Existing Annotations Review

GO Term	Evidence	Action	Reason
GO:0003857 (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity	IBA GO_REF:0000033	ACCEPT	Summary: Core C-terminal catalytic activity: NAD+-dependent oxidation of L-(3S)-3-hydroxyacyl-CoA to 3-oxoacyl-CoA (step 3 of peroxisomal beta-oxidation). Phylogenetically inferred and concordant with human experimental (IDA) evidence. Supporting Evidence: PMID:15060085 the main enzymes involved in beta-oxidation of C16DCA are SCOX, both LBP and DBP
GO:0005777 peroxisome	IBA GO_REF:0000033	ACCEPT	Summary: Core localization. EHHADH is a peroxisomal-matrix beta-oxidation enzyme; phylogenetic inference matches human IDA localization evidence. Supporting Evidence: file:human/EHHADH/EHHADH-uniprot.txt SUBCELLULAR LOCATION: Peroxisome
GO:0006635 fatty acid beta-oxidation	IBA GO_REF:0000033	ACCEPT	Summary: Core biological process. EHHADH catalyzes steps 2 and 3 of the peroxisomal fatty-acid beta-oxidation spiral. Phylogenetically inferred and supported by human IDA evidence. Supporting Evidence: file:human/EHHADH/EHHADH-uniprot.txt Catalyzes two of the four reactions of the
GO:0003824 catalytic activity	IEA GO_REF:0000002	MARK AS OVER ANNOTATED	Summary: Root-level catalytic activity term. Correct but uninformatively general; the specific hydratase (GO:0004300) and dehydrogenase (GO:0003857) activities are already annotated.
GO:0003857 (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity	IEA GO_REF:0000120	ACCEPT	Summary: IEA (ARBA/RHEA/EC) duplicate of the core C-terminal dehydrogenase activity; consistent with human IDA evidence. Supporting Evidence: PMID:15060085 the main enzymes involved in beta-oxidation of C16DCA are SCOX, both LBP and DBP
GO:0004165 delta(3)-delta(2)-enoyl-CoA isomerase activity	IEA GO_REF:0000120	KEEP AS NON CORE	Summary: Auxiliary activity of the N-terminal module (isomerizes 3-cis/3-trans double bonds to the 2-trans form for continued beta-oxidation of unsaturated fatty acids). Real by similarity to rat P07896, but not the defining core function and not supported by human experimental evidence; kept as non-core. Supporting Evidence: file:human/EHHADH/EHHADH-uniprot.txt Peroxisomal trifunctional enzyme possessing 2-enoyl-CoA
GO:0004300 enoyl-CoA hydratase activity	IEA GO_REF:0000120	ACCEPT	Summary: IEA (ARBA/RHEA/EC) duplicate of the core N-terminal hydratase activity; consistent with human IDA evidence. Supporting Evidence: PMID:15060085 the main enzymes involved in beta-oxidation of C16DCA are SCOX, both LBP and DBP
GO:0005777 peroxisome	IEA GO_REF:0000120	ACCEPT	Summary: IEA duplicate of the core peroxisomal localization; consistent with IDA evidence and the C-terminal SKL (PTS1) targeting signal. Supporting Evidence: file:human/EHHADH/EHHADH-uniprot.txt SUBCELLULAR LOCATION: Peroxisome
GO:0006631 fatty acid metabolic process	IEA GO_REF:0000002	MARK AS OVER ANNOTATED	Summary: Correct but general parent of the specific process fatty acid beta-oxidation (GO:0006635), which is already annotated with experimental evidence.
GO:0016491 oxidoreductase activity	IEA GO_REF:0000002	MARK AS OVER ANNOTATED	Summary: General parent term for the dehydrogenase activity. Correct but uninformative; the specific (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity is already annotated.
GO:0016509 long-chain (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity	IEA GO_REF:0000116	ACCEPT	Summary: Chain-length-specific (long-chain) form of the core C-terminal dehydrogenase activity; consistent with the human IDA annotation of the same term and with the measured long-chain substrate kinetics. Supporting Evidence: file:human/EHHADH/EHHADH-uniprot.txt KM=0.3 uM for (2E)-hexadecenedioyl-CoA
GO:0016616 oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor	IEA GO_REF:0000002	MARK AS OVER ANNOTATED	Summary: Intermediate parent term for the dehydrogenase activity. Correct but more general than the specific (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity already annotated.
GO:0018812 3-hydroxyacyl-CoA dehydratase activity	IEA GO_REF:0000116	MODIFY	Summary: This RHEA-mapped "dehydratase" term describes the same step-2 reaction as the curated enoyl-CoA hydratase activity (GO:0004300), which is already independently annotated by IDA for this enzyme. GO:0004300 is the appropriate curated term for EHHADH's N-terminal module; modify to it. Proposed replacements: enoyl-CoA hydratase activity Supporting Evidence: file:human/EHHADH/EHHADH-uniprot.txt note="Enoyl-CoA hydratase / isomerase"
GO:0070403 NAD+ binding	IEA GO_REF:0000002	KEEP AS NON CORE	Summary: NAD+ is the required cofactor of the C-terminal (3S)-3-hydroxyacyl-CoA dehydrogenase module (NAD(P)-binding Rossmann fold). Correct supporting activity; kept but non-core relative to the catalytic activities. Supporting Evidence: file:human/EHHADH/EHHADH-uniprot.txt note="3-hydroxyacyl-CoA dehydrogenase"
GO:0005515 protein binding	IPI PMID:25416956 A proteome-scale map of the human interactome network.	MARK AS OVER ANNOTATED	Summary: Bare "protein binding" from a high-throughput binary interactome map. Uninformative for molecular function and dominated by non-peroxisomal HT hits; over-annotated (not removed, per policy for IPI).
GO:0005515 protein binding	IPI PMID:31515488 Extensive disruption of protein interactions by genetic vari...	MARK AS OVER ANNOTATED	Summary: Bare "protein binding" from a high-throughput interactome/variant-disruption screen. Uninformative for molecular function; over-annotated.
GO:0005515 protein binding	IPI PMID:32296183 A reference map of the human binary protein interactome.	MARK AS OVER ANNOTATED	Summary: Bare "protein binding" from a reference binary interactome map (HuRI); large, largely non-peroxisomal partner set. Uninformative for molecular function; over-annotated.
GO:0005515 protein binding	IPI PMID:33961781 Dual proteome-scale networks reveal cell-specific remodeling...	MARK AS OVER ANNOTATED	Summary: Bare "protein binding" from a proteome-scale affinity-MS interactome map. Uninformative for molecular function; over-annotated.
GO:0005515 protein binding	IPI PMID:36217029 A proteome-scale map of the SARS-CoV-2-human contactome.	MARK AS OVER ANNOTATED	Summary: "Protein binding" from a SARS-CoV-2-human contactome (interaction with viral N protein, a xeno interaction). Not relevant to native EHHADH molecular function; over-annotated.
GO:0005515 protein binding	IPI PMID:40205054 Multimodal cell maps as a foundation for structural and func...	MARK AS OVER ANNOTATED	Summary: Bare "protein binding" from a multimodal cell-map/spatial-genomics interactome. Uninformative for molecular function; over-annotated.
GO:0005829 cytosol	IEA GO_REF:0000107	MARK AS OVER ANNOTATED	Summary: Cytosol annotation reflects only the transient pre-import (PTS1-folded) cargo state before peroxisomal translocation. The mature, functional enzyme resides in the peroxisomal matrix (IDA evidence). Not a genuine steady-state functional location; over-annotated.
GO:0006635 fatty acid beta-oxidation	IEA GO_REF:0000120	ACCEPT	Summary: IEA duplicate of the core fatty acid beta-oxidation process; consistent with human IDA evidence. Supporting Evidence: file:human/EHHADH/EHHADH-uniprot.txt Catalyzes two of the four reactions of the
GO:0016863 intramolecular oxidoreductase activity, transposing C=C bonds	IEA GO_REF:0000107	MARK AS OVER ANNOTATED	Summary: Parent term of the Delta-3,Delta-2-enoyl-CoA isomerase activity (GO:0004165), the auxiliary isomerase function of the N-terminal module. More general than the specific term already annotated; over-annotated.
GO:0019899 enzyme binding	IEA GO_REF:0000107	KEEP AS NON CORE	Summary: Ensembl-projected copy of the human catalase enzyme-binding annotation (PMID:16781659). The underlying interaction (EHHADH with catalase in the peroxisomal matrix) is specific and biologically plausible but is a binding activity, not a catalytic core function; kept as non-core, consistent with the IPI-supported annotation of the same term. Supporting Evidence: PMID:16781659 catalase physically interacts with L-bifunctional enzyme (L-BFE)
GO:0033540 fatty acid beta-oxidation using acyl-CoA oxidase	TAS Reactome:R-HSA-390247	ACCEPT	Summary: Precise process term: the peroxisomal (acyl-CoA-oxidase-initiated) variant of fatty-acid beta-oxidation in which EHHADH catalyzes the hydratase and dehydrogenase steps. Accurately captures the pathway context; accepted. Supporting Evidence: file:human/EHHADH/EHHADH-uniprot.txt Catalyzes two of the four reactions of the
GO:0003857 (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity	TAS Reactome:R-HSA-6809264	ACCEPT	Summary: Reactome TAS for the core C-terminal dehydrogenase activity (dehydrogenation of 3-hydroxyhexacosanoyl-CoA); consistent with human IDA evidence. Supporting Evidence: PMID:15060085 the main enzymes involved in beta-oxidation of C16DCA are SCOX, both LBP and DBP
GO:0018812 3-hydroxyacyl-CoA dehydratase activity	TAS Reactome:R-HSA-6809263	MODIFY	Summary: Reactome labels EHHADH's step-2 hydration reaction as a "dehydratase" activity. This is the same reaction captured by the curated enoyl-CoA hydratase activity (GO:0004300), already annotated by IDA. Modify to the curated hydratase term. Proposed replacements: enoyl-CoA hydratase activity Supporting Evidence: file:human/EHHADH/EHHADH-uniprot.txt note="Enoyl-CoA hydratase / isomerase"
GO:0003857 (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity	ISS GO_REF:0000024	ACCEPT	Summary: ISS (by similarity to rat P07896) for the core dehydrogenase activity; consistent with human IDA evidence. Supporting Evidence: PMID:15060085 the main enzymes involved in beta-oxidation of C16DCA are SCOX, both LBP and DBP
