Msed_0406 encodes 4-hydroxybutyrate--CoA ligase 1 (HBCL1), an AMP-forming class I adenylate-forming acyl-CoA synthetase (ANL superfamily) of the thermoacidophilic crenarchaeon Metallosphaera sedula. In the 3-hydroxypropionate/4-hydroxybutyrate autotrophic CO2-fixation cycle it activates 4-hydroxybutyrate to 4-hydroxybutyryl-CoA using ATP and CoA. This is the first step of the branch that converts 4-hydroxybutyrate, via 4-hydroxybutyryl-CoA dehydratase, crotonyl-CoA hydratase/3-hydroxybutyryl-CoA dehydrogenase and acetoacetyl-CoA thiolase, into two molecules of acetyl-CoA. A recombinant enzyme with these three partners converts 4-hydroxybutyrate to acetyl-CoA in vitro. The enzyme is promiscuous for short (C2-C5) carboxylic acids. Its catalytic efficiency in vitro is highest with propionate and butyrate, then valerate, acetate and 3-hydroxypropionate, and lowest with 4-hydroxybutyrate (Km about 2 mM). Even so, it is the physiologically relevant 4-hydroxybutyrate-CoA ligase: its rate on 4-hydroxybutyrate is about eightfold higher than that of its paralog Msed_0394 (HBCL2), which prefers butyrate and valerate. Both genes are constitutively expressed. It is a soluble, crystallised enzyme; the single predicted transmembrane segment lies within the conserved N-terminal domain.
Definition: Catalysis of the reaction: 4-hydroxybutanoate + ATP + CoA = 4-hydroxybutanoyl-CoA + AMP + diphosphate.
Justification: This is EC 6.2.1.40 (RHEA:23128), and GO has no term for it (OLS and QuickGO searches for 4-hydroxybutyrate and 4-hydroxybutanoyl-CoA ligase terms return none). The neighbouring step of the same cycle already has GO:0043955 3-hydroxypropionyl-CoA synthetase activity. The activity is required in both archaeal 4-hydroxybutyrate CO2-fixation cycles (3HP/4HB in Sulfolobales and Thaumarchaeota, DC/4HB in Thermoproteales and Desulfurococcales). 4HB is a hydroxy acid, not a fatty acid, so the parent should be CoA-ligase activity rather than short-chain fatty acid-CoA ligase activity.
Parent term: CoA-ligase activity
Supporting Evidence:
Definition: An autotrophic carbon dioxide fixation pathway in which acetyl-CoA is carboxylated twice by acetyl-CoA/propionyl-CoA carboxylase, and the products are reductively converted via 3-hydroxypropionate and succinyl-CoA to 4-hydroxybutyrate. 4-Hydroxybutyrate is then converted via 4-hydroxybutyryl-CoA dehydratase into two molecules of acetyl-CoA. The pathway occurs in autotrophic Sulfolobales and in ammonia-oxidizing Thaumarchaeota.
Justification: Same proposal as in the Msed_0709 review. GO:0043427 carbon fixation by 3-hydroxypropionate cycle is defined as the Chloroflexus cycle that releases glyoxylate via malyl-CoA cleavage. The archaeal 3HP/4HB cycle shares only the first half and regenerates acetyl-CoA via 4-hydroxybutyrate, and Msed_0406 acts in that 4HB branch.
