Msed_1456 encodes 3-hydroxypropionyl-CoA synthetase (EC 6.2.1.36) of the thermoacidophilic crenarchaeon Metallosphaera sedula. It is an AMP-forming acyl-CoA synthetase of the ANL (adenylate-forming) superfamily that works as a homotetramer of about 78 kDa subunits. It activates 3-hydroxypropionate to 3-hydroxypropionyl-CoA with ATP and CoA, releasing AMP and pyrophosphate. This is the first step of the reductive conversion of 3-hydroxypropionate to propionyl-CoA in the 3-hydroxypropionate/4-hydroxybutyrate autotrophic CO2-fixation cycle of the Sulfolobales. The enzyme has high affinity for 3-hydroxypropionate (Km 180 uM) and ATP (Km 45 uM), and uses propionate with similar catalytic efficiency. Acrylate is used about 10-fold less efficiently, and acetate and butyrate are poor substrates. Its activity is induced about eightfold under autotrophic growth. Although it falls in the acetyl-CoA synthetase sequence family, acetate activation in M. sedula is carried out mainly by a separate paralog. Recombinant M. sedula enzyme is soluble only when co-expressed with the next enzyme of the cycle, 3-hydroxypropionyl-CoA dehydratase (Msed_2001), with which it interacts in yeast two-hybrid assays.
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 and regenerates acetyl-CoA via 4-hydroxybutyrate, so its enzymes, including this 3-hydroxypropionyl-CoA synthetase, 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:0003987 acetyl-CoA synthetase activity | IEA GO_REF:0000120 | MARK AS OVER ANNOTATED | Summary: Family-level transfer of acetyl-CoA synthetase activity from the acetyl-CoA ligase family signature (IPR011904) and the PANTHER PTHR24095 acetyl-CoA synthetase node. Msed_1456 activates acetate only weakly. Reason: The purified native enzyme does activate acetate, so the term is not strictly false. However, acetate was a poor substrate, and the authors concluded that the enzyme is specific for 3-hydroxypropionate and propionate (PMID:18165310). A separate paralog, Msed_1353, is the M. sedula acetate/propionate-CoA synthetase (PMID:23258541). The row reflects the family name that this substrate-diverged member inherits from its founder, not the enzyme's physiological activity. Its specific activity is captured by GO:0043955. Supporting Evidence: PMID:18165310 Both the native enzyme from M. sedula and the recombinant enzyme from S. tokodaii not only activated 3-hydroxypropionate to its CoA ester but also activated propionate, acrylate, acetate, and butyrate; however, with the exception of propionate, the affinities for these substrates were reduced. PMID:18165310 The enzyme, therefore, is specific for 3-hydroxypropionate and propionate. PMID:23258541 Msed_1353 was active on propionate and acetate, but not on 4HB. |
| GO:0006085 acetyl-CoA biosynthetic process | IEA GO_REF:0000120 | REMOVE | Summary: Process term that follows from the family-level acetyl-CoA synthetase assignment. Msed_1456 does not make acetyl-CoA in vivo. Reason: The enzyme's product is 3-hydroxypropionyl-CoA, which is converted via acryloyl-CoA to propionyl-CoA in the first half of the 3HP/4HB cycle (PMID:18165310). Acetyl-CoA is regenerated in the second half of the cycle by the 4-hydroxybutyrate branch (PMID:18079405), in steps that Msed_1456 does not catalyse. The only route by which Msed_1456 could form acetyl-CoA is its weak acetate side-activity, which has no shown physiological role. A separate paralog (Msed_1353) activates acetate (PMID:23258541). The row is a family-level inference contradicted by the enzyme's substrate specificity and pathway position. Supporting Evidence: PMID:18165310 The further reductive conversion of 3-hydroxypropionate to propionyl-CoA requires three enzymatic steps: the activation of 3-hydroxypropionate to its CoA ester, dehydration to acrylyl-CoA, and reduction of acrylyl-CoA to propionyl-CoA. PMID:18079405 This intermediate was reduced to 4-hydroxybutyrate and converted into two acetyl-CoA molecules via 4-hydroxybutyryl-CoA dehydratase. |
