Msed_2055 encodes the coenzyme B12-binding (beta, small) subunit of the adenosylcobalamin-dependent methylmalonyl-CoA mutase (MCM) of the thermoacidophilic crenarchaeon Metallosphaera sedula. The 155-residue protein consists of a single Rossmann-type B12-binding domain, homologous to the C-terminal cofactor-binding domain of canonical bacterial and mitochondrial MCM alpha chains, and carries the conserved DXHXXG-SXL-GG motif whose histidine is the expected lower axial ligand to the cobalt of adenosylcobalamin. The substrate-binding TIM-barrel domain is supplied by the remotely encoded catalytic subunit Msed_0638. The recombinant mutase is soluble and active only when both subunits are coexpressed, and forms an alpha2beta2 heterotetramer that converts (R)-methylmalonyl-CoA to succinyl-CoA, a step in the 3-hydroxypropionate/4-hydroxybutyrate autotrophic CO2-fixation cycle. Msed_2055 transcription is induced under autotrophic growth. The overlapping downstream gene Msed_2056 encodes a MeaB/ArgK-like G-protein, a candidate cofactor-loading chaperone.
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. The archaeal 3HP/4HB cycle shares only the first half, which includes the methylmalonyl-CoA mutase step, and regenerates acetyl-CoA via 4-hydroxybutyrate. Its enzymes therefore have no specific process term. The same term is proposed in the Msed_0709 and Msed_0638 reviews.
Parent term: carbon fixation
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0016853 isomerase activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the MCM C-terminal B12-binding domain signature (IPR006159, Acid_CoA_mut_C) to the EC 5 isomerase class. Msed_2055 is that domain alone, as a separate subunit of the M. sedula alpha2beta2 mutase. Reason: The essence is right but the term is too general and is placed on the wrong unit. Methylmalonyl-CoA mutase (EC 5.4.99.2) is an isomerase, and Msed_2055 does part of the work, since it holds the adenosylcobalamin whose radical chemistry drives the rearrangement. However, the activity belongs to the alpha2beta2 complex. The substrate-binding TIM barrel is on Msed_0638, and active enzyme was obtained only when both subunits were coexpressed (PMID:22752162). This is the whole-complex-on-one-subunit pattern (ARCHAEA pattern 16). The replacement is the specific activity, GO:0004494 methylmalonyl-CoA mutase activity, which this review records in core_functions as contributes_to rather than as an independent activity. The subunit's own activity is cobalamin binding (GO:0031419). Proposed replacements: methylmalonyl-CoA mutase activity Supporting Evidence: PMID:22752162 soluble, active MCM was produced only if MCM-Ξ± and MCM-Ξ² were coexpressed PMID:22752162 MCM is a heterotetramer (Ξ±(2)Ξ²(2)) PMID:22803641 A variant arrangement has been reported for MCM from Metallosphaera sedula that exhibits a split organization (Fig. 2) with one subunit being homologous to the substrate-binding domain and the second, to the AdoCbl-binding domain in the Ξ±-subunit of the canonical MCM |
| GO:0031419 cobalamin binding | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the B12-binding domain signatures. Msed_2055 is a single B12-binding domain (PROSITE PS51332, residues 20-148) with the DXHXXG-SXL-GG motif (DGHDRG at 31-36, SIL at 78, GG at 108), and was named the coenzyme B12-binding subunit of the M. sedula mutase. Reason: This is the defining activity of the subunit. In split-subunit acyl-CoA mutases the B subunit binds adenosylcobalamin, and in class I B12-binding domains the histidine of DXHXXG is the axial ligand to cobalt (PMID:22803641). Han et al. identified that motif in M. sedula MCM-beta (PMID:22752162). The evidence is domain, motif and family based. No binding measurement on the isolated subunit is reported in the abstract, and the full text could not be read. Supporting Evidence: PMID:22752162 the gene for the coenzyme B(12)-binding subunit of MCM (MCM-Ξ²) is located remotely (Msed_2055) PMID:22752162 The conserved coenzyme B(12)-binding motif (DXHXXG-SXL-GG) was identified in M. sedula MCM-Ξ². PMID:22803641 subunit B (IcmB and HcmB, respectively) binds AdoCbl file:METS5/Msed_2055/Msed_2055-bioinformatics/RESULTS.md This matches the DXHXXG-SXL-GG motif reported for M. sedula MCM-beta |