Parent term: carbon fixation
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003987 acetyl-CoA synthetase activity | IDA PMID:23258541 Role of 4-hydroxybutyrate-CoA synthetase in the CO2 fixation... | KEEP AS NON CORE | Summary: Recombinant Msed_0406 ligates CoA to acetate in vitro (Km about 2 mM, Vmax 6 umol/min/mg), with kcat/Km about 13-fold below propionate. Reason: The activity was measured and is real, so the curator's IDA stands. It is not the physiological role. In the 3HP/4HB cycle acetyl-CoA is regenerated by thiolysis of acetoacetyl-CoA, not from free acetate, and the authors conclude the enzyme's cycle role is 4HB activation. M. sedula has a separate conserved acetate/propionate-CoA synthetase (Msed_1353). Tier choice per projects/ENZYME_SPECIFICITY.md - not promiscuous noise, because the acetate activity is a substantial part of a broadly promiscuous active site (the authors call the enzyme a promiscuous acetate/propionate ligase), not a trace activity far below Msed_1353; and not over-annotation, because the activity is a characterised property of the enzyme rather than an isolated assay of unknown relevance. Kept as a genuine secondary activity, non-core because Msed_1353 owns acetate activation in vivo. Supporting Evidence: PMID:23258541 Msed_0406 is also a promiscuous acetate/propionate ligase, although one that also shows activity on 4HB PMID:23258541 Conclusion: Msed_0406 is likely the physiologically relevant enzyme in the cycle. |
| GO:0003987 acetyl-CoA synthetase activity | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: Electronic mapping from the UniProt EC 6.2.1.1 / RHEA:23176 catalytic-activity line, which is itself based on the IDA data. Reason: Same claim as the IDA row. The in-vitro activity is real but peripheral to the enzyme's role in 4-hydroxybutyrate activation. Supporting Evidence: PMID:23258541 Msed_0406 is also a promiscuous acetate/propionate ligase, although one that also shows activity on 4HB |
| GO:0004321 fatty-acyl-CoA synthase activity | IEA GO_REF:0000118 | REMOVE | Summary: GO:0004321 is the fatty acid synthase reaction (EC 2.3.1.86, acetyl-CoA + n malonyl-CoA + NAD(P)H to long-chain acyl-CoA). Msed_0406 is an AMP-forming acid-CoA ligase (EC 6.2.1.-). Reason: Wrong activity. The term describes the multifunctional fatty acid synthase (a condensing acyltransferase/reductase system), not an acyl-CoA synthetase. The enzyme has the ANL-superfamily AMP-binding domain (PF00501/PF13193) and was shown to form acyl-CoA from acid, ATP and CoA. This looks like a "synthase" versus "synthetase" confusion propagated by TreeGrafter from PTHR43605 (ACSM-type ligases). The correct activities are captured by GO:0031955/GO:0050218 and the proposed 4-hydroxybutyrate-CoA ligase term. Supporting Evidence: PMID:23258541 For both enzymes, the production of 4HB-CoA from 4HB and CoA was confirmed using reversed-phase HPLC. |
| GO:0006633 fatty acid biosynthetic process | IEA GO_REF:0000118 | REMOVE | Summary: TreeGrafter propagation from mammalian medium-chain acyl-CoA synthetases (ACSM family). Msed_0406 activates short-chain acids for the 3HP/4HB cycle. Reason: There is no evidence for any role in fatty acid synthesis. Archaeal membrane lipids are isoprenoid ether lipids, and the enzyme's physiological substrate 4HB is channelled into acetyl-CoA regeneration in the CO2-fixation cycle. Activating a carboxylic acid to its CoA ester is not fatty acid biosynthesis; the propagation ignores the archaeal pathway context. Supporting Evidence: PMID:23258541 it is unlikely to have any other role in crenarchaeal metabolism outside of carbon fixation PMID:24532060 were produced recombinantly in Escherichia coli , combined in vitro , and shown to convert 4HB to acetyl-CoA |
