AUH is a mitochondrial-matrix enzyme of the enoyl-CoA hydratase/isomerase (crotonase) superfamily that assembles as a homohexamer. Its principal catalytic role is 3-methylglutaconyl-CoA hydratase activity (EC 4.2.1.18): it catalyzes the fifth step of the L-leucine degradation pathway, the reversible hydration of (E)-3-methylglutaconyl-CoA to (S)-3-hydroxy-3- methylglutaryl-CoA (HMG-CoA), with the hydration direction favored physiologically; the product HMG-CoA is then cleaved to acetyl-CoA and acetoacetate. AUH is the major human 3-methylglutaconyl-CoA hydratase, and loss of its activity causes the autosomal-recessive inborn error of leucine metabolism 3-methylglutaconic aciduria type I (MGCA1). The protein is synthesized as a precursor with an N-terminal mitochondrial transit peptide that is cleaved on import. AUH is also a genuinely bifunctional (moonlighting) protein: it was originally identified as an AU-rich element (ARE) RNA-binding protein that binds specifically to AU-rich sequences in the 3' untranslated regions of short-lived mRNAs (e.g. cytokine and proto-oncogene transcripts such as IL-3, GM-CSF, c-fos and c-myc), a function localized to a distinct region of the polypeptide separate from the catalytic site. In addition to its leucine-pathway role, the crotonase fold gives AUH broad in vitro activity on related short acyl-CoA substrates and an inferred itaconyl-CoA hydratase activity that may contribute to detoxification of macrophage-derived itaconate via the C5-dicarboxylate catabolism pathway.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005739 mitochondrion | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic (IBA) assignment of mitochondrial localization. AUH is a well-established mitochondrial-matrix enzyme with an N-terminal transit peptide; this is correct though less specific than the matrix annotation. Reason: Localization is consistent across orthologs and experimental data. Correct, but see the mitochondrial matrix (GO:0005759) annotation for the more precise compartment. Supporting Evidence: file:human/AUH/AUH-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion |
| GO:0004300 enoyl-CoA hydratase activity | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: Family-level (crotonase / enoyl-CoA hydratase) molecular function assigned by phylogeny. AUH does have measurable enoyl-CoA hydratase activity, but its physiological, specific activity is 3-methylglutaconyl-CoA hydratase. Reason: This is the superfamily-level activity; the historically observed enoyl-CoA hydratase activity of AUH is weak relative to its physiological 3-methylglutaconyl-CoA hydratase activity (GO:0004490). Retained as a correct but less-precise family annotation rather than as a core function. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Supporting Evidence: PMID:7892223 Sequence analysis revealed an unexpected homology to enoyl-CoA hydratase (EC 4.2.1.17), and the recombinant protein showed a low degree of the enzymatic activity. |
| GO:0004490 methylglutaconyl-CoA hydratase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic assignment of the core, specific molecular function of AUH: 3-methylglutaconyl-CoA hydratase activity in leucine degradation. Reason: This is the physiological catalytic function of AUH, concordant with direct biochemical (IDA) and disease evidence. Represents a core function. Supporting Evidence: PMID:16640564 giving strong evidence that the AUH gene encodes for the major human 3-MG-CoA hydratase in leucine degradation. |
| GO:0006635 fatty acid beta-oxidation | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: Phylogenetic assignment of a fatty-acid beta-oxidation role, propagated at the crotonase-superfamily level. AUH's physiological role is leucine catabolism, not fatty-acid beta-oxidation. Reason: AUH belongs to the enoyl-CoA hydratase/isomerase superfamily, many members of which act in fatty-acid beta-oxidation, so the IBA is propagated from FAO-acting relatives (the with/from includes P30084 ECHS1-type orthologs). The characterized physiological role of AUH is the leucine-degradation step 3-methylglutaconyl-CoA -> HMG-CoA, and there is no evidence AUH performs the enoyl-CoA hydratase step of mitochondrial fatty-acid beta-oxidation in vivo. This is a family-level over-propagation rather than a demonstrably wrong inference, so it is flagged rather than removed. Propagation Review Root cause: PROPAGATION BAD Failure modes: FUNCTIONAL DIVERGENCE Supporting Evidence: file:human/AUH/AUH-uniprot.txt Catalyzes the fifth step in the leucine degradation pathway, the reversible hydration of 3-methylglutaconyl-CoA (3-MG-CoA) to 3- hydroxy-3-methylglutaryl-CoA (HMG-CoA) |
