Msed_1700 encodes the aspartate-semialdehyde dehydrogenase (ASD, EC 1.2.1.11) of the thermoacidophilic crenarchaeon Metallosphaera sedula. It is a 348-residue member of the archaeal/fungal branch of the ASD family. ASD catalyses the NADPH-dependent reductive dephosphorylation of 4-phospho-L-aspartate to L-aspartate 4-semialdehyde, the branch-point intermediate for homoserine, threonine and methionine synthesis. The reaction goes through a covalent acyl-thioester intermediate on a catalytic cysteine (Cys150), with His242 as acid/base. The gene lies next to the genes for aspartokinase (Msed_1699) and threonine synthase (Msed_1698), and it carries the archaeal/fungal ASD conserved-domain models. It is about 69% identical to the asd gene product of Sulfolobus tokodaii but only about 39% identical to its M. sedula paralog Msed_0709. Msed_0709 evolved from an ancestral ASD into the malonyl-CoA/succinyl-CoA reductase of the 3-hydroxypropionate/4-hydroxybutyrate CO2-fixation cycle. By analogy with archaeal homologs, the enzyme is expected to be a cytoplasmic homotetramer (a dimer of dimers) that may accept NADH as well as NADPH. The protein has not been characterised experimentally, and its function is inferred from genomic context, sequence signatures and homology.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004073 aspartate-semialdehyde dehydrogenase (NADP+) activity | IEA GO_REF:0000118 | ACCEPT | Summary: TreeGrafter transfer of ASD activity from the PANTHER PTHR46718 ancestral node. For Msed_1700 this is the correct core activity. Reason: No experiment has tested Msed_1700, so this rests on three independent lines of non-experimental evidence. First, genomic context. The gene sits between the aspartokinase gene (Msed_1699) and the threonine synthase gene (Msed_1698). This is the conserved asd cluster of Sulfolobales (PMID:23325803), and Alber et al. 2006 used the same cluster to assign the true asd of S. tokodaii (NP_377174/ST1242). Second, orthology. Msed_1700 is 69% identical to ST1242 but only about 39-41% identical to the MCRs, including its paralog Msed_0709 (bioinformatics RESULTS.md). Third, signatures. It carries the archaeal/fungal ASD CDD models (cd18130, cd02315) rather than the MCR models carried by Msed_0709. It also keeps the catalytic cysteine and histidine (ACT_SITE 150 and 242), the residues the archaeal ASD structure shows are conserved (PMID:16225889). This is the ancestral, retained function of the duplicated pair, unlike the neofunctionalized Msed_0709, from which this term was removed. GO:0004073 is defined with NADP+. The M. jannaschii homolog also accepts NAD, but prefers NADPH. 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:16225889 However, the entire repertoire of functionally important active site amino acid residues is conserved, suggesting an identical catalytic mechanism but with lower catalytic efficiency. file:METS5/Msed_1700/Msed_1700-uniprot.txt CDD; cd18130; ASADH_C_arch_fung_like; 1. file:METS5/Msed_1700/Msed_1700-bioinformatics/RESULTS.md Msed_1700 is far closer to the S. tokodaii asd gene product (ST1242) than to either MCR. |
| GO:0008652 amino acid biosynthetic process | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the semialdehyde dehydrogenase dimerization domain (IPR012280). The statement is correct for Msed_1700 but general. Reason: For this gene the claim is true. ASD makes L-aspartate 4-semialdehyde for the aspartate-family amino acids, and Msed_1700 is the M. sedula ASD (see GO:0004073). The term is too general, because a more specific process is supported. GO:0009088 L-threonine biosynthetic process, which the gene already carries by TreeGrafter, is a descendant and is backed by the adjacent aspartokinase and threonine synthase genes. Note that the IPR012280 mapping itself does not discriminate. The same domain is on the MCR paralog Msed_0709, for which this term was removed. Proposed replacements: L-threonine biosynthetic process 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. |
| GO:0009088 L-threonine biosynthetic process | IEA GO_REF:0000118 | ACCEPT | Summary: TreeGrafter transfer of the ancestral ASD pathway role. ASD catalyses the second step from aspartate to threonine (aspartokinase, ASD, homoserine dehydrogenase, homoserine kinase, threonine synthase). Reason: ASD catalyses a step of the pathway itself, so it passes the participation test. In M. sedula the gene is flanked by Msed_1699 aspartokinase (the step before) and Msed_1698 threonine synthase (the last step), so the operon-like cluster supports this pathway assignment. ASD also supplies homoserine for methionine, so threonine is not its only output. No term covering the aspartate-semialdehyde branch is a more appropriate single choice. Sulfolobales make lysine by the alpha-aminoadipate (LysW) route rather than the diaminopimelate route, so a lysine term would not apply. Evidence is genomic context and homology only. 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. file:METS5/Msed_1700/Msed_1700-bioinformatics/RESULTS.md The sequence data place Msed_1700 with the archaeal/fungal ASDs, consistent with its genomic context (Msed_1698 threonine synthase, Msed_1699 aspartokinase). |
