Msed_1424 encodes the NADPH-dependent succinic semialdehyde reductase (SSR) of the thermoacidophilic crenarchaeon Metallosphaera sedula. It reduces succinate semialdehyde to 4-hydroxybutyrate in the second, acetyl-CoA-regenerating half of the 3-hydroxypropionate/4-hydroxybutyrate autotrophic CO2-fixation cycle of the Sulfolobales. The enzyme was purified from autotrophically grown cells, identified by peptide mass fingerprinting, and characterized as a recombinant protein. It is a homodimeric medium-chain, zinc-containing alcohol dehydrogenase with about two zinc ions per subunit, one at a catalytic site and one at a structural site. It strongly prefers NADPH over NADH, is specific for succinate semialdehyde (it does not reduce malonic semialdehyde or simple aliphatic aldehydes), and its activity is detectable only in autotrophically grown cells. The gene lies two genes downstream of the acryloyl-CoA reductase gene. The preceding step, reduction of succinyl-CoA to succinate semialdehyde, is catalysed by a different enzyme, the malonyl-CoA/succinyl-CoA reductase Msed_0709. Close homologs act as succinic semialdehyde reductases in other autotrophic Sulfolobales and in Thermoproteus.
Definition: Catalysis of the reaction: succinate semialdehyde + NADPH + H+ = 4-hydroxybutanoate + NADP+.
Justification: This reaction is RHEA:26381 (EC 1.1.1.n11), which has no GO term; Rhea writes its undirected master reaction in the oxidative direction, and the definition is given in the physiological (reductive) direction that the name follows. GO:0047577 4-hydroxybutyrate dehydrogenase activity covers only the NAD+ reaction (RHEA:23948). The NADPH-dependent enzyme catalyses the 4-hydroxybutyrate-forming step of the 3-hydroxypropionate/4-hydroxybutyrate and dicarboxylate/4-hydroxybutyrate cycles (M. sedula Msed_1424, T. neutrophilus Tneu_0419). An NAD(P)-agnostic grouping term could be considered as a common parent with GO:0047577.
Parent term: alcohol dehydrogenase (NADP+) activity
Supporting Evidence:
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 regenerates acetyl-CoA via 4-hydroxybutyrate, the branch to which Msed_1424 belongs. The same term is proposed in the Msed_0709 review.
Parent term: carbon fixation
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0008270 zinc ion binding | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the zinc-containing alcohol dehydrogenase signature (IPR002328, PROSITE PS00059). Msed_1424 is a medium-chain zinc alcohol dehydrogenase, and zinc was measured directly in the purified recombinant enzyme. Reason: The protein belongs to the zinc-containing alcohol dehydrogenase family, not the aldehyde dehydrogenase family, so the InterPro mapping is appropriate. Metal analysis of the purified enzyme found 1.9 mol of zinc per monomer and no other metal. The conserved catalytic-site ligands (Cys38, the GHE motif at 58-60, Cys165) and the structural-site cysteine cluster (Cys89, 92, 95, 103) are present (PMID:19684143), matching the UniProt binding-site features. EDTA did not remove the zinc, which the authors attribute to the thermostability of the enzyme rather than to a lack of zinc dependence. Zinc binding is the cofactor binding that underlies the catalytic activity, so the term is accepted. Supporting Evidence: PMID:19684143 Metal analysis (27 elements) of succinic semialdehyde reductase using plasma emission spectroscopy revealed the presence of 1.9 mol of zinc per mol of enzyme monomer, whereas other metals could not be detected. PMID:19684143 Apparently, both zinc binding sites are loaded with zinc since the enzyme contains 1.9 mol of zinc per mol of enzyme monomer. file:METS5/Msed_1424/Msed_1424-uniprot.txt SIMILARITY: Belongs to the zinc-containing alcohol dehydrogenase family. |
