Msed_0639 encodes the methylmalonyl-CoA epimerase (MCE) of the thermoacidophilic crenarchaeon Metallosphaera sedula. It is a 140-residue protein of the vicinal oxygen chelate (VOC, glyoxalase-like) fold. The recombinant enzyme is a homodimer that is activated by the divalent cations Ni2+, Co2+ and Mg2+, and has a high specific activity (218 umol/min/mg at 75 C). It interconverts (S)- and (R)-methylmalonyl-CoA. In the 3-hydroxypropionate/4-hydroxybutyrate autotrophic CO2-fixation cycle it converts the (S)-methylmalonyl-CoA formed by propionyl-CoA carboxylation into the (R)-epimer, which the adjacent-gene product, methylmalonyl-CoA mutase (Msed_0638 with Msed_2055), then rearranges to succinyl-CoA. Its transcription is induced under autotrophic growth. Single copies of the gene are conserved across Sulfolobaceae genomes.
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 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 review.
Parent term: carbon fixation
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004493 methylmalonyl-CoA epimerase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Msed_0639 is the methylmalonyl-CoA epimerase of M. sedula. The recombinant homodimeric enzyme is metal-activated, has a specific activity of 218 umol/min/mg at 75 C, and with MCM converts (S)-methylmalonyl-CoA to succinyl-CoA. Reason: The electronic annotation is confirmed by direct biochemistry on the recombinant M. sedula protein (PMID:22752162). The protein has the MCE-specific signatures (TIGR03081, IPR017515, cd07249). This activity is the core function of the gene. Supporting Evidence: PMID:22752162 MCE is a homodimer and MCM is a heterotetramer 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:0046491 L-methylmalonyl-CoA metabolic process | IEA GO_REF:0000118 | KEEP AS NON CORE | Summary: TreeGrafter assignment of the family-level process. The epimerase interconverts the two methylmalonyl-CoA epimers, so L-methylmalonyl-CoA is its substrate or product. Reason: The term is true, because the enzyme catalyses a reaction of L-methylmalonyl-CoA metabolism. The GO definition frames it around odd-chain fatty acid oxidation in animals, but in M. sedula the reaction is a step of autotrophic CO2 fixation, which is the gene's core process (GO:0015977, added below). The term is not a parent of carbon fixation, so it is kept as an accurate, non-core metabolite-level process term rather than marked too general. 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:0015977 carbon fixation | IDA PMID:22752162 Epimerase (Msed_0639) and mutase (Msed_0638 and Msed_2055) c... | NEW | Summary: MCE converts (S)-methylmalonyl-CoA, the product of propionyl-CoA carboxylation, into (R)-methylmalonyl-CoA for the mutase. This is a step of the 3-hydroxypropionate/4-hydroxybutyrate autotrophic CO2-fixation cycle of M. sedula, and Msed_0639 transcription is induced under autotrophic growth. Reason: The evidence goes beyond homology. The recombinant enzyme has the activity, performs the cycle step together with MCM, and its gene is significantly upregulated 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 activity is for the recombinant enzyme, 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: Which divalent metal occupies the VOC-fold active site of M. sedula MCE in vivo, given that recombinant enzyme was activated by Ni2+, Co2+ and Mg2+?
Q: Is the epimerase step rate-limiting at physiological temperature, given that (S)/(R)-methylmalonyl-CoA also epimerizes non-enzymatically?
Experiment: Delete Msed_0639 and compare growth on CO2/H2 with heterotrophic growth. Measure (S)-methylmalonyl-CoA accumulation and the residual rate of conversion to succinyl-CoA in extracts.
Hypothesis: Msed_0639 is required for efficient autotrophic growth of M. sedula.
Experiment: Determine the metal content of the native or anaerobically purified enzyme by ICP-MS, and test alanine substitutions of the predicted metal-ligand residues.
Hypothesis: MCE uses a specific divalent metal ion bound by conserved VOC-fold residues.
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