FOX2 (YKR009C) encodes the peroxisomal multifunctional enzyme type 2 (MFE-2), a ~100 kDa bifunctional protein that catalyses the second and third reactions of fatty acid beta-oxidation in Saccharomyces cerevisiae. Its C-terminal hydratase 2 (MaoC-like) domain hydrates trans-2-enoyl-CoA to (3R)-3-hydroxyacyl-CoA (2-enoyl-CoA hydratase 2, EC 4.2.1.119), and its two N-terminal short-chain dehydrogenase/reductase (SDR) domains, A and B, oxidise (3R)-3-hydroxyacyl-CoA to 3-ketoacyl-CoA with NAD+ ((3R)-3-hydroxyacyl-CoA dehydrogenase). Domain A prefers medium- and long-chain and domain B short-chain substrates, and both are needed for optimal growth on oleate. Contrary to earlier assumptions, the yeast enzyme lacks crotonase (hydratase 1), (3S)-3-hydroxyacyl-CoA dehydrogenase and 3-hydroxyacyl-CoA epimerase activities, so yeast beta-oxidation proceeds through the (3R)-hydroxyacyl-CoA stereoisomer. Fox2p is the only MFE in yeast and is essential for growth on fatty acids; it is a peroxisomal matrix protein carrying a PTS1 but can also be imported by Pex5p independently of that signal (and weakly by Pex9p).
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003857 (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity | IBA GO_REF:0000033 | MODIFY | Summary: IBA for (3S)-3-hydroxyacyl-CoA dehydrogenase from PANTHER node PTN002894972, whose only donor is yeast Fox2p itself (SGD:S000001717), i.e. it re-propagates the IDA below. The biochemistry shows the yeast MFE-2 dehydrogenase is (3R)-specific and that L-(3S)-3-hydroxyacyl-CoA dehydrogenase activity is absent. The correct term is the (3R)-specific dehydrogenase. Propagation Review Root cause: SOURCE BAD Proposed replacements: (3R)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity Supporting Evidence: PMID:1551874 demonstrated an absence of activities originally assigned to an MFP of S. cerevisiae (crotonase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyacyl-CoA epimerase), whereas two other activities were found: 2-enoyl-CoA hydratase 2 (converting trans-2-enoyl-CoA to D-3-hydroxyacyl-CoA) and D-3-hydroxyacyl CoA dehydrogenase (converting D-3-hydroxyacyl-CoA to 3-ketoacyl-CoA) |
| GO:0003857 (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity | IDA PMID:1551874 Peroxisomal multifunctional beta-oxidation protein of Saccha... | MODIFY | Summary: IDA from Hiltunen et al. 1992. The cited paper's own abstract states that L-3-hydroxyacyl-CoA dehydrogenase activity was absent from purified Fox2p and that the dehydrogenase present is D-3-hydroxyacyl-CoA ((3R)) dehydrogenase. The annotation captures the right reaction class with the wrong stereospecificity, most likely made before GO:0106386 existed. Replace with the (3R)-specific term. Proposed replacements: (3R)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity Supporting Evidence: PMID:1551874 demonstrated an absence of activities originally assigned to an MFP of S. cerevisiae (crotonase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyacyl-CoA epimerase), whereas two other activities were found: 2-enoyl-CoA hydratase 2 (converting trans-2-enoyl-CoA to D-3-hydroxyacyl-CoA) and D-3-hydroxyacyl CoA dehydrogenase (converting D-3-hydroxyacyl-CoA to 3-ketoacyl-CoA) PMID:1551874 The truncated form contained only the D-3-hydroxyacyl-CoA dehydrogenase activity |
| GO:0003857 (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity | RCA GO_REF:0000123 | MODIFY | Summary: YeastPathways RCA from SGD_PWY:PWY-5177, a pathway other than fatty acid beta-oxidation that YeastCyc links to an S-specific 3-hydroxyacyl-CoA dehydrogenase reaction. Fox2p has no (3S)-specific activity; where Fox2p acts it is the (3R)-specific dehydrogenase. Proposed replacements: (3R)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity Supporting Evidence: PMID:1551874 demonstrated an absence of activities originally assigned to an MFP of S. cerevisiae (crotonase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyacyl-CoA epimerase), whereas two other activities were found: 2-enoyl-CoA hydratase 2 (converting trans-2-enoyl-CoA to D-3-hydroxyacyl-CoA) and D-3-hydroxyacyl CoA dehydrogenase (converting D-3-hydroxyacyl-CoA to 3-ketoacyl-CoA) |
