Msed_2001 encodes the 3-hydroxypropionyl-CoA dehydratase (EC 4.2.1.116) of the thermoacidophilic crenarchaeon Metallosphaera sedula. It is a soluble enzyme of the enoyl-CoA hydratase (crotonase) family and works in the 3-hydroxypropionate/4-hydroxybutyrate autotrophic CO2-fixation cycle of the Sulfolobales. In the first half of the cycle it dehydrates 3-hydroxypropionyl-CoA to acryloyl-CoA, which acryloyl-CoA reductase then reduces to propionyl-CoA. The same active site reversibly hydrates crotonyl-CoA to (S)-3-hydroxybutyryl-CoA at a similar rate; it does not use the (R) stereoisomer. Quantitative proteomics and kinetics indicate that this promiscuous enzyme carries almost all of the cell's 3-hydroxypropionyl-CoA dehydratase activity and most of its crotonyl-CoA hydratase activity, the latter being a step of the second half of the cycle that regenerates acetyl-CoA. The crystal structure with CoA shows the conserved crotonase catalytic glutamates (Glu113, Glu133) and a small enoyl pocket lined by bulky aromatic residues, suited to short-chain substrates. Reported oligomeric states differ (monomer, hexamer, octamer). The enzyme is conserved in autotrophic Sulfolobales and is unrelated to the dehydratase domain of the Chloroflexus propionyl-CoA synthase.
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 of that route, uses unrelated enzymes for it, and regenerates acetyl-CoA via 4-hydroxybutyrate and crotonyl-CoA. Msed_2001 catalyses steps in both halves of the archaeal cycle. This proposal repeats the one in the Msed_0709 review.
Parent term: carbon fixation
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the enoyl-CoA hydratase/isomerase conserved-site signature (IPR018376) to the root molecular-function term catalytic activity. Reason: The term is correct but uninformative. The specific activity is known from purified native and recombinant enzyme (PMID:19429610, PMID:33472982), and the gene already carries GO:0043956 3-hydroxypropionyl-CoA dehydratase activity, a descendant of catalytic activity. The second physiological activity, crotonyl-CoA hydration, is captured by GO:0120092, proposed below as NEW. Proposed replacements: 3-hydroxypropionyl-CoA dehydratase activity (2E)-butenoyl-CoA hydratase activity Supporting Evidence: PMID:19429610 3-Hydroxypropionyl-CoA dehydratase (E.C.4.2.1.17) catalyzes the following reversible reaction: 3-hydroxypropionyl-CoA β acryloyl-CoA + H 2 O. PMID:33472982 we conclude that Msed_2001 is a promiscuous 3-hydroxypropionyl-CoA dehydratase/crotonyl-CoA hydratase that is responsible for both conversions in vivo. |
| GO:0006635 fatty acid beta-oxidation | IEA GO_REF:0000118 | MARK AS OVER ANNOTATED | Summary: TreeGrafter transfer of fatty acid beta-oxidation from PANTHER node PTN000235214. Msed_2001 is placed in subfamily PTHR11941:SF54, named for the mitochondrial short-chain enoyl-CoA hydratase (ECHS1), whose role is fatty acid beta-oxidation. Reason: This is a crotonase-family process carried over to an enzyme that was recruited into CO2 fixation. GO:0006635 is defined as the complete oxidation of a long-chain fatty acid. Msed_2001 has a small enoyl pocket suited to short-chain 3-hydroxyacyl-CoAs (PMID:30013155), and it has only been characterised on C3 and C4 substrates. Both of its activities are accounted for by the 3HP/4HB cycle, in which Msed_2001 carries 99% of the cell's 3-hydroxypropionyl-CoA dehydratase activity and 86% of its crotonyl-CoA hydratase activity (PMID:33472982). No study shows that M. sedula degrades fatty acids or that Msed_2001 takes part in doing so. In the related Sulfolobus acidocaldarius, beta-oxidation enzymes are encoded in a dedicated FadR-regulated