Msed_1423 encodes a 391-residue monofunctional (S)-3-hydroxybutyryl-CoA dehydrogenase of the thermoacidophilic crenarchaeon Metallosphaera sedula. It has an N-terminal Rossmann-fold NAD-binding domain followed by two 3-hydroxyacyl-CoA dehydrogenase C-terminal domains, the architecture of the bacterial Hbd/FadB'-type short-chain 3-hydroxyacyl-CoA dehydrogenases, and it lacks the enoyl-CoA hydratase domain of its bifunctional paralog Msed_0399. The purified recombinant enzyme oxidises (S)-3-hydroxybutyryl-CoA to acetoacetyl-CoA with NAD+. It has a very low Km for (S)-3-hydroxybutyryl-CoA (about 5 uM) and for NAD+ (about 30 uM). NADP+ is accepted only with a Km of about 5 mM, so the enzyme is effectively NAD+-specific, and it is inactive with the C8 substrate (S)-3-hydroxyoctanoyl-CoA. Its catalytic efficiency is about 20-fold higher than that of Msed_0399, its gene is up-regulated under autotrophic and mixotrophic growth, and it lies in a gene cluster with other enzymes of the 3-hydroxypropionate/4-hydroxybutyrate CO2-fixation cycle (succinic semialdehyde reductase Msed_1424 and acryloyl-CoA reductase Msed_1426). Homologs are present in almost all Sulfolobales and are missing only from the heterotrophic Sulfolobus acidocaldarius. Msed_1423 is therefore considered the main enzyme for the (S)-3-hydroxybutyryl-CoA dehydrogenase step in the acetyl-CoA-regenerating half of this cycle. The protein is presumed cytoplasmic.
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 of that route, uses unrelated enzymes for it, and regenerates acetyl-CoA via 4-hydroxybutyrate, crotonyl-CoA and (S)-3-hydroxybutyryl-CoA. Msed_1423 catalyses the (S)-3-hydroxybutyryl-CoA dehydrogenase step of that regeneration half, which has no counterpart in the Chloroflexus cycle. This proposal repeats the one in the Msed_0709 review.
Parent term: carbon fixation
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003857 (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity | IEA GO_REF:0000003 | ACCEPT | Summary: EC-to-GO mapping from the EC 1.1.1.35 number that the genome submission gave this protein. Reason: Confirmed by direct biochemistry on the purified recombinant protein. It catalyses the NAD-dependent oxidation of (S)-3-hydroxybutyryl-CoA to acetoacetyl-CoA with a Km of 5 uM, and its Km for NAD+ (0.03 mM) is about 170-fold lower than for NADP+ (5.1 mM), so its catalytic efficiency with NAD+ is about 160-fold higher (PMID:32218776, Table 3). GO:0003857 is defined as a (3S)-3-hydroxyacyl-CoA + NAD+ = a 3-oxoacyl-CoA + NADH + H+, which fits both the cofactor and the (S) substrate. GO:0008691 3-hydroxybutyryl-CoA dehydrogenase activity is not the right alternative, because GO defines it with NADP+ (EC 1.1.1.157). The enzyme is inactive with the C8 (S)-3-hydroxyacyl-CoA, so its substrate range is narrower than the generic term implies. GO has no NAD+-specific (S)-3-hydroxybutanoyl-CoA child, so GO:0003857 is as precise as GO allows. The (R) stereoisomer was not reported as tested for this enzyme. This is the core molecular function, and the same term is used for the paralog Msed_0399 and for the module's hbdh step. Supporting Evidence: PMID:32218776 The purified recombinant Msed_0389 and Msed_1423 catalyzed the NAD-dependent oxidation of (S)-3-hydroxybutyryl-CoA to acetoacetyl-CoA and were highly specific for their substrate (Km 2.6 and 5 ΞΌM, respectively; Table 3). PMID:32218776 Both enzymes were not active with (S)-3-hydroxyoctanoyl-CoA. PMID:34290692 Msed_1423 is the main (S)-3-hydroxybutyryl-CoA dehydrogenase in M. sedula with the highest Vmax value (96 U mgβ1 protein) and the lowest Km value (5 ΞΌM) to its substrate (Table 3). |
| GO:0006631 fatty acid metabolic process | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: InterPro2GO mapping of the 3-hydroxyacyl-CoA dehydrogenase NAD-binding (IPR006176) and C-terminal (IPR006108) domains to fatty acid metabolic process. Reason: This is a family-level transfer from the 3-hydroxyacyl-CoA dehydrogenase family, most of whose members act in fatty acid beta-oxidation. The evidence for Msed_1423 points the other way. It is inactive with (S)-3-hydroxyoctanoyl-CoA, unlike the bifunctional paralog Msed_0399, which is the only M. sedula 3-hydroxyacyl-CoA dehydrogenase active with C8 substrates (PMID:32218776). Its gene lies in a cluster with specific enzymes of the 3-hydroxypropionate/4-hydroxybutyrate cycle, whereas its paralog Msed_0389 clusters with beta-oxidation genes. The gene is up-regulated in autotrophic cells and is absent only from the heterotroph S. acidocaldarius. Its demonstrated physiological process is therefore autotrophic carbon fixation. The enzyme does handle a C4 acyl-CoA, and a minor role in short-chain acyl-CoA metabolism during heterotrophic growth has not been excluded, so the term is marked as an over-annotation rather than removed. Supporting Evidence: PMID:32218776 Importantly, Msed_0399 is the only 3-hydroxyacyl-CoA dehydrogenase that was active with C8 (3-hydroxy)acyl-CoAs (Tables 2, 3), further suggesting its participation in heterotrophic metabolism. PMID:32218776 The Msed_0389 gene clustered together with various Ξ²-oxidation genes in the M. sedula genome. In contrast, msed_1423 clustered with several genes encoding specific enzymes of the HP/HB cycle, suggesting its involvement in autotrophic CO2 fixation (Figure 2). |
