Enzyme Specificity Project
Overview
This project tracks genes where GO annotations fail to capture the correct enzyme specificity - either:
1. Too narrow: Annotation to one substrate when enzyme has broader specificity
2. Too broad: Generic enzyme class when specific activity is known
3. Wrong reaction: Incorrect reaction type based on domain classification
Enzyme specificity is crucial for accurate functional annotation. Misannotated specificity can:
- Mislead metabolic pathway reconstruction
- Cause errors in drug target identification
- Propagate incorrect annotations via IBA
Source: Presented at Gene Ontology Consortium Meeting, October 2025, Cambridge UK. See ai4curation/ai-gene-review.
Categories
1. Substrate Specificity Errors
The Problem: Enzyme annotated to specific substrate when it has broader or different specificity.
Example - LPL1 (C. albicans):
- Annotated: GO:0004622 (phosphatidylcholine lysophospholipase activity)
- Actual: Phospholipase B with three distinct activities:
1. sn-1/sn-2 fatty acid ester hydrolase
2. Lysophospholipase activity (on ALL glycerophospholipids, not just PC)
3. Transacylase activity
- Action: MODIFY → GO:0102545 (phospholipase B activity)
2. Reaction Mechanism Errors
The Problem: Wrong reaction type inferred from domain classification.
Example - PHYKPL (human):
- Domain: Aminotransferase III family
- Annotated: GO:0008483 (transaminase activity)
- Actual: Phospho-lyase (EC 4.2.3.134), NOT a transaminase
- Evidence: "Unlike AGXT2, AGXT2L1 and AGXT2L2 did not act as transaminases"
- Action: REMOVE transaminase; ADD GO:0016838 (carbon-oxygen lyase activity, acting on phosphates)
3. Cofactor Specificity Errors
The Problem: Enzyme annotated with wrong cofactor preference.
Examples:
- NAD+ vs NADP+ dependent dehydrogenases
- PLP-dependent enzymes with different reaction types
4. Non-Enzymatic Homologs
The Problem: Enzymatic activity annotated to proteins that have lost catalytic function.
Example - Epe1 (S. pombe) (see CONTESTED_FUNCTION.md):
- Annotated: Histone demethylase, oxidoreductase, dioxygenase
- Actual: Pseudo-enzyme with degenerate active site
- Action: REMOVE all enzymatic annotations
Featured Examples
LPL1 (Candida albicans)
UniProt: Q5AMS2
Species: CANAL
Status: COMPLETE
Specificity Issue:
- GO:0004622 (phosphatidylcholine lysophospholipase) - too narrow
- Enzyme acts on ALL glycerophospholipids, not just PC
- Has three distinct activities (hydrolase, lysophospholipase, transacylase)
Also: GO:0047372 (monoacylglycerol lipase activity) marked KEEP_AS_NON_CORE
- Likely substrate promiscuity, not primary function
PHYKPL (Homo sapiens)
UniProt: Q8IUZ5
Species: human
Status: COMPLETE
Specificity Issue:
- Classified in aminotransferase family → annotated with transaminase activity
- Biochemically demonstrated to be a phospho-lyase
- Different reaction mechanism entirely
Proposed New Terms:
- 5-phosphooxy-L-lysine phospho-lyase activity - specific MF for PHYKPL
- 5-phosphohydroxy-L-lysine catabolic process - specific BP
GND1 (Candida albicans)
UniProt: A0A1D8PFS4
Species: CANAL
Status: INITIALIZED (annotations pending review)
Notes: 6-phosphogluconate dehydrogenase with interesting dual localization (cytosol and peroxisome via alternative splicing). Review needed for specificity annotations.
