Msed_0148 encodes the biotin carboxyl carrier protein (BCCP, beta subunit, 18.6 kDa) of the acetyl-CoA/propionyl-CoA carboxylase of the thermoacidophilic crenarchaeon Metallosphaera sedula. This bifunctional biotin-dependent enzyme catalyses both carboxylations of the 3-hydroxypropionate/4-hydroxybutyrate autotrophic CO2-fixation cycle. The holoenzyme is a complex of about 560 kDa, probably (alpha beta gamma)4. Its other subunits are the biotin carboxylase (Msed_0147, encoded by the gene just upstream and translationally coupled to Msed_0148) and the carboxyltransferase (Msed_1375). The C-terminal biotinyl domain carries the covalently attached biotin prosthetic group. The biotin carboxylase subunit carboxylates this biotin with ATP and bicarbonate, and the carboxybiotin then delivers the carboxyl group to acetyl-CoA or propionyl-CoA at the carboxyltransferase site. As in other Sulfolobales, the residue after the biotinylated lysine is a serine rather than the usual methionine. The M. sedula biotin protein ligase (Msed_2010) biotinylates this subunit in vitro, and poor biotinylation by heterologous ligases limits the activity of the recombinant enzyme. The gene is strongly induced under autotrophic growth. Small biotinylated proteins of this kind are abundant in autotrophic Sulfolobales but absent from archaea that use other CO2-fixation pathways.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003989 acetyl-CoA carboxylase activity | IDA PMID:12581213 Characterization of acetyl-CoA/propionyl-CoA carboxylase in ... | NEW | Summary: GOA has no annotation for this protein. The purified complex that contains the 18.6 kDa biotin carrier subunit carboxylates acetyl-CoA to malonyl-CoA with ATP and bicarbonate. Reason: The BCCP subunit has no catalytic site of its own, but it carries the biotin cofactor that both half-reactions depend on. It is carboxylated by the biotin carboxylase subunit and donates the carboxyl group at the carboxyltransferase site. It therefore contributes to the activity of the complex (the cofactor case of the participation test) and does not enable it alone. GO has no carboxyl-carrier molecular-function term, so contributes_to the whole-enzyme activity is the most informative function annotation available. 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:27315782 BPL is responsible for attaching biotin to ACC-Ξ² (BCCP) to create active holoenzyme 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 at a rate similar to acetyl-CoA, the second CO2-fixing step of the cycle. Reason: The archaeal enzyme is bifunctional (M. sedula and the Acidianus brierleyi ortholog). The BCCP subunit carries the carboxybiotin for both carboxylations, so it contributes to the propionyl-CoA carboxylase activity of the complex as well. 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:12533469 the conserved biotin-binding site was located on the biotin carboxyl carrier protein |
| 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. BCCP carries the fixed carboxyl group between the two active sites of the enzyme. Reason: The protein does part of the work of the fixation step. Its biotin carries the activated bicarbonate-derived carboxyl group from the biotin carboxylase site to the acyl-CoA at the carboxyltransferase site, so it is a cofactor-carrying participant, not merely a requirement. The gene is among the most strongly induced under autotrophic growth. Comparator check - BCCPs are annotated to the processes that their carboxylases serve, for example E. coli AccB to fatty acid biosynthetic process and malonyl-CoA biosynthetic process. GO:0043427 is the Chloroflexus 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:9973333 They contained large amounts of a small biotin-carrying protein, which is most likely part of the acetyl-CoA and propionyl-CoA carboxylases. 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: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 molecular-function term for the carboxyl-carrier role of a BCCP (by analogy with GO:0000036 acyl carrier activity). Should GO add a "biotin carboxyl carrier activity" term, so that BCCP subunits need not be annotated only through contributes_to the whole-enzyme activity?
Q: GO:0009317 acetyl-CoA carboxylase complex is defined by fatty acid biosynthesis. Should it be generalized, or a new complex term made, to cover the archaeal bifunctional acetyl-CoA/propionyl-CoA carboxylase?
Experiment: Biotinylate recombinant Msed_0148 with Msed_2010 and map the modified residue by LC-MS/MS. Test K133A and S134M variants for biotinylation by Msed_2010 and by E. coli BirA, and test reconstituted holoenzyme activity with acetyl-CoA and propionyl-CoA.
Hypothesis: Lys133, in the AMKS motif, is the single biotinylation site of Msed_0148, and the serine at position +1 is what restricts biotinylation to Sulfolobales-type biotin protein ligases.
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