NadX (MJ0915) is an L-aspartate dehydrogenase (EC 1.4.1.21) that catalyzes the NAD(P)-dependent oxidative deamination of L-aspartate to iminoaspartate, the first committed step in the de novo NAD+ biosynthetic pathway from L-aspartate. This enzyme represents an alternative to the FAD-dependent L-aspartate oxidase (NadB) used in classical bacterial NAD biosynthesis pathways. NadX is a homodimeric protein with an N-terminal Rossmann fold NAD(P)-binding domain and a C-terminal catalytic domain. The iminoaspartate product is channeled to quinolinate synthase (NadA) for quinolinate biosynthesis. NadX homologs are found in many methanogenic archaea and some bacteria such as Thermotoga maritima.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
|
GO:0009435
NAD+ biosynthetic process
|
IEA
GO_REF:0000120 |
MODIFY |
Summary: NadX catalyzes the first step of de novo NAD+ biosynthesis from L-aspartate, producing iminoaspartate for the NadA quinolinate synthase reaction. This annotation is correct but could be more specific; GO:0034628 ('de novo' NAD+ biosynthetic process from L-aspartate) precisely describes the pathway route used by NadX.
Reason: The more specific term GO:0034628 ('de novo' NAD+ biosynthetic process from L-aspartate) precisely captures NadX's role in the aspartate-derived de novo pathway, distinguishing it from salvage pathways and the tryptophan-derived route.
Proposed replacements:
'de novo' NAD+ biosynthetic process from L-aspartate
Supporting Evidence:
PMID:12496312
two different enzymes, an oxidase and a dehydrogenase, may have evolved to catalyze the first step of NAD biosynthesis in prokaryotes
file:METJA/nadX/nadX-deep-research-bioreason-sft.md
BioReason SFT trace correctly identifies NAD biosynthesis pathway context
|
|
GO:0016491
oxidoreductase activity
|
IEA
GO_REF:0000002 |
MODIFY |
Summary: This InterPro2GO annotation from IPR005106 (Aspartate/homoserine dehydrogenase, NAD-binding domain) is correct but too general. NadX has a well-characterized specific activity: L-aspartate dehydrogenase [NAD(P)+] activity (GO:0033735). The general oxidoreductase term adds no information beyond what the more specific term already provides.
Reason: GO:0016491 (oxidoreductase activity) is far too general; GO:0033735 provides the precise molecular function. This is a typical InterPro2GO limitation where domain-level annotation yields only broad functional categories.
Proposed replacements:
L-aspartate dehydrogenase [NAD(P)+] activity
Supporting Evidence:
PMID:12496312
The enzymatic characterization of TM1643 revealed that it possesses NAD or NADP-dependent dehydrogenase activity toward l-aspartate but no aspartate oxidase activity
|
|
GO:0016639
oxidoreductase activity, acting on the CH-NH2 group of donors, NAD or NADP as acceptor
|
IEA
GO_REF:0000104 |
MODIFY |
Summary: This annotation correctly captures that NadX acts on the CH-NH2 group of L-aspartate using NAD or NADP as acceptor. However, the more specific child term GO:0033735 (L-aspartate dehydrogenase [NAD(P)+] activity) is available and precisely identifies the substrate specificity.
Reason: A more specific child term exists that exactly describes this enzyme's activity and substrate specificity.
Proposed replacements:
L-aspartate dehydrogenase [NAD(P)+] activity
Supporting Evidence:
PMID:16731057
The enzyme specifically utilized L-aspartate as the electron donor, while either NAD or NADP could serve as the electron acceptor
|
|
GO:0033735
L-aspartate dehydrogenase [NAD(P)+] activity
|
IEA
GO_REF:0000120 |
ACCEPT |
Summary: This is the most specific and accurate molecular function term for NadX. The enzyme catalyzes NAD(P)-dependent oxidative deamination of L-aspartate to iminoaspartate (which spontaneously decomposes to oxaloacetate + NH4+). Structural and biochemical studies on homologs from T. maritima and A. fulgidus have confirmed this activity.
Reason: GO:0033735 precisely matches the experimentally characterized enzymatic activity of NadX. Although the evidence for M. jannaschii NadX specifically is computational (IEA), the activity has been experimentally demonstrated for close homologs in T. maritima and A. fulgidus.
