ACAA2 (3-ketoacyl-CoA thiolase, mitochondrial; also called acetyl-CoA acyltransferase 2 or T1; EC 2.3.1.16) is a soluble enzyme of the mitochondrial matrix that catalyzes the fourth and final step of each cycle of the fatty acid beta-oxidation spiral. Using free coenzyme A, it performs the thiolytic cleavage of a 3-ketoacyl-CoA (3-oxoacyl-CoA) into acetyl-CoA and a fatty acyl-CoA shortened by two carbon atoms, preferring medium- to long-chain unbranched (straight-chain) substrates. The enzyme assembles as a homotetramer and belongs to the thiolase family of the thiolase-like superfamily, using an active-site cysteine (Cys92) to form an acyl-enzyme thioester intermediate and a second cysteine (Cys382) as the catalytic proton donor/acceptor. In the reverse direction it can condense two acetyl-CoA molecules into acetoacetyl-CoA (EC 2.3.1.9), and it also displays a slow intrinsic acyl-CoA thioesterase (hydrolase) side activity on various fatty acyl-CoAs. ACAA2 is a distinct gene product from HADHB (the long-chain thiolase of the membrane-bound mitochondrial trifunctional protein) and from ACAT1 (the mitochondrial acetoacetyl-CoA/ketone-body thiolase). It has additionally been reported to bind the pro-apoptotic protein BNIP3 and attenuate BNIP3-driven apoptosis, providing a possible link between mitochondrial fatty acid metabolism and apoptotic signaling.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003985 acetyl-CoA C-acetyltransferase activity | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Phylogenetic (IBA) propagation of acetyl-CoA C-acetyltransferase activity (EC 2.3.1.9), the acetoacetyl-CoA-forming/cleaving reaction. ACAA2 does possess this activity (demonstrated biochemically and structurally), but it is a secondary/biosynthetic capability; the core physiological role is the C-acyltransferase (thiolase) reaction on longer-chain 3-oxoacyl-CoAs. Keep, but mark as non-core. Reason: Supported by direct evidence (PMID:25478839) showing significant biosynthetic thiolase (acetoacetyl-CoA) activity, but the chain-shortening beta-oxidation thiolase reaction (GO:0003988) is the core function. |
| GO:0005739 mitochondrion | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Phylogenetic propagation of mitochondrial localization. Correct and consistent with experimental localization (PMID:18371312, HPA, mitochondrial proteome studies). A more precise term (mitochondrial matrix, GO:0005759) is also annotated; this broader term is acceptable. |
| GO:0006635 fatty acid beta-oxidation | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic propagation of the core biological process. ACAA2 catalyzes the final (thiolytic) step of each beta-oxidation cycle. Strongly supported and represents a core process for the gene. |
| GO:0003985 acetyl-CoA C-acetyltransferase activity | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: Electronic (ARBA/Rhea/EC) assertion of acetyl-CoA C-acetyltransferase activity (EC 2.3.1.9), the same biosynthetic acetoacetyl-CoA reaction captured by the IBA and IDA annotations. Redundant with the experimentally supported annotation; keep as non-core. |
| GO:0003986 acetyl-CoA hydrolase activity | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: Electronic (EC 3.1.2.1) assertion of acetyl-CoA hydrolase activity, an in-vitro thioesterase side reaction. PMID:25478839 confirmed low intrinsic acyl-CoA thioesterase activity (kcat ~0.02/s for octanoyl-CoA), so the activity is real but physiologically minor. Keep as non-core; the principal hydrolase substrate observed was butyryl-CoA, not acetyl-CoA specifically. |
| GO:0003988 acetyl-CoA C-acyltransferase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic (EC 2.3.1.16 / Rhea) assertion of acetyl-CoA C-acyltransferase activity. This is the core 3-ketoacyl-CoA thiolase molecular function of ACAA2 and is independently supported by direct assay (PMID:25478839). Accept. |
| GO:0005739 mitochondrion | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Electronic localization from the UniProt subcellular-location vocabulary. Correct; ACAA2 is a mitochondrial (matrix) protein. Non-core (matrix, GO:0005759, is the specific core location). |