GO:0003857 (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity	IDA PMID:15060085 Identification of the peroxisomal beta-oxidation enzymes inv...	ACCEPT	Summary: Primary experimental (IDA) evidence for the core C-terminal NAD+-dependent 3-hydroxyacyl-CoA dehydrogenase activity, assayed with recombinant human L-bifunctional protein. One of the two defining core molecular functions. Supporting Evidence: PMID:15060085 the main enzymes involved in beta-oxidation of C16DCA are SCOX, both LBP and DBP file:human/EHHADH/EHHADH-uniprot.txt a 3-oxoacyl-CoA + NADH +
GO:0004300 enoyl-CoA hydratase activity	ISS GO_REF:0000024	ACCEPT	Summary: ISS (by similarity to rat P07896) for the core N-terminal hydratase activity; consistent with human IDA evidence. Supporting Evidence: PMID:15060085 the main enzymes involved in beta-oxidation of C16DCA are SCOX, both LBP and DBP
GO:0004300 enoyl-CoA hydratase activity	IDA PMID:15060085 Identification of the peroxisomal beta-oxidation enzymes inv...	ACCEPT	Summary: Primary experimental (IDA) evidence for the core N-terminal 2-enoyl-CoA hydratase activity (hydration of trans-2-enoyl-CoA to L-(3S)-3-hydroxyacyl-CoA), assayed with recombinant human L-bifunctional protein. One of the two defining core molecular functions. Supporting Evidence: PMID:15060085 the main enzymes involved in beta-oxidation of C16DCA are SCOX, both LBP and DBP file:human/EHHADH/EHHADH-uniprot.txt a (3S)-3-hydroxyacyl-CoA = a (2E)-enoyl-CoA + H2O
GO:0006635 fatty acid beta-oxidation	IDA PMID:15060085 Identification of the peroxisomal beta-oxidation enzymes inv...	ACCEPT	Summary: Experimental (IDA) evidence that EHHADH (LBP) participates in peroxisomal beta-oxidation, specifically the degradation of long-chain dicarboxylic acids. Core biological process. Supporting Evidence: PMID:15060085 the main enzymes involved in beta-oxidation of C16DCA are SCOX, both LBP and DBP
GO:0016509 long-chain (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity	IDA PMID:15060085 Identification of the peroxisomal beta-oxidation enzymes inv...	ACCEPT	Summary: Experimental (IDA) evidence for the long-chain-specific form of the dehydrogenase activity, consistent with the measured long-chain/dicarboxylic substrate kinetics. A precise child of the core dehydrogenase activity. Supporting Evidence: file:human/EHHADH/EHHADH-uniprot.txt KM=0.3 uM for (2E)-hexadecenedioyl-CoA
GO:0004165 delta(3)-delta(2)-enoyl-CoA isomerase activity	ISS GO_REF:0000024	KEEP AS NON CORE	Summary: Auxiliary isomerase activity of the N-terminal module (by similarity to rat P07896). A real activity but not the defining core function; kept as non-core. Supporting Evidence: file:human/EHHADH/EHHADH-uniprot.txt Peroxisomal trifunctional enzyme possessing 2-enoyl-CoA
GO:0005777 peroxisome	ISS GO_REF:0000024	ACCEPT	Summary: ISS (by similarity) for peroxisomal localization; consistent with human IDA evidence and the C-terminal SKL (PTS1) signal. Supporting Evidence: file:human/EHHADH/EHHADH-uniprot.txt SUBCELLULAR LOCATION: Peroxisome
GO:0006635 fatty acid beta-oxidation	ISS GO_REF:0000024	ACCEPT	Summary: ISS (by similarity) for the core fatty acid beta-oxidation process; consistent with human IDA evidence. Supporting Evidence: file:human/EHHADH/EHHADH-uniprot.txt Catalyzes two of the four reactions of the
GO:0016863 intramolecular oxidoreductase activity, transposing C=C bonds	ISS GO_REF:0000024	MARK AS OVER ANNOTATED	Summary: Parent term of the Delta-3,Delta-2-enoyl-CoA isomerase activity (GO:0004165). More general than the specific isomerase term already annotated; over-annotated.
GO:0005829 cytosol	TAS Reactome:R-HSA-9033235	MARK AS OVER ANNOTATED	Summary: Cytosol annotation from the Reactome peroxisomal-protein-import pathway, representing the transient pre-import cargo state (PEX5-bound). The mature functional enzyme is in the peroxisomal matrix; over-annotated as a steady-state functional location.
GO:0005829 cytosol	TAS Reactome:R-HSA-9033236	MARK AS OVER ANNOTATED	Summary: Cytosol annotation from the Reactome peroxisomal-import docking/translocation step; represents the transient pre-import cargo state, not a steady-state functional location. Over-annotated.
GO:0005782 peroxisomal matrix	TAS Reactome:R-HSA-6809263	ACCEPT	Summary: Peroxisomal matrix is the precise functional location of this soluble matrix beta-oxidation enzyme; more specific than "peroxisome" and consistent with IDA evidence. Accepted. Supporting Evidence: file:human/EHHADH/EHHADH-uniprot.txt SUBCELLULAR LOCATION: Peroxisome
GO:0005782 peroxisomal matrix	TAS Reactome:R-HSA-6809264	ACCEPT	Summary: Peroxisomal matrix location (Reactome), consistent with the enzyme being a soluble matrix beta-oxidation protein; precise and supported. Accepted. Supporting Evidence: file:human/EHHADH/EHHADH-uniprot.txt SUBCELLULAR LOCATION: Peroxisome
GO:0005782 peroxisomal matrix	TAS Reactome:R-HSA-9033235	ACCEPT	Summary: Peroxisomal matrix location (destination of the Reactome import pathway); the correct mature functional compartment for EHHADH. Accepted. Supporting Evidence: file:human/EHHADH/EHHADH-uniprot.txt SUBCELLULAR LOCATION: Peroxisome
GO:0019899 enzyme binding	IPI PMID:16781659 Molecular organization of peroxisomal enzymes: protein-prote...	KEEP AS NON CORE	Summary: Specific enzyme-binding annotation supported by IPI with catalase (CAT, P04040): yeast two-hybrid, affinity purification and co-immunoprecipitation showed L-bifunctional enzyme binds catalase, proposed to help localize catalase at the peroxisomal H2O2 production site. Biologically plausible peroxisomal-matrix interaction, but a binding activity rather than a catalytic core function; kept as non-core. Supporting Evidence: PMID:16781659 catalase physically interacts with L-bifunctional enzyme (L-BFE)
GO:0005777 peroxisome	IDA PMID:9053548 Immunocytochemical localization of peroxisomal proteins in h...	ACCEPT	Summary: Experimental (IDA) immunocytochemical localization of the peroxisomal beta-oxidation enzymes (including the bifunctional protein) to peroxisomes in human liver. Core localization. Supporting Evidence: PMID:9053548 subcellular localization of peroxisomal proteins (catalase, the three beta-oxidation enzymes
GO:0005777 peroxisome	IDA PMID:2895531 Immunocytochemical demonstration of peroxisomal enzymes in h...	ACCEPT	Summary: Experimental (IDA) immunocytochemistry localizing the bifunctional protein (enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase) to peroxisomes of human kidney proximal tubular epithelial cells. Core localization. Supporting Evidence: PMID:2895531 bifunctional protein (enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase) PMID:2895531 immunoreactive peroxisomes were distinctly visualized in proximal tubular epithelial cells
GO:0005777 peroxisome	IDA PMID:1651711 Import of human bifunctional enzyme into peroxisomes of huma...	ACCEPT	Summary: Experimental (IDA) evidence that the human bifunctional enzyme is imported into peroxisomes via its C-terminal SKL (PTS1) signal; deletion of the last nine residues abolishes import. Core localization. Supporting Evidence: PMID:1651711 SKL, located at the carboxyl-terminus of human bifunctional enzyme appears to be the targeting signal
GO:0003857 (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity	NAS PMID:8188243 cDNA cloning of the human peroxisomal enoyl-CoA hydratase: 3...	ACCEPT	Summary: Author-stated (NAS) from the cDNA-cloning paper identifying the enzyme as the peroxisomal enoyl-CoA hydratase:3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme. Corroborated by later experimental (IDA) evidence for the dehydrogenase activity. Supporting Evidence: PMID:8188243 cDNA cloning of the human peroxisomal enoyl-CoA hydratase: 3-hydroxyacyl-CoA
GO:0006635 fatty acid beta-oxidation	NAS PMID:8188243 cDNA cloning of the human peroxisomal enoyl-CoA hydratase: 3...	ACCEPT	Summary: Author-stated (NAS): the bifunctional enzyme is "one of the four enzymes of the peroxisomal beta-oxidation pathway". Corroborated by experimental (IDA) evidence. Core biological process. Supporting Evidence: PMID:8188243 one of the four enzymes of the peroxisomal beta-oxidation pathway
GO:0004300 enoyl-CoA hydratase activity	NAS PMID:8188243 cDNA cloning of the human peroxisomal enoyl-CoA hydratase: 3...	ACCEPT	Summary: Author-stated (NAS) from the cDNA-cloning paper identifying the enzyme as the peroxisomal enoyl-CoA hydratase:3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme. Corroborated by later experimental (IDA) evidence for the hydratase activity. Supporting Evidence: PMID:8188243 cDNA cloning of the human peroxisomal enoyl-CoA hydratase: 3-hydroxyacyl-CoA
GO:0005777 peroxisome	NAS PMID:8188243 cDNA cloning of the human peroxisomal enoyl-CoA hydratase: 3...	ACCEPT	Summary: Author-stated (NAS): the enzyme carries the C-terminal SKL peroxisomal targeting signal. Corroborated by experimental (IDA) peroxisomal localization. Core localization. Supporting Evidence: PMID:8188243 The tripeptide SKL at the carboxy terminus, known to serve as a peroxisomal targeting signal, is present

Core Functions

N-terminal 2-enoyl-CoA hydratase that catalyzes step 2 of the peroxisomal fatty-acid beta-oxidation spiral, hydrating trans-2-enoyl-CoA to L-(3S)-3-hydroxyacyl-CoA (L stereochemistry, distinguishing it from the D-bifunctional protein HSD17B4). Acts on medium/long-chain and dicarboxylic-acid (omega-oxidation-product) substrates.

