Msed_1426 encodes the NADPH-dependent acryloyl-CoA reductase (EC 1.3.1.84) of the thermoacidophilic crenarchaeon Metallosphaera sedula. It reduces acryloyl-CoA to propionyl-CoA, the last of three reactions that convert 3-hydroxypropionate to propionyl-CoA in the 3-hydroxypropionate/4-hydroxybutyrate autotrophic CO2-fixation cycle of the Sulfolobales; the two preceding reactions are catalysed by 3-hydroxypropionyl-CoA synthetase (Msed_1456) and 3-hydroxypropionyl-CoA dehydratase (Msed_2001). The enzyme was enriched from autotrophically grown cells and identified by peptide mass fingerprinting; its activity is more than ten-fold higher in autotrophic than in heterotrophic cells and appears to limit the rate of 3-hydroxypropionate reduction. It is a soluble member of the medium-chain zinc-containing alcohol dehydrogenase family. The recombinant Sulfolobus tokodaii ortholog is a monomer that contains about one zinc per subunit, uses NADPH but not NADH, and does not reduce crotonyl-CoA, an intermediate of the second half of the cycle. Recombinant M. sedula enzyme, obtained by co-expression with the neighbouring succinic semialdehyde reductase Msed_1424, has comparable kinetics. The archaeal enzyme is unrelated to the reductase domain of the trifunctional propionyl-CoA synthase of Chloroflexus, indicating independent evolution of the two 3-hydroxypropionate-based cycles.
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 cycle shares only the first half with it, and even there uses separate, unrelated enzymes (such as Msed_1426) in place of the Chloroflexus trifunctional propionyl-CoA synthase. The same term is proposed in the Msed_0709 review.
Parent term: carbon fixation
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004022 alcohol dehydrogenase (NAD+) activity | IEA GO_REF:0000118 | REMOVE | Summary: TreeGrafter transfer of the ancestral zinc alcohol dehydrogenase activity from PANTHER node PTN000191633 (PTHR42940). Msed_1426 is a medium-chain zinc dehydrogenase family member, but it reduces the C=C double bond of an enoyl-CoA with NADPH, not an alcohol/aldehyde couple with NAD+. Reason: The activity is contradicted on two counts. The characterized reaction is acryloyl-CoA + NADPH + H+ = propionyl-CoA + NADP+ (EC 1.3.1.84, acting on a CH-CH group), not oxidation of an alcohol (EC 1.1.1.1, acting on CH-OH). And the enzyme does not use NADH as electron donor at all (PMID:19429610). No alcohol dehydrogenase activity has been reported for Msed_1426 or its orthologs. This is a family-level over-annotation of a neofunctionalized zinc-ADH-family enzyme (the same family as the succinic semialdehyde reductase Msed_1424, which is a genuine NADP-dependent alcohol dehydrogenase). The correct activity is already captured by GO:0043957. Supporting Evidence: PMID:19429610 Acryloyl-CoA reductase (E.C. 1.3.1.x) catalyzes the following irreversible reaction: acryloyl-CoA + NADPH + H + → propionyl-CoA + NADP + . PMID:19429610 The enzyme does not require cofactors and does not use NADH as an electron donor. |
| GO:0005737 cytoplasm | IEA GO_REF:0000118 | ACCEPT | Summary: The enzyme is soluble, lacks signal peptide and transmembrane segments, and acts in a cytoplasmic CO2-fixation pathway with soluble CoA-ester intermediates. Reason: Acryloyl-CoA reductase activity was measured in, and enriched from, the soluble (100,000 x g supernatant) fraction of M. sedula extracts (PMID:19429610). In a prokaryote without internal compartments, cytoplasm is the correct and most precise location. Supporting Evidence: PMID:19429610 Based on the acryloyl-CoA-dependent oxidation of NADPH in the spectrophotometric assay, the enzyme that catalyzes acryloyl-CoA reduction to propionyl-CoA was enriched from extract of autotrophically grown cells. |
