Msed_0656 encodes an acetoacetyl-CoA beta-ketothiolase (acetyl-CoA C-acetyltransferase, EC 2.3.1.9) of the thermoacidophilic crenarchaeon Metallosphaera sedula. It is a 395-residue member of the thiolase family with the conserved thiolase N- and C-terminal domains. In the 3-hydroxypropionate/4-hydroxybutyrate autotrophic CO2-fixation cycle of the Sulfolobales, it catalyses the final step, the CoA-dependent thiolytic cleavage of acetoacetyl-CoA into two acetyl-CoA. One acetyl-CoA re-enters the cycle as the CO2 acceptor and the other is released for biosynthesis. The gene was first assigned by its similarity to bacterial thiolases and by its induction under autotrophic growth. The recombinant enzyme cleaves acetoacetyl-CoA with a Km of 0.18 mM and a high specific activity (about 140 U/mg at 70 C), making it the fastest enzyme of the acetyl-CoA-regenerating branch, and it supports in vitro reconstitution of the conversion of 4-hydroxybutyrate to acetyl-CoA. M. sedula has several other thiolase paralogs, at least one of which is also induced in autotrophy and may contribute to the reaction.
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: GO:0043427 carbon fixation by 3-hydroxypropionate cycle is defined as the Chloroflexus cycle that releases glyoxylate via malyl-CoA cleavage. Msed_0656 catalyses the acetyl-CoA-regenerating final step of the archaeal cycle, which has no counterpart in that term. This proposal repeats the one in the Msed_0709 review.
Parent term: carbon fixation
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003985 acetyl-CoA C-acetyltransferase activity | IEA GO_REF:0000003 | ACCEPT | Summary: EC2GO mapping from the EC 2.3.1.9 assignment in the EMBL entry. Reason: GO:0003985 is defined as 2 acetyl-CoA = CoA + acetoacetyl-CoA (verified in OLS). Recombinant Msed_0656 catalyses the CoA-dependent cleavage of acetoacetyl-CoA, with a Km of 0.18 mM and about 140 U/mg (PMID:24532060). This is the thiolytic direction of the same reversible reaction and the direction that operates in the 3HP/4HB cycle. The electronic annotation therefore agrees with direct biochemical evidence on this protein. It is the core activity. Supporting Evidence: PMID:24532060 For the Ξ²-ketothiolase, enzyme activity was measured using the discontinuous DTNB assay to measure the substrate-dependent disappearance of CoA at 70Β°C. PMID:24532060 Msed_0656 on acetoacetyl-CoA, 0.18 mM PMID:21169482 Therefore, these similar genes most likely code for beta-ketothiolase, catalyzing the last step in the two carbon fixation cycles. |
| GO:0016746 acyltransferase activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the thiolase-like superfamily (IPR016039). Reason: The term is correct but general. GO:0016746 is an ancestor of GO:0003985 acetyl-CoA C-acetyltransferase activity (OLS). That specific activity is shown for the recombinant enzyme (PMID:24532060), and the gene already carries it, so the row is refined to it. Proposed replacements: acetyl-CoA C-acetyltransferase activity Supporting Evidence: PMID:24532060 Msed_0656 on acetoacetyl-CoA, 0.18 mM |
| GO:0016747 acyltransferase activity, transferring groups other than amino-acyl groups | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the thiolase family and the thiolase N-terminal, C-terminal and conserved-site signatures. Reason: The term is correct but general. GO:0016747 is the parent of acetyltransferase activity and an ancestor of GO:0003985 (OLS), which is shown experimentally for Msed_0656 (PMID:24532060) and already carried by the gene. The row is refined to GO:0003985. Proposed replacements: acetyl-CoA C-acetyltransferase activity Supporting Evidence: PMID:24532060 Msed_0656 on acetoacetyl-CoA, 0.18 mM |
| GO:0015977 carbon fixation | IDA PMID:24532060 Conversion of 4-hydroxybutyrate to acetyl coenzyme A and its... | NEW | Summary: Msed_0656 catalyses the last step of the 3HP/4HB autotrophic CO2-fixation cycle, the cleavage of acetoacetyl-CoA into two acetyl-CoA, which regenerates the CO2 acceptor. The branch was reconstituted in vitro with Msed_0656 and three other recombinant enzymes. Reason: The enzyme catalyses a step of the cycle itself, so it passes the participation test. Comparators in the same role (catalysts of individual cycle steps), namely Msed_0406, Msed_1424, Msed_0709 and Msed_2001, carry GO:0015977 in their reviews. GO:0043427 is the Chloroflexus glyoxylate-forming cycle and is not used. GO:0015977 is the most precise correct existing term, and the 3HP/4HB-cycle child term from the Msed_0709 review is repeated below. As a caveat, paralogs such as Msed_0386 may share the reaction in vivo (PMID:32218776), and there is no genetic evidence. Supporting Evidence: PMID:24532060 were produced recombinantly in Escherichia coli , combined in vitro , and shown to convert 4HB to acetyl-CoA PMID:32218776 in addition to the characterized Ξ²-ketothiolase Msed_0656 gene PMID:21169482 In M. sedula , Msed_0656 exhibited upregulation under autotrophic conditions |
Loading supporting contentβ¦
Download this section (compressed HTML)Q: How is the acetoacetyl-CoA thiolase flux of autotrophic M. sedula divided between Msed_0656 and its paralogs (Msed_0386, Msed_0396, Msed_1647, Msed_1290, Msed_0270, Msed_0271)?
Q: Does Msed_0656 also supply acetoacetyl-CoA for the archaeal mevalonate pathway, as the ARBA "Isoprene biosynthesis" keyword implies, or does a different paralog do so?
Experiment: Quantify the thiolase paralogs by proteomics in autotrophic cells. Assay recombinant Msed_0386 and Msed_0396 kinetically, and if genetics allow, delete Msed_0656 in a tractable Sulfolobales host and measure acetoacetyl-CoA thiolase activity and autotrophic growth.
Hypothesis: Msed_0656 is the main acetoacetyl-CoA thiolase of the autotrophic cycle.
Loading supporting contentβ¦
Download this section (compressed HTML)Loading supporting contentβ¦
Download this section (compressed HTML)