Supporting Evidence:
PMID:12496312
The enzymatic characterization of TM1643 revealed that it possesses NAD or NADP-dependent dehydrogenase activity toward l-aspartate but no aspartate oxidase activity
PMID:16731057
Characterization revealed the enzyme to be a highly thermostable L-aspartate dehydrogenase
|
|
GO:0050661
NADP binding
|
IEA
GO_REF:0000120 |
KEEP AS NON CORE |
Summary: NadX binds NADP as one of its two cofactor options. The A. fulgidus homolog has a Km of 0.32 mM for NADP. While NADP binding is implicit in the GO:0033735 term (which specifies NAD(P)+), this annotation provides explicit documentation of NADP cofactor binding capability.
Reason: NADP binding is already implicit in GO:0033735 (L-aspartate dehydrogenase [NAD(P)+] activity). This annotation is not wrong but is redundant with the more specific MF term. Keeping as non-core for completeness.
Supporting Evidence:
PMID:16731057
The Km values for NAD and NADP were 0.11 and 0.32 mM, respectively
|
|
GO:0051287
NAD binding
|
IEA
GO_REF:0000104 |
KEEP AS NON CORE |
Summary: NadX binds NAD as its preferred cofactor. The A. fulgidus homolog has a Km of 0.11 mM for NAD, and crystal structures show NAD bound in the Rossmann fold domain. As with NADP binding, this is implicit in GO:0033735 but provides explicit documentation.
Reason: NAD binding is already implicit in GO:0033735 (L-aspartate dehydrogenase [NAD(P)+] activity). Keeping as non-core for completeness. Kinetic data from the A. fulgidus homolog suggest a slight preference for NAD over NADP.
Supporting Evidence:
PMID:17651440
The crystal structure of the highly thermostable L-aspartate dehydrogenase (L-aspDH; EC 1.4.1.21) from the hyperthermophilic archaeon Archaeoglobus fulgidus was determined in the presence of NAD and a substrate analog, citrate
PMID:16731057
The Km values for L-aspartate were 0.19 and 4.3 mM when NAD or NADP, respectively, served as the electron acceptor
|
|
GO:0005737
cytoplasm
|
IEA
GO_REF:0000120 |
NEW |
Summary: NadX is a soluble enzyme with no predicted transmembrane segments or signal peptides, consistent with cytoplasmic localization. As a central metabolic enzyme in NAD biosynthesis, cytoplasmic localization is expected for this archaeal protein.
Reason: Cytoplasmic localization is consistent with the enzyme's soluble nature and its role in the cytoplasmic de novo NAD biosynthesis pathway. No transmembrane domains are predicted.
Supporting Evidence:
PMID:16731057
a homodimeric protein with a molecular mass of about 48 kDa
|
Q: Has L-aspartate dehydrogenase activity been directly demonstrated for the M. jannaschii NadX protein (Q58325), or is all experimental evidence from homologs (T. maritima TM1643, A. fulgidus)?
Suggested experts: Toshihisa Ohshima, Liang Tong
Q: Is there experimental evidence for physical interaction between NadX and NadA (quinolinate synthase) in M. jannaschii to channel the unstable iminoaspartate intermediate?
Experiment: Express and purify recombinant M. jannaschii NadX. Perform kinetic characterization measuring NAD- and NADP-dependent oxidation of L-aspartate by monitoring NADH/NADPH production spectrophotometrically at 340 nm. Determine Km values for L-aspartate, NAD, and NADP.
Hypothesis: The M. jannaschii NadX (Q58325) has L-aspartate dehydrogenase activity with similar kinetic parameters to the characterized A. fulgidus homolog.
Type: enzyme kinetics
Experiment: Perform co-purification or co-immunoprecipitation experiments with tagged NadX and NadA from M. jannaschii. Use cross-linking mass spectrometry to identify interaction surfaces. Test whether coupled NadX-NadA reactions show enhanced quinolinate production compared to sequential reactions.
Hypothesis: NadX physically interacts with NadA to channel the unstable iminoaspartate intermediate in M. jannaschii.