| GO:0016208 AMP binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro2GO mapping from the acetyl-CoA ligase family. Msed_1456 is an AMP-forming acyl-CoA synthetase whose reaction passes through an acyl-adenylate and releases AMP. Reason: Binding of the adenylate moiety is part of the catalytic mechanism of this AMP-forming enzyme, whose reaction yields AMP and pyrophosphate (PMID:18165310). It is a mechanistic feature of the ligase activity, not a separate function, so it is kept as non-core. Supporting Evidence: PMID:18165310 The first enzyme, 3-hydroxypropionyl-CoA synthetase, catalyzes the following reaction: 3-hydroxypropionate + ATP + CoA β 3-hydroxypropionyl-CoA + AMP + PP i . PMID:23258541 These enzymes use a two-step mechanism in which first an acyl-AMP intermediate is formed (with release of pyrophosphate) followed by displacement of AMP by CoA |
| GO:0043427 carbon fixation by 3-hydroxypropionate cycle | IDA PMID:18165310 3-Hydroxypropionyl-coenzyme A synthetase from Metallosphaera... | MODIFY | Summary: The experimental basis is sound. The enzyme was purified from autotrophically grown M. sedula, catalyses a step of the CO2-fixation cycle, and is induced eightfold under autotrophy. However, the GO term names a different pathway. Reason: GO:0043427 is defined (OLS, checked 2026-10-31) as the cycle that fixes two CO2 to form glyoxylate, with malyl-CoA cleaved to acetyl-CoA and glyoxylate. That is the Chloroflexus 3-hydroxypropionate bicycle. The same paper reports that M. sedula lacks L-malyl-CoA lyase and that the Sulfolobaceae pathway diverges after succinyl-CoA. The crenarchaeal pathway is the 3-hydroxypropionate/4-hydroxybutyrate cycle, which regenerates acetyl-CoA via 4-hydroxybutyrate (PMID:18079405). The curators' evidence that Msed_1456 takes part in autotrophic CO2 fixation is not in question. Only the pathway term is mis-scoped. GO has no 3HP/4HB-cycle term, so the row is modified to the proposed new term, as in the Msed_0709 review, and to the existing parent GO:0015977 carbon fixation until that term exists. Proposed replacements: carbon fixation by 3-hydroxypropionate/4-hydroxybutyrate cycle carbon fixation Supporting Evidence: PMID:18165310 3-Hydroxypropionyl-CoA synthetase is up-regulated eightfold in autotrophically versus heterotrophically grown M. sedula , supporting its proposed role during CO 2 fixation in this archaeon and possibly other members of the Sulfolobaceae family. PMID:18165310 l -Malyl-CoA lyase activity was not detected in cell extracts of M. sedula ( 14 ). PMID:18079405 This intermediate was reduced to 4-hydroxybutyrate and converted into two acetyl-CoA molecules via 4-hydroxybutyryl-CoA dehydratase. |
| GO:0043427 carbon fixation by 3-hydroxypropionate cycle | IEA GO_REF:0000117 | MODIFY | Summary: ARBA machine-learning transfer of the same pathway term as the IDA row. Reason: For consistency with the IDA row, this row is modified for the same reason. The enzyme takes part in autotrophic CO2 fixation, but GO:0043427 is defined as the glyoxylate-forming Chloroflexus cycle, not the crenarchaeal 3HP/4HB cycle. Proposed replacements: carbon fixation by 3-hydroxypropionate/4-hydroxybutyrate cycle carbon fixation Supporting Evidence: 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. |