| GO:0016746 acyltransferase activity | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: InterPro2GO mapping to the high-level parent term acyltransferase activity. Correct in direction but far less informative than the specific thiolase MF (GO:0003988) that is already annotated with experimental evidence. Over-general. Reason: Redundant generic ancestor of the experimentally supported GO:0003988; carries no information beyond the specific thiolase activity term. |
| GO:0016747 acyltransferase activity, transferring groups other than amino-acyl groups | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: InterPro2GO mapping to an intermediate (still generic) acyltransferase parent. As with GO:0016746, this is a non-informative ancestor of the specific thiolase activity GO:0003988 already annotated with direct evidence. Reason: Over-general ancestor of GO:0003988; the specific thiolase MF term should be used. |
| GO:0052815 medium-chain fatty acyl-CoA hydrolase activity | IEA GO_REF:0000116 | KEEP AS NON CORE | Summary: Rhea-mapped assertion of medium-chain (C6-C12) acyl-CoA hydrolase activity, derived from the in-vitro thioesterase side reactions of T1. PMID:25478839 confirmed low acyl-CoA thioesterase activity across chain lengths (kcat ~0.02/s). The activity is real but a minor side reaction, not the core function. Keep as non-core. |
| GO:0052816 long-chain fatty acyl-CoA hydrolase activity | IEA GO_REF:0000116 | KEEP AS NON CORE | Summary: Rhea-mapped long-chain acyl-CoA hydrolase (thioesterase) activity from the same low-level in-vitro side reactions. Physiologically minor relative to the thiolase function. Keep as non-core. |
| GO:0141126 short-chain fatty acyl-CoA hydrolase activity | IEA GO_REF:0000116 | KEEP AS NON CORE | Summary: Rhea-mapped short-chain acyl-CoA hydrolase activity. PMID:25478839 noted the fastest hydrolysis rate was for butyryl-CoA (a short-chain substrate), so this is the best-supported of the hydrolase terms, but it remains a slow side reaction. Keep as non-core. |
| GO:0006635 fatty acid beta-oxidation | IEA GO_REF:0000041 | ACCEPT | Summary: UniPathway-based electronic assertion of involvement in fatty acid beta-oxidation, the core process. Consistent with the IBA annotation and with UniProt PATHWAY ("Lipid metabolism; fatty acid beta-oxidation"). Accept. |
| GO:0005739 mitochondrion | IDA GO_REF:0000052 | KEEP AS NON CORE | Summary: Direct immunofluorescence (Human Protein Atlas) localization to mitochondrion. Correct and consistent with all other localization evidence. Non-core (matrix, GO:0005759, is the specific core location). |
| GO:0003986 acetyl-CoA hydrolase activity | EXP PMID:25478839 The crystal structure of human mitochondrial 3-ketoacyl-CoA ... | KEEP AS NON CORE | Summary: Experimental annotation from the crystallographic/biochemical study, which demonstrated intrinsic acyl-CoA thioesterase (hydrolase) activity of human T1. The activity is genuine but very slow (kcat ~0.02/s) and is described as a possible side reaction producing acetate, not the core role. Keep as non-core. Reason: Direct evidence supports the activity, but it is a minor in-vitro side reaction relative to the thiolase function. Supporting Evidence: PMID:25478839 Solution studies confirm that hT1 has low acyl-CoA thioesterase activity for fatty acyl-CoA substrates. |
| GO:0005739 mitochondrion | HTP PMID:34800366 Quantitative high-confidence human mitochondrial proteome an... | KEEP AS NON CORE | Summary: High-throughput localization from a high-confidence human mitochondrial proteome study. Corroborates the well-established mitochondrial localization. Non-core (matrix, GO:0005759, is the specific core location). |
| GO:0003985 acetyl-CoA C-acetyltransferase activity | IDA PMID:25478839 The crystal structure of human mitochondrial 3-ketoacyl-CoA ... | KEEP AS NON CORE | Summary: Direct-assay evidence that T1 has significant biosynthetic (acetoacetyl-CoA synthesizing/cleaving) thiolase activity, i.e. acetyl-CoA C-acetyltransferase (EC 2.3.1.9). The activity is real and well measured (KM 9.2 uM for acetoacetyl-CoA), but it is a secondary capability; the chain-shortening thiolase reaction (GO:0003988) is the core function. Keep as non-core. Reason: Experimentally demonstrated, but secondary to the core C-acyltransferase thiolase activity in beta-oxidation. Supporting Evidence: PMID:25478839 It is also shown that T1 has significant biosynthetic thiolase activity, which is predicted to be of physiological importance. |
