Msed_1321 encodes the 4-hydroxybutyryl-CoA dehydratase (EC 4.2.1.120) of the thermoacidophilic crenarchaeon Metallosphaera sedula. This is the key enzyme of the 4-hydroxybutyrate part of the 3-hydroxypropionate/4-hydroxybutyrate autotrophic CO2-fixation cycle of the Sulfolobales. It dehydrates 4-hydroxybutyryl-CoA to crotonyl-CoA, which is then hydrated, oxidised and cleaved into two acetyl-CoA. The 507-residue protein belongs to the HpaB/PvcC/4-hydroxybutyryl-CoA dehydratase family, which has an acyl-CoA dehydrogenase-like fold. By homology with the clostridial enzyme, it is expected to carry FAD and a [4Fe-4S] cluster and to use radical chemistry. The recombinant enzyme, produced anaerobically in Escherichia coli, is a homotetramer with a Km of 0.15 mM for 4-hydroxybutyryl-CoA and a low specific activity (about 2.2 U/mg at 70 C). Together with 4-hydroxybutyrate-CoA ligase, it is rate-limiting for the acetyl-CoA-regenerating branch. Unlike the clostridial enzyme, it is relatively oxygen tolerant, with a half-life of about 4 days in air. Its transcript is strongly induced (about 27-fold) under autotrophic growth. The submitter's name, vinylacetyl-CoA Delta-isomerase, refers to a partial reaction documented for the clostridial homolog but not tested for this protein.
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, and it has no 4-hydroxybutyrate branch. Msed_1321 catalyses the 4-hydroxybutyryl-CoA dehydratase step that defines 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:0016627 oxidoreductase activity, acting on the CH-CH group of donors | IEA GO_REF:0000002 | REMOVE | Summary: InterPro2GO mapping from the acyl-CoA dehydrogenase/oxidase-like superfamily signatures (IPR009100 N-terminal and middle domain, IPR036250 C-terminal domain, IPR046373 middle domain) to oxidoreductase activity acting on CH-CH donors. Reason: These are fold-level superfamily signatures that are shared by acyl-CoA dehydrogenases, acyl-CoA oxidases, HpaB-type flavin monooxygenases and the 4-hydroxybutyryl-CoA dehydratases. The family-specific InterPro entries (IPR004925, IPR024719, IPR024674 HpaB/PvcC/4-BUDH) carry no such mapping. The net reaction catalysed by Msed_1321 is the dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA (PMID:24532060). This is a hydro-lyase reaction (GO:0043721, under GO:0016836) with no net electron transfer, so it is not a CH-CH oxidoreduction. In the clostridial paradigm, FAD only transiently oxidises the substrate within a radical mechanism and is regenerated each turnover. No CH-CH dehydrogenase activity has been reported for Msed_1321 or for its characterised homologs. This is a superfamily-fold over-propagation that contradicts the known reaction class. Supporting Evidence: PMID:24532060 The reaction rate for the subsequent transformation, the dehydration by 4-hydroxybutyryl-CoA dehydratase, is also low (2.2 ΞΌmol min β1 mg β1 ), and hence, these two reactions form the rate-limiting steps for the acetyl-CoA branch. PMID:21169482 4-Hydroxybutyryl-CoA dehydratase, which catalyzes a mechanistically intriguing elimination of water, is well conserved and rightly can be considered the key enzyme of these two cycles. |
| GO:0050393 vinylacetyl-CoA delta-isomerase activity | IEA GO_REF:0000003 | KEEP AS NON CORE | Summary: EC2GO mapping from the submitter-assigned EC 5.3.3.3 (vinylacetyl-CoA Delta-isomerase) in the EMBL entry, which is also the source of the protein name. Reason: The isomerase name was carried over from the clostridial paradigm enzyme (AbfD of Clostridium aminobutyricum). IUBMB (EC 4.2.1.120) and Rhea describe AbfD as having an intrinsic vinylacetyl-CoA Delta3-Delta2-isomerase activity as well as the dehydratase activity, because both reactions run through the same radical intermediates. The isomerase activity has not been assayed for Msed_1321. Hawkins et al. measured only 4-hydroxybutyryl-CoA-dependent crotonyl-CoA formation (PMID:24532060). The isomerisation of vinylacetyl-CoA is not a step of the 3HP/4HB cycle. The activity is plausible as a conserved partial reaction of the dehydratase active site, so the row is kept, but as non-core. The physiological activity is GO:0043721 4-hydroxybutanoyl-CoA dehydratase activity, added below. The clostridial primary paper (Scherf and Buckel 1993) could not be cached during this review, because NCBI E-utilities were unavailable. Supporting Evidence: PMID:24532060 This was likely due to the oxygen sensitivity of the enzyme, based on properties of a well-studied homolog from an anaerobic bacterium, Clostridium aminobutyricum |
