Msed_0638 encodes the catalytic (alpha, large) subunit of the coenzyme B12-dependent methylmalonyl-CoA mutase (MCM) of the thermoacidophilic crenarchaeon Metallosphaera sedula. The 553-residue protein consists of the substrate-binding TIM-barrel domain of the MCM/isobutyryl-CoA mutase family and lacks the C-terminal B12-binding domain that is fused to it in bacterial and mitochondrial MCMs. That domain is supplied by a separate, remotely encoded subunit, Msed_2055 (MCM-beta), which carries the DXHXXG-SXL-GG cobalamin-binding motif. The recombinant enzyme is soluble and active only when both subunits are coexpressed, and forms an alpha2beta2 heterotetramer that converts (R)-methylmalonyl-CoA to succinyl-CoA. Together with the adjacent methylmalonyl-CoA epimerase gene Msed_0639, it converts the (S)-methylmalonyl-CoA formed by propionyl-CoA carboxylation into succinyl-CoA, closing the 3-hydroxypropionate half of the 3-hydroxypropionate/4-hydroxybutyrate autotrophic CO2-fixation cycle. Its transcription is induced under autotrophic growth.
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 epimerase and mutase steps, and regenerates acetyl-CoA via 4-hydroxybutyrate. Its enzymes therefore have no specific process term. The same term is proposed in the Msed_0709 review.
Parent term: carbon fixation
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | TOO GENERAL | Summary: InterPro2GO mapping from the cobalamin-dependent enzyme catalytic domain superfamily (IPR016176) to the root molecular-function term. Reason: True but uninformative. The specific activity, methylmalonyl-CoA mutase activity (GO:0004494), is accepted in this review and entails this term. The recombinant alpha2beta2 enzyme containing Msed_0638 converts methylmalonyl-CoA to succinyl-CoA (PMID:22752162). Supporting Evidence: PMID:22752162 soluble, active MCM was produced only if MCM-Ξ± and MCM-Ξ² were coexpressed |
| GO:0004494 methylmalonyl-CoA mutase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Msed_0638 is the catalytic subunit (MCM-alpha) of the M. sedula methylmalonyl-CoA mutase. Recombinant MCM, coexpressed with the B12-binding subunit Msed_2055, is an alpha2beta2 heterotetramer with a specific activity of 2.2 umol/min/mg at 75 C, and together with the epimerase Msed_0639 converts (S)-methylmalonyl-CoA to succinyl-CoA. Reason: The electronic annotation is confirmed by direct biochemistry on the M. sedula protein (PMID:22752162). Msed_0638 carries the substrate-binding TIM barrel that holds the active site, so it is the catalytic subunit. Activity requires the partner subunit Msed_2055, which supplies the B12-binding domain; on its own Msed_0638 did not give soluble, active enzyme. The activity therefore belongs to the heterotetramer, and core_functions records it as a contribution to the complex activity. The term itself is the correct and most specific activity term. Supporting Evidence: PMID:22752162 the gene encoding MCE (Msed_0639) is adjacent to that encoding the catalytic subunit of MCM-Ξ± (Msed_0638) PMID:22752162 soluble, active MCM was produced only if MCM-Ξ± and MCM-Ξ² were coexpressed 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. |
| GO:0016866 intramolecular transferase activity | IEA GO_REF:0000002 | TOO GENERAL | Summary: InterPro2GO mapping from the MCM alpha/beta catalytic domain (IPR006099) to the EC 5.4 parent class, intramolecular transferase activity. Reason: Correct but uninformative. Methylmalonyl-CoA mutase is an intramolecular transferase (EC 5.4.99.2), and the specific term GO:0004494, accepted in this review, entails it. Supporting Evidence: 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. |
| GO:0031419 cobalamin binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro2GO mapping from the MCM catalytic-domain signatures. Msed_0638 consists of the TIM-barrel domain only. In the M. sedula enzyme the B12-binding domain, with the conserved DXHXXG-SXL-GG motif, is a separate subunit (Msed_2055). Reason: In MCM-family enzymes, adenosylcobalamin binds at the interface between the TIM-barrel domain, which contacts the corrin ring and the 5'-deoxyadenosyl group, and the Rossmann-type B12-binding domain, which supplies the lower axial histidine. In M. sedula that second domain is the separate subunit Msed_2055, which carries the B12-binding motif (PMID:22752162). Msed_0638 therefore contributes to cofactor binding within the heterotetramer, but is not the B12-binding subunit. This is inferred from the domain architecture and from family structures. No cobalamin-binding measurement exists for the isolated M. sedula subunit, which was not soluble alone. The term is kept as a non-core, cofactor-level annotation and is not proposed as the defining function. Supporting Evidence: PMID:22752162 The conserved coenzyme B(12)-binding motif (DXHXXG-SXL-GG) was identified in M. sedula MCM-Ξ². file:METS5/Msed_0638/Msed_0638-uniprot.txt Methylmalonyl-CoA mutase alpha/beta chain catalytic |
| GO:0015977 carbon fixation | IDA PMID:22752162 Epimerase (Msed_0639) and mutase (Msed_0638 and Msed_2055) c... | NEW | Summary: MCM catalyses the rearrangement of (R)-methylmalonyl-CoA to succinyl-CoA, a step of the 3-hydroxypropionate/4-hydroxybutyrate autotrophic CO2-fixation cycle of M. sedula. Transcription of Msed_0638 is induced under autotrophic growth. Reason: The evidence goes beyond homology. Msed_0638 is the catalytic subunit of the recombinantly verified M. sedula mutase, and with the epimerase it converts the cycle intermediate (S)-methylmalonyl-CoA to succinyl-CoA. Its transcription rises significantly under autotrophic compared with heterotrophic growth (PMID:22752162). The enzyme catalyses a step of the cycle, so it passes the participation test. Comparators in the same role (catalysts of individual cycle steps), namely Msed_0709, Msed_1424, Msed_0406 and 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 cited enzyme activity is for the recombinant complex, not a measurement in cell extracts. 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. PMID:18079405 In this system, one acetyl-CoA and two bicarbonate molecules were reductively converted via 3-hydroxypropionate to succinyl-CoA. |
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Download this section (compressed HTML)Q: Does the MeaB/ArgK-like G-protein Msed_2056, whose ORF overlaps the MCM-beta gene Msed_2055, act as the cofactor-loading and repair chaperone of the M. sedula mutase, as MeaB does for bacterial MCM?
Q: Should GO represent the M. sedula mutase as a heteromeric complex, with Msed_0638 (catalytic) and Msed_2055 (B12-binding) each annotated as contributing to methylmalonyl-CoA mutase activity, given that neither subunit is active alone?
Experiment: Delete Msed_0638 or Msed_2055 with a Sulfolobales genetic system, and compare growth on CO2/H2 with growth on peptides or yeast extract. Test for accumulation of methylmalonyl-CoA under autotrophic conditions.
Hypothesis: Methylmalonyl-CoA mutase (Msed_0638/Msed_2055) is required for autotrophic, but not heterotrophic, growth of M. sedula.
Experiment: Coexpress Msed_0638, Msed_2055 and Msed_2056. Measure MCM activity, adenosylcobalamin loading and the rate of inactivation during turnover, with and without GTP.
Hypothesis: Msed_2056 increases MCM activity or protects it from inactivation.
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