gene cluster (PMID:30948713). By contrast, Msed_2001 is the cycle enzyme conserved across autotrophic Sulfolobales. Crotonyl-CoA hydration is the same chemistry as the last round of beta-oxidation, and activity is still present in heterotrophically grown cells (PMID:19429610). A minor catabolic role therefore cannot be excluded, so the row is marked as an over-annotation rather than removed. Supporting Evidence: PMID:30013155 Ms3HPCD has a tightly formed Ξ±3 helix near the active site, and bulky aromatic residues are located at the enoyl-group binding site, resulting in the enzyme having an optimal substrate binding site for accepting short-chain 3-hydroxyacyl-CoA as a substrate. PMID:33472982 Msed_2001 is responsible for 99% of 3-hydroxypropionyl-CoA dehydratase activity and for 86% of crotonyl-CoA hydratase activity in M. sedula PMID:30948713 acting as a repressor of a 30-kb gene cluster comprising 23 open reading frames encoding lipases and Ξ²-oxidation enzymes. |
| GO:0043956 3-hydroxypropionyl-CoA dehydratase activity | IDA PMID:19429610 3-hydroxypropionyl-coenzyme A dehydratase and acryloyl-coenz... | ACCEPT | Summary: The dehydratase was purified from autotrophically grown M. sedula and identified by peptide mass fingerprinting as Msed_2001. The recombinant His-tagged enzyme dehydrated 3-hydroxypropionyl-CoA to acryloyl-CoA (Km 60 uM, kcat about 96 per second at 65 C). Reason: Direct biochemical evidence from native and recombinant enzyme. The activity was confirmed with independently produced enzyme by Loder et al. (PMID:27771364) and Liu et al. (PMID:33472982). It is the defining activity of the gene in the 3HP/4HB cycle. GO:0043956 is defined as 3-hydroxypropionyl-CoA = acryloyl-CoA + H2O (EC 4.2.1.116, RHEA:26518). Supporting Evidence: PMID:19429610 The best hit was the Msed_2001 gene annotated as an enoyl-CoA hydratase gene (100% identity and 41% coverage) coding for a 28.3-kDa (259 amino acids) protein. PMID:19429610 3-Hydroxypropionyl-CoA dehydratase (E.C.4.2.1.17) catalyzes the following reversible reaction: 3-hydroxypropionyl-CoA β acryloyl-CoA + H 2 O. |
| GO:0043956 3-hydroxypropionyl-CoA dehydratase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Combined automated annotation from the UniProt catalytic-activity line (RHEA:26518, EC 4.2.1.116). Reason: Same claim as the IDA row. The UniProt catalytic-activity annotation is itself based on experiment (PMID:19429610), so the electronic row agrees with the direct evidence. Supporting Evidence: PMID:19429610 3-Hydroxypropionyl-CoA dehydratase (E.C.4.2.1.17) catalyzes the following reversible reaction: 3-hydroxypropionyl-CoA β acryloyl-CoA + H 2 O. |
| GO:0120092 (2E)-butenoyl-CoA hydratase activity | IDA PMID:33472982 Convergent Evolution of a Promiscuous 3-Hydroxypropionyl-CoA... | NEW | Summary: Recombinant Msed_2001 reversibly hydrates crotonyl-CoA to (S)-3-hydroxybutyryl-CoA. It is about as active on (S)-3-hydroxybutyryl-CoA as on 3-hydroxypropionyl-CoA and is inactive on the (R) isomer. Kinetics and quantitative proteomics show it carries most of the cell's crotonyl-CoA hydratase activity in autotrophic growth. Reason: This is a second physiological activity, not a side activity. Crotonyl-CoA hydration is a step of the acetyl-CoA-regenerating half of the 3HP/4HB cycle, and Liu et al. assign about 86% of that flux in M. sedula to Msed_2001 (PMID:33472982). GO:0120092 is defined as 3-hydroxybutanoyl-CoA = (2E)-butenoyl-CoA + H2O and does not specify stereochemistry. Its Rhea cross-references include RHEA:26558, the (3S) reaction, which Rhea links to EC 4.2.1.116. GO:0120092 is neither an ancestor nor a descendant of GO:0043956. Supporting Evidence: PMID:19429610 3-Hydroxypropionyl-CoA dehydratase acted nearly equally as well on ( S )-3-hydroxybutyryl-CoA as on 3-hydroxypropionyl-CoA. PMID:19429610 The enzyme did not convert the ( R )-stereoisomer of 3-hydroxybutyryl-CoA. PMID:33472982 we conclude that Msed_2001 is a promiscuous 3-hydroxypropionyl-CoA dehydratase/crotonyl-CoA hydratase that is responsible for both conversions in vivo. |
