Echs1 (CG6543; FlyBase FBgn0033879) is the Drosophila melanogaster ortholog of human ECHS1, a short-chain enoyl-CoA hydratase of the crotonase superfamily (EC 4.2.1.17). Like its human counterpart, it is predicted to act in the mitochondrion, where it catalyzes the second step of the fatty acid beta-oxidation spiral: the reversible addition of water across the double bond of a short-/medium-chain 2-trans-enoyl-CoA to yield the corresponding (3S)-3-hydroxyacyl-CoA. The enzyme carries the canonical crotonase-like fold (Pfam ECH_1; InterPro enoyl-CoA hydratase/isomerase signature) and is encoded as two isoforms with an N-terminal region resembling a mitochondrial targeting presequence. Beyond fatty acid oxidation, Echs1 participates in branched-chain amino acid catabolism, most notably valine catabolism, hydrating the reactive valine-pathway intermediate methacrylyl-CoA. Loss of Echs1 in Drosophila causes accumulation of valine-pathway intermediates, ectopic protein lysine methacrylation, abnormal mitochondrial morphology and age-dependent eye degeneration, and the null larval phenotype can be rescued by expression of a human ECHS1 transgene, establishing conserved enzymatic function between the fly and human enzymes.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: Root-level molecular function term assigned electronically from an InterPro domain match (IPR018376, the enoyl-CoA hydratase/isomerase conserved site). Echs1 is indeed a catalytic enzyme, but this term is uninformative and fully subsumed by the specific enoyl-CoA hydratase activity annotation. Reason: "catalytic activity" is a high-level placeholder superseded by GO:0004300 enoyl-CoA hydratase activity, the defining EC 4.2.1.17 function of this crotonase-family enzyme. |
| GO:0004300 enoyl-CoA hydratase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic assignment of the defining EC 4.2.1.17 activity (ARBA:ARBA00086629 | EC:4.2.1.17). This is the core molecular function of Echs1: hydration of a 2-trans-enoyl-CoA to the (3S)-3-hydroxyacyl-CoA, the second step of the beta-oxidation spiral. The fly protein carries the crotonase-like fold and is the clear ortholog of human ECHS1, whose enoyl-CoA hydratase activity is experimentally established, and the fly Echs1-null phenotype is rescued by a human ECHS1 transgene (Mele et al. 2025). Reason: Enoyl-CoA hydratase activity (EC 4.2.1.17) is the central, conserved function of Echs1; the EC/ARBA-based electronic annotation is correct and grounded by ortholog biochemistry (PMID:26251176) and by functional complementation of the fly mutant by human ECHS1. An independent OpenScientist analysis (run blinded to this review action as a neutral function-assignment hypothesis, focused on substrate breadth) strongly supports the assignment and, importantly, indicates the broad substrate range is conserved rather than narrowed in the fly - both catalytic glutamates and the active-site/substrate-binding residues are 100% conserved with human ECHS1, so the same short-chain and branched-chain enoyl-CoA panel (including the valine-pathway substrate methacrylyl-CoA) is expected. This is corroborated in vivo by methacrylyl-CoA accumulation (elevated lysine methacrylation) upon Echs1 loss in flies, and underpins the separate valine-catabolism core function. Supporting Evidence: PMID:26251176 Human ECHS1 catalyses the hydration of five substrates via different metabolic pathways, with the highest specificity for crotonyl-CoA and the lowest specificity for tiglyl-CoA. file:DROME/Echs1/Echs1-hypotheses/function-hypothesis-go-0004300/openscientist.md the enzymatic activity framework that is expected to be shared by the Drosophila ortholog based on the complete conservation of catalytic and substrate-binding residues |
| GO:0005739 mitochondrion | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic (IBA) inference that Echs1 is active in the mitochondrion, consistent with the crotonase-family enoyl-CoA hydratases of the beta-oxidation spiral, which are matrix enzymes. Directly corroborated by experimental IDA/HDA mitochondrial localization for the fly protein (PMID:40056416, PMID:19317464). Reason: Mitochondrion is the correct and core site of action for a beta-oxidation enoyl-CoA hydratase; the phylogenetic call agrees with organism-matched experimental localization evidence. |
| GO:0006635 fatty acid beta-oxidation | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic (IBA) inference that Echs1 participates in fatty acid beta-oxidation, the pathway in which its EC 4.2.1.17 enoyl-CoA hydratase activity catalyzes the second (hydration) step for short-/medium-chain substrates. Reason: Fatty acid beta-oxidation is a core biological process for Echs1; the phylogenetic call matches the conserved enzymatic role of the ECHS1 ortholog group. |
| GO:0019477 L-lysine catabolic process | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Orthology-based (ISS) transfer of L-lysine catabolic process. As in human ECHS1, the connection to lysine catabolism is indirect β through the enzyme's action on the shared crotonyl-CoA pool (crotonyl-CoA is also a lysine-degradation intermediate) rather than through a dedicated lysine-degradation reaction. Reason: Retained as a valid non-core annotation, mirroring the human ortholog assessment. Echs1's link to lysine catabolism is via consumption of the shared crotonyl-CoA metabolite, not a canonical lysine-specific step, so it is peripheral to the core beta-oxidation and valine-catabolism functions. |
