Msed_0399 encodes a 70-kDa bifunctional enzyme of the thermoacidophilic crenarchaeon Metallosphaera sedula. It has an N-terminal NAD+-dependent (S)-3-hydroxyacyl-CoA dehydrogenase region and a C-terminal enoyl-CoA hydratase (crotonase) domain, and it belongs to the same multifunctional-enzyme family as bacterial FadB. The purified native and recombinant enzyme hydrates crotonyl-CoA to (S)-3-hydroxybutyryl-CoA and oxidises that product to acetoacetyl-CoA. It uses NAD+ but not NADP+, and it does not act on the (R) stereoisomer. Both activities also work on C8 substrates, and the enzyme is most active with (E)-2-octenoyl-CoA. It was first identified as the crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase of the 3-hydroxypropionate/4-hydroxybutyrate CO2-fixation cycle, and with three other enzymes it converts 4-hydroxybutyrate to acetyl-CoA in vitro. Later kinetic, proteomic and comparative-genomic work showed that it is not the main enzyme for either step in vivo. The separate dehydrogenase Msed_1423 and the promiscuous hydratase Msed_2001 carry most of the flux, and the Msed_0399 gene is missing from several autotrophic Sulfolobales. The protein is abundant in both autotrophically and heterotrophically grown cells, and its gene lies in a cluster of beta-oxidation-like genes. Its main physiological role may therefore lie in short- to medium-chain acyl-CoA (fatty acid) metabolism, but this has not been tested in vivo.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping of the enoyl-CoA hydratase/isomerase conserved-site signature (IPR018376) to the root term catalytic activity. Reason: The term is correct but uninformative. Both activities of the protein have been measured on purified native and recombinant enzyme (PMID:21169482, PMID:32218776). The specific terms are GO:0003857 (already on the gene) for the dehydrogenase and GO:0018812 3-hydroxyacyl-CoA dehydratase activity for the C-terminal crotonase domain that carries this signature. Proposed replacements: (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity 3-hydroxyacyl-CoA dehydratase activity Supporting Evidence: PMID:21169482 The native enzyme preparation catalyzed both hydratase and dehydrogenase reactions PMID:32218776 In hydratase reaction, Msed_0399 was promiscuous and could hydrate not only crotonyl-CoA, but also (E)-2-octenoyl-CoA and acrylyl-CoA (Table 2). |
| GO:0003857 (3S)-3-hydroxyacyl-CoA dehydrogenase (NAD+) activity | IEA GO_REF:0000120 | ACCEPT | Summary: Combined automated annotation (ARBA and PANTHER TreeGrafter node PTN009069900) of NAD+-dependent (3S)-3-hydroxyacyl-CoA dehydrogenase activity, EC 1.1.1.35. Reason: Confirmed by direct biochemistry. The enzyme purified from M. sedula and the recombinant protein oxidise (S)-3-hydroxybutyryl-CoA with NAD+ and do not use NADP+ or the (R) isomer (PMID:21169482). The recombinant protein also oxidises the C8 (S)-3-hydroxyacyl-CoA (PMID:32218776). GO:0003857 is defined as a (3S)-3-hydroxyacyl-CoA + NAD+ = a 3-oxoacyl-CoA + NADH + H+, which fits both the cofactor and the stereochemistry. GO:0008691 3-hydroxybutyryl-CoA dehydrogenase activity would be wrong, because GO defines it with NADP+ (EC 1.1.1.157), although the original paper uses that EC number. This is the core molecular function. Its in-vivo share of the 3HP/4HB-cycle step is minor, because Msed_1423 is the main (S)-3-hydroxybutyryl-CoA dehydrogenase (PMID:32218776). That bears on the process role, not on the activity. Supporting Evidence: PMID:21169482 Substrates were indeed crotonyl-CoA ( K m 0.3 mM), ( S )-3-hydroxybutyryl-CoA ( K m 0.2 mM), and NAD + ( K m 0.25 mM). PMID:21169482 NADP + , acryloyl-CoA, 3-hydroxypropionyl-CoA, and the ( R ) stereoisomer of 3-hydroxybutyryl-CoA were not transformed. PMID:32218776 Similarly, it acted as dehydrogenase with both C4 and C8 (S)-3-hydroxyacyl-CoAs (Table 2). |