| GO:0006637 acyl-CoA metabolic process | IEA GO_REF:0000118 | KEEP AS NON CORE | Summary: The enzyme forms acyl-CoA thioesters (4-hydroxybutyryl-CoA, propionyl-CoA, butyryl-CoA, acetyl-CoA) from the free acids. Reason: True at a general level, since the product of every reaction is an acyl-CoA. GO has no 4-hydroxybutyryl-CoA-specific metabolic process term, and the physiological process is better captured by carbon fixation (proposed NEW). Kept as a broad, non-core process. Supporting Evidence: PMID:23258541 For both enzymes, the production of 4HB-CoA from 4HB and CoA was confirmed using reversed-phase HPLC. |
| GO:0015645 fatty acid ligase activity | IEA GO_REF:0000118 | MODIFY | Summary: Correct but general parent of the short-chain acid-CoA ligase activities measured for Msed_0406. Reason: The enzyme ligates CoA to C2-C5 acids only. The specific supported descendant is GO:0031955 short-chain fatty acid-CoA ligase activity (whose child GO:0050218 propionate-CoA ligase activity the gene already carries). Proposed replacements: short-chain fatty acid-CoA ligase activity Supporting Evidence: PMID:23258541 Both Msed_0394 and Msed_0406 were found to have activity on a broad range of small organic acid substrates of up to five carbons in length. |
| GO:0016020 membrane | IEA GO_REF:0000044 | REMOVE | Summary: Derived from a UniProt sequence-analysis (ECO:0000255) single-pass membrane prediction for residues 105-125. Reason: The predicted helix lies within the conserved N-terminal domain of the ANL fold, just before the ATP-binding P-loop (204-212); it is a buried hydrophobic segment, not a membrane anchor. The recombinant enzyme was purified from the soluble, heat-treated cell-free extract and by gel filtration, and its full-length structure has been solved by X-ray crystallography (PDB 8BIQ, 8BIT) without a membrane. ANL-superfamily acyl-CoA synthetases are soluble cytoplasmic enzymes. No experimental data place it in a membrane. Supporting Evidence: file:METS5/Msed_0406/Msed_0406-uniprot.txt SUBCELLULAR LOCATION: Membrane {ECO:0000255}; Single-pass membrane protein {ECO:0000255}. PMID:23258541 Soluble, cell-free extract was heated to 65 file:METS5/Msed_0406/Msed_0406-uniprot.txt PDB; 8BIQ; X-ray; 2.80 A; A/B/C/D=2-564. |
| GO:0031956 medium-chain fatty acid-CoA ligase activity | IDA PMID:23258541 Role of 4-hydroxybutyrate-CoA synthetase in the CO2 fixation... | MODIFY | Summary: The IDA rests on the measured butyrate (C4) and valerate (C5) activities, which UniProt records as EC 6.2.1.2 (RHEA:48340). GO defines medium-chain fatty acids as having 6-12 carbons. Reason: The activities are real and the curator is right that the enzyme ligates CoA to butyrate and valerate. But GO:0031956 is defined for C6-C12 acids, and the full text reports activity only up to C5. Butyrate and valerate fall under GO:0031955 short-chain fatty acid-CoA ligase activity (fewer than 6 carbons). This is a lateral correction caused by the EC 6.2.1.2 name (butanoate--CoA ligase, also called medium-chain acyl-CoA ligase) mapping to the GO medium-chain term. Like acetate and propionate, butyrate is not a physiological substrate in the cycle, so the replacement is non-core. Proposed replacements: short-chain fatty acid-CoA ligase activity Supporting Evidence: PMID:23258541 Both Msed_0394 and Msed_0406 were found to have activity on a broad range of small organic acid substrates of up to five carbons in length. |
| GO:0031956 medium-chain fatty acid-CoA ligase activity | IEA GO_REF:0000120 | MODIFY | Summary: Electronic mapping from EC 6.2.1.2 / RHEA:48340 (a medium-chain fatty acid + ATP + CoA), applied by ARBA00086692 and the UniProt catalytic-activity line. Reason: Same claim as the IDA row. The measured substrates (butyrate, valerate) are short-chain acids under the GO definitions, so the correct term is GO:0031955. Proposed replacements: short-chain fatty acid-CoA ligase activity Supporting Evidence: PMID:23258541 Both Msed_0394 and Msed_0406 were found to have activity on a broad range of small organic acid substrates of up to five carbons in length. |
