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.
| 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 |
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Download this section (compressed HTML)Q: 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?
Q: 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?
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.
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