| GO:0003857 (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity | RCA GO_REF:0000123 | MODIFY | Summary: YeastPathways RCA from YEAST-FAO-PWY, which models the yeast pathway with the generic (3S)-hydroxyacyl intermediate (EC 1.1.1.35). Yeast beta-oxidation goes through (3R)-hydroxyacyl-CoA, so the step Fox2p catalyses is the (3R)-specific dehydrogenation. Proposed replacements: (3R)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity Supporting Evidence: PMID:1551874 demonstrated an absence of activities originally assigned to an MFP of S. cerevisiae (crotonase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyacyl-CoA epimerase), whereas two other activities were found: 2-enoyl-CoA hydratase 2 (converting trans-2-enoyl-CoA to D-3-hydroxyacyl-CoA) and D-3-hydroxyacyl CoA dehydrogenase (converting D-3-hydroxyacyl-CoA to 3-ketoacyl-CoA) PMID:1551874 beta-oxidation of fatty acids in S. cerevisiae follows a previously unknown stereochemical course, namely it occurs via a D-3-hydroxyacyl-CoA intermediate |
| GO:0004300 enoyl-CoA hydratase activity | IBA GO_REF:0000033 | MODIFY | Summary: IBA for enoyl-CoA hydratase (EC 4.2.1.17-defined parent) from node PTN002894972, seeded only by Fox2p. Fox2p has hydratase 2 activity producing the (3R)-hydroxyacyl-CoA and lacks crotonase (hydratase 1) activity. GO:0080023 (2E)-enoyl-CoA hydratase activity, which is (3R)-specific, is the exact term. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Proposed replacements: (2E)-enoyl-CoA hydratase activity Supporting Evidence: PMID:1551874 demonstrated an absence of activities originally assigned to an MFP of S. cerevisiae (crotonase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyacyl-CoA epimerase), whereas two other activities were found: 2-enoyl-CoA hydratase 2 (converting trans-2-enoyl-CoA to D-3-hydroxyacyl-CoA) and D-3-hydroxyacyl CoA dehydrogenase (converting D-3-hydroxyacyl-CoA to 3-ketoacyl-CoA) |
| GO:0004300 enoyl-CoA hydratase activity | IDA PMID:1551874 Peroxisomal multifunctional beta-oxidation protein of Saccha... | MODIFY | Summary: IDA (Hiltunen et al. 1992): purified Fox2p has 2-enoyl-CoA hydratase 2 activity (trans-2-enoyl-CoA to D-3-hydroxyacyl-CoA) and no crotonase activity. The parent term is correct but the (3R)-specific child is the accurate description. Proposed replacements: (2E)-enoyl-CoA hydratase activity Supporting Evidence: PMID:1551874 demonstrated an absence of activities originally assigned to an MFP of S. cerevisiae (crotonase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyacyl-CoA epimerase), whereas two other activities were found: 2-enoyl-CoA hydratase 2 (converting trans-2-enoyl-CoA to D-3-hydroxyacyl-CoA) and D-3-hydroxyacyl CoA dehydrogenase (converting D-3-hydroxyacyl-CoA to 3-ketoacyl-CoA) |
| GO:0004300 enoyl-CoA hydratase activity | IEA GO_REF:0000117 | MODIFY | Summary: IEA from an ARBA rule for enoyl-CoA hydratase. Correct in class but should be the (3R)-specific hydratase 2 term, as for the experimental annotation. Proposed replacements: (2E)-enoyl-CoA hydratase activity Supporting Evidence: file:yeast/FOX2/FOX2-uniprot.txt Reaction=a (3R)-3-hydroxyacyl-CoA = a (2E)-enoyl-CoA + H2O |
| GO:0004300 enoyl-CoA hydratase activity | RCA GO_REF:0000123 | MODIFY | Summary: YeastPathways RCA (YEAST-FAO-PWY) assigning Fox2p to the EC 4.2.1.17 hydration step that produces (3S)-3-hydroxyacyl-CoA. Fox2p performs the hydration step but with the opposite stereochemistry (hydratase 2, EC 4.2.1.119). Proposed replacements: (2E)-enoyl-CoA hydratase activity Supporting Evidence: PMID:1551874 demonstrated an absence of activities originally assigned to an MFP of S. cerevisiae (crotonase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyacyl-CoA epimerase), whereas two other activities were found: 2-enoyl-CoA hydratase 2 (converting trans-2-enoyl-CoA to D-3-hydroxyacyl-CoA) and D-3-hydroxyacyl CoA dehydrogenase (converting D-3-hydroxyacyl-CoA to 3-ketoacyl-CoA) PMID:1551874 beta-oxidation of fatty acids in S. cerevisiae follows a previously unknown stereochemical course, namely it occurs via a D-3-hydroxyacyl-CoA intermediate |
