Msed_1993 encodes the NADPH-dependent malonic semialdehyde reductase of the thermoacidophilic crenarchaeon Metallosphaera sedula. It reduces malonate semialdehyde (3-oxopropanoate) to 3-hydroxypropionate, the second reductive step of the 3-hydroxypropionate/4-hydroxybutyrate autotrophic CO2-fixation cycle of the Sulfolobales, immediately after malonyl-CoA reduction by the malonyl-CoA/succinyl-CoA reductase Msed_0709. The enzyme was partially purified from autotrophically grown cells, identified by peptide mass fingerprinting, and characterized as a recombinant protein. It is a homotrimeric, metal-independent member of the 3-hydroxyacyl-CoA dehydrogenase family with an N-terminal Rossmann-fold dinucleotide-binding domain. It prefers NADPH (NADH supports about 20% of the activity) and is specific for malonate semialdehyde; succinate semialdehyde and simple aliphatic aldehydes are not reduced. Its activity is about five-fold higher in autotrophically than in heterotrophically grown cells. Expressed together with the cycle's carboxylase and malonyl-CoA reductase, it allowed engineered Pyrococcus furiosus to make 3-hydroxypropionate from CO2.
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 its malonic semialdehyde reductase (Msed_1993) is unrelated to the Chloroflexus enzyme. The same term is proposed in the Msed_0709 review.
Parent term: carbon fixation
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0006631 fatty acid metabolic process | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: InterPro2GO mapping from the 3-hydroxyacyl-CoA dehydrogenase family signatures, which assume a role in fatty acid beta-oxidation. Msed_1993 is a neofunctionalized family member that reduces free malonate semialdehyde in autotrophic CO2 fixation. Reason: The family-level mapping is right for canonical 3-hydroxyacyl-CoA dehydrogenases, which oxidize 3-hydroxyacyl-CoA thioesters in beta-oxidation. Msed_1993 has not been shown to act on any acyl-CoA. Its characterized substrate is the free aldehyde malonate semialdehyde, and the reaction runs in the reductive, anabolic direction in the 3HP/4HB cycle (PMID:19684143). The product, 3-hydroxypropionate, is a C3 hydroxy acid, so the term is not strictly impossible, but it does not describe the role of the enzyme. The same treatment was given to the fatty acid beta-oxidation row on the sibling 3-hydroxypropionyl-CoA dehydratase Msed_2001. The process is captured by carbon fixation (NEW below). Supporting Evidence: PMID:19684143 Malonic semialdehyde reductase, a member of the 3-hydroxyacyl-CoA dehydrogenase family, reduces malonic semialdehyde with NADPH to 3-hydroxypropionate. PMID:19684143 Also, the NADPH specificity suggests that they function as reductases in an anabolic pathway rather than as dehydrogenases in a catabolic pathway. |
| GO:0016491 oxidoreductase activity | IEA GO_REF:0000120 | MODIFY | Summary: Correct but uninformative. The enzyme catalyses malonate semialdehyde + NADPH + H+ = 3-hydroxypropionate + NADP+, measured with native and recombinant enzyme. Reason: A specific GO term exists for the measured reaction. GO:0035527 3-hydroxypropionate dehydrogenase (NADP+) activity is defined as 3-hydroxypropanoate + NADP+ = 3-oxopropanoate + H+ + NADPH; 3-oxopropanoate is malonate semialdehyde, and GO:0035527 is a descendant of GO:0016491 via GO:0016616. The physiological direction is reduction, which the reversible GO definition covers. Proposed replacements: 3-hydroxypropionate dehydrogenase (NADP+) activity Supporting Evidence: PMID:19684143 The malonic semialdehyde reductase gene encoding the Msed_1993 protein was cloned into the expression vector pT7-7 ( 43 ) and expressed in E. coli Rosetta 2(DE3). PMID:19684143 The apparent K m for malonic semialdehyde was 70 Β± 10 ΞΌM, and the apparent K m for NADPH was 70 Β± 20 ΞΌM. |
| GO:0016616 oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor | IEA GO_REF:0000002 | MODIFY | Summary: Correct class of activity (3-hydroxypropionate/malonate semialdehyde is a CH-OH/C=O couple with NAD(P) as acceptor), but more general than the evidence supports. Reason: GO:0035527 3-hydroxypropionate dehydrogenase (NADP+) activity is a direct is_a child of this term and describes exactly the reaction measured for recombinant Msed_1993 (PMID:19684143). The enzyme prefers NADPH, so the NADP+-specific child is chosen rather than GO:0047565 3-hydroxypropionate dehydrogenase (NAD+) activity; NADH supports only about 20% of the activity. Proposed replacements: 3-hydroxypropionate dehydrogenase (NADP+) activity Supporting Evidence: PMID:19684143 NADH (20% activity) could partially substitute for NADPH (100%). PMID:19684143 Malonic semialdehyde reductase, a member of the 3-hydroxyacyl-CoA dehydrogenase family, reduces malonic semialdehyde with NADPH to 3-hydroxypropionate. |
