Msed_0709 encodes the NADPH-dependent malonyl-CoA/succinyl-CoA reductase (MCR) of the thermoacidophilic crenarchaeon Metallosphaera sedula, a soluble homotetrameric enzyme of the 3-hydroxypropionate/4-hydroxybutyrate autotrophic CO2-fixation cycle of the Sulfolobales. It reduces malonyl-CoA, the product of acetyl-CoA carboxylation, to malonate semialdehyde with release of CoA (EC 1.2.1.75), and with similar efficiency reduces succinyl-CoA to succinate semialdehyde (EC 1.2.1.76) in the second half of the cycle that regenerates acetyl-CoA. The activity is NADPH-specific, thiol-dependent (a catalytic cysteine forms an acyl-enzyme thioester) and about 10-fold induced under autotrophic growth. MCR is a paralog of aspartate-semialdehyde dehydrogenase (about 40% identity) that arose by gene duplication and neofunctionalization; structures of the Sulfolobus tokodaii ortholog show a remodelled substrate pocket and a single bispecific site that is occupied in turn by NADP+ and CoA. It is unrelated to the bifunctional malonyl-CoA reductase of Chloroflexus, and has been used to engineer CO2-dependent 3-hydroxypropionate production in Pyrococcus furiosus.
Definition: Catalysis of the reaction: malonyl-CoA + NADPH + H+ = 3-oxopropanoate (malonate semialdehyde) + CoA + NADP+.
Justification: This is EC 1.2.1.75 (RHEA:26446), and GO has no term for it; ec2go has no mapping for EC 1.2.1.75. GO:0018478 (EC 1.2.1.18) is a different, decarboxylating reaction. The activity is the key reductive step of the 3-hydroxypropionate/4-hydroxybutyrate cycle in Sulfolobales. Any new term should also cover the unrelated Chloroflexus enzyme's first half-reaction.
Parent term: oxidoreductase activity, acting on the aldehyde or oxo group of donors, NAD or NADP as acceptor
Supporting Evidence:
Definition: Catalysis of the reaction: succinyl-CoA + NADPH + H+ = succinate semialdehyde + CoA + NADP+.
Justification: This is EC 1.2.1.76, succinate-semialdehyde dehydrogenase (acylating), and GO has no term for it. Existing succinate-semialdehyde dehydrogenase terms (GO:0009013, GO:0036243) describe the non-acylating reaction that forms succinate. The activity is required in the 3-hydroxypropionate/4-hydroxybutyrate and dicarboxylate/4-hydroxybutyrate cycles.
Parent term: oxidoreductase activity, acting on the aldehyde or oxo group of donors, NAD or NADP as acceptor
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 3HP/4HB cycle shares only the first half and regenerates acetyl-CoA via 4-hydroxybutyrate, so its enzymes currently have no specific process term.
Parent term: carbon fixation
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004073 aspartate-semialdehyde dehydrogenase (NADP+) activity | IEA GO_REF:0000118 | REMOVE | Summary: TreeGrafter transfer of the ancestral aspartate-semialdehyde dehydrogenase (ASD) activity from the PANTHER PTHR46718 node onto MCR, a neofunctionalized ASD paralog. Reason: Msed_0709 is the malonyl-CoA/succinyl-CoA reductase, identified by purification from M. sedula and by peptide mass fingerprinting (PMID:17041055, PMID:19684143). It shares only about 40% identity with ASD, and the genuine asd gene of Sulfolobales sits elsewhere in the genome next to threonine synthase and aspartokinase genes (PMID:23325803). In M. sedula that paralog is Msed_1700 (A4YHF3), which neighbours Msed_1698 threonine synthase and Msed_1699 aspartokinase and matches the archaeal/fungal ASD CDD models (cd18130, cd02315), whereas Msed_0709 matches the MCR-type models (cd23940, cd24150). Both paralogs carry NCBIfam TIGR00978 (asd_EA), so that signature does not separate them (see genes/METS5/Msed_1700/Msed_1700-bioinformatics/RESULTS.md). Structural analysis of the ortholog shows the substrate pocket was remodelled to accommodate malonyl/succinyl groups that lack the ammonium group of aspartate. No ASD activity has been reported for MCR. The PANTHER subfamily (SF1) does not separate the MCR clade from true ASDs, so the ancestral activity is propagated to the neofunctionalized paralog. This is a paralog over-annotation. Supporting Evidence: PMID:23325803 The two paralogous MCR and ASD enzymes show ∼40% amino acid sequence identity in the same organism, and their genes mcr and asd have different chromosomal locations. PMID:23325803 The asd gene clusters together with threonine synthase, aspartokinase, and ornithine carbamoyltransferase, among other genes, which is in line with its role in the biosynthesis of amino acids of the aspartate family. PMID:23325803 The modified polypeptide surrounding around the absent ammonium group in malonate/succinate compared with aspartate provides the structural basis for engineering a methylmalonyl-CoA reductase PMID:19684143 Succinyl-CoA reduction to succinic semialdehyde is catalyzed by malonyl-CoA/succinyl-CoA reductase, a promiscuous NADPH-dependent enzyme that is a paralogue of aspartate semialdehyde dehydrogenase. |
