_ANNOTATED. Interactors confirmed in the UniProt INTERACTION block.
[file:human/ADO/ADO-uniprot.txt "Q96SZ5; P40123: CAP2"]

Missing GO term (proposed)

GO currently has no dedicated molecular-function term for "oxygen-dependent
protein/peptidyl N-terminal cysteine dioxygenase activity" (the RHEA:70895 reaction:
N-terminal L-cysteinyl-[protein] + O2 = N-terminal S-hydroxy-S-oxy-L-cysteinyl-[protein]).
The nearest existing terms are GO:0047800 (cysteamine dioxygenase activity, the
small-molecule reaction), GO:0017172 (cysteine dioxygenase activity, the CDO1
reaction), and the BP term GO:0018171 (peptidyl-cysteine oxidation). This second
core catalytic activity of ADO is therefore proposed as a new MF term and, in the
interim, modeled through the process terms GO:0018171 (peptidyl-cysteine oxidation)
and GO:0031648 (protein destabilization) plus GO:0071456 (cellular response to hypoxia).

Key references

PMID:17581819 — discovery of ADO as the mammalian cysteamine dioxygenase (mouse
Gm237 / human C10orf22); RNAi in HepG2 reduces hypotaurine. full_text_available: false (abstract only).
PMID:29752763 — Cys220–Tyr222 cross-linked cofactor; mutagenesis of Tyr87/Tyr222/Tyr223. Full text.
PMID:31273118 (Science) — ADO is the N-terminal cysteine dioxygenase of the Cys/N-degron
pathway; RGS4/5, IL-32 substrates; O2 sensor; high KmO2. Full text.
PMID:32601061 — non-heme iron center; monodentate thiol substrate binding; ferrous form. Full text.
PMID:34508780 — first crystal structure (7REI); 3-His iron center; monomer; open,
loop-rich cavity for protein substrates; structural basis for oxygen sensing. Full text.
Reactome R-HSA-6814153 — "ADO oxidises 2AET to HTAU" (cytosolic).
PMID:28514442, 32296183, 33961781 — large-scale interactome maps (bare protein-binding IPI).
PMID:34800366 — HTP mitochondrial proteome (ADO listed; localization not corroborated by UniProt).

2026-10-24 ORGANELLES batch 2 (mitochondrion rows)

Rows on GO:0005739: IBA GO_REF:0000033 (was MARK_AS_OVER_ANNOTATED), IEA
GO_REF:0000107 Ensembl projection from mouse Q6PDY2 (was REMOVE), HTP PMID:34800366
(was MARK_AS_OVER_ANNOTATED). The earlier review called all three co-purification
and stated that UniProt places ADO in the cytosol. No sub-compartment rows.

Checked:
- UniProt Q96SZ5 has no SUBCELLULAR LOCATION comment at all, so no curated UniProt
location contradicts the mitochondrion; the cytosol call is Reactome TAS plus the
N-degron substrates.
- PAINT (interpro/panther/PTHR22966/PTHR22966-paint.tsv): PTN000539917 GO:0005739
IBD seeded by MGI:MGI:2685083 (mouse Ado).
- Mouse Q6PDY2 GO:0005739 (QuickGO): HDA PMID:18614015 (MitoCarta 2008, MGI) and IDA
PMID:37630723 (FlyBase). So the Ensembl projection is not circular.
- PMID:37630723 (full text): nuclear/cytoplasmic/mitochondrial fractionation of
primary inguinal adipocytes and DE 2.3 brown adipocytes PMID:37630723.
- PMID:34800366 (MitoCoP): built to exclude contaminants PMID:34800366. ADO itself is in the supplementary data only.
- Sequence: N-terminus MPRDNMASLIQRIARQACLTFRGS is Arg-rich, but UniProt annotates
no transit peptide and no TM; MobiDB-lite disorder at 21-48.

Outcome: root; all three rows changed to KEEP_AS_NON_CORE (dual cytosolic and
mitochondrial distribution; cytosol stays core). The REMOVE on the IEA row was a
consistency violation with the other rows and is no longer justified. No
sub-compartment row: the processed-looking lower band is a single line, with no TM
and no annotated presequence. Reference reviews for PMID:34800366, GO_REF:0000033
and GO_REF:0000107 updated; PMID:37630723 added; suggested experiment extended with
protease protection. This reverses an earlier call and should be double-checked.