Molecular Function:

enoyl-CoA hydratase activity

Directly Involved In:

fatty acid beta-oxidation fatty acid beta-oxidation using acyl-CoA oxidase

Cellular Locations:

peroxisomal matrix

Supporting Evidence:

PMID:15060085
the main enzymes involved in beta-oxidation of C16DCA are SCOX, both LBP and DBP
file:human/EHHADH/EHHADH-uniprot.txt
a (3S)-3-hydroxyacyl-CoA = a (2E)-enoyl-CoA + H2O

C-terminal NAD+-dependent L-3-hydroxyacyl-CoA dehydrogenase that catalyzes step 3 of the peroxisomal beta-oxidation spiral, oxidizing L-(3S)-3-hydroxyacyl-CoA to 3-oxoacyl-CoA with reduction of NAD+ to NADH. The long-chain-specific activity (GO:0016509) is a precise child.

Molecular Function:

(3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity

Directly Involved In:

fatty acid beta-oxidation fatty acid beta-oxidation using acyl-CoA oxidase

Cellular Locations:

peroxisomal matrix

Supporting Evidence:

PMID:15060085
the main enzymes involved in beta-oxidation of C16DCA are SCOX, both LBP and DBP
file:human/EHHADH/EHHADH-uniprot.txt
a 3-oxoacyl-CoA + NADH +

References

GO_REF:0000002

Gene Ontology annotation through association of InterPro records with GO terms

GO_REF:0000024

Manual transfer of experimentally-verified manual GO annotation data to orthologs by curator judgment of sequence similarity

GO_REF:0000033

Annotation inferences using phylogenetic trees

GO_REF:0000107

Automatic transfer of experimentally verified manual GO annotation data to orthologs using Ensembl Compara

GO_REF:0000116

Automatic Gene Ontology annotation based on Rhea mapping

GO_REF:0000120

Combined Automated Annotation using Multiple IEA Methods

PMID:15060085

Identification of the peroxisomal beta-oxidation enzymes involved in the degradation of long-chain dicarboxylic acids.

Using recombinant human L-bifunctional protein (LBP) and patient fibroblasts, shows that peroxisomes (not mitochondria) beta-oxidize C16 dicarboxylic acid and that LBP is one of the main enzymes involved; first demonstration of a specific physiological function for LBP.

"the main enzymes involved in beta-oxidation of C16DCA are SCOX, both LBP and DBP"

PMID:1651711

Import of human bifunctional enzyme into peroxisomes of human hepatoma cells in vitro.

The C-terminal SKL tripeptide is the peroxisomal targeting signal (PTS1) directing import of the human bifunctional enzyme; deletion of the last nine residues prevents import.

"SKL, located at the carboxyl-terminus of human bifunctional enzyme appears to be the targeting signal"

PMID:16781659

Molecular organization of peroxisomal enzymes: protein-protein interactions in the membrane and in the matrix.

Yeast two-hybrid, affinity purification and co-immunoprecipitation show that catalase physically interacts with L-bifunctional enzyme (EHHADH), which may help localize catalase at the peroxisomal H2O2 production site.

"catalase physically interacts with L-bifunctional enzyme (L-BFE)"

PMID:25416956

A proteome-scale map of the human interactome network.

PMID:2895531

Immunocytochemical demonstration of peroxisomal enzymes in human kidney biopsies.

Immunocytochemistry localizes the bifunctional protein (enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase) to peroxisomes of human kidney proximal tubular epithelial cells.

"bifunctional protein (enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase)"

PMID:31515488

Extensive disruption of protein interactions by genetic variants across the allele frequency spectrum in human populations.

PMID:32296183

A reference map of the human binary protein interactome.

PMID:33961781

Dual proteome-scale networks reveal cell-specific remodeling of the human interactome.

PMID:36217029

A proteome-scale map of the SARS-CoV-2-human contactome.

PMID:40205054

Multimodal cell maps as a foundation for structural and functional genomics.

PMID:8188243

cDNA cloning of the human peroxisomal enoyl-CoA hydratase: 3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme and localization to chromosome 3q26.3-3q28: a free left Alu Arm is inserted in the 3' noncoding region.

Reports the full-length human cDNA for the peroxisomal enoyl-CoA hydratase:3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme (chromosome 3q26.3-3q28); the enzyme is one of the four peroxisomal beta-oxidation enzymes, carries a C-terminal SKL PTS1 signal, and is expressed most strongly in liver and kidney.

"one of the four enzymes of the peroxisomal beta-oxidation pathway"

PMID:9053548

Immunocytochemical localization of peroxisomal proteins in human liver and kidney.

Immunocytochemistry localizes the three peroxisomal beta-oxidation enzymes (including the bifunctional protein) to peroxisomes in human liver.

"subcellular localization of peroxisomal proteins (catalase, the three beta-oxidation enzymes"

Reactome:R-HSA-390247

Beta-oxidation of very long chain fatty acids

Reactome:R-HSA-6809263

EHHADH hydrates trans-2,3-dehydrohexacosanoyl-CoA

Reactome:R-HSA-6809264

EHHADH dehydrogenates 3-hydroxyhexacosanoyl-CoA

Reactome:R-HSA-9033235

Cargo of PEX5S,L translocates from the cytosol to the peroxisomal matrix

Reactome:R-HSA-9033236

PEX5S,L:Cargo binds PEX13:PEX14:PEX2:PEX10:PEX12 (Docking and Translocation Module)

Suggested Experiments

Experiment: Quantify accumulation of medium/long-chain dicarboxylic acyl-CoAs and their beta-oxidation flux in EHHADH-null vs HSD17B4-null cells to define the non-redundant substrate niche of LBP.

Experiment: Compare peroxisomal versus mitochondrial localization and downstream metabolic/transport phenotypes of wild-type EHHADH versus the p.E3K FRTS3 variant in proximal tubule cell models.

📚 Additional Documentation

Notes

(EHHADH-notes.md)

EHHADH (Q08426, ECHP_HUMAN) — Gene Review Notes

Identity and overview

EHHADH is the human peroxisomal L-bifunctional protein (LBP; also "peroxisomal
bifunctional enzyme", PBE/PBFE; "multifunctional enzyme 1", MFE1). It is a single
723-residue polypeptide encoded on chromosome 3q26.3-3q28
PMID:8188243.

The protein carries two (arguably three) catalytic activities of the peroxisomal
fatty-acid β-oxidation spiral in one chain:
- an N-terminal 2-enoyl-CoA hydratase / Δ3,Δ2-enoyl-CoA isomerase module
(EC 4.2.1.17; EC 5.3.3.8), and
- a C-terminal L-3-hydroxyacyl-CoA dehydrogenase module (EC 1.1.1.35, NAD+-dependent).

UniProt annotates the domain architecture directly:
REGION 1..282 "Enoyl-CoA hydratase / isomerase" and
REGION 283..572 "3-hydroxyacyl-CoA dehydrogenase"
[file:human/EHHADH/EHHADH-uniprot.txt "REGION 1..282"],
[file:human/EHHADH/EHHADH-uniprot.txt "REGION 283..572"].

UniProt FUNCTION: "Peroxisomal trifunctional enzyme possessing 2-enoyl-CoA
hydratase, 3-hydroxyacyl-CoA dehydrogenase, and delta 3, delta 2-enoyl-CoA isomerase
activities. Catalyzes two of the four reactions of the long chain fatty acids
peroxisomal beta-oxidation pathway"
[file:human/EHHADH/EHHADH-uniprot.txt "Peroxisomal trifunctional enzyme possessing 2-enoyl-CoA"].

Reaction chemistry (steps 2 and 3 of peroxisomal β-oxidation)

The hydratase step: a (3S)-3-hydroxyacyl-CoA = a (2E)-enoyl-CoA + H2O (EC 4.2.1.17,
RHEA:16105) [file:human/EHHADH/EHHADH-uniprot.txt "a (3S)-3-hydroxyacyl-CoA = a (2E)-enoyl-CoA + H2O"].
The dehydrogenase step: a (3S)-3-hydroxyacyl-CoA + NAD(+) = a 3-oxoacyl-CoA + NADH +
H(+) (EC 1.1.1.35, RHEA:22432)
[file:human/EHHADH/EHHADH-uniprot.txt "a 3-oxoacyl-CoA + NADH +"].
Note the L (3S) stereochemistry: EHHADH generates/uses the (3S)-3-hydroxyacyl-CoA
intermediate, in contrast to the D-bifunctional protein HSD17B4, which uses the
opposite chirality: "With HSD17B4, catalyzes the hydration of trans-2-enoyl-CoA and
the dehydrogenation of 3-hydroxyacyl-CoA, but with opposite chiral specificity"
[file:human/EHHADH/EHHADH-uniprot.txt "the dehydrogenation of 3-hydroxyacyl-CoA, but with opposite chiral"].