| GO:0004300 enoyl-CoA hydratase activity | IEA GO_REF:0000117 | MODIFY | Summary: ARBA machine-learning annotation of enoyl-CoA hydratase activity, from the C-terminal crotonase (enoyl-CoA hydratase/isomerase) domain. Reason: The activity is real. Native and recombinant enzyme hydrate crotonyl-CoA (PMID:21169482), and the recombinant enzyme also hydrates (E)-2-octenoyl-CoA and acrylyl-CoA (PMID:32218776). All of these are (2E)-enoyl-CoAs, so the specific child GO:0018812 3-hydroxyacyl-CoA dehydratase activity is supported. GO:0120092 (2E)-butenoyl-CoA hydratase activity covers only the C4 reaction, and the enzyme is more active on C8. GO:0080023 is the (3R) reaction, and the enzyme is (S)-specific. The hydratase is only a minor contributor to crotonyl-CoA hydration in autotrophic cells (about 8% of the activity; Msed_2001 supplies about 86%, PMID:33472982). The activity is therefore kept, but its share of the cycle flux is small. A low in-vivo flux share is not grounds for removing a demonstrated activity. Proposed replacements: 3-hydroxyacyl-CoA dehydratase activity Supporting Evidence: PMID:32218776 In hydratase reaction, Msed_0399 was promiscuous and could hydrate not only crotonyl-CoA, but also (E)-2-octenoyl-CoA and acrylyl-CoA (Table 2). PMID:33472982 the latter probably with some contribution of Msed_0399 (8.2%) |
| GO:0006631 fatty acid metabolic process | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: Combined automated annotation from ARBA and the 3-hydroxyacyl-CoA dehydrogenase domains (IPR006108, IPR006176). Reason: This broad term fits the enzyme's chemistry and its likely physiology. The protein acts on C4 and C8 acyl-CoAs and is most active with (E)-2-octenoyl-CoA. It is the only M. sedula 3-hydroxyacyl-CoA dehydrogenase tested that is active on C8. Its gene lies in a cluster of beta-oxidation homologs. The authors therefore suggest that its main role is in fatty acid metabolism rather than CO2 fixation (PMID:32218776). The activity is present at similar levels in heterotrophically grown cells (PMID:21169482). Growth of M. sedula on fatty acids has not been shown, so the role rests on biochemistry and gene context rather than on in-vivo data. For that reason the term is kept but not treated as core. 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 In fact, msed_0399 is located in the cluster with the genes homologous to various Ξ²-oxidation genes (Figure 2A) and may thus be primarily involved in, e.g., fatty acid metabolism rather than CO2 fixation (Table 2). PMID:21169482 The expected bifunctional hydratase/dehydrogenase activity was present in similar amounts in extracts of autotrophically and heterotrophically grown cells of M. sedula |
| GO:0006635 fatty acid beta-oxidation | IEA GO_REF:0000120 | UNDECIDED | Summary: Combined automated annotation from PANTHER node PTN009069900 (subfamily PTHR23309:SF49, named peroxisomal bifunctional enzyme) and the UniPathway fatty acid beta-oxidation pathway. Reason: This is a family-level transfer from the FadB/EHHADH multifunctional-enzyme family, but it cannot be dismissed as a pure over-annotation. Msed_0399 hydrates (E)-2-octenoyl-CoA and oxidises (S)-3-hydroxyoctanoyl-CoA, its gene clusters with beta-oxidation homologs, and Liu et al. propose that it takes part in beta-oxidation (PMID:32218776). Some Sulfolobales degrade fatty acids with a dedicated beta-oxidation gene cluster (PMID:30948713). Against this, GO:0006635 is defined as the complete oxidation of a long-chain fatty acid. No substrate longer than C8 has been tested, and growth of M. sedula on fatty acids has not been shown (PMID:32218776). Until chain-length range and in-vivo involvement are