| GO:0050218 propionate-CoA ligase activity | IDA PMID:23258541 Role of 4-hydroxybutyrate-CoA synthetase in the CO2 fixation... | KEEP AS NON CORE | Summary: Propionate is the best in-vitro substrate of Msed_0406 (Vmax 15.1 umol/min/mg, Km 0.38 mM, kcat/Km about 43,000 M-1 s-1). Reason: A well-measured activity, so the IDA stands. It is not a cycle step, however. In the 3HP/4HB cycle propionyl-CoA is formed from 3-hydroxypropionyl-CoA by dehydration and reduction, not from free propionate. The authors and the follow-up reconstitution assign Msed_0406 its cycle role as the 4HB-CoA ligase. Kept as a real but non-core in-vitro activity. Supporting Evidence: PMID:23258541 It appears that Msed_0406 is primarily a promiscuous propionate-CoA synthetase. PMID:23258541 Next, 3HP is converted and reduced to propionyl-CoA, carboxylated a second time |
| GO:0050218 propionate-CoA ligase activity | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: Electronic mapping from the UniProt EC 6.2.1.17 / RHEA:20373 catalytic-activity line, based on the IDA data. Reason: Same claim as the IDA row; real in-vitro activity, peripheral to the physiological role. Supporting Evidence: PMID:23258541 It appears that Msed_0406 is primarily a promiscuous propionate-CoA synthetase. |
| GO:0015977 carbon fixation | IDA PMID:24532060 Conversion of 4-hydroxybutyrate to acetyl coenzyme A and its... | NEW | Summary: Msed_0406 catalyses the activation of 4-hydroxybutyrate to 4-hydroxybutyryl-CoA, a step of the 3-hydroxypropionate/4-hydroxybutyrate CO2-fixation cycle, and with the three downstream enzymes reconstitutes 4HB-to-acetyl-CoA conversion in vitro. Reason: The enzyme itself performs a step of the cycle, so it passes the participation test. Comparable catalytic steps of the same cycle (Msed_0709, malonyl-CoA/succinyl-CoA reductase) are given the same term in this project. GO:0043427 carbon fixation by 3-hydroxypropionate cycle is defined as the Chloroflexus glyoxylate-forming bicycle and does not include the 4HB branch, so it is not used. GO:0015977 is the most precise correct existing term, and a specific child term is proposed. The assignment rests on biochemistry and kinetics; there is no genetic evidence yet, and the paralog Msed_0394 may contribute a little. Supporting Evidence: PMID:24532060 were produced recombinantly in Escherichia coli , combined in vitro , and shown to convert 4HB to acetyl-CoA PMID:23258541 Conclusion: Msed_0406 is likely the physiologically relevant enzyme in the cycle. PMID:18079405 This intermediate was reduced to 4-hydroxybutyrate and converted into two acetyl-CoA molecules via 4-hydroxybutyryl-CoA dehydratase. |
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Download this section (compressed HTML)Q: Should GO record every measured in-vitro substrate of a promiscuous archaeal acyl-CoA ligase as an enables annotation? For Msed_0406 the best in-vitro substrate (propionate) is not a cycle intermediate, and the physiological substrate (4HB) is the poorest one and has no GO term, so the annotation set currently describes the enzyme by its non-physiological activities only.
Q: Should the EC 6.2.1.2 (RHEA:48340) to GO:0031956 mapping be reviewed? EC 6.2.1.2 covers butanoate and other C4-C11 acids, while GO defines medium-chain as C6-C12, so butyrate- and valerate-only activities are mapped to a term whose definition excludes them.
Q: Is the GO:0004321 fatty-acyl-CoA synthase activity (EC 2.3.1.86) assignment on PANTHER node PTN008683229 (PTHR43605, ACSM-type acyl-CoA synthetases) an error to be corrected in PAINT?
Experiment: Delete Msed_0406 and Msed_0394 singly and together, for example with a Sulfolobales genetic system, and compare growth on H2/CO2 with heterotrophic growth on peptides. Measure 4HB-CoA ligase activity and 4HB accumulation in the mutant extracts.
Hypothesis: Msed_0406, but not Msed_0394, is required for autotrophic growth of M. sedula.
Experiment: Using the 8BIQ structure, mutate candidate pocket residues (for example Ala249, Lys250, Ala321) and determine kcat/Km for 4HB, propionate and butyrate.
Hypothesis: Residues lining the acyl pocket determine the 4HB versus propionate preference of Msed_0406.
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