| GO:0043721 4-hydroxybutanoyl-CoA dehydratase activity | IDA PMID:24532060 Conversion of 4-hydroxybutyrate to acetyl coenzyme A and its... | NEW | Summary: Recombinant His-tagged Msed_1321 was produced and purified anaerobically. Its activity was assayed at 70 C in a coupled assay. Msed_0406 generated 4-hydroxybutyryl-CoA from 4HB, and crotonyl-CoA formation was read out through the NAD+-dependent Msed_0399 hydratase/dehydrogenase. The Km for 4-hydroxybutyryl-CoA was 0.15 mM and the rate was 2.2 U/mg. Reason: This is the defining activity of the gene and is missing from GOA. GO:0043721 is defined as 4-hydroxybutanoyl-CoA = (2E)-butenoyl-CoA + H2O (verified in OLS, under hydro-lyase activity). The evidence is direct, from the purified recombinant M. sedula enzyme, and not only by analogy to AbfD. The reaction was initiated by adding the dehydratase, and the enzyme supported reconstitution of the whole 4HB-to-acetyl-CoA branch. The dehydratase activity of autotrophic cell extracts was already attributed to this gene (PMID:21169482). Its paralog Msed_1220 lacks catalytic residues. Supporting Evidence: PMID:24532060 This approach resulted in the production of soluble, active enzyme that was purified to electrophoretic homogeneity after metal affinity chromatography. PMID:24532060 The reaction mixture was preheated for 5 min at 70Β°C to allow accumulation of 4HB-CoA, and then the reaction was initiated by the addition of purified 4-hydroxybutyryl-CoA dehydratase. PMID:24532060 Msed_1321 on 4-hydroxybutyryl-CoA, 0.15 mM PMID:21169482 Cell extracts of autotrophic Sulfolobales , Thermoproteales , and Desulfurococcales contained high 4-hydroxybutyryl-CoA dehydratase activity |
| GO:0015977 carbon fixation | IDA PMID:24532060 Conversion of 4-hydroxybutyrate to acetyl coenzyme A and its... | NEW | Summary: Msed_1321 catalyses the 4-hydroxybutyryl-CoA dehydration step of the acetyl-CoA-regenerating branch of the 3HP/4HB autotrophic CO2-fixation cycle. The branch was reconstituted in vitro with Msed_1321 and three other recombinant enzymes, and the gene is about 27-fold induced in autotrophy. Reason: The enzyme catalyses a step of the cycle itself, so it passes the participation test. Comparators in the same role (catalysts of individual cycle steps), namely Msed_0406, Msed_1424, Msed_0709 and Msed_2001, carry GO:0015977 in their reviews. GO:0043427 carbon fixation by 3-hydroxypropionate cycle is the Chloroflexus glyoxylate-forming cycle, which has no 4-hydroxybutyrate branch, so it is not used. GO:0015977 is the most precise correct existing term, and the 3HP/4HB-cycle child term proposed in the Msed_0709 review is repeated below. The in vivo role rests on biochemistry, reconstitution and expression; there is no genetic evidence. Supporting Evidence: PMID:24532060 were produced recombinantly in Escherichia coli , combined in vitro , and shown to convert 4HB to acetyl-CoA PMID:24532060 4-hydroxybutyryl-CoA dehydratase (Msed_1321; 27-fold) PMID:18079405 This intermediate was reduced to 4-hydroxybutyrate and converted into two acetyl-CoA molecules via 4-hydroxybutyryl-CoA dehydratase. |
Loading supporting contentβ¦
Download this section (compressed HTML)Q: Does Msed_1321 have vinylacetyl-CoA Delta-isomerase activity, as the clostridial AbfD does? Should UniProt rename the entry 4-hydroxybutyryl-CoA dehydratase (EC 4.2.1.120), with EC 5.3.3.3 kept only as a secondary activity?
Q: Does the M. sedula enzyme contain FAD and a [4Fe-4S] cluster like AbfD, and what structural features account for its much greater oxygen tolerance?
Q: Should InterPro2GO stop mapping the acyl-CoA dehydrogenase-like fold superfamilies (IPR009100, IPR036250, IPR046373) to GO:0016627, given that they also cover the 4-hydroxybutyryl-CoA dehydratase and HpaB monooxygenase families, which are not CH-CH oxidoreductases?
Experiment: Assay purified recombinant Msed_1321 with vinylacetyl-CoA, and follow crotonyl-CoA formation by HPLC. Determine flavin and iron/acid-labile sulfide content, and record EPR and UV-visible spectra.
Hypothesis: Msed_1321 is bifunctional, with vinylacetyl-CoA Delta-isomerase activity as well as 4-hydroxybutyryl-CoA dehydratase activity, and carries FAD and a [4Fe-4S] cluster.
Experiment: Delete or knock down Msed_1321 in M. sedula, or its ortholog in a genetically tractable autotrophic Sulfolobales relative. Compare autotrophic and heterotrophic growth and measure 4-hydroxybutyryl-CoA dehydratase activity in extracts.
Hypothesis: Msed_1321 is essential for autotrophic growth.
Loading supporting contentβ¦
Download this section (compressed HTML)Loading supporting contentβ¦
Download this section (compressed HTML)