| GO:0008652 amino acid biosynthetic process | IEA GO_REF:0000002 | REMOVE | Summary: InterPro2GO mapping from the semialdehyde dehydrogenase dimerization domain (IPR012280), which is shared by ASD and its MCR paralog. Reason: The dimerization domain signature does not discriminate aspartate-semialdehyde dehydrogenase from its neofunctionalized paralog. Msed_0709 acts in autotrophic CO2 fixation, reducing malonyl-CoA and succinyl-CoA. No role in amino acid biosynthesis has been shown, and the aspartate-family amino acid pathway uses the separate asd paralog (Msed_1700 in M. sedula). Supporting Evidence: PMID:23325803 The mcr gene occurs only in autotrophic Sulfolobales underlining its role in the carbon fixation cycle and its neighbor genes are generally not conserved among different species. PMID:19684143 The gene for malonyl-CoA/succinyl-CoA reductase (Msed_0709) is located between genes encoding hypothetical proteins; no known gene of the 3-hydroxypropionate/4-hydroxybutyrate cycle is located nearby. |
| GO:0009088 L-threonine biosynthetic process | IEA GO_REF:0000118 | REMOVE | Summary: TreeGrafter transfer of the ancestral ASD pathway role (threonine biosynthesis) to the MCR paralog. Reason: This propagation shares its cause with the GO:0004073 row. MCR does not catalyse the aspartate-semialdehyde step of the aspartate-family pathway. In Sulfolobales that step belongs to the separate asd paralog, which clusters with threonine synthase and aspartokinase (Msed_1700 in M. sedula). MCR's physiological role is in the 3-hydroxypropionate/4-hydroxybutyrate CO2-fixation cycle. Supporting Evidence: PMID:23325803 The asd gene clusters together with threonine synthase, aspartokinase, and ornithine carbamoyltransferase, among other genes, which is in line with its role in the biosynthesis of amino acids of the aspartate family. PMID:17041055 Extracts of Metallosphaera sedula contained NADPH-specific malonyl-CoA reductase activity that was 10-fold up-regulated under autotrophic growth conditions. |
| GO:0016620 oxidoreductase activity, acting on the aldehyde or oxo group of donors, NAD or NADP as acceptor | IEA GO_REF:0000002 | ACCEPT | Summary: Correct parent-level activity. MCR interconverts malonyl-CoA plus NADPH and malonate semialdehyde plus CoA plus NADP+ (EC 1.2.1.75), and does the same for succinyl-CoA and succinate semialdehyde (EC 1.2.1.76). Written in the oxidative direction, both are CoA-acylating aldehyde dehydrogenase reactions with NADP+ as acceptor. Reason: The term is correct, and it is the most specific existing GO term for MCR's catalytic activity. GO has no term for EC 1.2.1.75 or EC 1.2.1.76 (the GO ec2go mapping has no entry for either, and a QuickGO search found none). GO:0018478 malonate-semialdehyde dehydrogenase (acetylating) is a different, decarboxylating reaction. Specific child terms are proposed in proposed_new_terms. Until GO creates them, this term is as precise as GO allows, so it is accepted as the core activity term. Supporting Evidence: PMID:17041055 The enzyme catalyzes the following reaction: malonyl-CoA + NADPH + H+ --> malonate-semialdehyde + CoA + NADP+. PMID:19684143 These data showed that malonyl-CoA reductase is a promiscuous enzyme and acts nearly equally well with malonyl-CoA and succinyl-CoA. |
| GO:0046983 protein dimerization activity | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: MCR is a homotetramer, and the C-terminal domain mediates subunit contacts as in the semialdehyde dehydrogenase family. Reason: Self-association into a homotetramer is established for the enzyme (UniProt SUBUNIT from PMID:17041055; ortholog structure in PMID:23325803). Oligomerization is structural and not the enzyme's biological role, so the term is kept as non-core. Supporting Evidence: PMID:23325803 MCR is a homotetramer. file:METS5/Msed_0709/Msed_0709-uniprot.txt SUBUNIT: Homotetramer. |