| GO:0016620 oxidoreductase activity, acting on the aldehyde or oxo group of donors, NAD or NADP as acceptor | IEA GO_REF:0000002 | MODIFY | Summary: Correct parent-level activity from InterPro2GO. The ASD reaction is a phosphorylating aldehyde dehydrogenase reaction with NADP+ as acceptor. Reason: The term is correct but general. Its is_a descendant GO:0004073 aspartate-semialdehyde dehydrogenase (NADP+) activity, which the gene already carries by TreeGrafter, is supported for Msed_1700. Unlike the MCR paralog Msed_0709, where GO lacks a specific term for EC 1.2.1.75, here a specific GO activity term exists. Proposed replacements: aspartate-semialdehyde dehydrogenase (NADP+) activity Supporting Evidence: file:METS5/Msed_1700/Msed_1700-uniprot.txt SIMILARITY: Belongs to the aspartate-semialdehyde dehydrogenase family. |
| GO:0046983 protein dimerization activity | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: The C-terminal dimerization domain mediates subunit contacts. ASDs of the archaeal/fungal branch form GAPDH-like homotetramers (dimers of dimers). Reason: Self-association is well established for archaeal/fungal-branch ASDs, including the M. jannaschii enzyme. In the fungal enzyme, tetramerization is required for activity. It has not been shown for Msed_1700 itself, but the domain and branch membership make it very likely. Oligomerization is structural rather than the protein's biological role, so the term is kept as non-core. Supporting Evidence: PMID:26869335 Further, our structural analyses and native PAGE results with other fungal ASADHs demonstrated that ASADH tetramerization is universal in the archaeal/fungal branch. PMID:26869335 Crystal packing analysis was performed for the structures of caASADH and mjASADH, which also identified the presence of GAPDH-like homotetramers in each of these structures. |
| GO:0050661 NADP binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: NADP(H) is the cofactor of the ASD reaction defined by GO:0004073. The archaeal M. jannaschii homolog binds NADP+ and prefers NADPH. Reason: The claim is correct. NADP+ is the acceptor in the GO:0004073 reaction, and the closest characterised archaeal ASD shows a clear preference for NADPH. Cofactor binding is part of the catalytic activity rather than a separate function, so it is kept as non-core. No direct binding data exist for Msed_1700. Supporting Evidence: PMID:23325803 This difference rationalizes its dual specificity for NADPH and NADH as electron donor, albeit still with a clear preference for NADPH ( 18 ). PMID:23325803 ADP as major component for NADP + binding has also been experimentally verified by ASD of M. jannaschii structurally related to MCR ( 18 ). |
| GO:0051287 NAD binding | IEA GO_REF:0000002 | UNDECIDED | Summary: This is a generic InterPro2GO mapping from the semialdehyde dehydrogenase Rossmann (NAD-binding) domain, IPR000534. Archaeal-branch ASDs can be dual-specific, but Msed_1700's cofactor use is unmeasured. Reason: The archaeal M. jannaschii ASD uses both NAD and NADP, with a preference for NADPH. So, unlike the strictly NADPH-specific MCR paralog Msed_0709, where this row was removed, NAD binding is plausible for an archaeal ASD. Whether M. sedula ASD binds or uses NAD(H) has not been tested, and the IPR000534 mapping is a domain-level default that does not record cofactor specificity. The evidence supports neither keeping nor removing the row. Supporting Evidence: PMID:16225889 A new coenzyme-binding conformation and dual NAD/NADP coenzyme specificity further distinguish this archaeal branch from the bacterial ASADHs. |
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Download this section (compressed HTML)Q: Should UniProt ARBA rules ARBA00067001 (name) and ARBA00052213 (EC 1.2.1.75 catalytic activity) gain a condition that excludes ASD orthologs? The archaeal/fungal ASD CDD models cd18130/cd02315 would work as a negative condition, or the MCR models cd23940/cd24150 as a positive one. NCBIfam TIGR00978 does not work, because it is present on both paralogs.
Q: Does Msed_1700 retain any malonyl-CoA or succinyl-CoA reductase side activity, and does it use NADH as well as NADPH, like the M. jannaschii ASD?
Experiment: Express Msed_1700 in E. coli, purify it by heat treatment and affinity chromatography, and assay ASD activity in both directions (aspartate semialdehyde plus phosphate with NADP+ or NAD+; 4-phospho-L-aspartate generated by aspartokinase with NADPH). In parallel, assay malonyl-CoA and succinyl-CoA reduction with NADPH, using Msed_0709 as a positive control.
Hypothesis: Msed_1700 is an aspartate-semialdehyde dehydrogenase without significant malonyl-CoA reductase activity.
Experiment: Delete or knock down Msed_1700 (or its Sulfolobus acidocaldarius ortholog, which can be manipulated genetically) and test growth in defined medium with and without threonine, methionine and homoserine supplements.
Hypothesis: Msed_1700 is essential for growth without exogenous threonine and methionine.
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