| GO:0046872 metal ion binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro2GO mapping from the B12-binding domain signatures, reflecting coordination of the cobalt of cobalamin by the conserved motif histidine (His33 in Msed_2055). Reason: Not false. In base-off/His-on binding the motif histidine is the axial ligand to the cobalt atom (PMID:22803641), so the protein does contact a metal. But the cobalt is bound only as part of the corrin cofactor, and cobalamin binding (GO:0031419, accepted here) is the informative statement. A free-ion refinement such as cobalt ion binding is not proposed, because no evidence exists that the subunit binds cobalt outside the cofactor. Kept as a cofactor-level, non-core term, as in the human MTR review. Supporting Evidence: PMID:22803641 Cobalamin is bound to all these enzymes in the βbase-off/His-onβ conformation, in which the side chain of the histidine in a conserved DXHXXG motif, serves as the axial ligand to cobalt file:METS5/Msed_2055/Msed_2055-bioinformatics/RESULTS.md His33 is the candidate lower axial ligand to cobalt |
| GO:0015977 carbon fixation | IDA PMID:22752162 Epimerase (Msed_0639) and mutase (Msed_0638 and Msed_2055) c... | NEW | Summary: The Msed_0638/Msed_2055 mutase rearranges (R)-methylmalonyl-CoA to succinyl-CoA, a step of the 3-hydroxypropionate/4-hydroxybutyrate CO2-fixation cycle of M. sedula. Msed_2055 transcription is induced under autotrophic growth. Reason: Participation test. The mutase step is catalysed by the alpha2beta2 complex, and Msed_2055 supplies the cofactor-binding domain that the step depends on, since active enzyme needed both subunits (PMID:22752162). This is the cofactor case of participation, not mere necessity. Comparator check. The catalytic partner Msed_0638 and other single-step catalysts of the cycle (Msed_0709, Msed_1424, Msed_0406, Msed_2001) carry GO:0015977 in their reviews. GO:0043427 is the Chloroflexus glyoxylate-forming cycle and is not used, and a 3HP/4HB-cycle child term is proposed below. Caveats are that no genetic evidence exists, and that the activity was measured for the recombinant complex. Supporting Evidence: PMID:22752162 The expression of all three genes was significantly upregulated under autotrophic compared to heterotrophic growth conditions, implying a role in CO(2) fixation. PMID:22752162 The two enzymes together catalyzed the two-step conversion of (S)-methylmalonyl-CoA to succinyl-CoA, consistent with their proposed role in the 3-HP/4-HB cycle. |
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Download this section (compressed HTML)Q: Should the InterPro2GO mapping from IPR006159 (MCM C-terminal B12-binding domain) to isomerase activity be restricted to fused MCM chains, or changed to contributes_to, given that in split-subunit mutases such as M. sedula Msed_2055 and IcmB the matching protein is a non-catalytic cofactor-binding subunit?
Q: Does the MeaB/ArgK-like G-protein Msed_2056, whose ORF overlaps Msed_2055, load adenosylcobalamin onto the beta subunit and protect the mutase from inactivation, as MeaB does for bacterial MCM?
Experiment: Purify Msed_2055 alone or as the reconstituted complex, and measure adenosylcobalamin binding by UV-visible and EPR spectroscopy. Compare wild type with an H33 mutant, and test whether the H33 mutant complex retains mutase activity.
Hypothesis: Msed_2055 binds adenosylcobalamin through His33 in the base-off/His-on mode.
Experiment: Coexpress Msed_0638, Msed_2055 and Msed_2056, and measure activity, cofactor occupancy and inactivation rate during turnover, with and without GTP.
Hypothesis: Msed_2056 increases cofactor loading or the stability of the Msed_0638/Msed_2055 mutase.
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