| GO:0050104 L-gulonate 3-dehydrogenase activity | IEA GO_REF:0000118 | REMOVE | Summary: TreeGrafter transfer from PANTHER node PTN000592773 (subfamily PTHR48075:SF1, lambda-crystallin homolog). L-gulonate 3-dehydrogenase (EC 1.1.1.45) is the activity of lambda-crystallin/CRYL1 in the eukaryotic uronate pathway. Reason: Msed_1993 has a different, experimentally established activity, the NADPH- dependent reduction of the C3 oxo acid malonate semialdehyde (PMID:19684143). Its substrate range is narrow; succinate semialdehyde and C2-C5 aliphatic aldehydes are not used. L-gulonate, a C6 sugar acid, was not tested, and there is no evidence of a glucuronate/uronate pathway in which such an activity would act in this autotrophic archaeon. The authors note the protein cannot be classified from its sequence. The term is a paralog-subfamily transfer of a eukaryotic activity onto a neofunctionalized archaeal enzyme (ARCHAEA annotation pattern 1), and should be replaced by GO:0035527. Supporting Evidence: PMID:19684143 Succinic semialdehyde, acetaldehyde, butyraldehyde, propionaldehyde, or glutaraldehyde did not serve as a substrate PMID:19684143 This enzyme cannot be classified unambiguously, and no divalent metal ion was required. |
| GO:0070403 NAD+ binding | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the 3-hydroxyacyl-CoA dehydrogenase NAD-binding domain (IPR006176). The domain is a Rossmann fold that binds the nicotinamide cofactor, but the physiological cofactor of Msed_1993 is NADPH. Reason: Cofactor binding by the N-terminal Rossmann domain is real, but the NAD+ specificity of the term is wrong for this enzyme. Recombinant Msed_1993 uses NADPH (apparent Km 70 uM), with NADH giving only 20% of the activity, and it functions in the reductive direction, so it binds NADPH rather than NAD+ physiologically. The lateral correction is GO:0050661 NADP binding, which covers both oxidized and reduced forms. Proposed replacements: NADP binding Supporting Evidence: PMID:19684143 This enzyme, annotated as a putative 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35), exhibits a Rossmann fold NAD(P)H/NAD(P) + binding domain at the N terminus PMID:19684143 NADH (20% activity) could partially substitute for NADPH (100%). |
| GO:0015977 carbon fixation | IDA PMID:19684143 Malonic semialdehyde reductase, succinic semialdehyde reduct... | NEW | Summary: Msed_1993 catalyses the reduction of malonate semialdehyde to 3-hydroxypropionate, 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. It was purified from autotrophic cells, its activity is about five-fold up-regulated under autotrophic growth, and its gene is near the 3-hydroxypropionyl-CoA dehydratase gene Msed_2001. When it was co-expressed with the cycle's carboxylase and malonyl-CoA reductase in Pyrococcus furiosus, the strain fixed CO2 into 3-hydroxypropionate (PMID:23530213). This matches the treatment of the other catalytic steps of the cycle (Msed_0709, Msed_1424, Msed_2001), which carry the same term. GO:0043427 carbon fixation by 3-hydroxypropionate cycle describes the Chloroflexus glyoxylate-forming cycle and is not used. The 3HP/4HB-cycle child term is proposed with the same wording as in the Msed_0709 review. Supporting Evidence: PMID:19684143 The specific activity of this conversion in autotrophically grown cells was 1.8 ΞΌmol min β1 mg β1 protein, whereas in heterotrophically grown cells the specific activity was 0.35 ΞΌmol min β1 mg β1 protein. PMID:19684143 The best hit was with the gene gi|146304741 (100% identity, 54% coverage), annotated as 3-hydroxyacyl-CoA dehydrogenase (NAD binding) gene, encoding a 35.3-kDa protein (Msed_1993; 314 amino acids). PMID:23530213 E3 (malonate semialdehyde reductase, Msed_1993) |
Loading supporting contentβ¦
Download this section (compressed HTML)Q: Should PANTHER PTHR48075:SF1 (lambda-crystallin homolog) be split so that the Sulfolobales malonic semialdehyde reductase clade no longer receives L-gulonate 3-dehydrogenase activity by TreeGrafter?
Q: Does Msed_1993 retain any 3-hydroxyacyl-CoA dehydrogenase or L-gulonate 3-dehydrogenase activity, or has the acyl-CoA binding site been lost?
Experiment: Delete Msed_1993 with a Sulfolobales genetic system and compare growth on CO2/H2 with growth on yeast extract, with and without 3-hydroxypropionate supplementation.
Hypothesis: Msed_1993 is required for autotrophic but not heterotrophic growth of M. sedula.
Experiment: Assay recombinant Msed_1993 with (S)-3-hydroxybutyryl-CoA/acetoacetyl-CoA and with L-gulonate/3-dehydro-L-gulonate in both directions, with NAD(H) and NADP(H), and solve its structure with malonate semialdehyde or 3-hydroxypropionate bound.
Hypothesis: Msed_1993 does not act on 3-hydroxyacyl-CoA thioesters or on L-gulonate.
Loading supporting contentβ¦
Download this section (compressed HTML)Loading supporting contentβ¦
Download this section (compressed HTML)