Type: protein-protein interaction
The architecture begins with an N-terminal dinucleotide-binding module: IPR036291 (NAD(P)-binding domain superfamily, residues 1–146) overlaps IPR005106 (Aspartate/homoserine dehydrogenase, NAD-binding domain, residues 8–121), establishing a Rossmann-like fold that positions the pyrophosphate and ribose of NADP(H) for hydride transfer. This is followed by a catalytic core: IPR002811 (Aspartate dehydrogenase domain, residues 168–253), which houses the active-site residues that bind L-aspartate and stabilize the oxaloacetate intermediate. Three family-level signatures span nearly the full polypeptide—IPR011182 (L-aspartate dehydrogenase family, residues 1–267), IPR020626 (L-aspartate dehydrogenase, prokaryotic family, residues 2–265), and IPR022487 (L-aspartate dehydrogenase, archaeal family, residues 27–264)—together specifying a prokaryotic/archaeal L-aspartate dehydrogenase scaffold. The juxtaposition of an N-terminal Rossmann-like NADP-binding region with a C-terminal aspartate dehydrogenase catalytic domain causes a reversible redox chemistry on L-aspartate’s C2, using NADP+ as electron acceptor to form oxaloacetate and NH4+, or using NAD+ as donor to oxidize oxaloacetate back to L-aspartate. The archaeal/prokaryotic family context typically enforces dimerization, where each protomer contributes to the stability of the partner’s active site; this explains protein homodimerization activity (GO:0042803). The presence of both NADP binding (GO:0050661) and NAD binding (GO:0051287) follows directly from the dual-cofactor Rossmann features and the known bidirectionality of the reaction.
At the process level, the NADP-dependent oxidative direction supplies oxaloacetate that feeds into the shikimate pathway, where oxaloacetate condenses with phosphoenolpyruvate to form phosphoenolpyruvate-derived intermediates en route to chorismate. Thus, the enzyme’s redox activity causally supports the chorismate biosynthetic process (GO:0009423) by provisioning oxaloacetate under NADP+–favored conditions. The archaeal family signature and lack of transmembrane segments indicate a soluble enzyme. In prokaryotic/archaeal cells, such central metabolic dehydrogenases operate in the cytoplasm, aligning with a cytoplasmic localization (GO:0005737).
Mechanistically, the N-terminal Rossmann-like domain binds NADP+ with preference, orienting the nicotinamide for hydride acceptance, while the C-terminal catalytic domain binds L-aspartate and promotes hydride transfer to NADP+, yielding oxaloacetate and releasing ammonium. The archaeal family context implies a homodimer that stabilizes the catalytic geometry and may create a metabolon interface. In this role, the enzyme likely associates with quinolinate synthetase (nadA) to channel aspartate-derived flux toward NAD biosynthesis, and with aspartate carbamoyltransferase regulatory and catalytic chains (pyrI, pyrB) to balance aspartate partitioning between pyrimidine and purine branches. Interactions with aspartate aminotransferase (aspB1) would buffer oxaloacetate/aspartate pools, while contacts with adenylosuccinate synthetase (purA), argininosuccinate synthetase (argG), and asparagine synthetase (asnB) would coordinate nitrogen and aspartate utilization. Association with a pantothenate metabolism flavoprotein (dfp) and a conserved hypothetical protein could stabilize a broader central-metabolism assembly that tunes cofactor availability and flux directionality according to cellular redox state.
## Functional Summary
A soluble archaeal dehydrogenase that forms a homodimer and uses a Rossmann-like dinucleotide-binding module to bind NADP+ preferentially alongside a catalytic domain that oxidizes L-aspartate to oxaloacetate with release of ammonium, or reduces oxaloacetate to L-aspartate with NAD+. By supplying oxaloacetate under NADP+-favored conditions, it feeds the shikimate pathway toward chorismate and coordinates aspartate flux with neighboring metabolic nodes in the cytoplasm.
## UniProt Summary
Catalyzes the reversible oxidation of L-aspartate to oxaloacetate. Has a preference for NADP(+) as a hydrogen acceptor. Plays a role in the chorismate biosynthesis pathway.
## InterPro Domains
- IPR011182: L-aspartate dehydrogenase (family) [1-267]
- IPR036291: NAD(P)-binding domain superfamily (homologous_superfamily) [1-146]
- IPR020626: L-aspartate dehydrogenase, prokaryotic (family) [2-265]
- IPR005106: Aspartate/homoserine dehydrogenase, NAD-binding (domain) [8-121]
- IPR022487: L-aspartate dehydrogenase, archaeal (family) [27-264]
- IPR002811: Aspartate dehydrogenase (domain) [168-253]
## GO Term Predictions
### Molecular Function
### Biological Process
### Cellular Component
NadX catalyzes the first committed step in the de novo NAD+ biosynthetic pathway from L-aspartate in archaea and some bacteria. It performs the NAD(P)-dependent oxidative deamination of L-aspartate to iminoaspartate, which is the substrate for the next enzyme in the pathway, quinolinate synthase (NadA).