tested, the row is left undecided. The broader GO:0006631 is kept as non-core. Supporting Evidence: PMID:32218776 Interestingly, the enzyme was most active with octenoyl-CoA, thus suggesting that Msed_0399 may participate in Ξ²-oxidation, in addition to autotrophic CO2 fixation. PMID:32218776 Although the growth of M. sedula on fatty acids has not been shown, some Sulfolobales are capable to use these compounds as a sole carbon source PMID:30948713 acting as a repressor of a 30-kb gene cluster comprising 23 open reading frames encoding lipases and Ξ²-oxidation enzymes. |
| GO:0016491 oxidoreductase activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping of the 3-hydroxyacyl-CoA dehydrogenase C-terminal domain (IPR006108) to oxidoreductase activity. Reason: Correct but too general. The specific dehydrogenase activity is established (NAD+-dependent oxidation of (S)-3-hydroxyacyl-CoAs, PMID:21169482) 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:21169482 Substrates were indeed crotonyl-CoA ( K m 0.3 mM), ( S )-3-hydroxybutyryl-CoA ( K m 0.2 mM), and NAD + ( K m 0.25 mM). |
| 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 is NAD+-specific (NADP+ is not used) and acts on (S)-3-hydroxyacyl-CoAs (PMID:21169482). GO:0003857, the 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:21169482 NADP + , acryloyl-CoA, 3-hydroxypropionyl-CoA, and the ( R ) stereoisomer of 3-hydroxybutyryl-CoA were not transformed. |
| 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, residues 2-181) to NAD+ binding. Reason: Correct for this protein. NAD+ is the cosubstrate (Km 0.25 mM) and NADP+ is not used. The enzyme was also eluted from a dye-affinity column with NAD+ (PMID:21169482). Unlike the default NAD-binding mapping on the NADPH-specific Msed_0709, this mapping is right. Cofactor binding is part of the dehydrogenase activity, not a separate role, so the term is non-core. Supporting Evidence: PMID:21169482 The enzyme was eluted with 1 ml of 0.25 mM NAD + in buffer A. PMID:21169482 NADP + , acryloyl-CoA, 3-hydroxypropionyl-CoA, and the ( R ) stereoisomer of 3-hydroxybutyryl-CoA were not transformed. |
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Download this section (compressed HTML)Q: Should Msed_0399 be annotated to carbon fixation (GO:0015977, or the proposed 'carbon fixation by 3-hydroxypropionate/4-hydroxybutyrate cycle') when Liu et al. estimate that it carries a small flux at best, and the gene is absent from several autotrophic Sulfolobales? The orthologous fusion protein is the sole candidate for these steps in the dicarboxylate/4-hydroxybutyrate cycle of Ignicoccus and Pyrobaculum.
Q: Is Msed_0399 a beta-oxidation enzyme in M. sedula? What is its chain-length limit beyond C8, and is its gene cluster induced on fatty acids?
Q: UniProt's ARBA00008750 SIMILARITY line places the enoyl-CoA hydratase in the N-terminal section, but the Pfam domains (and the original paper) place it C-terminally. Should the rule's positional text be corrected for archaeal fusions with the reversed (dehydrogenase-first) domain order?
Experiment: Delete the Msed_0399 ortholog in a genetically tractable Sulfolobales species (or in M. sedula if genetics allow). Compare autotrophic growth and growth on fatty acids, and measure crotonyl-CoA hydratase and (S)-3-hydroxybutyryl-CoA dehydrogenase activities in extracts.
Hypothesis: Msed_0399 is dispensable for autotrophic growth but contributes to fatty acid catabolism.
Experiment: Assay recombinant Msed_0399 with C10 to C16 (E)-2-enoyl-CoAs and (S)-3-hydroxyacyl-CoAs to define its chain-length range. Use quantitative proteomics to test whether it is induced on fatty acids.
Hypothesis: Msed_0399 acts on long-chain acyl-CoAs.
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