Msed_0147 encodes the biotin carboxylase (alpha) subunit of the acetyl-CoA/propionyl-CoA carboxylase of the thermoacidophilic crenarchaeon Metallosphaera sedula. This biotin-dependent enzyme is the carboxylating enzyme of the 3-hydroxypropionate/4-hydroxybutyrate autotrophic CO2-fixation cycle of the Sulfolobales. The purified enzyme is a stable complex of about 560 kDa, probably (alpha beta gamma)4. It is made of this 57 kDa biotin carboxylase, the 18.6 kDa biotin carboxyl carrier protein (Msed_0148) and the 57 kDa carboxyltransferase (Msed_1375). The single enzyme carboxylates acetyl-CoA to malonyl-CoA and propionyl-CoA to (S)-methylmalonyl-CoA at similar rates, using ATP and bicarbonate. Within the complex the alpha subunit performs the first half-reaction: the ATP-dependent carboxylation of the biotin carried on the beta subunit (biotin carboxylase, EC 6.3.4.14). It has an N-terminal ATP-grasp biotin carboxylation domain and a C-terminal BC domain. The three subunit genes are strongly induced under autotrophic growth. Because archaea have no fatty acids, the enzyme serves CO2 fixation rather than lipid synthesis. Together with the reductase steps that follow, the M. sedula enzyme has been expressed in Pyrococcus furiosus to make 3-hydroxypropionate from CO2.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000166 nucleotide binding | IEA GO_REF:0000104 | MODIFY | Summary: UniRule-derived nucleotide binding on the biotin carboxylase subunit, which binds ATP in its ATP-grasp domain. Reason: The term is correct but less specific than the evidence supports. The nucleotide bound is ATP, the substrate of the biotin carboxylase half-reaction. In the archaeal acyl-CoA carboxylase the ATP-binding residues lie in this subunit, and the gene already carries GO:0005524 ATP binding in this review. MODIFY to that descendant. Proposed replacements: ATP binding Supporting Evidence: PMID:12533469 The ATP-binding residues were found in the biotin carboxylase subunit file:METS5/Msed_0147/Msed_0147-uniprot.txt /note="ATP-grasp" |
| GO:0004075 biotin carboxylase activity | IEA GO_REF:0000003 | ACCEPT | Summary: EC-to-GO mapping from the UniProt EC 6.3.4.14 on the alpha subunit. This is the first half-reaction of the biotin-dependent carboxylase, the ATP-dependent carboxylation of biotin-BCCP. Reason: Msed_0147 is the alpha (biotin carboxylase) subunit of the purified M. sedula acetyl-CoA/propionyl-CoA carboxylase. Biotin carboxylase activity is the subunit's own catalytic activity, so it is annotated with enables, unlike the whole-enzyme acyl-CoA carboxylase activities, to which the subunit only contributes. The protein carries the complete biotin-carboxylation domain (PS50979) with its ATP-grasp subdomain. The gene is induced 7- to 30-fold under autotrophic growth with the other two subunit genes, and expression of the three genes in P. furiosus gives ATP- and bicarbonate-dependent carboxylase activity. No isolated-subunit assay of the M. sedula BC has been published, so the half-reaction assignment rests on the domain architecture and on the subunit composition of the purified enzyme. Supporting Evidence: PMID:12581213 The enzyme consists of two large subunits of 57 kDa each representing biotin carboxylase (alpha) and carboxytransferase (gamma), respectively, and a small 18.6 kDa biotin carrier protein (beta). PMID:24532060 especially the genes encoding acetyl-CoA/propionyl-CoA carboxylase (ACCΞ±Ξ²Ξ³) (Msed_0147, Msed_0148, Msed_1375; 7- to 30-fold) file:METS5/Msed_0147/Msed_0147-uniprot.txt /note="Biotin carboxylation" |
| GO:0005524 ATP binding | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: ATP binding by the ATP-grasp domain of the biotin carboxylase subunit. Reason: ATP is the phosphoryl donor of the biotin carboxylase half-reaction, and in the archaeal acyl-CoA carboxylase the ATP-binding residues are on the BC subunit. The term is as specific as warranted. It describes substrate binding that is part of the core biotin carboxylase activity rather than a separate function, so it is kept as non-core. Supporting Evidence: PMID:12533469 The ATP-binding residues were found in the biotin carboxylase subunit, whereas the conserved biotin-binding site was located on the biotin carboxyl carrier protein. PMID:23530213 E1 carboxylates acetyl-CoA using bicarbonate and requires ATP. |
| GO:0046872 metal ion binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro2GO mapping from the ATP-grasp domain (IPR011761), whose members bind Mg2+ (or Mn2+) together with ATP. Reason: ATP-grasp enzymes use divalent cations bound to the ATP phosphates, and the carboxylase assays used MgATP. The bound ion has not been identified for the M. sedula enzyme. The UniProt Co(2+) cofactor line comes from an ARBA rule with no M. sedula data. No more specific ion term is justified, so the general term is kept as non-core. Supporting Evidence: PMID:9973333 Extracts catalyzed the CO2-, MgATP-, and NADPH-dependent conversion of acetyl-CoA to 3-hydroxypropionate via malonyl-CoA |