This enzyme represents an alternative to the FAD-dependent L-aspartate oxidase (NadB) used in the classical bacterial NAD biosynthesis pathway (e.g., in E. coli). The key discovery paper is Yang et al. (2003), who identified this novel enzymatic activity in the Thermotoga maritima homolog TM1643 PMID:12496312.
L-aspartate + NAD(P)+ + H2O -> iminoaspartate -> (spontaneous) oxaloacetate + NH4+ + NAD(P)H
The iminoaspartate product is unstable in aqueous solution and spontaneously hydrolyzes to oxaloacetate and ammonia. The biologically relevant product is iminoaspartate, which is channeled to NadA for quinolinate synthesis before it can decompose.
The enzyme can use either NAD+ or NADP+ as electron acceptor. According to PubMed, kinetic characterization of the A. fulgidus homolog showed Km values for L-aspartate of 0.19 mM (with NAD+) and 4.3 mM (with NADP+), and Km values for NAD and NADP of 0.11 and 0.32 mM respectively [PMID:16731057, DOI:10.1016/j.bbapap.2006.04.006 "The enzyme specifically utilized L-aspartate as the electron donor, while either NAD or NADP could serve as the electron acceptor"].
According to PubMed, the crystal structure of the T. maritima homolog (TM1643) revealed an N-terminal Rossmann fold domain with a bound NAD+ cofactor and a C-terminal alpha+beta domain, with the active site at the interface between the two domains [PMID:12496312, DOI:10.1074/jbc.M211892200 "The structure reveals the presence of an N-terminal Rossmann fold domain with a bound NAD(+) cofactor and a C-terminal alpha+beta domain"].
The crystal structure of the A. fulgidus homolog in complex with NAD and citrate (substrate analog) was determined at 1.9 A resolution, showing a dimeric enzyme with a closed conformation upon substrate binding [PMID:17651440, DOI:10.1111/j.1742-4658.2007.05961.x "The dimeric structure of A. fulgidus L-aspDH was refined at a resolution of 1.9 A...each subunit consists of two domains separated by a deep cleft containing an active site"].
The enzyme forms a homodimer. According to PubMed, the A. fulgidus homolog was characterized as "a homodimeric protein with a molecular mass of about 48 kDa" [PMID:16731057, DOI:10.1016/j.bbapap.2006.04.006].
The enzyme is highly thermostable, consistent with the hyperthermophilic lifestyle of M. jannaschii and related archaea. The A. fulgidus homolog showed an optimum temperature of about 80 degrees C and little loss of activity after incubation for 1 h at up to 80 degrees C PMID:16731057.
The de novo NAD+ biosynthetic pathway from L-aspartate in prokaryotes proceeds:
The discovery that NadX provides an alternative to NadB was a significant finding in comparative genomics PMID:12496312.
Within the archaeal domain, homologs of L-aspartate dehydrogenase occur in many methanogenic species, but not in Thermococcales or Sulfolobales [PMID:16731057, DOI:10.1016/j.bbapap.2006.04.006 "Within the archaeal domain, homologues of this enzyme occurred in many Methanogenic species, but not in Thermococcales or Sulfolobales species"]. Some bacteria (e.g., Thermotoga maritima) also have NadX instead of NadB.
The M. jannaschii genome was sequenced in 1996 [PMID:8688087, DOI:10.1126/science.273.5278.1058 "Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii"]. The nadX gene (MJ0915) was identified based on homology. In T. maritima, TM1643 was found in an operon with two other genes encoding enzymes involved in NAD biosynthesis PMID:12496312.
The UniProt entry includes a note about a role in chorismate biosynthesis. However, the primary literature does not support a direct role for NadX in chorismate biosynthesis. The enzyme's established function is exclusively in NAD+ biosynthesis, converting L-aspartate to iminoaspartate for quinolinate synthesis. While oxaloacetate (the spontaneous decomposition product) could theoretically enter other metabolic pathways, the biological function of NadX is specifically to produce iminoaspartate for NAD biosynthesis, not to supply oxaloacetate for the shikimate pathway.