| GO:0050661 NADP binding | IEA GO_REF:0000002 | ACCEPT | Summary: MCR is NADPH-specific, and the ortholog was crystallized in complex with NADP+. Reason: NADPH is the obligate reductant. The M. sedula extract activity was NADPH-specific, and the S. tokodaii ortholog structure shows NADP+ bound in a bispecific NADP+/CoA site. The cofactor-binding term is accurate and informative about specificity. Supporting Evidence: PMID:17041055 The enzyme was specific for NADPH (Km = 25 microM) and malonyl-CoA (Km = 40 microM). PMID:23325803 Here, we present the crystal structure of Sulfolobus tokodaii malonyl-CoA reductase in the substrate-free state and in complex with NADP + and CoA. |
| GO:0051287 NAD binding | IEA GO_REF:0000002 | REMOVE | Summary: This is a generic InterPro2GO mapping from the semialdehyde dehydrogenase NAD-binding (Rossmann) domain signature, which covers NAD- and NADP-using family members alike. Reason: Both the native M. sedula activity and the recombinant ortholog were NADPH-specific. The structure shows a site shaped for the 2'-phosphate of NADP+, which is shared with the 3'-phosphate of CoA. No NAD(H) binding or use has been reported. The IPR000534 mapping is a domain-level default that does not reflect this enzyme's cofactor specificity. Supporting Evidence: PMID:17041055 Extracts of Metallosphaera sedula contained NADPH-specific malonyl-CoA reductase activity PMID:23325803 The template-governed cofactor shaping implicates the same binding site for the 3′- and 2′-ribose phosphate group of CoA and NADP + , respectively |
| GO:0015977 carbon fixation | IDA PMID:17041055 Malonyl-coenzyme A reductase in the modified 3-hydroxypropio... | NEW | Summary: MCR catalyses the reduction of malonyl-CoA and of succinyl-CoA, two reductive steps of the 3-hydroxypropionate/4-hydroxybutyrate autotrophic CO2-fixation cycle of M. sedula. Its activity is about 10-fold induced under autotrophic growth. Reason: The enzyme performs two of the cycle's steps directly, so it passes the participation test. The comparable enzymes are other catalytic steps of autotrophic carbon-fixation cycles, such as RuBisCO and the 3-hydroxypropionate-cycle enzymes, which are annotated to carbon-fixation terms. GO:0043427 carbon fixation by 3-hydroxypropionate cycle is defined as the Chloroflexus glyoxylate-forming cycle, a different pathway that uses an unrelated malonyl-CoA reductase. It is therefore not used here, and a specific 3HP/4HB-cycle child term is proposed. The parent term GO:0015977 is the most precise correct existing term. Supporting Evidence: PMID:17041055 Extracts of Metallosphaera sedula contained NADPH-specific malonyl-CoA reductase activity that was 10-fold up-regulated under autotrophic growth conditions. 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. PMID:19684143 Succinyl-CoA reduction to succinic semialdehyde is catalyzed by malonyl-CoA/succinyl-CoA reductase, a promiscuous NADPH-dependent enzyme that is a paralogue of aspartate semialdehyde dehydrogenase. |
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Download this section (compressed HTML)Q: Does MCR retain any residual aspartate-semialdehyde dehydrogenase activity, and conversely does the true ASD paralog Msed_1700 (A4YHF3, which ARBA names a malonyl-CoA reductase) have any malonyl-CoA or succinyl-CoA reductase activity?
Q: Should PANTHER PTHR46718 be split so that the Sulfolobales MCR clade forms its own subfamily? That would stop TreeGrafter propagating ASD activity and threonine biosynthesis to MCR orthologs.
Q: What is the physiological significance, if any, of the small RNA that co-purifies with native MCR?
Experiment: Construct a Msed_0709 deletion, for example with a Sulfolobales genetic system, and compare growth on CO2/H2 with growth on yeast extract or peptides. Test complementation with Msed_0709 and with the ASD paralog Msed_1700.
Hypothesis: Msed_0709 is required for autotrophic but not heterotrophic growth of Metallosphaera sedula.
Experiment: Purify recombinant Msed_0709 and Msed_1700. Assay both with malonyl-CoA, succinyl-CoA and L-aspartyl-4-phosphate (forward direction), and with aspartate semialdehyde (reverse direction), and determine kcat/Km for each pair.
Hypothesis: The MCR and ASD paralogs have non-overlapping substrate specificities.
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