| GO:0008270 zinc ion binding | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the zinc-containing alcohol dehydrogenase signature. Zinc was measured in the recombinant S. tokodaii ortholog, and the catalytic and structural zinc ligands are conserved in Msed_1426. Reason: Metal analysis of the 76%-identical S. tokodaii acryloyl-CoA reductase found 0.8 mol Zn2+ per monomer and no other metal (PMID:19429610). The catalytic GHE motif with flanking Cys38 and Cys146, and a structural-site motif, are conserved, matching the UniProt zinc binding-site features. EDTA did not inactivate the enzyme, which the authors attribute to poor zinc accessibility or thermostability. Zinc binding is the cofactor binding that underlies the catalytic activity. Supporting Evidence: PMID:19429610 a metal analysis (27 elements) of acryloyl-CoA reductase by plasma emission spectroscopy revealed the presence of 0.8 mol of Zn 2+ per mol of enzyme monomer, whereas other metals could not be detected. PMID:19429610 An alignment of the acryloyl-CoA reductase with the well-studied horse liver alcohol dehydrogenase shows the presence of the strictly conserved sequence motif GHE of the catalytic zinc binding side (amino acid position 59 to 61) with two flanking cysteines (amino acids 38 and 146). |
| GO:0016491 oxidoreductase activity | IEA GO_REF:0000002 | MODIFY | Summary: Correct but uninformative; the specific activity, acryloyl-CoA reductase (NADPH), is established and already annotated. Reason: GO:0043957 acryloyl-CoA reductase (NADPH) activity, a descendant of oxidoreductase activity, is supported by direct biochemical evidence (PMID:19429610, PMID:27771364) and is already carried by the gene. It is the appropriate specific replacement. Proposed replacements: acryloyl-CoA reductase (NADPH) activity Supporting Evidence: PMID:19429610 Acryloyl-CoA reductase, a member of the zinc-containing alcohol dehydrogenase family, reduces acryloyl-CoA with NADPH to propionyl-CoA. |
| GO:0043957 acryloyl-CoA reductase (NADPH) activity | EXP PMID:19429610 3-hydroxypropionyl-coenzyme A dehydratase and acryloyl-coenz... | ACCEPT | Summary: The NADPH-dependent acryloyl-CoA reductase was enriched over 200-fold from autotrophic M. sedula and identified by peptide mass fingerprinting as Msed_1426. The recombinant 76%-identical S. tokodaii ortholog reduced acryloyl-CoA with NADPH (Km 3 uM acryloyl-CoA, 36 uM NADPH). Reason: This is the defining activity of the gene. GO:0043957 is defined as acryloyl-CoA + NADPH + H+ = propionyl-CoA + NADP+, exactly the reaction measured. The activity of the native M. sedula enzyme is direct; the detailed kinetics are from the S. tokodaii ortholog. Recombinant M. sedula Msed_1426 itself was later purified and assayed by an independent group (PMID:27771364), which confirms the assignment. Supporting Evidence: PMID:19429610 Reinterpretation of the fingerprint mass spectrometry data identified the same gene (Msed_1426) in M. sedula as a best hit (100% identity and 73% coverage). PMID:19429610 Acryloyl-CoA reductase (E.C. 1.3.1.x) catalyzes the following irreversible reaction: acryloyl-CoA + NADPH + H + → propionyl-CoA + NADP + . PMID:27771364 Note that although the KM for the recombinant versions of ACR from M. sedula and S. tokodaii were comparable, this is the first report of a KM for the M. sedula enzyme |
| GO:0043957 acryloyl-CoA reductase (NADPH) activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic mapping from the Swiss-Prot catalytic activity (RHEA:26454, EC 1.3.1.84), which is itself based on PMID:19429610. Reason: Same claim as the EXP row, which is well supported. The Rhea reaction (propanoyl-CoA + NADP+ = acryloyl-CoA + NADPH + H+) maps correctly to GO:0043957. Supporting Evidence: file:METS5/Msed_1426/Msed_1426-uniprot.txt Reaction=propanoyl-CoA + NADP(+) = acryloyl-CoA + NADPH + H(+); |