Source: nadX-deep-research-bioreason-sft.md
The BioReason SFT functional summary describes NadX as:
A soluble archaeal dehydrogenase that forms a homodimer and uses a Rossmann-like dinucleotide-binding module to bind NADP+ preferentially alongside a catalytic domain that oxidizes L-aspartate to oxaloacetate with release of ammonium, or reduces oxaloacetate to L-aspartate with NAD+.
This description is partially correct but contains several inaccuracies:
Incorrect product identification. The summary states the enzyme "oxidizes L-aspartate to oxaloacetate." In reality, the direct enzymatic product is iminoaspartate, not oxaloacetate. Oxaloacetate is the spontaneous decomposition product of iminoaspartate in aqueous solution. This distinction is biologically critical because the relevant product in vivo is iminoaspartate, which is channeled to quinolinate synthase (NadA) before it can decompose. Yang et al. (2003, PMID:12496312) explicitly state: "The product of the aspartate dehydrogenase activity is also iminoaspartate."
NADP+ preference claim is unsupported. The summary claims the enzyme binds "NADP+ preferentially." Kinetic data from the A. fulgidus homolog (PMID:16731057) actually show a lower Km for NAD (0.11 mM) than NADP (0.32 mM), and a much lower Km for L-aspartate when NAD is the acceptor (0.19 mM vs 4.3 mM), suggesting NAD may be the preferred cofactor in vivo.
False claim about chorismate biosynthesis. The summary states NadX "feeds the shikimate pathway toward chorismate." This is incorrect. NadX functions in NAD+ biosynthesis, not chorismate biosynthesis. The biological product (iminoaspartate) feeds into quinolinate synthesis via NadA. While oxaloacetate (the decomposition product) could theoretically enter other pathways, there is no evidence that NadX functions in the shikimate pathway, and this claim appears to be a hallucination or over-interpretation.
Homodimer claim is correct. The A. fulgidus homolog was characterized as a homodimeric protein of ~48 kDa (PMID:16731057), and the crystal structure confirms dimeric architecture (PMID:17651440).
Core structural description is broadly correct. The Rossmann-like NAD(P)-binding N-terminal domain and the C-terminal catalytic domain are accurately described, consistent with the crystal structure (PMID:12496312).
The thinking trace contains additional problematic claims, including speculative interactions with "quinolinate synthetase (nadA)," "aspartate carbamoyltransferase regulatory and catalytic chains (pyrI, pyrB)," "aspartate aminotransferase (aspB1)," and several others. These protein-protein interactions are entirely speculative and not supported by any published evidence.
The claim about "protein homodimerization activity (GO:0042803)" in the thinking trace is an over-annotation. The enzyme forms a homodimer, but GO:0042803 implies homodimerization is itself a molecular function of the protein rather than a structural feature. GO curation guidelines would not annotate a simple homodimeric enzyme with this term.
The InterPro2GO annotation (GO_REF:0000002) assigns GO:0016491 (oxidoreductase activity) based on IPR005106 (Aspartate/homoserine dehydrogenase, NAD-binding domain). This is a very general annotation that correctly identifies the enzyme as an oxidoreductase but provides no information about substrate specificity or biological context.
BioReason goes substantially beyond InterPro2GO by:
- Correctly identifying the specific substrate (L-aspartate)
- Correctly identifying the homodimeric quaternary structure
- Correctly describing the two-domain architecture (Rossmann fold + catalytic domain)
- Identifying the biological pathway context (NAD biosynthesis)
However, BioReason also introduces errors not present in InterPro2GO:
- The false chorismate biosynthesis claim is entirely novel (not from InterPro2GO)
- The misidentification of the product as oxaloacetate rather than iminoaspartate
- Speculative protein-protein interactions with no literature support
The BioReason functional summary is clearly not a simple recapitulation of InterPro2GO. It synthesizes domain architecture information with pathway context, but introduces biological errors in the process. The InterPro2GO annotation, while uninformative, is at least not wrong. The more informative annotations in the GOA file (GO:0033735 from GO_REF:0000120, and the BP term GO:0009435) come from UniRule/HAMAP rather than InterPro2GO, and BioReason's functional summary covers similar ground to these UniRule annotations but with lower precision.
