Msed_1375 encodes the carboxyltransferase (gamma) subunit of the acetyl-CoA/propionyl-CoA carboxylase of the thermoacidophilic crenarchaeon Metallosphaera sedula. This biotin-dependent enzyme catalyses both carboxylations of the 3-hydroxypropionate/4-hydroxybutyrate autotrophic CO2-fixation cycle: acetyl-CoA to malonyl-CoA and propionyl-CoA to (S)-methylmalonyl-CoA, at similar rates. The purified holoenzyme is a complex of about 560 kDa, probably (alpha beta gamma)4. It is made of the biotin carboxylase (Msed_0147), the biotin carboxyl carrier protein (Msed_0148) and this 57 kDa carboxyltransferase, which is encoded at a separate locus. The protein is a single-chain carboxyltransferase of the propionyl-CoA carboxylase beta type, with N- and C-terminal CoA carboxyltransferase domains on a crotonase fold. In the complex it performs the second half-reaction: it transfers the carboxyl group from carboxybiotin on the carrier subunit to the acyl-CoA. Sequence analysis of the Acidianus brierleyi ortholog places the acyl-CoA and carboxybiotin binding sites in this subunit, so it is the subunit that sets the dual acetyl-CoA/propionyl-CoA specificity. The gene is strongly induced under autotrophic growth.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004658 propionyl-CoA carboxylase activity | IEA GO_REF:0000118 | MARK AS OVER ANNOTATED | Summary: TreeGrafter transfer of propionyl-CoA carboxylase activity from the PANTHER PCC beta-chain family node (PTHR43842) onto the carboxyltransferase subunit. Reason: The M. sedula enzyme has propionyl-CoA carboxylase activity, but the full reaction (ATP + propionyl-CoA + bicarbonate to methylmalonyl-CoA) is a property of the alpha-beta-gamma complex, not of this polypeptide. The gamma subunit carries out only the carboxyl-transfer half-reaction. It cannot use ATP or carboxylate biotin itself, and it needs the biotinylated carrier subunit. Assigning the whole-enzyme activity to the subunit alone overstates its function. The row is not removed, because the subunit does take part in the activity. Its contribution is recorded in core_functions as contributes_to GO:0004658, beside the equal acetyl-CoA carboxylase activity (NEW row below), which this PCC-beta family node does not capture. The subunit's own activity is a carboxyltransferase, proposed as GO:0016743 on the ligase row. This follows the treatment of the bacterial AccD carboxyltransferase subunit in genes/PSEPK/accD. 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:12533469 The acyl-CoA-binding site and the carboxybiotin-binding site were found in the carboxyltransferase subunit. |
| GO:0016874 ligase activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the CoA carboxyltransferase C-terminal domain (IPR011763) to the generic ligase class, reflecting the EC 6.4.1 classification of the whole carboxylase. Reason: The ATP-coupled ligation in the carboxylase reaction belongs to the biotin carboxylase subunit. The CoA carboxyltransferase domains of this subunit catalyse a carboxyl transfer from carboxybiotin to acetyl-CoA or propionyl-CoA, which is a transferase reaction (UniProt keyword Transferase). For this subunit, the ligase term describes the complex, not the subunit's own chemistry. This is a lateral correction. The subunit-level activity is best captured by GO:0016743 carboxyl- or carbamoyltransferase activity, because GO has no specific term for the acyl-CoA carboxytransferase half-reaction. The whole-enzyme ligase activities are captured by the contributes_to rows. Proposed replacements: carboxyl- or carbamoyltransferase activity Supporting Evidence: PMID:12533469 The acyl-CoA-binding site and the carboxybiotin-binding site were found in the carboxyltransferase subunit. PMID:12533469 The ATP-binding residues were found in the biotin carboxylase subunit file:METS5/Msed_1375/Msed_1375-uniprot.txt /note="CoA carboxyltransferase C-terminal" |
| GO:0003989 acetyl-CoA carboxylase activity | IDA PMID:12581213 Characterization of acetyl-CoA/propionyl-CoA carboxylase in ... | NEW | Summary: The purified complex that contains the gamma subunit carboxylates acetyl-CoA to malonyl-CoA as well as it carboxylates propionyl-CoA. Reason: GOA records only the propionyl-CoA activity for this subunit, but the archaeal enzyme is bifunctional. Because the activity belongs to the complex, the row carries contributes_to. The gamma subunit, which carries the acyl-CoA binding site, contributes the carboxyl transfer to acetyl-CoA in the first CO2-fixing step of the cycle. 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: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 cycle. The gamma subunit makes the C-C bond that attaches the fixed carboxyl group to the acyl-CoA. Reason: The subunit catalyses the bond-forming step of both carboxylations, so it does the work of the fixation step and passes the participation test. The comparators are the other catalytic proteins of the cycle, such as Msed_0709, which carry GO:0015977 in their reviews. GO:0043427 is the Chloroflexus glyoxylate-forming cycle and is not used. A specific 3HP/4HB-cycle child term 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 especially the genes encoding acetyl-CoA/propionyl-CoA carboxylase (ACCΞ±Ξ²Ξ³) (Msed_0147, Msed_0148, Msed_1375; 7- to 30-fold) |
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Download this section (compressed HTML)Q: GO has no term for the acyl-CoA carboxytransferase half-reaction of biotin-dependent carboxylases (carboxybiotin-[carrier protein] + acetyl-CoA or propionyl-CoA = biotin-[carrier protein] + malonyl-CoA or methylmalonyl-CoA; EC 2.1.3.15 for acetyl-CoA). Should one be created as a child of GO:0016743, by analogy with GO:0004075 for the biotin carboxylase half-reaction?
Q: The PANTHER PCC beta-chain node (PTN000523576) propagates propionyl-CoA carboxylase activity with enables to carboxyltransferase subunits. Should TreeGrafter and PAINT annotations of CT subunits use contributes_to? And should the archaeal bifunctional clade also carry acetyl-CoA carboxylase activity?
Q: Which residues of the gamma subunit allow it to accept acetyl-CoA and propionyl-CoA equally, when bacterial AccD/AccA and mitochondrial PCCB are substrate-selective?
Experiment: Express soluble Msed_1375 (for example in P. furiosus or Sulfolobus acidocaldarius) and assay the reverse half-reaction, malonyl-CoA or methylmalonyl-CoA + free biotin to acyl-CoA + carboxybiotin, with kinetic constants for both acyl-CoAs.
Hypothesis: The gamma subunit alone catalyses carboxyl transfer between a carboxylated acyl-CoA and biotin, and its own substrate preference matches that of the holoenzyme.
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