Mitochondrial fatty acid β-oxidation (acyl-chain-length specificity)
Species: human · Status: COMPLETE · Module: MODULE:fatty_acid_beta_oxidation
A clean, paralog-rich worked example of substrate (acyl-chain-length)
specificity. The four-step β-oxidation spiral (dehydrogenase → hydratase →
3-hydroxyacyl-CoA dehydrogenase → thiolase) is run by families of chain-length-
specific isozymes; the curation principle is use the chain-length-specific MF
term where one exists, and fall back to the general term where it does not
("not all MF have them"). Reviewed across all ten human enzymes:
| Step | Gene | UniProt | MF term | Chain-length specificity |
|---|---|---|---|---|
| ① dehydrogenase | ACADVL | P49748 | GO:0017099 |
very-long/long-chain-specific |
| ① dehydrogenase | ACAD9 | Q9H845 | GO:0017099 |
VLC + complex I assembly factor |
| ① dehydrogenase | ACADM | P11310 | GO:0070991 |
medium-chain-specific |
| ① dehydrogenase | ACADS | P16219 | GO:0016937 |
short-chain-specific |
| ② hydratase | HADHA | P40939 | GO:0004300 |
long-chain (no LC-specific MF; in MTP) |
| ② hydratase | ECHS1 | P30084 | GO:0004300 |
short/medium (no SC-specific MF) |
| ③ 3-OH-acyl-CoA DH | HADHA | P40939 | GO:0016509 |
long-chain-specific (in MTP) |
| ③ 3-OH-acyl-CoA DH | HADH | Q16836 | GO:0003857 |
short/medium (no SC-specific MF) |
| ④ thiolase | HADHB | P55084 | GO:0003988 |
long-chain (in MTP; no chain-specific MF) |
| ④ thiolase | ACAA2 | P42765 | GO:0003988 |
medium/long straight-chain (no chain-specific MF) |
| ④ thiolase | ACAT1 | P24752 | GO:0003985 |
acetoacetyl-CoA (C4; ketone-body/Ile, not the spiral) |
Specificity errors caught:
- Too broad → specific (MODIFY): HADHA's generic 3-hydroxyacyl-CoA
dehydrogenase annotations modified to the long-chain-specific GO:0016509.
- Wrong chain-length (over-annotation): long-chain GO:0016509 annotated on
HADH, which is the short/medium-chain enzyme — that activity is HADHA's.
- Wrong subunit / activity (REMOVE): thiolase MF (GO:0003985) on HADHA —
thiolase is the HADHB subunit's activity; the cited paper itself shows the
α-cDNA yields only hydratase + dehydrogenase.
- Cross-gene mis-attribution (REMOVE): cholesterol O-acyltransferase +
ER localization on ACAT1 — these came from a SOAT1/SOAT2 paper via the
historical "ACAT1" nickname collision; mitochondrial T2 has no sterol activity.
- Paralog cross-transfer (REMOVE): several IEA/ISS terms on ACADVL were
propagated from mouse LCAD (Acadl), including a self-contradictory
"negative regulation of fatty acid oxidation".
Reaction-specificity / mapping gap (links to RHEA project): the GO→RHEA
chaining check on the module flags ① → ② as a break — step ① makes
(2E)-enoyl-CoA, but the hydratase MF GO:0004300 maps only to RHEA:20724 (the
(3E)-enoyl-CoA variant), not the canonical (2E) crotonase RHEA:16105. The
chemistry is correct; the gap is in the GO:0004300 → RHEA mapping. See
projects/RHEA/RHEA-EC-SPECIFICITY.md and the module's
modules/fatty_acid_beta_oxidation/RESULTS.md.
Genes for Review
Priority 1: Completed
| Gene | Species | Issue | Status |
|---|---|---|---|
| LPL1 | CANAL | Substrate specificity too narrow | COMPLETE |
| PHYKPL | human | Wrong reaction mechanism | COMPLETE |
| eryCIII | SACEN | Wrong donor: GO:0008194 UDP-glycosyltransferase, but uses TDP-D-desosamine |
COMPLETE |
eryCIII (cofactor/donor specificity error, BGC project): the desosaminyl transferase
EryCIII (EC 2.4.1.278) was IEA-annotated GO:0008194 UDP-glycosyltransferase activity, but it
transfers TDP-D-desosamine (a dTDP-sugar), not a UDP-sugar (PMID:15303858). MODIFY to the
accurate, IDA-supported GO:0016758 hexosyltransferase activity. See genes/SACEN/eryCIII/.