| GO:0006574 L-valine catabolic process | IMP PMID:39727068 Valine Restriction Extends Survival in a Drosophila Model of... | ACCEPT | Summary: Direct experimental (IMP) evidence in Drosophila that Echs1 acts in valine catabolism β a fly ECHS1-deficiency model is rescued by dietary valine restriction, confirming that the phenotype arises from a block in the valine-degradation pathway where Echs1 hydrates the intermediate methacrylyl-CoA. Reason: A core biological process for Echs1, established here by an organism-matched genetic/dietary-modifier experiment and consistent with the human ECHS1 role in valine catabolism. Supporting Evidence: PMID:40056416 Elevated lysine methacrylation (Kmea) is observed in both HIBCH- and ECHS1-deficient cells and fly tissues. |
| GO:0005739 mitochondrion | IDA PMID:40056416 Ectopic protein lysine methacrylation contributes to defects... | ACCEPT | Summary: Direct experimental (IDA) localization of the Drosophila Echs1 protein to the mitochondrion, consistent with its role as a matrix beta-oxidation and valine-catabolism enzyme and with the abnormal mitochondrial morphology seen upon its loss. Reason: Organism-matched experimental evidence for the core (mitochondrial) site of action; the strongest of the several mitochondrial localization annotations for this gene. |
| GO:0006574 L-valine catabolic process | IMP PMID:40056416 Ectopic protein lysine methacrylation contributes to defects... | ACCEPT | Summary: Direct experimental (IMP) evidence that loss of Echs1 in Drosophila disrupts valine catabolism β deficiency causes accumulation of the valine-pathway intermediate methacrylyl-CoA and ectopic protein lysine methacrylation, mitochondrial dysfunction, and age-dependent eye degeneration. Duplicates the PMID:39727068 IMP call from an independent line of evidence. Reason: Core biological process, independently supported by an organism-matched loss-of-function experiment linking Echs1 to the valine-degradation pathway. Supporting Evidence: PMID:40056416 Elevated lysine methacrylation (Kmea) is observed in both HIBCH- and ECHS1-deficient cells and fly tissues. |
| GO:0043956 3-hydroxypropionyl-CoA dehydratase activity | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Orthology-based (ISS) transfer of 3-hydroxypropionyl-CoA dehydratase activity (hydration/dehydration of acryloyl-CoA), one of the five enoyl-CoA substrate reactions measured for purified human ECHS1. This is a valid specific child of the general enoyl-CoA hydratase activity, reflecting the enzyme's substrate promiscuity across valine/propionate-pathway thioesters. Reason: A genuine but non-core substrate-specific activity; the core molecular function of Echs1 is the general enoyl-CoA hydratase activity (GO:0004300), with acryloyl-CoA being one of several accepted substrates by orthology to human ECHS1. |
| GO:0004300 enoyl-CoA hydratase activity | ISS GO_REF:0000024 | ACCEPT | Summary: Orthology-based (ISS) assignment of the defining EC 4.2.1.17 enoyl-CoA hydratase activity, duplicating the IEA (EC/ARBA) annotation above. This is the central conserved molecular function of Echs1. Reason: The core molecular function of Echs1; the ISS orthology transfer independently corroborates the EC-based electronic annotation and is grounded by the human ortholog biochemistry (PMID:26251176) and by functional rescue of the fly null by human ECHS1. Supporting Evidence: PMID:26251176 Human ECHS1 catalyses the hydration of five substrates via different metabolic pathways, with the highest specificity for crotonyl-CoA and the lowest specificity for tiglyl-CoA. |
| GO:0006635 fatty acid beta-oxidation | ISS GO_REF:0000024 | ACCEPT | Summary: Orthology-based (ISS) transfer of fatty acid beta-oxidation, duplicating the IBA call above. Echs1 catalyzes the second (hydration) step of the mitochondrial beta-oxidation spiral for short-/medium-chain substrates. Reason: Core biological process for Echs1, consistent across phylogenetic (IBA) and orthology (ISS) inference and with the conserved enzymatic role of the ECHS1 family. |
| GO:0005739 mitochondrion | HDA PMID:19317464 Mapping organelle proteins and protein complexes in Drosophi... | ACCEPT | Summary: High-throughput direct assay (HDA) localization of Echs1 to the mitochondrion from a Drosophila organelle-proteomics survey, corroborating the low-throughput IDA and phylogenetic mitochondrial calls. Reason: Organism-matched proteomic localization evidence consistent with the core mitochondrial site of action; adds independent support for the mitochondrion annotation. |
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Download this section (compressed HTML)Q: Does Drosophila Echs1 exhibit the same broad enoyl-CoA substrate range as human ECHS1 (crotonyl-CoA, acryloyl-CoA, methacrylyl-CoA, 3-methylcrotonyl-CoA, tiglyl-CoA), or is its substrate preference tuned differently in the fly?
Q: Is the mitochondrial matrix localization of Echs1 experimentally confirmed in Drosophila, and is the predicted N-terminal targeting presequence cleaved?
Experiment: Assay hydratase kinetics of purified recombinant Drosophila Echs1 against a panel of short-chain and branched-chain enoyl-CoA substrates (crotonyl-CoA, methacrylyl-CoA, acryloyl-CoA, 3-methylcrotonyl-CoA, tiglyl-CoA) to confirm EC 4.2.1.17 activity and compare substrate specificity with the human ortholog.
Hypothesis: Fly Echs1 is a bona fide short-chain enoyl-CoA hydratase with a substrate profile conserved with human ECHS1, explaining functional rescue of the fly null by a human ECHS1 transgene.
Experiment: Perform metabolomic tracing of valine and fatty-acid carbon in Echs1-null versus control Drosophila to determine which accumulating thioester (methacrylyl-CoA vs beta-oxidation enoyl-CoAs) most strongly drives ectopic protein lysine methacrylation and mitochondrial dysfunction.
Hypothesis: Accumulation of the valine-pathway intermediate methacrylyl-CoA, rather than the beta-oxidation block per se, is the principal driver of the Echs1-deficiency phenotype in flies.
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