The enzyme acts on a chain-length range; UniProt lists catalytic activity entries for
C6 (hexanoyl), C10 (decanoyl), C16 (hexadecanoyl) and the C16 dicarboxylic (hexadecanedioyl)
series. Kinetic parameters were measured for the dicarboxylic/long-chain substrates:
KM=0.3 uM for (2E)-hexadecenedioyl-CoA and KM=10.4 uM for (2E)-hexadecenoyl-CoA
[file:human/EHHADH/EHHADH-uniprot.txt "KM=0.3 uM for (2E)-hexadecenedioyl-CoA"].

Direct enzymatic evidence (PMID:15060085, Ferdinandusse et al. 2004)

This is the key experimental paper (abstract-only in cache; full_text_available: false).
Using recombinant human LBP expressed in a yeast fox2 (DBP) deletion mutant plus patient
fibroblast studies, it showed that peroxisomes (not mitochondria) β-oxidize C16
dicarboxylic acid, and that "the main enzymes involved in beta-oxidation of C16DCA are
SCOX, both LBP and DBP, and sterol carrier protein X"
PMID:15060085.
Importantly it states: "This is the first indication of a specific function for LBP,
which has remained elusive until now"
PMID:15060085.
This paper is the IDA source in GOA for enoyl-CoA hydratase activity (GO:0004300),
(3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity (GO:0003857), long-chain (3S)-3-
hydroxyacyl-CoA dehydrogenase (NAD+) activity (GO:0016509), and fatty acid β-oxidation
(GO:0006635). The dicarboxylic-acid/omega-oxidation-product role and the C16DCA KM are
the defining physiological niche distinguishing EHHADH from HSD17B4/DBP.

Subcellular localization

Peroxisomal matrix protein; imported via a C-terminal PTS1 (type-1 peroxisomal
targeting) tripeptide SKL at residues 721-723 (MOTIF 721..723 "Microbody targeting signal") [file:human/EHHADH/EHHADH-uniprot.txt "Microbody targeting signal"].
PMID:1651711 demonstrated PTS1-dependent import: "A tripeptide sequence, SKL, located
at the carboxyl-terminus of human bifunctional enzyme appears to be the targeting
signal for the peroxisomal importation"
PMID:1651711,
and deletion of the last nine residues blocks import
PMID:1651711.
Immunocytochemistry localized the bifunctional protein to peroxisomes of human kidney
proximal tubules PMID:2895531,
PMID:2895531, and of human liver
PMID:9053548.

Tissue expression

Liver and kidney, strongly in terminal proximal tubule segments:
"Liver and kidney. Strongly expressed in the terminal segments of the proximal tubule.
Lower amounts seen in the brain"
[file:human/EHHADH/EHHADH-uniprot.txt "Strongly expressed in the"]. Northern analysis
originally showed highest expression in liver and kidney
PMID:8188243.

Disease

Fanconi renotubular syndrome 3 (FRTS3, MIM:615605), autosomal dominant. The disease
variant is not caused by loss of β-oxidation but by mistargeting of EHHADH: the
p.E3K variant is "mistargeted to mitochondria; results in impaired mitochondrial
oxidative phosphorylation and defects in the transport of fluids across the epithelium
of renal proximal tubular cells"
[file:human/EHHADH/EHHADH-uniprot.txt "mitochondria; results in impaired mitochondrial oxidative"]. UniProt DISEASE:
"generalized dysfunction of the proximal kidney tubule resulting in decreased solute
and water reabsorption ... FRTS3 inheritance is autosomal dominant"
[file:human/EHHADH/EHHADH-uniprot.txt "generalized"]. (Source PMID:24401050, not in cache.)
The enzyme is absent in generalized peroxisome-biogenesis disorders: "Absent in patients
suffering with peroxisomal disorders such as Zellweger syndrome, neonatal
adrenoleukodystrophy and infantile Refsum disease"
[file:human/EHHADH/EHHADH-uniprot.txt "Absent in patients suffering with peroxisomal disorders"].

Regulation / PTM

Enzyme activity is enhanced by acetylation: "Enzyme activity enhanced by acetylation"
[file:human/EHHADH/EHHADH-uniprot.txt "Enzyme activity enhanced by acetylation"];
"Acetylated, leading to enhanced enzyme activity"
[file:human/EHHADH/EHHADH-uniprot.txt "Acetylated, leading to enhanced enzyme activity"]
(sites Lys-165/171/346/584; source PMID:20167786, not in cache). EHHADH is a classic
PPARα target gene (peroxisome-proliferator inducible), consistent with its being one of
the four "classic" inducible peroxisomal β-oxidation enzymes, though that regulatory
detail is not quoted from the UniProt file here.

Interactions

Catalase (CAT, P04040) — enzyme binding, IPI, PMID:16781659. Yeast two-hybrid +
affinity purification + co-IP showed L-bifunctional enzyme interacts with catalase:
"catalase physically interacts with L-bifunctional enzyme (L-BFE)"
PMID:16781659; proposed to help localize catalase at
the site of H2O2 production. This is a biologically plausible peroxisomal-matrix
interaction (catalase detoxifies the H2O2 produced by acyl-CoA oxidase upstream in the
same pathway).
The large set of GO:0005515 "protein binding" IPI annotations
(PMID:25416956, PMID:31515488, PMID:32296183, PMID:33961781, PMID:36217029,
PMID:40205054) derive from high-throughput interactome maps (Y2H HuRI / affinity-MS /
spatial proteomics / SARS-CoV-2 contactome). The IntAct partner lists in the UniProt
entry (AARS2, ACTB, ACTG1, DES, MID1, SSNA1, TPP2, catalase-family, many TRIMs, etc.)
are largely non-peroxisomal and non-specific; these are uninformative for EHHADH's
molecular function and are marked over-annotated (bare "protein binding" is
discouraged in core functions). PMID:36217029 is a SARS-CoV-2 N-protein interaction
(xeno), of no relevance to native EHHADH function.

Core function summary

Two (three) catalytic MFs, all peroxisomal-matrix, all in the fatty-acid β-oxidation
spiral:
1. enoyl-CoA hydratase activity (GO:0004300) — hydrates 2-trans-enoyl-CoA to
L-(3S)-3-hydroxyacyl-CoA (step 2). IDA PMID:15060085.
2. (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity (GO:0003857) — NAD+-dependent
oxidation of L-(3S)-3-hydroxyacyl-CoA to 3-oxoacyl-CoA (step 3). IDA PMID:15060085;
requires NAD+ binding (GO:0070403). The long-chain-specific variant GO:0016509 is a
more precise child (IDA PMID:15060085).
3. Δ3,Δ2-enoyl-CoA isomerase activity (GO:0004165) — auxiliary activity for
unsaturated-fatty-acid oxidation (ISS/IEA, by similarity to rat P07896).

Core BP: fatty acid beta-oxidation (GO:0006635), specifically the peroxisomal
acyl-CoA-oxidase-initiated variant (GO:0033540); physiological niche is degradation of
medium/long-chain dicarboxylic acids (omega-oxidation products) and the branched-chain
substrate 2-methyl-2E-butenoyl-CoA. Core CC: peroxisome (GO:0005777) / peroxisomal
matrix (GO:0005782).

Annotation-review decisions (rationale)

Experimental MF/BP/CC annotations sourced to PMID:15060085 (hydratase, dehydrogenase,
long-chain dehydrogenase, β-oxidation) and the IDA peroxisome-localization annotations
(PMID:9053548, PMID:2895531, PMID:1651711) → ACCEPT (core).
IBA (GO_REF:0000033) for dehydrogenase, peroxisome, β-oxidation → ACCEPT (phylogenetically
well-supported, matches experimental).
ISS/IEA duplicates of the two core activities and peroxisome/β-oxidation → ACCEPT
(consistent with experimental).
GO:0004165 Δ3,Δ2-enoyl-CoA isomerase (ISS/IEA) and GO:0016863 (parent) → KEEP_AS_NON_CORE:
a real auxiliary activity of the N-terminal module (by similarity), but not the defining
core function; supported only by similarity, not human IDA.
GO:0018812 "3-hydroxyacyl-CoA dehydratase activity" (RHEA IEA + Reactome TAS) → MODIFY
to GO:0004300. The RHEA/Reactome mapping labels the hydratase reaction as a "dehydratase";
GO:0004300 (enoyl-CoA hydratase) is the correct, curated term for this enzyme's step-2
activity and is already independently annotated by IDA.
Broad IEA parent terms (GO:0003824 catalytic activity; GO:0016491 oxidoreductase activity;
GO:0016616 oxidoreductase acting on CH-OH … NAD/NADP; GO:0006631 fatty acid metabolic
process) → MARK_AS_OVER_ANNOTATED: correct but uninformatively general; better specific
terms are already present.
GO:0070403 NAD+ binding (IEA:InterPro) → ACCEPT as non-core supporting activity of the
dehydrogenase module.
GO:0005829 cytosol (IEA:Ensembl and Reactome TAS) → MARK_AS_OVER_ANNOTATED: the cytosol
annotations reflect the transient pre-import (PTS1-folded) cargo state in the Reactome
peroxisomal-import pathway; the mature functional enzyme is peroxisomal-matrix, and IDA
evidence localizes it to peroxisomes. Not a genuine steady-state functional location.
GO:0019899 enzyme binding IPI PMID:16781659 (catalase) → KEEP_AS_NON_CORE: a specific,
biologically plausible peroxisomal interaction, but not a molecular function per se.
The IEA (Ensembl-projected) enzyme-binding duplicate → MARK_AS_OVER_ANNOTATED.
All bare GO:0005515 "protein binding" IPI (6 HT-interactome PMIDs) → MARK_AS_OVER_ANNOTATED
(per policy, never REMOVE an IPI); uninformative and dominated by non-peroxisomal HT hits.
NAS annotations (PMID:8188243) for the two activities, β-oxidation, peroxisome → ACCEPT:
author-stated but corroborated by experimental annotations.
Reactome TAS for the two activities and peroxisomal-matrix/cytosol → ACCEPT (activities/matrix)
or MARK_AS_OVER_ANNOTATED (cytosol import-intermediate).