| GO:0016491 oxidoreductase activity | IEA GO_REF:0000002 | MODIFY | Summary: Correct but very general. The enzyme catalyses succinate semialdehyde + NADPH + H+ = 4-hydroxybutanoate + NADP+ (RHEA:26381), an NADP-dependent alcohol dehydrogenase reaction run in the reductive direction. Reason: The activity has been measured on the purified native and recombinant enzyme (PMID:19684143), so a much more specific term is supported. The authors classify the enzyme as EC 1.1.1.2, which GO represents as GO:0008106 alcohol dehydrogenase (NADP+) activity. 4-Hydroxybutanoate is a primary alcohol and succinate semialdehyde its aldehyde, so the reaction is an instance of that term. GO:0047577 4-hydroxybutyrate dehydrogenase activity is not suitable, because it is defined with NAD+ as the acceptor, and this enzyme strongly prefers NADPH (apparent Km 6 uM, versus 180 uM for NADH). GO has no term for the NADP+ reaction (RHEA:26381 has no GO cross-reference), so a child term is proposed in proposed_new_terms. Note that UniProt names this protein succinate-semialdehyde dehydrogenase (acetylating), EC 1.2.1.76, which is the succinyl-CoA reductase reaction. That is the preceding step of the cycle, and it is catalysed by Msed_0709, not by Msed_1424; the replacement term deliberately does not follow the UniProt name. Proposed replacements: alcohol dehydrogenase (NADP+) activity Supporting Evidence: PMID:19684143 Succinic semialdehyde is then reduced with NADPH to 4-hydroxybutyrate by succinic semialdehyde reductase, an enzyme belonging to the Zn-dependent alcohol dehydrogenase family. PMID:19684143 Succinic semialdehyde reductase (EC 1.1.1.2) (Fig. 1 ) strongly prefers NADPH as the electron donor and does not act on malonic semialdehyde. PMID:19684143 the apparent K m for succinic semialdehyde (determined with NADPH) was 52 Β± 8 ΞΌM, the apparent K m for NADPH was 6 Β± 2 ΞΌM, and the apparent K m for NADH was 180 Β± 25 ΞΌM. |
| GO:0015977 carbon fixation | IDA PMID:19684143 Malonic semialdehyde reductase, succinic semialdehyde reduct... | NEW | Summary: Msed_1424 catalyses the reduction of succinate semialdehyde to 4-hydroxybutyrate, a step of the 3-hydroxypropionate/4-hydroxybutyrate autotrophic CO2-fixation cycle of M. sedula. Its activity is detectable only in autotrophically grown cells. Reason: The enzyme itself catalyses a step of the cycle, so it passes the participation test. The native enzyme was purified from autotrophic cells and identified as Msed_1424. Its activity is absent from heterotrophically grown cells, and the gene sits next to the acryloyl-CoA reductase gene of the same cycle. This matches the treatment of the other catalytic steps of the cycle, such as Msed_0709, which carries the same term. GO:0043427 carbon fixation by 3-hydroxypropionate cycle is defined as the Chloroflexus glyoxylate-forming cycle, which has no 4-hydroxybutyrate branch. It is therefore not used here; the parent term GO:0015977 is the most precise correct existing term. A 3HP/4HB-cycle child term is proposed, using the same wording as the Msed_0709 review. Supporting Evidence: PMID:19684143 The best hit was with the gene gi|146304190 (100% identity, 65% coverage) annotated as a Zn-dependent alcohol dehydrogenase gene encoding a 37.9-kDa protein (Msed_1424; 360 amino acids). PMID:19684143 No (<0.01 ΞΌmol min β1 mg β1 of protein) succinic semialdehyde-dependent oxidation of NADPH or NADH was observed with heterotrophically grown cells. 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: Can UniProt correct A4YGN0's name, EC and catalytic activity from succinate-semialdehyde dehydrogenase (acetylating), EC 1.2.1.76 (RHEA:26450), to succinic semialdehyde reductase (NADPH), RHEA:26381, as shown by PMID:19684143? Do Sulfolobales orthologs named by rules carry the same misassignment?
Q: Should GO create a term for RHEA:26381 (NADPH-dependent succinate semialdehyde reduction to 4-hydroxybutyrate) rather than leaving such enzymes at GO:0008106 alcohol dehydrogenase (NADP+) activity?
Q: Is the structural zinc essential for activity or only for stability, given that EDTA did not remove zinc from this thermostable enzyme?
Experiment: Delete Msed_1424 with a Sulfolobales genetic system and compare growth on CO2/H2 with growth on yeast extract. Test whether 4-hydroxybutyrate supplementation or complementation with Msed_1424 restores autotrophic growth.
Hypothesis: Msed_1424 is required for autotrophic but not heterotrophic growth of M. sedula.
Experiment: Mutate the catalytic-site ligands (Cys38, His59, Cys165) and the structural-site cysteines (Cys89, 92, 95, 103) of recombinant Msed_1424. Measure succinate semialdehyde reductase activity, zinc content and thermal stability of each mutant.
Hypothesis: Both zinc sites are needed, the catalytic site for activity and the structural site for stability.
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