The thinking trace demonstrates a systematic domain-by-domain analysis that is methodologically sound. It correctly maps InterPro domain boundaries and structural features. However, the reasoning goes off track in two areas:
The chorismate pathway connection is fabricated. The trace states "the NADP-dependent oxidative direction supplies oxaloacetate that feeds into the shikimate pathway, where oxaloacetate condenses with phosphoenolpyruvate." This confuses the role of oxaloacetate in the shikimate pathway (oxaloacetate is not a shikimate pathway intermediate; the pathway starts from erythrose 4-phosphate and phosphoenolpyruvate).
The extensive protein-protein interaction predictions (with nadA, pyrI, pyrB, aspB1, purA, argG, asnB, dfp) appear to be fabricated metabolic network reasoning without any supporting evidence. While NadX and NadA do participate in the same pathway, none of these specific physical interactions have been demonstrated.
The trace correctly uses the archaeal family signatures (IPR022487) to contextualize the enzyme taxonomically.
id: Q58325
gene_symbol: nadX
product_type: PROTEIN
status: COMPLETE
taxon:
id: NCBITaxon:243232
label: Methanocaldococcus jannaschii (strain ATCC 43067 / DSM 2661 / JAL-1 / JCM
10045 / NBRC 100440)
description: >-
NadX (MJ0915) is an L-aspartate dehydrogenase (EC 1.4.1.21) that catalyzes the
NAD(P)-dependent oxidative deamination of L-aspartate to iminoaspartate, the first
committed step in the de novo NAD+ biosynthetic pathway from L-aspartate. This enzyme
represents an alternative to the FAD-dependent L-aspartate oxidase (NadB) used in
classical bacterial NAD biosynthesis pathways. NadX is a homodimeric protein with an
N-terminal Rossmann fold NAD(P)-binding domain and a C-terminal catalytic domain. The
iminoaspartate product is channeled to quinolinate synthase (NadA) for quinolinate
biosynthesis. NadX homologs are found in many methanogenic archaea and some bacteria
such as Thermotoga maritima.
existing_annotations:
# ================== NAD+ BIOSYNTHETIC PROCESS ==================
- term:
id: GO:0009435
label: NAD+ biosynthetic process
evidence_type: IEA
original_reference_id: GO_REF:0000120
review:
summary: >-
NadX catalyzes the first step of de novo NAD+ biosynthesis from L-aspartate,
producing iminoaspartate for the NadA quinolinate synthase reaction. This
annotation is correct but could be more specific; GO:0034628 ('de novo' NAD+
biosynthetic process from L-aspartate) precisely describes the pathway route
used by NadX.
action: MODIFY
reason: >-
The more specific term GO:0034628 ('de novo' NAD+ biosynthetic process from
L-aspartate) precisely captures NadX's role in the aspartate-derived de novo
pathway, distinguishing it from salvage pathways and the tryptophan-derived
route.
proposed_replacement_terms:
- id: GO:0034628
label: "'de novo' NAD+ biosynthetic process from L-aspartate"
supported_by:
- reference_id: PMID:12496312
supporting_text: "two different enzymes, an oxidase and a dehydrogenase, may have evolved to catalyze the first step of NAD biosynthesis in prokaryotes"
- reference_id: file:METJA/nadX/nadX-deep-research-bioreason-sft.md
supporting_text: "BioReason SFT trace correctly identifies NAD biosynthesis pathway context"
# ================== OXIDOREDUCTASE ACTIVITY (InterPro2GO) ==================
- term:
id: GO:0016491
label: oxidoreductase activity
evidence_type: IEA
original_reference_id: GO_REF:0000002
review:
summary: >-
This InterPro2GO annotation from IPR005106 (Aspartate/homoserine dehydrogenase,
NAD-binding domain) is correct but too general. NadX has a well-characterized
specific activity: L-aspartate dehydrogenase [NAD(P)+] activity (GO:0033735).