QA re-review — 2026-07-23

Conservative QA pass of EHHADH-ai-review.yaml (50 annotations). No edits made; the
review is biologically and curatorially sound and validates clean.

Validation: ✓ Valid under default, --strict, --verbose, and --terms. No
warnings, no PENDING.
Same-term action consistency: verified by hand across all recurring terms — each has a
single consistent action across evidence types (GO:0003857 ACCEPT×6, GO:0004300 ACCEPT×4,
GO:0006635 ACCEPT×5, GO:0005777 ACCEPT×6, GO:0005515 MARK_AS_OVER_ANNOTATED×6,
GO:0018812 MODIFY×2, GO:0005829 MARK_AS_OVER_ANNOTATED×3, GO:0016863 over-annotated×2,
GO:0004165 KEEP_AS_NON_CORE×2, GO:0019899 KEEP_AS_NON_CORE×2, GO:0005782 ACCEPT×3).
protein binding (GO:0005515): all 6 HT-interactome IPIs are MARK_AS_OVER_ANNOTATED
(none ACCEPT); correct per IPI-never-REMOVE policy.
core_functions: two catalytic MFs (GO:0004300 hydratase, GO:0003857 dehydrogenase) are
captured with specific curated terms, not over-generalized; ids all in correct GO aspect.
Note (left as-is): the GO:0016509 long-chain-dehydrogenase annotations (IEA + IDA) use
the verbatim supporting_text "KM=0.3 uM for (2E)-hexadecenedioyl-CoA", which is an
enoyl-CoA (hydratase-substrate) KM from UniProt rather than a dehydrogenase-substrate
KM. It is verbatim, demonstrates long-chain/dicarboxylic substrate handling, and the
ACCEPT action is independently justified by the PMID:15060085 IDA; UniProt has no
long-chain dehydrogenase-specific KM to substitute, so no change was warranted.