| GO:0043955 3-hydroxypropionyl-CoA synthetase activity | IDA PMID:18165310 3-Hydroxypropionyl-coenzyme A synthetase from Metallosphaera... | ACCEPT | Summary: The native enzyme was purified 95-fold from autotrophically grown M. sedula and shown to catalyse ATP- and CoA-dependent activation of 3-hydroxypropionate with release of AMP and pyrophosphate. Reason: This is direct biochemical evidence for the enzyme's defining activity, measured on the M. sedula protein with full kinetic characterization (PMID:18165310). It was confirmed independently by kinetic measurements on recombinant Msed_1456 (PMID:27771364). This is the core molecular function. Supporting Evidence: PMID:18165310 The enzyme was purified 95-fold to a specific activity of 18 ΞΌmol min β1 mg protein β1 from autotrophically grown M. sedula cells. PMID:18165310 The enzyme had a high affinity for ATP and 3-hydroxypropionate, with apparent K m values of 45 ΞΌM for ATP and 180 ΞΌM for 3-hydroxypropionate. |
| GO:0043955 3-hydroxypropionyl-CoA synthetase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic assignment of the same activity from the EC 6.2.1.36 / RHEA:26534 annotation and ARBA. Reason: The row agrees with the IDA row for the same term and with the experimentally determined reaction (PMID:18165310). Supporting Evidence: PMID:18165310 Both the native enzyme from M. sedula and the recombinant enzyme from S. tokodaii not only activated 3-hydroxypropionate to its CoA ester |
| GO:0050218 propionate-CoA ligase activity | IDA PMID:18165310 3-Hydroxypropionyl-coenzyme A synthetase from Metallosphaera... | NEW | Summary: The purified M. sedula enzyme activates propionate with about the same catalytic efficiency as 3-hydroxypropionate. Reason: The kcat/Km values for propionate and 3-hydroxypropionate were similar, and the authors concluded that the enzyme is specific for both substrates (PMID:18165310). An independent comparison also notes equal activity on propionate and 3HP (PMID:23258541). Unlike the weak acetate activity, this is a major activity of the enzyme. It is recorded as a demonstrated molecular function. Its physiological role is unclear, because in the cycle propionyl-CoA is made from acryloyl-CoA rather than from free propionate, so it is not treated as core. Supporting Evidence: PMID:18165310 The specificities ( k cat / K m ) of the enzyme with respect to 3-hydroxypropionate and propionate were similar but were decreased about 10-fold for acrylate (Table 4 ). PMID:23258541 Similarly, Msed_1456, which catalyzes the ligation of CoA to 3HP in the 3HP/4HB pathway, has equal activity on propionate and 3HP |
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Download this section (compressed HTML)Q: Should GO:0043427 be restricted explicitly to the Chloroflexus bicycle (or made a grouping term) and a sibling term created for the 3HP/4HB cycle? Existing GO:0043427 rows on Sulfolobales enzymes would then need to be moved.
Q: Does free propionate activation by Msed_1456 have a physiological role, for example in scavenging propionate during heterotrophic or mixotrophic growth?
Q: Should the acetyl-CoA ligase family signature IPR011904 / TIGR02188 continue to map to acetyl-CoA synthetase activity and acetyl-CoA biosynthesis for crenarchaeal members whose substrate has diverged, such as 3-hydroxypropionyl-CoA synthetase?
Experiment: Co-purify native or co-expressed HPCS and HPCD and test complex formation by size-exclusion chromatography with multi-angle light scattering or cross-linking mass spectrometry. Compare coupled-assay kinetics with free enzymes and with added exogenous 3-hydroxypropionyl-CoA to look for channelling.
Hypothesis: Msed_1456 and 3-hydroxypropionyl-CoA dehydratase (Msed_2001) form a stable complex that channels 3-hydroxypropionyl-CoA.
Experiment: Delete or knock down Msed_1456 with a Sulfolobales genetic system and compare growth on H2/CO2 with growth on yeast extract or peptides, with complementation.
Hypothesis: Msed_1456 is required for autotrophic but not heterotrophic growth of M. sedula.
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