| GO:0003723 RNA binding | IEA GO_REF:0000117 | ACCEPT | Summary: Electronic (ARBA) assignment of RNA binding. This is the parent of the more specific, experimentally supported mRNA 3'-UTR (AU-rich element) binding of AUH. Reason: AUH is a bona fide RNA-binding protein (originally identified as an AU-specific RNA-binding protein). The electronic RNA-binding term is correct though less specific than GO:0003730 (mRNA 3'-UTR binding). Supporting Evidence: PMID:7892223 In vitro, the recombinant protein bound specifically to AU-rich transcripts |
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: Root-level catalytic-activity term assigned electronically from an InterPro crotonase domain. Uninformative given the specific hydratase annotations. Reason: GO:0003824 is the most general molecular-function catalytic term and adds no information beyond the specific methylglutaconyl-CoA hydratase activity (GO:0004490). Retained but flagged as an over-general InterPro-to-GO mapping. Supporting Evidence: file:human/AUH/AUH-uniprot.txt CDD; cd06558; crotonase-like; 1. |
| GO:0004300 enoyl-CoA hydratase activity | IEA GO_REF:0000117 | MARK AS OVER ANNOTATED | Summary: Electronic (ARBA) assignment of the family-level enoyl-CoA hydratase activity; duplicate in aspect of the IBA enoyl-CoA hydratase annotation. Reason: Correct at the superfamily level but less precise than the physiological 3-methylglutaconyl-CoA hydratase activity; flagged as a family-level over-annotation for the same reasons as the IBA enoyl-CoA hydratase term. Supporting Evidence: PMID:7892223 Sequence analysis revealed an unexpected homology to enoyl-CoA hydratase (EC 4.2.1.17), and the recombinant protein showed a low degree of the enzymatic activity. |
| GO:0004490 methylglutaconyl-CoA hydratase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic assignment (RHEA:21536 / EC 4.2.1.18) of the core specific molecular function, mirroring the experimental IDA and IBA annotations. Reason: Correctly captures the physiological 3-methylglutaconyl-CoA hydratase activity (EC 4.2.1.18) and matches the UniProt catalytic-activity record. Supporting Evidence: file:human/AUH/AUH-uniprot.txt Reaction=(3S)-3-hydroxy-3-methylglutaryl-CoA = 3-methyl-(2E)- |
| GO:0005739 mitochondrion | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic assignment of mitochondrial localization (UniProt SubCell SL-0173). Correct. Reason: Consistent with the transit peptide, experimental proteomics, and Reactome; the mitochondrial matrix annotation (GO:0005759) is the more specific form. Supporting Evidence: file:human/AUH/AUH-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion |
| GO:0009083 branched-chain amino acid catabolic process | IEA GO_REF:0000117 | ACCEPT | Summary: Electronic (ARBA) assignment of branched-chain amino acid catabolism, the parent process of leucine (a branched-chain amino acid) degradation. Reason: Leucine is a branched-chain amino acid, so AUH's leucine-degradation role is correctly a branched-chain amino acid catabolic process. Correct but less specific than L-leucine catabolic process (GO:0006552). Supporting Evidence: file:human/AUH/AUH-uniprot.txt Catalyzes the fifth step in the leucine degradation pathway |
| GO:0050011 itaconyl-CoA hydratase activity | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: Electronic assignment (RHEA:13785 / EC 4.2.1.56) of itaconyl-CoA hydratase activity, a secondary crotonase-family activity attributed to AUH in itaconate detoxification. Reason: AUH is reported to catalyze the reversible hydration between itaconyl-CoA and citramalyl-CoA in the C5-dicarboxylate pathway. This is a genuine but secondary/context-dependent activity (evidence is inferential, ECO:0000303), not the core physiological function; kept as non-core. Supporting Evidence: PMID:29056341 itaconyl-CoA and citramalyl-CoA via the previously reported reversible hydration |