| GO:0005777 peroxisome | IBA GO_REF:0000033 | ACCEPT | Summary: IBA for peroxisome from the MFE-2 family node (donors include yeast Fox2p, worm DHS-28, Aspergillus and Dictyostelium MFE-2). Correct. Supporting Evidence: PMID:30131444 a multifunctional enzyme from the Ξ²-oxidation pathway (Fox2p) and catalase A (Cta1p), both of which contain PTS1, can be imported independently of this signal |
| GO:0005777 peroxisome | IDA PMID:30131444 The budding yeast Pex5p receptor directs Fox2p and Cta1p int... | ACCEPT | Summary: IDA (Rymer et al. 2018): Fox2p is imported into peroxisomes by Pex5p, either via its PTS1 or via a PTS1-independent route involving the Pex5p N-terminal region. Supporting Evidence: PMID:30131444 a multifunctional enzyme from the Ξ²-oxidation pathway (Fox2p) and catalase A (Cta1p), both of which contain PTS1, can be imported independently of this signal |
| GO:0005777 peroxisome | IEA GO_REF:0000044 | ACCEPT | Summary: IEA from UniProt subcellular location (SL-0204); consistent with experimental data. Supporting Evidence: file:yeast/FOX2/FOX2-uniprot.txt SUBCELLULAR LOCATION: Peroxisome |
| GO:0005829 cytosol | RCA GO_REF:0000123 | REMOVE | Summary: YeastPathways RCA (PWY-5177) placing the reaction in the cytosol. Fox2p is a peroxisomal matrix enzyme; no cytosolic activity is reported. Supporting Evidence: PMID:30131444 a multifunctional enzyme from the Ξ²-oxidation pathway (Fox2p) and catalase A (Cta1p), both of which contain PTS1, can be imported independently of this signal |
| GO:0005829 cytosol | RCA GO_REF:0000123 | REMOVE | Summary: YeastPathways RCA (YEAST-FAO-PWY) placing fatty acid oxidation in the cytosol. Yeast beta-oxidation is peroxisomal and Fox2p is imported into peroxisomes; the cytosol assignment is a conversion artefact. Supporting Evidence: PMID:30131444 a multifunctional enzyme from the Ξ²-oxidation pathway (Fox2p) and catalase A (Cta1p), both of which contain PTS1, can be imported independently of this signal |
| GO:0006635 fatty acid beta-oxidation | IBA GO_REF:0000033 | ACCEPT | Summary: IBA for fatty acid beta-oxidation from the MFE-2 node; Fox2p catalyses the hydration and dehydrogenation steps of the cycle. Supporting Evidence: file:yeast/FOX2/FOX2-uniprot.txt Converts trans-2-enoyl-CoA via D-3-hydroxyacyl-CoA to |
| GO:0006635 fatty acid beta-oxidation | IEA GO_REF:0000041 | ACCEPT | Summary: IEA from UniPathway UPA00659; correct. Supporting Evidence: file:yeast/FOX2/FOX2-uniprot.txt PATHWAY: Lipid metabolism; fatty acid beta-oxidation |
| GO:0006635 fatty acid beta-oxidation | IMP PMID:10497229 Yeast peroxisomal multifunctional enzyme: (3R)-hydroxyacyl-C... | ACCEPT | Summary: IMP (Qin et al. 1999): fox2 cells expressing MFE-2 with both SDR domains inactivated fail to grow on oleate, and single-domain mutants grow more slowly; Fox2p dehydrogenase activity is required for beta-oxidation. Supporting Evidence: PMID:10497229 fox-2 cells transformed with pYE352:: ScMFE-2(aDelta) and pYE352::ScMFE-2(bDelta) grew slower than cells transformed with pYE352::ScMFE-2, whereas cells transformed with pYE352::ScMFE-2(aDeltabDelta) failed to grow PMID:10497229 The data show that the dehydrogenase domains of yeast MFE-2 have different substrate specificities required to allow the yeast to propagate optimally on fatty acids as the carbon source |
| GO:0006635 fatty acid beta-oxidation | IMP PMID:1551874 Peroxisomal multifunctional beta-oxidation protein of Saccha... | ACCEPT | Summary: IMP (Hiltunen et al. 1992): FOX2 was cloned by complementation of a fox2 beta-oxidation mutant and the purified product carries the hydratase 2 and (3R)-dehydrogenase activities of the cycle. Supporting Evidence: PMID:1551874 was isolated from a genomic library via functional complementation of a fox2 mutant strain PMID:1551874 beta-oxidation of fatty acids in S. cerevisiae follows a previously unknown stereochemical course, namely it occurs via a D-3-hydroxyacyl-CoA intermediate |
| GO:0016616 oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor | IEA GO_REF:0000117 | MODIFY | Summary: ARBA IEA for oxidoreductase acting on CH-OH donors with NAD(P). Correct but very general; the specific activity is NAD+-dependent (3R)-3-hydroxyacyl-CoA dehydrogenase. Proposed replacements: (3R)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity Supporting Evidence: file:yeast/FOX2/FOX2-uniprot.txt Reaction=a (3R)-3-hydroxyacyl-CoA + NAD(+) = a 3-oxoacyl-CoA + NADH + |