| GO:0015977 carbon fixation | IDA PMID:19429610 3-hydroxypropionyl-coenzyme A dehydratase and acryloyl-coenz... | NEW | Summary: Msed_2001 catalyses two steps of the 3-hydroxypropionate/4-hydroxybutyrate autotrophic CO2-fixation cycle of M. sedula. These are 3-hydroxypropionyl-CoA dehydration in the first half and crotonyl-CoA hydration in the second half. Reason: The enzyme catalyses two of the cycle's steps itself, so it passes the participation test. Comparators in the same role (catalysts of cycle steps) carry carbon-fixation process terms. The 3-hydroxypropionyl-CoA synthetase Msed_1456, the preceding enzyme, has GO:0043427 by IDA (PMID:18165310) in GOA, and the Msed_0709 review adds GO:0015977 for malonyl-CoA reductase. GO:0043427 carbon fixation by 3-hydroxypropionate cycle is defined as the Chloroflexus glyoxylate-forming cycle, an independently evolved pathway that uses unrelated enzymes for this step (PMID:19429610). It is therefore not used here, consistent with the Msed_0709 review. A specific 3HP/4HB-cycle child term is proposed instead, and the parent GO:0015977 is the most precise correct existing term. Supporting Evidence: PMID:19429610 In M. sedula the reaction sequence is catalyzed by three enzymes. PMID:33472982 we conclude that Msed_2001 is a promiscuous 3-hydroxypropionyl-CoA dehydratase/crotonyl-CoA hydratase that is responsible for both conversions in vivo. PMID:18079405 This intermediate was reduced to 4-hydroxybutyrate and converted into two acetyl-CoA molecules via 4-hydroxybutyryl-CoA dehydratase. |
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Download this section (compressed HTML)Q: UniProt annotates the preceding enzyme, Msed_1456, to GO:0043427 carbon fixation by 3-hydroxypropionate cycle by IDA, and ARBA spreads the term across archaea. Yet the term is defined as the Chloroflexus glyoxylate-forming cycle. Should GO broaden GO:0043427, or create a separate 3HP/4HB-cycle term, so that enzymes of the two cycles are annotated consistently?
Q: What is the true oligomeric state of Msed_2001? Gel filtration gave a monomer, the crystal structure and size exclusion gave a hexamer, and Loder et al. reported an octamer. Does it form a complex with 3-hydroxypropionyl-CoA synthetase (Msed_1456) in vivo?
Q: Should PANTHER separate the archaeal 3-hydroxypropionyl-CoA dehydratase clade from the ECHS1-named subfamily PTHR11941:SF54, so that TreeGrafter stops propagating fatty acid beta-oxidation to it?
Experiment: Delete Msed_2001 in M. sedula or a genetically tractable Sulfolobales relative, for example Sulfolobus acidocaldarius with a reconstituted pathway or its ortholog in a related autotroph. Compare autotrophic and heterotrophic growth, and measure 3-hydroxypropionyl-CoA dehydratase and crotonyl-CoA hydratase activities in extracts.
Hypothesis: Msed_2001 is required for autotrophic growth of M. sedula and is the main crotonyl-CoA hydratase in vivo.
Experiment: Test recombinant Msed_2001 with C6 to C16 trans-2-enoyl-CoAs to define its chain-length limit. Compare Msed_2001 protein levels and growth of a deletion strain on fatty acids versus peptides.
Hypothesis: Msed_2001 contributes to fatty acid or butyrate catabolism during heterotrophic growth.
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