| GO:0170035 obsolete L-amino acid catabolic process | IEA GO_REF:0000117 | REMOVE | Summary: Electronic (ARBA) assignment to a GO term that is now obsolete. Reason: GO:0170035 is an obsolete term (verified via QuickGO: isObsolete=true). Obsolete electronic annotations should not be retained; the substantive biology (leucine/branched-chain amino acid catabolism) is captured by GO:0006552 and GO:0009083. Supporting Evidence: file:human/AUH/AUH-uniprot.txt Catalyzes the fifth step in the leucine degradation pathway |
| GO:0006552 L-leucine catabolic process | IEA GO_REF:0000041 | ACCEPT | Summary: UniPathway-derived electronic assignment of L-leucine catabolism, the core biological process for AUH. Reason: Matches the experimentally supported (IMP) leucine-catabolism role and the UniProt pathway record (L-leucine degradation, step 3/3 to HMG-CoA). Core biological process. Supporting Evidence: file:human/AUH/AUH-uniprot.txt PATHWAY: Amino-acid degradation; L-leucine degradation |
| GO:0005759 mitochondrial matrix | TAS Reactome:R-HSA-9914271 | ACCEPT | Summary: Reactome-asserted mitochondrial matrix localization, the specific subcompartment where the soluble leucine-degradation enzymes act. Reason: AUH is a soluble matrix enzyme (imported via a cleaved transit peptide); the matrix is the correct, specific compartment and represents the core location. Supporting Evidence: file:human/AUH/AUH-uniprot.txt TRANSIT 1..67 |
| GO:0050011 itaconyl-CoA hydratase activity | TAS PMID:29056341 The Human Knockout Gene CLYBL Connects Itaconate to Vitamin ... | KEEP AS NON CORE | Summary: Author-stated (TAS) itaconyl-CoA hydratase activity: AUH catalyzes the reversible hydration converting itaconyl-CoA to citramalyl-CoA in the C5-dicarboxylate / itaconate-detoxification pathway. Reason: The itaconate paper attributes the itaconyl-CoA <-> citramalyl-CoA hydration to methylglutaconyl-CoA hydratase (AUH). This is a real but secondary activity of the crotonase fold, distinct from the core leucine-pathway role; kept as non-core. Supporting Evidence: PMID:29056341 reaction catalyzed by methylglutaconyl-CoA hydratase (AUH) |
| GO:0110052 toxic metabolite repair | TAS PMID:29056341 The Human Knockout Gene CLYBL Connects Itaconate to Vitamin ... | KEEP AS NON CORE | Summary: Author-stated (TAS) involvement in toxic-metabolite repair: the C5-dicarboxylate pathway (in which AUH's itaconyl-CoA hydratase activity participates) detoxifies macrophage-derived itaconate, a B12-poisoning metabolite. Reason: This process annotation is tied to AUH's secondary itaconyl-CoA hydratase activity in itaconate detoxification, not to its core leucine-degradation role; retained as non-core. Supporting Evidence: PMID:29056341 itaconate, an anti-microbial metabolite |
| GO:0006552 L-leucine catabolic process | IMP PMID:16640564 Biochemical characterization of human 3-methylglutaconyl-CoA... | ACCEPT | Summary: Experimental (IMP) evidence that AUH functions in L-leucine catabolism: the disease-associated A240V variant produces an enzyme with only ~9% of wild-type 3-methylglutaconyl-CoA hydratase activity, linking loss of AUH function to a leucine-degradation defect (MGCA1). Reason: Direct genotype-phenotype and enzymatic evidence place AUH in the leucine degradation pathway; this is a core biological process. The curator had the full text linking the MGCA1 mutation to reduced hydratase activity. Supporting Evidence: PMID:16640564 MGA1 is caused by reduced or absent 3-methylglutaconyl-coenzyme A (3-MG-CoA) hydratase activity within the leucine degradation pathway. |
| GO:0005739 mitochondrion | HTP PMID:34800366 Quantitative high-confidence human mitochondrial proteome an... | ACCEPT | Summary: High-throughput proteomics (HTP) detection of AUH in the human mitochondrial proteome. Reason: Consistent with all other localization evidence (transit peptide, Reactome, IBA/IEA). Mitochondrial localization is well established; matrix (GO:0005759) is the more specific compartment. Supporting Evidence: file:human/AUH/AUH-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion |
| GO:0004490 methylglutaconyl-CoA hydratase activity | IDA PMID:16640564 Biochemical characterization of human 3-methylglutaconyl-CoA... | ACCEPT | Summary: Direct biochemical assay (IDA) of purified AUH gene product demonstrating 3-methylglutaconyl-CoA hydratase activity with (E)-3-MG-CoA as the best substrate. This is the strongest evidence for the core molecular function. Reason: Purified recombinant AUH shows robust hydratase activity on (E)-3-MG-CoA (Vmax 3.9 U/mg, Km 8.3 uM), establishing it as the major human 3-methylglutaconyl-CoA hydratase. Core molecular function. Supporting Evidence: PMID:16640564 The best substrates were (E)-3-MG-CoA (V(max) = 3.9 U.mg(-1), K(m) = 8.3 microM, k(cat) = 5.1 s(-1)) |
| GO:0005759 mitochondrial matrix | TAS Reactome:R-HSA-70785 | ACCEPT | Summary: Reactome-asserted mitochondrial matrix localization associated with the 3-methylglutaconyl-CoA hydration reaction. Reason: Duplicate-in-aspect of the other matrix annotation; correct and specific compartment for this soluble matrix enzyme. Supporting Evidence: file:human/AUH/AUH-uniprot.txt TRANSIT 1..67 |
| GO:0003730 mRNA 3'-UTR binding | IDA PMID:7892223 AUH, a gene encoding an AU-specific RNA binding protein with... | ACCEPT | Summary: Direct experimental evidence (IDA) that AUH binds AU-rich elements in mRNA 3' untranslated regions: it was affinity-purified on an AUUUA matrix and the recombinant protein bound specifically to AU-rich 3'UTR transcripts (IL-3, GM-CSF, c-fos, c-myc). This is the moonlighting RNA-binding function. Reason: Establishes AUH's second, genuine molecular function as an AU-rich element (ARE) RNA-binding protein, on a domain distinct from the catalytic site. Represents a core molecular function separate from the hydratase activity. Supporting Evidence: PMID:7892223 AU-rich elements within the 3' untranslated region of transcripts of lymphokines and some protooncogenes serve as signal for rapid mRNA degradation. PMID:7892223 In vitro, the recombinant protein bound specifically to AU-rich transcripts, including those for interleukin 3, granulocyte/macrophage colony-stimulating factor, c-fos, and c-myc. |
| GO:0004300 enoyl-CoA hydratase activity | IDA PMID:7892223 AUH, a gene encoding an AU-specific RNA binding protein with... | MARK AS OVER ANNOTATED | Summary: Direct assay (IDA) showing that recombinant AUH has enoyl-CoA hydratase activity, though only a low degree. Historically this was the first demonstration of the catalytic (crotonase-family) activity of AUH. Reason: The observed enoyl-CoA hydratase activity is the family-level activity and is weak; the physiologically relevant, specific activity is 3-methylglutaconyl-CoA hydratase (GO:0004490). Retained as a correct but less-precise molecular-function annotation rather than a core function. Supporting Evidence: PMID:7892223 the recombinant protein showed a low degree of the enzymatic activity |
| GO:0005739 mitochondrion | ISS GO_REF:0000024 | ACCEPT | Summary: Sequence-similarity (ISS) transfer of mitochondrial localization from the rat ortholog (Q62651). Correct. Reason: Concordant with transit peptide, proteomics, Reactome and phylogenetic evidence for mitochondrial localization. Supporting Evidence: file:human/AUH/AUH-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion |
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Download this section (compressed HTML)Q: Is the AU-rich element (ARE) RNA-binding activity of AUH functional in the mitochondrial matrix, or does a cytosolic/precursor pool of AUH mediate mRNA regulation? How is the moonlighting RNA-binding role coordinated with the matrix-localized enzymatic role?
Q: Does AUH contribute physiologically to itaconate detoxification (itaconyl-CoA hydratase activity) in human macrophages in vivo, and is this activity relevant to the clinical phenotype of MGCA1 patients?
Experiment: Quantify AUH-dependent ARE-mediated mRNA turnover in cells depleted of AUH versus rescued with hydratase-dead or RNA-binding-dead (K105/K109/K113) mutants, to dissect the moonlighting RNA-binding function from catalysis.
Experiment: Assay itaconyl-CoA hydratase activity of purified human AUH and measure citramalyl-CoA flux in AUH-knockout macrophages challenged with itaconate to test the proposed role in C5-dicarboxylate / itaconate detoxification.
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