📄 View Raw YAML

id: Q08426
gene_symbol: EHHADH
product_type: PROTEIN
status: INITIALIZED
taxon:
  id: NCBITaxon:9606
  label: Homo sapiens
description: >-
  EHHADH is the peroxisomal L-bifunctional protein (LBP; also peroxisomal bifunctional
  enzyme PBE/PBFE, multifunctional enzyme 1 MFE1), a single 723-residue polypeptide that
  carries two catalytic activities of the peroxisomal fatty-acid beta-oxidation spiral in
  one chain. Its N-terminal module has 2-enoyl-CoA hydratase activity (EC 4.2.1.17) that
  hydrates 2-trans-enoyl-CoA to L-(3S)-3-hydroxyacyl-CoA (step 2), together with an
  auxiliary Delta-3,Delta-2-enoyl-CoA isomerase activity (EC 5.3.3.8) used in unsaturated
  fatty-acid oxidation. Its C-terminal module has NAD+-dependent L-3-hydroxyacyl-CoA
  dehydrogenase activity (EC 1.1.1.35) that oxidizes the (3S)-3-hydroxyacyl-CoA intermediate
  to 3-oxoacyl-CoA (step 3). EHHADH thus performs the second and third reactions of the
  peroxisomal beta-oxidation pathway with L-stereochemistry, in contrast to the D-bifunctional
  protein HSD17B4, which uses the opposite chirality and handles very-long-chain, branched-chain
  and bile-acid substrates. EHHADH is comparatively more important for medium/long-chain
  substrates and for the degradation of dicarboxylic acids (omega-oxidation products). The
  mature enzyme is a peroxisomal-matrix protein imported via a C-terminal PTS1 (SKL) targeting
  signal, and it is expressed most strongly in liver and in the proximal tubule of the kidney.
  A dominant EHHADH variant (p.E3K) that mistargets the protein to mitochondria causes Fanconi
  renotubular syndrome 3.
alternative_products:
- name: '1'
  id: Q08426-1
- name: '2'
  id: Q08426-2
  sequence_note: VSP_042811
existing_annotations:
- term:
    id: GO:0003857
    label: (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  qualifier: enables
  review:
    summary: >-
      Core C-terminal catalytic activity: NAD+-dependent oxidation of L-(3S)-3-hydroxyacyl-CoA
      to 3-oxoacyl-CoA (step 3 of peroxisomal beta-oxidation). Phylogenetically inferred and
      concordant with human experimental (IDA) evidence.
    action: ACCEPT
    supported_by:
    - reference_id: PMID:15060085
      supporting_text: "the main enzymes involved in \nbeta-oxidation of C16DCA are SCOX, both LBP and DBP"
- term:
    id: GO:0005777
    label: peroxisome
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  qualifier: is_active_in
  review:
    summary: >-
      Core localization. EHHADH is a peroxisomal-matrix beta-oxidation enzyme; phylogenetic
      inference matches human IDA localization evidence.
    action: ACCEPT
    supported_by:
    - reference_id: file:human/EHHADH/EHHADH-uniprot.txt
      supporting_text: "SUBCELLULAR LOCATION: Peroxisome"
- term:
    id: GO:0006635
    label: fatty acid beta-oxidation
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  qualifier: involved_in
  review:
    summary: >-
      Core biological process. EHHADH catalyzes steps 2 and 3 of the peroxisomal fatty-acid
      beta-oxidation spiral. Phylogenetically inferred and supported by human IDA evidence.
    action: ACCEPT
    supported_by:
    - reference_id: file:human/EHHADH/EHHADH-uniprot.txt
      supporting_text: "Catalyzes two of the four reactions of the"
- term:
    id: GO:0003824
    label: catalytic activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  qualifier: enables
  review:
    summary: >-
      Root-level catalytic activity term. Correct but uninformatively general; the specific
      hydratase (GO:0004300) and dehydrogenase (GO:0003857) activities are already annotated.
    action: MARK_AS_OVER_ANNOTATED
- term:
    id: GO:0003857
    label: (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  qualifier: enables
  review:
    summary: >-
      IEA (ARBA/RHEA/EC) duplicate of the core C-terminal dehydrogenase activity; consistent
      with human IDA evidence.
    action: ACCEPT
    supported_by:
    - reference_id: PMID:15060085
      supporting_text: "the main enzymes involved in \nbeta-oxidation of C16DCA are SCOX, both LBP and DBP"
- term:
    id: GO:0004165
    label: delta(3)-delta(2)-enoyl-CoA isomerase activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  qualifier: enables
  review:
    summary: >-
      Auxiliary activity of the N-terminal module (isomerizes 3-cis/3-trans double bonds to
      the 2-trans form for continued beta-oxidation of unsaturated fatty acids). Real by
      similarity to rat P07896, but not the defining core function and not supported by human
      experimental evidence; kept as non-core.
    action: KEEP_AS_NON_CORE
    supported_by:
    - reference_id: file:human/EHHADH/EHHADH-uniprot.txt
      supporting_text: "Peroxisomal trifunctional enzyme possessing 2-enoyl-CoA"
- term:
    id: GO:0004300
    label: enoyl-CoA hydratase activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  qualifier: enables
  review:
    summary: >-
      IEA (ARBA/RHEA/EC) duplicate of the core N-terminal hydratase activity; consistent with
      human IDA evidence.
    action: ACCEPT
    supported_by:
    - reference_id: PMID:15060085
      supporting_text: "the main enzymes involved in \nbeta-oxidation of C16DCA are SCOX, both LBP and DBP"
- term:
    id: GO:0005777
    label: peroxisome
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  qualifier: located_in
  review:
    summary: >-
      IEA duplicate of the core peroxisomal localization; consistent with IDA evidence and the
      C-terminal SKL (PTS1) targeting signal.
    action: ACCEPT
    supported_by:
    - reference_id: file:human/EHHADH/EHHADH-uniprot.txt
      supporting_text: "SUBCELLULAR LOCATION: Peroxisome"
- term:
    id: GO:0006631
    label: fatty acid metabolic process
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  qualifier: involved_in
  review:
    summary: >-
      Correct but general parent of the specific process fatty acid beta-oxidation (GO:0006635),
      which is already annotated with experimental evidence.
    action: MARK_AS_OVER_ANNOTATED
- term:
    id: GO:0016491
    label: oxidoreductase activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  qualifier: enables
  review:
    summary: >-
      General parent term for the dehydrogenase activity. Correct but uninformative; the specific
      (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity is already annotated.
    action: MARK_AS_OVER_ANNOTATED
- term:
    id: GO:0016509
    label: long-chain (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000116
  qualifier: enables
  review:
    summary: >-
      Chain-length-specific (long-chain) form of the core C-terminal dehydrogenase activity;
      consistent with the human IDA annotation of the same term and with the measured long-chain
      substrate kinetics.
    action: ACCEPT
    supported_by:
    - reference_id: file:human/EHHADH/EHHADH-uniprot.txt
      supporting_text: "KM=0.3 uM for (2E)-hexadecenedioyl-CoA"
- term:
    id: GO:0016616
    label: oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP
      as acceptor
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  qualifier: enables
  review:
    summary: >-
      Intermediate parent term for the dehydrogenase activity. Correct but more general than the
      specific (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity already annotated.
    action: MARK_AS_OVER_ANNOTATED
- term:
    id: GO:0018812
    label: 3-hydroxyacyl-CoA dehydratase activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000116
  qualifier: enables
  review:
    summary: >-
      This RHEA-mapped "dehydratase" term describes the same step-2 reaction as the curated
      enoyl-CoA hydratase activity (GO:0004300), which is already independently annotated by
      IDA for this enzyme. GO:0004300 is the appropriate curated term for EHHADH's N-terminal
      module; modify to it.
    action: MODIFY
    proposed_replacement_terms:
    - id: GO:0004300
      label: enoyl-CoA hydratase activity
    supported_by:
    - reference_id: file:human/EHHADH/EHHADH-uniprot.txt
      supporting_text: "note=\"Enoyl-CoA hydratase / isomerase\""
- term:
    id: GO:0070403
    label: NAD+ binding
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  qualifier: enables
  review:
    summary: >-
      NAD+ is the required cofactor of the C-terminal (3S)-3-hydroxyacyl-CoA dehydrogenase module
      (NAD(P)-binding Rossmann fold). Correct supporting activity; kept but non-core relative to
      the catalytic activities.
    action: KEEP_AS_NON_CORE
    supported_by:
    - reference_id: file:human/EHHADH/EHHADH-uniprot.txt
      supporting_text: "note=\"3-hydroxyacyl-CoA dehydrogenase\""
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:25416956
  qualifier: enables
  review:
    summary: >-
      Bare "protein binding" from a high-throughput binary interactome map. Uninformative for
      molecular function and dominated by non-peroxisomal HT hits; over-annotated (not removed,
      per policy for IPI).
    action: MARK_AS_OVER_ANNOTATED
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:31515488
  qualifier: enables
  review:
    summary: >-
      Bare "protein binding" from a high-throughput interactome/variant-disruption screen.
      Uninformative for molecular function; over-annotated.
    action: MARK_AS_OVER_ANNOTATED
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:32296183
  qualifier: enables
  review:
    summary: >-
      Bare "protein binding" from a reference binary interactome map (HuRI); large, largely
      non-peroxisomal partner set. Uninformative for molecular function; over-annotated.
    action: MARK_AS_OVER_ANNOTATED
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:33961781
  qualifier: enables
  review:
    summary: >-
      Bare "protein binding" from a proteome-scale affinity-MS interactome map. Uninformative
      for molecular function; over-annotated.
    action: MARK_AS_OVER_ANNOTATED
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:36217029
  qualifier: enables
  review:
    summary: >-
      "Protein binding" from a SARS-CoV-2-human contactome (interaction with viral N protein,
      a xeno interaction). Not relevant to native EHHADH molecular function; over-annotated.
    action: MARK_AS_OVER_ANNOTATED
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:40205054
  qualifier: enables
  review:
    summary: >-
      Bare "protein binding" from a multimodal cell-map/spatial-genomics interactome. Uninformative
      for molecular function; over-annotated.
    action: MARK_AS_OVER_ANNOTATED
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: IEA
  original_reference_id: GO_REF:0000107
  qualifier: located_in
  review:
    summary: >-
      Cytosol annotation reflects only the transient pre-import (PTS1-folded) cargo state before
      peroxisomal translocation. The mature, functional enzyme resides in the peroxisomal matrix
      (IDA evidence). Not a genuine steady-state functional location; over-annotated.
    action: MARK_AS_OVER_ANNOTATED
- term:
    id: GO:0006635
    label: fatty acid beta-oxidation
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  qualifier: involved_in
  review:
    summary: >-
      IEA duplicate of the core fatty acid beta-oxidation process; consistent with human IDA
      evidence.
    action: ACCEPT
    supported_by:
    - reference_id: file:human/EHHADH/EHHADH-uniprot.txt
      supporting_text: "Catalyzes two of the four reactions of the"
- term:
    id: GO:0016863
    label: intramolecular oxidoreductase activity, transposing C=C bonds
  evidence_type: IEA
  original_reference_id: GO_REF:0000107
  qualifier: enables
  review:
    summary: >-
      Parent term of the Delta-3,Delta-2-enoyl-CoA isomerase activity (GO:0004165), the auxiliary
      isomerase function of the N-terminal module. More general than the specific term already
      annotated; over-annotated.
    action: MARK_AS_OVER_ANNOTATED
- term:
    id: GO:0019899
    label: enzyme binding
  evidence_type: IEA
  original_reference_id: GO_REF:0000107
  qualifier: enables
  review:
    summary: >-
      Ensembl-projected copy of the human catalase enzyme-binding annotation (PMID:16781659). The
      underlying interaction (EHHADH with catalase in the peroxisomal matrix) is specific and
      biologically plausible but is a binding activity, not a catalytic core function; kept as
      non-core, consistent with the IPI-supported annotation of the same term.
    action: KEEP_AS_NON_CORE
    supported_by:
    - reference_id: PMID:16781659
      supporting_text: "catalase physically \ninteracts with L-bifunctional enzyme (L-BFE)"
- term:
    id: GO:0033540
    label: fatty acid beta-oxidation using acyl-CoA oxidase
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-390247
  qualifier: involved_in
  review:
    summary: >-
      Precise process term: the peroxisomal (acyl-CoA-oxidase-initiated) variant of fatty-acid
      beta-oxidation in which EHHADH catalyzes the hydratase and dehydrogenase steps. Accurately
      captures the pathway context; accepted.
    action: ACCEPT
    supported_by:
    - reference_id: file:human/EHHADH/EHHADH-uniprot.txt
      supporting_text: "Catalyzes two of the four reactions of the"
- term:
    id: GO:0003857
    label: (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-6809264
  qualifier: enables
  review:
    summary: >-
      Reactome TAS for the core C-terminal dehydrogenase activity (dehydrogenation of
      3-hydroxyhexacosanoyl-CoA); consistent with human IDA evidence.
    action: ACCEPT
    supported_by:
    - reference_id: PMID:15060085
      supporting_text: "the main enzymes involved in \nbeta-oxidation of C16DCA are SCOX, both LBP and DBP"
- term:
    id: GO:0018812
    label: 3-hydroxyacyl-CoA dehydratase activity
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-6809263
  qualifier: enables
  review:
    summary: >-
      Reactome labels EHHADH's step-2 hydration reaction as a "dehydratase" activity. This is the
      same reaction captured by the curated enoyl-CoA hydratase activity (GO:0004300), already
      annotated by IDA. Modify to the curated hydratase term.
    action: MODIFY
    proposed_replacement_terms:
    - id: GO:0004300
      label: enoyl-CoA hydratase activity
    supported_by:
    - reference_id: file:human/EHHADH/EHHADH-uniprot.txt
      supporting_text: "note=\"Enoyl-CoA hydratase / isomerase\""
- term:
    id: GO:0003857
    label: (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity
  evidence_type: ISS
  original_reference_id: GO_REF:0000024
  qualifier: enables
  review:
    summary: >-
      ISS (by similarity to rat P07896) for the core dehydrogenase activity; consistent with human
      IDA evidence.
    action: ACCEPT
    supported_by:
    - reference_id: PMID:15060085
      supporting_text: "the main enzymes involved in \nbeta-oxidation of C16DCA are SCOX, both LBP and DBP"
- term:
    id: GO:0003857
    label: (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity
  evidence_type: IDA
  original_reference_id: PMID:15060085
  qualifier: enables
  review:
    summary: >-
      Primary experimental (IDA) evidence for the core C-terminal NAD+-dependent
      3-hydroxyacyl-CoA dehydrogenase activity, assayed with recombinant human L-bifunctional
      protein. One of the two defining core molecular functions.
    action: ACCEPT
    supported_by:
    - reference_id: PMID:15060085
      supporting_text: "the main enzymes involved in \nbeta-oxidation of C16DCA are SCOX, both LBP and DBP"
    - reference_id: file:human/EHHADH/EHHADH-uniprot.txt
      supporting_text: "a 3-oxoacyl-CoA + NADH +"
- term:
    id: GO:0004300
    label: enoyl-CoA hydratase activity
  evidence_type: ISS
  original_reference_id: GO_REF:0000024
  qualifier: enables
  review:
    summary: >-
      ISS (by similarity to rat P07896) for the core N-terminal hydratase activity; consistent
      with human IDA evidence.
    action: ACCEPT
    supported_by:
    - reference_id: PMID:15060085
      supporting_text: "the main enzymes involved in \nbeta-oxidation of C16DCA are SCOX, both LBP and DBP"
- term:
    id: GO:0004300
    label: enoyl-CoA hydratase activity
  evidence_type: IDA
  original_reference_id: PMID:15060085
  qualifier: enables
  review:
    summary: >-
      Primary experimental (IDA) evidence for the core N-terminal 2-enoyl-CoA hydratase activity
      (hydration of trans-2-enoyl-CoA to L-(3S)-3-hydroxyacyl-CoA), assayed with recombinant human
      L-bifunctional protein. One of the two defining core molecular functions.
    action: ACCEPT
    supported_by:
    - reference_id: PMID:15060085
      supporting_text: "the main enzymes involved in \nbeta-oxidation of C16DCA are SCOX, both LBP and DBP"
    - reference_id: file:human/EHHADH/EHHADH-uniprot.txt
      supporting_text: "a (3S)-3-hydroxyacyl-CoA = a (2E)-enoyl-CoA + H2O"
- term:
    id: GO:0006635
    label: fatty acid beta-oxidation
  evidence_type: IDA
  original_reference_id: PMID:15060085
  qualifier: involved_in
  review:
    summary: >-
      Experimental (IDA) evidence that EHHADH (LBP) participates in peroxisomal beta-oxidation,
      specifically the degradation of long-chain dicarboxylic acids. Core biological process.
    action: ACCEPT
    supported_by:
    - reference_id: PMID:15060085
      supporting_text: "the main enzymes involved in \nbeta-oxidation of C16DCA are SCOX, both LBP and DBP"
- term:
    id: GO:0016509
    label: long-chain (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity
  evidence_type: IDA
  original_reference_id: PMID:15060085
  qualifier: enables
  review:
    summary: >-
      Experimental (IDA) evidence for the long-chain-specific form of the dehydrogenase activity,
      consistent with the measured long-chain/dicarboxylic substrate kinetics. A precise child of
      the core dehydrogenase activity.
    action: ACCEPT
    supported_by:
    - reference_id: file:human/EHHADH/EHHADH-uniprot.txt
      supporting_text: "KM=0.3 uM for (2E)-hexadecenedioyl-CoA"
- term:
    id: GO:0004165
    label: delta(3)-delta(2)-enoyl-CoA isomerase activity
  evidence_type: ISS
  original_reference_id: GO_REF:0000024
  qualifier: enables
  review:
    summary: >-
      Auxiliary isomerase activity of the N-terminal module (by similarity to rat P07896). A real
      activity but not the defining core function; kept as non-core.
    action: KEEP_AS_NON_CORE
    supported_by:
    - reference_id: file:human/EHHADH/EHHADH-uniprot.txt
      supporting_text: "Peroxisomal trifunctional enzyme possessing 2-enoyl-CoA"
- term:
    id: GO:0005777
    label: peroxisome
  evidence_type: ISS
  original_reference_id: GO_REF:0000024
  qualifier: located_in
  review:
    summary: >-
      ISS (by similarity) for peroxisomal localization; consistent with human IDA evidence and the
      C-terminal SKL (PTS1) signal.
    action: ACCEPT
    supported_by:
    - reference_id: file:human/EHHADH/EHHADH-uniprot.txt
      supporting_text: "SUBCELLULAR LOCATION: Peroxisome"
- term:
    id: GO:0006635
    label: fatty acid beta-oxidation
  evidence_type: ISS
  original_reference_id: GO_REF:0000024
  qualifier: involved_in
  review:
    summary: >-
      ISS (by similarity) for the core fatty acid beta-oxidation process; consistent with human IDA
      evidence.
    action: ACCEPT
    supported_by:
    - reference_id: file:human/EHHADH/EHHADH-uniprot.txt
      supporting_text: "Catalyzes two of the four reactions of the"
- term:
    id: GO:0016863
    label: intramolecular oxidoreductase activity, transposing C=C bonds
  evidence_type: ISS
  original_reference_id: GO_REF:0000024
  qualifier: enables
  review:
    summary: >-
      Parent term of the Delta-3,Delta-2-enoyl-CoA isomerase activity (GO:0004165). More general
      than the specific isomerase term already annotated; over-annotated.
    action: MARK_AS_OVER_ANNOTATED
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-9033235
  qualifier: located_in
  review:
    summary: >-
      Cytosol annotation from the Reactome peroxisomal-protein-import pathway, representing the
      transient pre-import cargo state (PEX5-bound). The mature functional enzyme is in the
      peroxisomal matrix; over-annotated as a steady-state functional location.
    action: MARK_AS_OVER_ANNOTATED
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-9033236
  qualifier: located_in
  review:
    summary: >-
      Cytosol annotation from the Reactome peroxisomal-import docking/translocation step; represents
      the transient pre-import cargo state, not a steady-state functional location. Over-annotated.
    action: MARK_AS_OVER_ANNOTATED
- term:
    id: GO:0005782
    label: peroxisomal matrix
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-6809263
  qualifier: located_in
  review:
    summary: >-
      Peroxisomal matrix is the precise functional location of this soluble matrix beta-oxidation
      enzyme; more specific than "peroxisome" and consistent with IDA evidence. Accepted.
    action: ACCEPT
    supported_by:
    - reference_id: file:human/EHHADH/EHHADH-uniprot.txt
      supporting_text: "SUBCELLULAR LOCATION: Peroxisome"
- term:
    id: GO:0005782
    label: peroxisomal matrix
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-6809264
  qualifier: located_in
  review:
    summary: >-
      Peroxisomal matrix location (Reactome), consistent with the enzyme being a soluble matrix
      beta-oxidation protein; precise and supported. Accepted.
    action: ACCEPT
    supported_by:
    - reference_id: file:human/EHHADH/EHHADH-uniprot.txt
      supporting_text: "SUBCELLULAR LOCATION: Peroxisome"
- term:
    id: GO:0005782
    label: peroxisomal matrix
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-9033235
  qualifier: located_in
  review:
    summary: >-
      Peroxisomal matrix location (destination of the Reactome import pathway); the correct mature
      functional compartment for EHHADH. Accepted.
    action: ACCEPT
    supported_by:
    - reference_id: file:human/EHHADH/EHHADH-uniprot.txt
      supporting_text: "SUBCELLULAR LOCATION: Peroxisome"
- term:
    id: GO:0019899
    label: enzyme binding
  evidence_type: IPI
  original_reference_id: PMID:16781659
  qualifier: enables
  review:
    summary: >-
      Specific enzyme-binding annotation supported by IPI with catalase (CAT, P04040): yeast
      two-hybrid, affinity purification and co-immunoprecipitation showed L-bifunctional enzyme
      binds catalase, proposed to help localize catalase at the peroxisomal H2O2 production site.
      Biologically plausible peroxisomal-matrix interaction, but a binding activity rather than a
      catalytic core function; kept as non-core.
    action: KEEP_AS_NON_CORE
    supported_by:
    - reference_id: PMID:16781659
      supporting_text: "catalase physically \ninteracts with L-bifunctional enzyme (L-BFE)"
- term:
    id: GO:0005777
    label: peroxisome
  evidence_type: IDA
  original_reference_id: PMID:9053548
  qualifier: located_in
  review:
    summary: >-
      Experimental (IDA) immunocytochemical localization of the peroxisomal beta-oxidation enzymes
      (including the bifunctional protein) to peroxisomes in human liver. Core localization.
    action: ACCEPT
    supported_by:
    - reference_id: PMID:9053548
      supporting_text: "subcellular localization of peroxisomal proteins (catalase, the \nthree beta-oxidation enzymes"
- term:
    id: GO:0005777
    label: peroxisome
  evidence_type: IDA
  original_reference_id: PMID:2895531
  qualifier: located_in
  review:
    summary: >-
      Experimental (IDA) immunocytochemistry localizing the bifunctional protein (enoyl-CoA
      hydratase, 3-hydroxyacyl-CoA dehydrogenase) to peroxisomes of human kidney proximal tubular
      epithelial cells. Core localization.
    action: ACCEPT
    supported_by:
    - reference_id: PMID:2895531
      supporting_text: "bifunctional protein \n(enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase)"
    - reference_id: PMID:2895531
      supporting_text: "immunoreactive peroxisomes were \ndistinctly visualized in proximal tubular epithelial cells"
- term:
    id: GO:0005777
    label: peroxisome
  evidence_type: IDA
  original_reference_id: PMID:1651711
  qualifier: located_in
  review:
    summary: >-
      Experimental (IDA) evidence that the human bifunctional enzyme is imported into peroxisomes
      via its C-terminal SKL (PTS1) signal; deletion of the last nine residues abolishes import.
      Core localization.
    action: ACCEPT
    supported_by:
    - reference_id: PMID:1651711
      supporting_text: "SKL, located at the carboxyl-terminus of human bifunctional \nenzyme appears to be the targeting signal"
- term:
    id: GO:0003857
    label: (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity
  evidence_type: NAS
  original_reference_id: PMID:8188243
  qualifier: enables
  review:
    summary: >-
      Author-stated (NAS) from the cDNA-cloning paper identifying the enzyme as the peroxisomal
      enoyl-CoA hydratase:3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme. Corroborated by