| GO:0016491 oxidoreductase activity | IEA GO_REF:0000120 | MODIFY | Summary: Combined automated annotation (InterPro IPR006108 and PANTHER TreeGrafter node PTN000592963) of the generic term oxidoreductase activity. Reason: Correct but too general. The specific activity, NAD+-dependent oxidation of (S)-3-hydroxybutyryl-CoA, has been measured on the purified enzyme (PMID:32218776) and is already on the gene as GO:0003857, a descendant of this term. Proposed replacements: (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity Supporting Evidence: PMID:32218776 The purified recombinant Msed_0389 and Msed_1423 catalyzed the NAD-dependent oxidation of (S)-3-hydroxybutyryl-CoA to acetoacetyl-CoA |
| GO:0016616 oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping of IPR006108 to CH-OH oxidoreductase activity with NAD or NADP as acceptor. Reason: Correct but too general. The enzyme oxidises the CH-OH group of (S)-3-hydroxybutyryl-CoA and strongly prefers NAD+ over NADP+ (PMID:32218776). GO:0003857, a direct child of this term and already on the gene, is the precise term. Proposed replacements: (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity Supporting Evidence: PMID:32218776 The purified recombinant Msed_0389 and Msed_1423 catalyzed the NAD-dependent oxidation of (S)-3-hydroxybutyryl-CoA to acetoacetyl-CoA |
| GO:0070403 NAD+ binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro2GO mapping of the 3-hydroxyacyl-CoA dehydrogenase NAD-binding domain (IPR006176, Pfam PF02737 at residues 3-177) to NAD+ binding. Reason: Correct for this protein. NAD+ is the cosubstrate, with an apparent Km of 0.03 mM, against 5.1 mM for NADP+ (PMID:32218776, Table 3). Unlike the default NAD-binding mapping on the NADPH-specific Msed_0709, this mapping is right. Binding of the cosubstrate is part of the dehydrogenase activity rather than a separate role, so the term is non-core, as for the paralog Msed_0399. Supporting Evidence: PMID:32218776 The purified recombinant Msed_0389 and Msed_1423 catalyzed the NAD-dependent oxidation of (S)-3-hydroxybutyryl-CoA to acetoacetyl-CoA |
| GO:0015977 carbon fixation | IDA PMID:32218776 Enzymes Catalyzing Crotonyl-CoA Conversion to Acetoacetyl-Co... | NEW | Summary: Msed_1423 catalyses the (S)-3-hydroxybutyryl-CoA dehydrogenase step in the acetyl-CoA-regenerating half 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 role in the cycle rests on kinetics of the purified enzyme, up-regulation in autotrophic cells, clustering with the cycle genes Msed_1424 and Msed_1426, and its near-universal distribution in autotrophic Sulfolobales (PMID:32218776). It is inferred rather than shown with a mutant. Comparators in the same role (catalysts of cycle steps) carry this term in their reviews; for example Msed_2001 (crotonyl-CoA hydratase) and Msed_0709 (malonyl-CoA/succinyl-CoA reductase) both have GO:0015977 as NEW. GO:0043427 carbon fixation by 3-hydroxypropionate cycle is defined as the Chloroflexus glyoxylate-forming cycle, which does not include this step, so it is not used. A specific 3HP/4HB-cycle child term is proposed instead, and the parent GO:0015977 is the most precise correct existing term. Supporting Evidence: PMID:32218776 show that Msed_1423 is the main (S)-3-hydroxybutyryl-CoA dehydrogenase in the HP/HB cycle in Sulfolobales. PMID:32218776 This protein is clustered together with two specific enzymes of the HP/HB cycle, succinic semialdehyde reductase Msed_1424 and acryloyl-CoA reductase Msed_1426 (Figure 2B), further supporting its involvement in autotrophic CO2 fixation. PMID:33472982 in M. sedula, the (S)-3-hydroxybutyryl-CoA dehydrogenase reaction is primarily catalyzed by Msed_1423 (which is universally found in autotrophic Sulfolobales) |
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Download this section (compressed HTML)Q: Msed_1423 accepts NADP+ with a similar Vmax but a 170-fold higher Km (5.1 mM versus 0.03 mM for NAD+). Is the enzyme strictly NAD+-dependent in vivo, given cellular NAD+/NADP+ ratios in M. sedula, so that GO:0003857 rather than GO:0008691 (NADP+-defined) is the correct activity term for the cycle step?
Q: PANTHER places Msed_1423 in PTHR48075:SF5 (3-hydroxybutyryl-CoA dehydrogenase), and InterPro2GO maps its domains to fatty acid metabolic process. Should the IPR006108/IPR006176 to GO:0006631 mapping be restricted, since many archaeal members of this family are short-chain enzymes of autotrophic cycles that do not act on fatty acids longer than C4?
Experiment: Delete the Msed_1423 ortholog in a genetically tractable autotrophic Sulfolobales species and compare autotrophic and heterotrophic growth, measuring (S)-3-hydroxybutyryl-CoA dehydrogenase activity in extracts of the mutant.
Hypothesis: Msed_1423 is required for autotrophic growth but not for heterotrophic growth.
Experiment: Assay purified Msed_1423 with (R)-3-hydroxybutyryl-CoA and with C6 (S)-3-hydroxyhexanoyl-CoA to define its stereospecificity and chain-length limit.
Hypothesis: Msed_1423 is specific for the (S) stereoisomer and for C4 substrates.
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