| GO:0003989 acetyl-CoA carboxylase activity | IDA PMID:12581213 Characterization of acetyl-CoA/propionyl-CoA carboxylase in ... | NEW | Summary: The purified alpha-beta-gamma complex that contains Msed_0147 carboxylates acetyl-CoA to malonyl-CoA, the first CO2-fixing step of the 3-hydroxypropionate/4-hydroxybutyrate cycle. Reason: GOA has no acyl-CoA carboxylase activity row for this subunit. The overall reaction (ATP + acetyl-CoA + bicarbonate to malonyl-CoA) needs all three subunits. The biotin carboxylase subunit carries out its first half, so the correct claim is contributes_to, not enables. Supporting Evidence: PMID:12581213 These reactions were studied in the thermophilic and acidophilic Metallosphaera sedula and are shown to be catalyzed by one single large enzyme, which acts equally well on acetyl-CoA and propionyl-CoA. PMID:23530213 E1 (acetyl/propionyl-CoA carboxylase, encoded by Msed_0147, Msed_0148, and Msed_1375) |
| GO:0004658 propionyl-CoA carboxylase activity | IDA PMID:12581213 Characterization of acetyl-CoA/propionyl-CoA carboxylase in ... | NEW | Summary: The same purified complex carboxylates propionyl-CoA to (S)-methylmalonyl-CoA, the second CO2-fixing step of the cycle, at a rate similar to acetyl-CoA. Reason: The archaeal enzyme is bifunctional, as shown for the purified M. sedula enzyme and for the Acidianus brierleyi ortholog. Like the acetyl-CoA activity, the propionyl-CoA carboxylase activity belongs to the complex, and the BC subunit contributes the biotin carboxylation half. Supporting Evidence: PMID:12581213 The apparent Km values were 0.06 mm for acetyl-CoA, 0.07 mm for propionyl-CoA, 0.04 mm for ATP and 0.3 mm for bicarbonate. PMID:19684143 This enzyme is the second promiscuous enzyme in this cycle; the other is acetyl-CoA/propionyl-CoA carboxylase PMID:12533469 This result showed that A. brierleyi acyl-CoA carboxylase is a bifunctional enzyme in the modified 3-hydroxypropionate cycle. |
| GO:0015977 carbon fixation | IDA PMID:12581213 Characterization of acetyl-CoA/propionyl-CoA carboxylase in ... | NEW | Summary: The acetyl-CoA/propionyl-CoA carboxylase is the CO2-fixing enzyme of the 3-hydroxypropionate/4-hydroxybutyrate autotrophic cycle of M. sedula. The BC subunit performs the ATP-dependent bicarbonate activation step of both carboxylations. Reason: The subunit does part of the work of the process. It catalyses the carboxylation of biotin, which brings the fixed bicarbonate into the cycle, so it passes the participation test. The comparators are the other catalytic proteins of this cycle, such as the malonyl-CoA/succinyl-CoA reductase Msed_0709, which carry GO:0015977 in their reviews. GO:0043427 is the Chloroflexus glyoxylate-forming cycle and is not used. A specific child term, carbon fixation by 3-hydroxypropionate/4-hydroxybutyrate cycle, is proposed in the Msed_0709 review. Until it exists, GO:0015977 is the most precise correct term. Supporting Evidence: PMID:18079405 We discovered that an autotrophic member of the archaeal order Sulfolobales, Metallosphaera sedula, fixed CO2 with acetyl-coenzyme A (acetyl-CoA)/propionyl-CoA carboxylase as the key carboxylating enzyme. PMID:12581213 Acetyl-CoA/propionyl-CoA carboxylase is considered the main CO2 fixation enzyme of autotrophic members of Sulfolobaceae and the sequenced genomes of these Archaea contain the respective genes. PMID:24532060 Among the most highly upregulated genes for the HTR versus ACL contrast were those directly involved in CO 2 fixation in the 3HP/4HB pathway |
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Download this section (compressed HTML)Q: GO:0009317 acetyl-CoA carboxylase complex is defined as catalysing the first step in long-chain fatty acid biosynthesis, and GO has no propionyl-CoA carboxylase complex term. Should the definition be generalized, or a term created for the archaeal bifunctional acetyl-CoA/propionyl-CoA carboxylase complex, so that its three subunits can be annotated to a specific complex?
Q: Which divalent cation does the M. sedula biotin carboxylase use in vivo? The UniProt Co(2+) cofactor statement comes from an ARBA rule, not from data on this enzyme.
Experiment: Express Msed_0147 alone and assay bicarbonate-dependent ATP hydrolysis with free D-biotin and with BCCP (Msed_0148) biotinylated in vitro by Msed_2010. Measure ADP or phosphate formation, with and without acetyl-CoA.
Hypothesis: The isolated alpha subunit catalyses ATP-dependent carboxylation of free biotin or of the biotinylated beta subunit, independently of the carboxyltransferase.
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