| GO:0018812 3-hydroxyacyl-CoA dehydratase activity | RCA GO_REF:0000123 | MODIFY | Summary: YeastPathways RCA (PWY-5177) for 3-hydroxyacyl-CoA dehydratase, a stereo-unspecified parent of the hydratase 2 activity. Fox2p's activity is the (3R)-specific (2E)-enoyl-CoA hydratase. Proposed replacements: (2E)-enoyl-CoA hydratase activity Supporting Evidence: PMID:1551874 demonstrated an absence of activities originally assigned to an MFP of S. cerevisiae (crotonase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyacyl-CoA epimerase), whereas two other activities were found: 2-enoyl-CoA hydratase 2 (converting trans-2-enoyl-CoA to D-3-hydroxyacyl-CoA) and D-3-hydroxyacyl CoA dehydrogenase (converting D-3-hydroxyacyl-CoA to 3-ketoacyl-CoA) |
| GO:0019395 fatty acid oxidation | RCA GO_REF:0000123 | ACCEPT | Summary: YeastPathways RCA for fatty acid oxidation (YEAST-FAO-PWY); correct parent of beta-oxidation, in which Fox2p acts. Supporting Evidence: file:yeast/FOX2/FOX2-uniprot.txt Converts trans-2-enoyl-CoA via D-3-hydroxyacyl-CoA to |
| GO:0046395 carboxylic acid catabolic process | RCA GO_REF:0000123 | MARK AS OVER ANNOTATED | Summary: YeastPathways RCA from PWY-5177 for carboxylic acid catabolic process. Fox2p does act in a carboxylic acid catabolic process (fatty acid beta-oxidation, already annotated), but this row derives from a YeastCyc pathway other than beta-oxidation with no yeast evidence that Fox2p participates in it; the very general term adds nothing beyond fatty acid beta-oxidation. Supporting Evidence: file:yeast/FOX2/FOX2-uniprot.txt Converts trans-2-enoyl-CoA via D-3-hydroxyacyl-CoA to |
| GO:0080023 (2E)-enoyl-CoA hydratase activity | IEA GO_REF:0000120 | ACCEPT | Summary: IEA from Rhea RHEA:26526 / EC 4.2.1.119; exactly matches the experimentally demonstrated hydratase 2 activity (trans-2-enoyl-CoA to (3R)-3-hydroxyacyl-CoA). Supporting Evidence: file:yeast/FOX2/FOX2-uniprot.txt Reaction=a (3R)-3-hydroxyacyl-CoA = a (2E)-enoyl-CoA + H2O PMID:1551874 demonstrated an absence of activities originally assigned to an MFP of S. cerevisiae (crotonase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyacyl-CoA epimerase), whereas two other activities were found: 2-enoyl-CoA hydratase 2 (converting trans-2-enoyl-CoA to D-3-hydroxyacyl-CoA) and D-3-hydroxyacyl CoA dehydrogenase (converting D-3-hydroxyacyl-CoA to 3-ketoacyl-CoA) |
| GO:0106386 (3R)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity | IEA GO_REF:0000116 | ACCEPT | Summary: IEA from Rhea RHEA:32711; NAD+-dependent (3R)-3-hydroxyacyl-CoA dehydrogenase, matching the D-3-hydroxyacyl-CoA dehydrogenase activity of purified Fox2p and of its SDR domains A and B. Supporting Evidence: file:yeast/FOX2/FOX2-uniprot.txt Reaction=a (3R)-3-hydroxyacyl-CoA + NAD(+) = a 3-oxoacyl-CoA + NADH + PMID:1551874 The truncated form contained only the D-3-hydroxyacyl-CoA dehydrogenase activity |
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Download this section (compressed HTML)Q: Should the YeastPathways fatty acid oxidation pathway (and the GO-CAMs derived from it) be corrected to the (3R)-hydroxyacyl-CoA route (EC 4.2.1.119 and 3R-specific dehydrogenase) for S. cerevisiae, given that Fox2p lacks crotonase and (3S)-dehydrogenase activities?
Q: Which YeastPathways pathway is behind the PWY-5177 rows on Fox2p, and is there any yeast evidence that Fox2p acts outside fatty acid beta-oxidation?
Experiment: Assay stereospecific hydratase and dehydrogenase activities (with (3R)- and (3S)-substrates) in purified peroxisomes from wild type and fox2 deletion strains grown on oleate.
Hypothesis: Fox2p is the only source of 2-enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activity in oleate-grown yeast peroxisomes.
3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase; multifunctional enzyme of the peroxisomal fatty acid beta-oxidation pathway; mutation is functionally complemented by human HSD17B4
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