      later experimental (IDA) evidence for the dehydrogenase activity.
    action: ACCEPT
    supported_by:
    - reference_id: PMID:8188243
      supporting_text: "cDNA cloning of the human peroxisomal enoyl-CoA hydratase: 3-hydroxyacyl-CoA"
- term:
    id: GO:0006635
    label: fatty acid beta-oxidation
  evidence_type: NAS
  original_reference_id: PMID:8188243
  qualifier: involved_in
  review:
    summary: >-
      Author-stated (NAS): the bifunctional enzyme is "one of the four enzymes of the peroxisomal
      beta-oxidation pathway". Corroborated by experimental (IDA) evidence. Core biological process.
    action: ACCEPT
    supported_by:
    - reference_id: PMID:8188243
      supporting_text: "one \nof the four enzymes of the peroxisomal beta-oxidation pathway"
- term:
    id: GO:0004300
    label: enoyl-CoA hydratase activity
  evidence_type: NAS
  original_reference_id: PMID:8188243
  qualifier: enables
  review:
    summary: >-
      Author-stated (NAS) from the cDNA-cloning paper identifying the enzyme as the peroxisomal
      enoyl-CoA hydratase:3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme. Corroborated by later
      experimental (IDA) evidence for the hydratase activity.
    action: ACCEPT
    supported_by:
    - reference_id: PMID:8188243
      supporting_text: "cDNA cloning of the human peroxisomal enoyl-CoA hydratase: 3-hydroxyacyl-CoA"
- term:
    id: GO:0005777
    label: peroxisome
  evidence_type: NAS
  original_reference_id: PMID:8188243
  qualifier: located_in
  review:
    summary: >-
      Author-stated (NAS): the enzyme carries the C-terminal SKL peroxisomal targeting signal.
      Corroborated by experimental (IDA) peroxisomal localization. Core localization.
    action: ACCEPT
    supported_by:
    - reference_id: PMID:8188243
      supporting_text: "The tripeptide SKL at the carboxy \nterminus, known to serve as a peroxisomal targeting signal, is present"
core_functions:
- description: >-
    N-terminal 2-enoyl-CoA hydratase that catalyzes step 2 of the peroxisomal fatty-acid
    beta-oxidation spiral, hydrating trans-2-enoyl-CoA to L-(3S)-3-hydroxyacyl-CoA (L
    stereochemistry, distinguishing it from the D-bifunctional protein HSD17B4). Acts on
    medium/long-chain and dicarboxylic-acid (omega-oxidation-product) substrates.
  supported_by:
  - reference_id: PMID:15060085
    supporting_text: "the main enzymes involved in \nbeta-oxidation of C16DCA are SCOX, both LBP and DBP"
  - reference_id: file:human/EHHADH/EHHADH-uniprot.txt
    supporting_text: "a (3S)-3-hydroxyacyl-CoA = a (2E)-enoyl-CoA + H2O"
  molecular_function:
    id: GO:0004300
    label: enoyl-CoA hydratase activity
  directly_involved_in:
  - id: GO:0006635
    label: fatty acid beta-oxidation
  - id: GO:0033540
    label: fatty acid beta-oxidation using acyl-CoA oxidase
  locations:
  - id: GO:0005782
    label: peroxisomal matrix
- description: >-
    C-terminal NAD+-dependent L-3-hydroxyacyl-CoA dehydrogenase that catalyzes step 3 of the
    peroxisomal beta-oxidation spiral, oxidizing L-(3S)-3-hydroxyacyl-CoA to 3-oxoacyl-CoA with
    reduction of NAD+ to NADH. The long-chain-specific activity (GO:0016509) is a precise child.
  supported_by:
  - reference_id: PMID:15060085
    supporting_text: "the main enzymes involved in \nbeta-oxidation of C16DCA are SCOX, both LBP and DBP"
  - reference_id: file:human/EHHADH/EHHADH-uniprot.txt
    supporting_text: "a 3-oxoacyl-CoA + NADH +"
  molecular_function:
    id: GO:0003857
    label: (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity
  directly_involved_in:
  - id: GO:0006635
    label: fatty acid beta-oxidation
  - id: GO:0033540
    label: fatty acid beta-oxidation using acyl-CoA oxidase
  locations:
  - id: GO:0005782
    label: peroxisomal matrix
proposed_new_terms: []
suggested_questions:
- question: >-
    What is the physiological substrate spectrum that distinguishes EHHADH (LBP) from HSD17B4
    (DBP) in vivo, given that both can catalyze the peroxisomal hydratase/dehydrogenase steps
    with opposite chirality?
- question: >-
    By what mechanism does the FRTS3 p.E3K mistargeting of EHHADH to mitochondria impair
    mitochondrial oxidative phosphorylation and proximal-tubule solute transport, if the enzyme's
    peroxisomal catalytic function is retained?
suggested_experiments:
- description: >-
    Quantify accumulation of medium/long-chain dicarboxylic acyl-CoAs and their beta-oxidation
    flux in EHHADH-null vs HSD17B4-null cells to define the non-redundant substrate niche of LBP.
- description: >-
    Compare peroxisomal versus mitochondrial localization and downstream metabolic/transport
    phenotypes of wild-type EHHADH versus the p.E3K FRTS3 variant in proximal tubule cell models.
references:
- id: GO_REF:0000002
  title: Gene Ontology annotation through association of InterPro records with GO
    terms
  findings: []
- id: GO_REF:0000024
  title: Manual transfer of experimentally-verified manual GO annotation data to orthologs
    by curator judgment of sequence similarity
  findings: []
- id: GO_REF:0000033
  title: Annotation inferences using phylogenetic trees
  findings: []
- id: GO_REF:0000107
  title: Automatic transfer of experimentally verified manual GO annotation data to
    orthologs using Ensembl Compara
  findings: []
- id: GO_REF:0000116
  title: Automatic Gene Ontology annotation based on Rhea mapping
  findings: []
- id: GO_REF:0000120
  title: Combined Automated Annotation using Multiple IEA Methods
  findings: []
- id: PMID:15060085
  title: Identification of the peroxisomal beta-oxidation enzymes involved in the
    degradation of long-chain dicarboxylic acids.
  findings:
  - statement: >-
      Using recombinant human L-bifunctional protein (LBP) and patient fibroblasts, shows that
      peroxisomes (not mitochondria) beta-oxidize C16 dicarboxylic acid and that LBP is one of
      the main enzymes involved; first demonstration of a specific physiological function for LBP.
    reference_section_type: ABSTRACT
    supporting_text: "the main enzymes involved in \nbeta-oxidation of C16DCA are SCOX, both LBP and DBP"
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      Abstract-only in cache (full_text_available: false). PMID/title verified against PubMed;
      it is the GOA IDA source for EHHADH's hydratase and dehydrogenase activities and for its
      dicarboxylic-acid beta-oxidation role. Anchored by the GOA IDA evidence code.
- id: PMID:1651711
  title: Import of human bifunctional enzyme into peroxisomes of human hepatoma cells
    in vitro.
  findings:
  - statement: >-
      The C-terminal SKL tripeptide is the peroxisomal targeting signal (PTS1) directing import
      of the human bifunctional enzyme; deletion of the last nine residues prevents import.
    reference_section_type: ABSTRACT
    supporting_text: "SKL, located at the carboxyl-terminus of human bifunctional \nenzyme appears to be the targeting signal"
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: Abstract-only; GOA IDA source for peroxisomal localization. Verified against PubMed.
- id: PMID:16781659
  title: 'Molecular organization of peroxisomal enzymes: protein-protein interactions
    in the membrane and in the matrix.'
  findings:
  - statement: >-
      Yeast two-hybrid, affinity purification and co-immunoprecipitation show that catalase
      physically interacts with L-bifunctional enzyme (EHHADH), which may help localize catalase
      at the peroxisomal H2O2 production site.
    reference_section_type: ABSTRACT
    supporting_text: "catalase physically \ninteracts with L-bifunctional enzyme (L-BFE)"
  reference_review:
    relevance: MEDIUM
    correctness: VERIFIED
    review_notes: Abstract-only; supports the enzyme-binding (catalase) IPI annotation. Verified against PubMed.
- id: PMID:25416956
  title: A proteome-scale map of the human interactome network.
  findings: []
  reference_review:
    relevance: LOW
    correctness: VERIFIED
    review_notes: >-
      High-throughput binary interactome map; source of bare "protein binding" (GO:0005515) IPI.
      Uninformative for EHHADH molecular function.
- id: PMID:2895531
  title: Immunocytochemical demonstration of peroxisomal enzymes in human kidney biopsies.
  findings:
  - statement: >-
      Immunocytochemistry localizes the bifunctional protein (enoyl-CoA hydratase,
      3-hydroxyacyl-CoA dehydrogenase) to peroxisomes of human kidney proximal tubular epithelial
      cells.
    reference_section_type: ABSTRACT
    supporting_text: "bifunctional protein \n(enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase)"
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: Abstract-only; GOA IDA source for peroxisomal (kidney proximal tubule) localization.
- id: PMID:31515488
  title: Extensive disruption of protein interactions by genetic variants across the
    allele frequency spectrum in human populations.
  findings: []
  reference_review:
    relevance: LOW
    correctness: VERIFIED
    review_notes: High-throughput interactome/variant screen; source of bare "protein binding" IPI. Uninformative.
- id: PMID:32296183
  title: A reference map of the human binary protein interactome.
  findings: []
  reference_review:
    relevance: LOW
    correctness: VERIFIED
    review_notes: HuRI reference binary interactome; source of bare "protein binding" IPI with a large, mostly non-peroxisomal partner set.
- id: PMID:33961781
  title: Dual proteome-scale networks reveal cell-specific remodeling of the human
    interactome.
  findings: []
  reference_review:
    relevance: LOW
    correctness: VERIFIED
    review_notes: Proteome-scale affinity-MS interactome; source of bare "protein binding" IPI. Uninformative.
- id: PMID:36217029
  title: A proteome-scale map of the SARS-CoV-2-human contactome.
  findings: []
  reference_review:
    relevance: NONE
    correctness: VERIFIED
    review_notes: >-
      SARS-CoV-2-human contactome; "protein binding" IPI reflects interaction with the viral N
      protein (xeno), not relevant to native EHHADH function.
- id: PMID:40205054
  title: Multimodal cell maps as a foundation for structural and functional genomics.
  findings: []
  reference_review:
    relevance: LOW
    correctness: VERIFIED
    review_notes: Multimodal cell-map/spatial-genomics interactome; source of bare "protein binding" IPI. Uninformative.
- id: PMID:8188243
  title: 'cDNA cloning of the human peroxisomal enoyl-CoA hydratase: 3-hydroxyacyl-CoA
    dehydrogenase bifunctional enzyme and localization to chromosome 3q26.3-3q28:
    a free left Alu Arm is inserted in the 3'' noncoding region.'
  findings:
  - statement: >-
      Reports the full-length human cDNA for the peroxisomal enoyl-CoA hydratase:3-hydroxyacyl-CoA
      dehydrogenase bifunctional enzyme (chromosome 3q26.3-3q28); the enzyme is one of the four
      peroxisomal beta-oxidation enzymes, carries a C-terminal SKL PTS1 signal, and is expressed
      most strongly in liver and kidney.
    reference_section_type: ABSTRACT
    supporting_text: "one \nof the four enzymes of the peroxisomal beta-oxidation pathway"
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: Abstract-only; cDNA-cloning paper, GOA NAS source for the two activities, beta-oxidation and localization.
- id: PMID:9053548
  title: Immunocytochemical localization of peroxisomal proteins in human liver and
    kidney.
  findings:
  - statement: >-
      Immunocytochemistry localizes the three peroxisomal beta-oxidation enzymes (including the
      bifunctional protein) to peroxisomes in human liver.
    reference_section_type: ABSTRACT
    supporting_text: "subcellular localization of peroxisomal proteins (catalase, the \nthree beta-oxidation enzymes"
  reference_review:
    relevance: MEDIUM
    correctness: VERIFIED
    review_notes: Abstract-only; GOA IDA source for peroxisomal (liver) localization.
- id: Reactome:R-HSA-390247
  title: Beta-oxidation of very long chain fatty acids
  findings: []
- id: Reactome:R-HSA-6809263
  title: EHHADH hydrates trans-2,3-dehydrohexacosanoyl-CoA
  findings: []
- id: Reactome:R-HSA-6809264
  title: EHHADH dehydrogenates 3-hydroxyhexacosanoyl-CoA
  findings: []
- id: Reactome:R-HSA-9033235
  title: Cargo of PEX5S,L translocates from the cytosol to the peroxisomal matrix
  findings: []
- id: Reactome:R-HSA-9033236
  title: PEX5S,L:Cargo binds PEX13:PEX14:PEX2:PEX10:PEX12 (Docking and Translocation
    Module)
  findings: []

EHHADH

Gene Description