| GO:0003988 acetyl-CoA C-acyltransferase activity | IDA PMID:25478839 The crystal structure of human mitochondrial 3-ketoacyl-CoA ... | ACCEPT | Summary: Direct-assay annotation of the core 3-ketoacyl-CoA thiolase activity (EC 2.3.1.16) confirmed by the structural/biochemical characterization of human T1 as a degradative tetrameric thiolase. This is THE core molecular function of ACAA2 in beta-oxidation. Accept. Reason: Core molecular function; supported by direct enzymatic and structural data. Supporting Evidence: PMID:25478839 The structures confirm the tetrameric quaternary structure of this degradative thiolase. |
| GO:0047617 fatty acyl-CoA hydrolase activity | IMP PMID:25478839 The crystal structure of human mitochondrial 3-ketoacyl-CoA ... | KEEP AS NON CORE | Summary: Annotation of fatty acyl-CoA hydrolase (thioesterase) activity, based on the observed intrinsic hydrolysis of fatty acyl-CoAs (and supported by mutagenesis of the catalytic Cys92/Cys382 that reduced hydrolase activity). A genuine but slow side reaction; keep as non-core. Reason: Direct evidence supports a real but minor in-vitro thioesterase side activity. Supporting Evidence: PMID:25478839 owing to the intrinsic acyl-CoA thioesterase activity of hT1 |
| GO:0005759 mitochondrial matrix | TAS Reactome:R-HSA-8874745 | ACCEPT | Summary: Reactome-asserted localization to the mitochondrial matrix. This is the most precise and correct localization for ACAA2, a soluble matrix thiolase (distinct from the inner-membrane MTP thiolase HADHB). Accept as core localization. |
| GO:0071456 cellular response to hypoxia | IDA PMID:18371312 Acetyl-Coenzyme A acyltransferase 2 attenuates the apoptotic... | KEEP AS NON CORE | Summary: Based on the BNIP3 study, where ACAA2 abolished apoptosis induced by hypoxia-driven BNIP3 overexpression. The connection to hypoxia is indirect (via BNIP3, a hypoxia-induced pro-apoptotic factor) and rests on a single overexpression study. Keep but mark as non-core/contextual. Reason: Indirect, single-study (overexpression) evidence; not a core function of the thiolase. Supporting Evidence: PMID:18371312 the apoptosis induced by over-expressed BNIP3 via transfection or hypoxia treatment was abolished by ACAA2 |
| GO:1902109 negative regulation of mitochondrial membrane permeability involved in apoptotic process | IDA PMID:18371312 Acetyl-Coenzyme A acyltransferase 2 attenuates the apoptotic... | KEEP AS NON CORE | Summary: Based on ACAA2 abolishing BNIP3-induced apoptosis and mitochondrial damage in HepG2 and U-2 OS cells. A plausible secondary (moonlighting) role linking fatty acid metabolism to apoptosis, but from a single overexpression study. Keep as non-core. Reason: Real but single-study, overexpression-based; not the core catalytic function. Supporting Evidence: PMID:18371312 the apoptosis induced by over-expressed BNIP3 via transfection or hypoxia treatment was abolished by ACAA2 |
| GO:0003723 RNA binding | HDA PMID:22658674 Insights into RNA biology from an atlas of mammalian mRNA-bi... | KEEP AS NON CORE | Summary: Detected in a proteome-wide mRNA-interactome capture screen in HeLa cells that identified many metabolic enzymes as RNA-binding. There is no gene-specific functional validation of RNA binding for ACAA2 and no proposed mechanism; this is a high-throughput moonlighting candidate, not a characterized function. Keep as non-core. Reason: Orthogonal high-throughput detection only; no functional follow-up specific to ACAA2. Supporting Evidence: PMID:22658674 shedding light on RBPs in disease, RNA-binding enzymes of intermediary metabolism |
| GO:1901029 negative regulation of mitochondrial outer membrane permeabilization involved in apoptotic signaling pathway | IDA PMID:18371312 Acetyl-Coenzyme A acyltransferase 2 attenuates the apoptotic... | KEEP AS NON CORE | Summary: Same BNIP3 evidence as the related membrane-permeability term. ACAA2 counteracts BNIP3-driven mitochondrial outer-membrane permeabilization and apoptosis. A secondary contextual role from a single overexpression study; keep as non-core. Reason: Single overexpression study; secondary to the core thiolase function. Supporting Evidence: PMID:18371312 ACAA2 was also found to co-localize with BNIP3 in mitochondria. |