| GO:0043958 acryloyl-CoA reductase (NADH) activity | IDA PMID:19429610 3-hydroxypropionyl-coenzyme A dehydratase and acryloyl-coenz... | MODIFY | Summary: The cited paper shows acryloyl-CoA reductase activity, but it states explicitly that the enzyme does not use NADH. The NADH-specific term describes a different, flavin-containing Clostridium propionicum enzyme of propionate fermentation. Reason: The full text of PMID:19429610 is cached and was read. Every activity assay in it uses NADPH, and the authors state twice that NADH is not used as electron donor; they also contrast the Metallosphaera enzyme with the unrelated NADH-dependent Clostridium acryloyl-CoA reductase. GO:0043958 is defined as propanoyl-CoA + NAD+ = acryloyl-CoA + H+ + NADH (L-alanine fermentation). The essence of the annotation (acryloyl-CoA reductase activity) is correct, but the cofactor is wrong, so this is a lateral correction to GO:0043957 rather than a removal. It is probably a term-selection slip. Proposed replacements: acryloyl-CoA reductase (NADPH) activity Supporting Evidence: PMID:19429610 The enzyme did not act on either NADH or crotonyl-CoA. PMID:19429610 The enzyme does not require cofactors and does not use NADH as an electron donor. PMID:19429610 An NADH-dependent, oxygen-sensitive acryloyl-CoA reductase has been studied in Clostridium propionicum that is involved in propionate fermentation |
| GO:0015977 carbon fixation | IDA PMID:19429610 3-hydroxypropionyl-coenzyme A dehydratase and acryloyl-coenz... | NEW | Summary: Msed_1426 catalyses the reduction of acryloyl-CoA to propionyl-CoA, a step of the 3-hydroxypropionate/4-hydroxybutyrate autotrophic CO2-fixation cycle of M. sedula. Reason: The enzyme itself catalyses a step of the cycle, so it passes the participation test. Its activity is more than ten-fold up-regulated in autotrophic cells and appears rate-limiting for 3-hydroxypropionate reduction in extracts (PMID:19429610), and it was used as the acr step in the kinetic reconstruction of the cycle (PMID:27771364). Sibling catalytic steps (Msed_0709, Msed_1424, Msed_2001) carry the same term. GO:0043427 carbon fixation by 3-hydroxypropionate cycle describes the Chloroflexus glyoxylate-forming cycle, whose acryloyl-CoA reduction is done by an unrelated domain of propionyl-CoA synthase, so it is not used. A 3HP/4HB-cycle child term is proposed with the Msed_0709 wording. Supporting Evidence: PMID:19429610 The reductase activity may limit the conversion rate and clearly is more than 10-fold downregulated in heterotrophically grown cells. PMID:19429610 These results are consistent with the proposed function in autotrophic CO 2 fixation. PMID:27771364 The genes hpcs (Msed_1456), hpcd (Msed_2001), acr (Msed_1426), mcr (Msed_0709), and ssr (Msed_1424) were amplified from genomic DNA using the primers listed in Supplementary Table S1. |
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Download this section (compressed HTML)Q: Can the UniProtKB IDA annotation of A4YGN2 to GO:0043958 acryloyl-CoA reductase (NADH) activity be corrected to GO:0043957 (NADPH), given that PMID:19429610 states the enzyme does not use NADH?
Q: Should TreeGrafter stop propagating alcohol dehydrogenase (NAD+) activity from PTN000191633 to the archaeal acryloyl-CoA reductase clade (InterPro IPR053496, NCBIfam NF041172)?
Q: Why is Msed_1426 soluble and active only when co-expressed with Msed_1424 in E. coli; does it need a partner or folding factor in vivo?
Experiment: Delete Msed_1426 with a Sulfolobales genetic system and compare growth on CO2/H2 with growth on yeast extract; test complementation and accumulation of 3-hydroxypropionate or acrylate.
Hypothesis: Msed_1426 is required for autotrophic but not heterotrophic growth of M. sedula.
Experiment: Mutate Cys38, His60 and Cys146 of recombinant Msed_1426, measure zinc content by ICP-OES and acryloyl-CoA reductase activity, and solve a structure with acryloyl-CoA or propionyl-CoA bound.
Hypothesis: The catalytic zinc site is required for acryloyl-CoA reduction.
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