Priority 1: Completed — fatty acid β-oxidation (acyl-chain-length specificity)
| Gene | Species | Issue | Status |
|---|---|---|---|
| ACADVL | human | Chain-length-specific MF; mouse-LCAD paralog cross-transfer removed | COMPLETE |
| ACAD9 | human | VLC dehydrogenase + complex I assembly factor | COMPLETE |
| ACADM | human | Medium-chain-specific MF (GO:0070991) |
COMPLETE |
| ACADS | human | Short-chain-specific MF (GO:0016937) |
COMPLETE |
| HADHA | human | Generic→long-chain-specific MODIFY; thiolase MF removed (HADHB's) | COMPLETE |
| HADHB | human | Long-chain thiolase (MTP β subunit) | COMPLETE |
| ECHS1 | human | General hydratase MF (no SC-specific term) | COMPLETE |
| HADH | human | Short/medium MF; long-chain GO:0016509 over-annotation flagged |
COMPLETE |
| ACAT1 | human | SOAT1/cholesterol mis-attribution removed | COMPLETE |
| ACAA2 | human | Straight-chain 3-ketoacyl-CoA thiolase | COMPLETE |
Priority 2: Pending
| Gene | Species | Issue | Status |
|---|---|---|---|
| GND1 | CANAL | Review pending | INITIALIZED |
Curation Principles
- Verify substrate range: Don't assume specificity from one characterized reaction
- Check reaction mechanism: Domain family doesn't guarantee mechanism
- Distinguish promiscuity from core function: Secondary activities → KEEP_AS_NON_CORE
- Propose specific terms: When existing terms are too broad or narrow
STATUS
Completed Reviews
- [x] CANAL/LPL1 - Phospholipase B specificity
- [x] human/PHYKPL - Phospho-lyase vs transaminase
- [x] human β-oxidation acyl-chain-length set (ACADVL, ACAD9, ACADM, ACADS, HADHA, HADHB, ECHS1, HADH, ACAT1, ACAA2)
Pending Reviews
- [ ] CANAL/GND1 - 6-phosphogluconate dehydrogenase
Last updated: 2026-06-30
NOTES
2026-01-22
Project Creation
Created project to track enzyme specificity annotation issues.
LPL1 Key Points:
- Phospholipase B enzymes have three activities in one protein
- Current annotation (GO:0004622) captures only lysophospholipase on PC
- Better term: GO:0102545 (phospholipase B activity)
- Also: GO:0047372 (monoacylglycerol lipase) kept as non-core (likely promiscuous)
PHYKPL Key Points:
- Domain classification misleading (aminotransferase family)
- Biochemically: "did not act as transaminases"
- Functions as phospho-lyase (EC 4.2.3.134)
- Needs specific GO term for its reaction
2026-06-30
Added the mitochondrial fatty acid β-oxidation acyl-chain-length specificity set.
Reviewed all ten human β-oxidation enzymes as a paralog-rich worked example of
substrate-chain-length specificity (a flavour of categories 1 "too broad" and
"wrong substrate"). Curation principle confirmed: use the chain-length-specific
MF where it exists (VLCAD GO:0017099, MCAD GO:0070991, SCAD GO:0016937,
long-chain 3-OH-acyl-CoA DH GO:0016509) and fall back to the general term where
none exists (the hydratases ECHS1/HADHA → GO:0004300; the straight-chain
thiolases HADHB/ACAA2 → GO:0003988; short/medium HADH → GO:0003857).
Specificity errors removed/modified: HADHA generic→long-chain MODIFY and the
mis-attributed thiolase MF removed; the long-chain GO:0016509 over-annotation
on short-chain HADH flagged; ACAT1's SOAT1/cholesterol terms removed
(nickname collision); ACADVL's mouse-LCAD paralog cross-transfers removed.
Cross-link: the cross-species MODULE:fatty_acid_beta_oxidation and its
GO→RHEA chaining check (now generalized into the module tooling) surface a
reaction-specificity mapping gap — GO:0004300 maps to RHEA:20724 (the (3E)
variant) rather than the canonical (2E) crotonase RHEA:16105. Logged for the
RHEA project (projects/RHEA/RHEA-EC-SPECIFICITY.md).