| GO:0005515 protein binding | IPI PMID:18371312 Acetyl-Coenzyme A acyltransferase 2 attenuates the apoptotic... | KEEP AS NON CORE | Summary: IPI annotation recording the physical interaction with BNIP3 (UniProtKB:Q12983), confirmed by yeast two-hybrid, pull-down and co-immunoprecipitation. The interaction is real and well supported, but "protein binding" is an uninformative molecular-function term and is not a core function. Keep as non-core (the biological consequence is captured by the apoptosis-regulation process terms). Reason: Documents a verified BNIP3 interaction, but the bare protein-binding MF term is uninformative and should not be treated as a core function. Supporting Evidence: PMID:18371312 The interaction between BNIP3 and ACAA2 was confirmed by pull-down and co-immunoprecipitation assays. |
| GO:0005739 mitochondrion | IDA PMID:18371312 Acetyl-Coenzyme A acyltransferase 2 attenuates the apoptotic... | KEEP AS NON CORE | Summary: Direct localization to mitochondria from the BNIP3 study (co-localization imaging). Consistent with all other localization evidence. Non-core (matrix, GO:0005759, is the specific core location). Supporting Evidence: PMID:18371312 ACAA2 was also found to co-localize with BNIP3 in mitochondria. |
| GO:0003988 acetyl-CoA C-acyltransferase activity | NAS PMID:8241273 Cloning and sequence analysis of a full length cDNA encoding... | ACCEPT | Summary: Author-statement (NAS) assignment of the thiolase activity from the original cDNA cloning paper, which identified the gene as human mitochondrial 3-oxoacyl-CoA thiolase. The activity assignment is correct (matches the core MF) though the cited paper is sequence/expression-based rather than an enzyme assay. Accept; corroborated by the later direct-assay annotation. Supporting Evidence: PMID:8241273 The cDNA sequence of human mitochondrial 3-oxoacyl-CoA thiolase was determined |
| GO:0005739 mitochondrion | NAS PMID:8241273 Cloning and sequence analysis of a full length cDNA encoding... | KEEP AS NON CORE | Summary: Author-statement mitochondrial localization from the cloning paper (the protein is named "human mitochondrial 3-oxoacyl-CoA thiolase" and carries a mitochondrial transit sequence). Correct. Non-core (matrix, GO:0005759, is the specific core location). |
| GO:0006695 cholesterol biosynthetic process | NAS PMID:8241273 Cloning and sequence analysis of a full length cDNA encoding... | REMOVE | Summary: NAS annotation linking ACAA2 to cholesterol biosynthesis. The cited cloning paper (PMID:8241273) describes only cDNA sequencing, rat homology, and Northern expression; its abstract makes no claim about cholesterol biosynthesis. The assertion is also biologically implausible: ACAA2 is a mitochondrial-matrix beta-oxidation (catabolic) thiolase, whereas cholesterol synthesis proceeds via the cytosolic acetoacetyl-CoA thiolase/HMG-CoA pathway. This is most likely a confusion with the cytosolic/peroxisomal thiolases. Remove. Reason: Unsupported by the cited reference and contradicted by the established catabolic, mitochondrial role of ACAA2; cholesterol biosynthesis uses cytosolic thiolase/HMG-CoA enzymes, not mitochondrial ACAA2. |
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Download this section (compressed HTML)Q: Is ACAA2's biosynthetic acetoacetyl-CoA-forming (acetyl-CoA C-acetyltransferase) activity physiologically relevant in vivo, or is it primarily a reflection of the reversibility of the thiolase reaction measured in vitro?
Q: Is the anti-apoptotic, BNIP3-attenuating role of ACAA2 a genuine endogenous function or an artifact of overexpression, and does it depend on the catalytic activity of the enzyme?
Q: Does ACAA2 have any bona fide RNA-binding function, or is its appearance in mRNA-interactome capture a non-functional consequence of CoA/nucleotide-binding surfaces?
Experiment: Determine the chain-length substrate specificity of recombinant human ACAA2 with a panel of straight-chain 3-ketoacyl-CoA substrates (C4-C16) to define its physiological substrate range relative to HADHB and ACAT1.
Experiment: Use ACAA2 knockout/knockdown in human hepatocytes followed by acylcarnitine and organic-acid profiling to test the in-vivo contribution of ACAA2 to beta-oxidation flux and the proposed acetate side-production.
Experiment: Test whether catalytically dead ACAA2 (C92A/C382A) still attenuates BNIP3-induced apoptosis, to determine whether the apoptosis-modulating role is independent of thiolase catalysis.
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