The general oxidoreductase term adds no information beyond what the more specific
term already provides.
action: MODIFY
reason: >-
GO:0016491 (oxidoreductase activity) is far too general; GO:0033735 provides
the precise molecular function. This is a typical InterPro2GO limitation where
domain-level annotation yields only broad functional categories.
proposed_replacement_terms:
- id: GO:0033735
label: L-aspartate dehydrogenase [NAD(P)+] activity
supported_by:
- reference_id: PMID:12496312
supporting_text: "The enzymatic characterization of TM1643 revealed that it possesses NAD or NADP-dependent dehydrogenase activity toward l-aspartate but no aspartate oxidase activity"
# ================== OXIDOREDUCTASE ACTIVITY (CH-NH2 group) ==================
- term:
id: GO:0016639
label: oxidoreductase activity, acting on the CH-NH2 group of donors, NAD or NADP
as acceptor
evidence_type: IEA
original_reference_id: GO_REF:0000104
review:
summary: >-
This annotation correctly captures that NadX acts on the CH-NH2 group of
L-aspartate using NAD or NADP as acceptor. However, the more specific child
term GO:0033735 (L-aspartate dehydrogenase [NAD(P)+] activity) is available
and precisely identifies the substrate specificity.
action: MODIFY
reason: >-
A more specific child term exists that exactly describes this enzyme's
activity and substrate specificity.
proposed_replacement_terms:
- id: GO:0033735
label: L-aspartate dehydrogenase [NAD(P)+] activity
supported_by:
- reference_id: PMID:16731057
supporting_text: "The enzyme specifically utilized L-aspartate as the electron donor, while either NAD or NADP could serve as the electron acceptor"
# ================== L-ASPARTATE DEHYDROGENASE ACTIVITY ==================
- term:
id: GO:0033735
label: L-aspartate dehydrogenase [NAD(P)+] activity
evidence_type: IEA
original_reference_id: GO_REF:0000120
review:
summary: >-
This is the most specific and accurate molecular function term for NadX.
The enzyme catalyzes NAD(P)-dependent oxidative deamination of L-aspartate
to iminoaspartate (which spontaneously decomposes to oxaloacetate + NH4+).
Structural and biochemical studies on homologs from T. maritima and A. fulgidus
have confirmed this activity.
action: ACCEPT
reason: >-
GO:0033735 precisely matches the experimentally characterized enzymatic
activity of NadX. Although the evidence for M. jannaschii NadX specifically
is computational (IEA), the activity has been experimentally demonstrated
for close homologs in T. maritima and A. fulgidus.
supported_by:
- reference_id: PMID:12496312
supporting_text: "The enzymatic characterization of TM1643 revealed that it possesses NAD or NADP-dependent dehydrogenase activity toward l-aspartate but no aspartate oxidase activity"
- reference_id: PMID:16731057
supporting_text: "Characterization revealed the enzyme to be a highly thermostable L-aspartate dehydrogenase"
# ================== NADP BINDING ==================
- term:
id: GO:0050661
label: NADP binding
evidence_type: IEA
original_reference_id: GO_REF:0000120
review:
summary: >-
NadX binds NADP as one of its two cofactor options. The A. fulgidus homolog
has a Km of 0.32 mM for NADP. While NADP binding is implicit in the
GO:0033735 term (which specifies NAD(P)+), this annotation provides explicit
documentation of NADP cofactor binding capability.
action: KEEP_AS_NON_CORE
reason: >-
NADP binding is already implicit in GO:0033735 (L-aspartate dehydrogenase
[NAD(P)+] activity). This annotation is not wrong but is redundant with the
more specific MF term. Keeping as non-core for completeness.
supported_by:
- reference_id: PMID:16731057
supporting_text: "The Km values for NAD and NADP were 0.11 and 0.32 mM, respectively"
# ================== NAD BINDING ==================
- term:
id: GO:0051287
label: NAD binding
evidence_type: IEA
original_reference_id: GO_REF:0000104
review:
summary: >-
NadX binds NAD as its preferred cofactor. The A. fulgidus homolog has a Km
of 0.11 mM for NAD, and crystal structures show NAD bound in the Rossmann fold
domain. As with NADP binding, this is implicit in GO:0033735 but provides
explicit documentation.
action: KEEP_AS_NON_CORE
reason: >-
NAD binding is already implicit in GO:0033735 (L-aspartate dehydrogenase
[NAD(P)+] activity). Keeping as non-core for completeness. Kinetic data from
the A. fulgidus homolog suggest a slight preference for NAD over NADP.
supported_by:
- reference_id: PMID:17651440
supporting_text: "The crystal structure of the highly thermostable L-aspartate dehydrogenase (L-aspDH; EC 1.4.1.21) from the hyperthermophilic archaeon Archaeoglobus fulgidus was determined in the presence of NAD and a substrate analog, citrate"
- reference_id: PMID:16731057
supporting_text: "The Km values for L-aspartate were 0.19 and 4.3 mM when NAD or NADP, respectively, served as the electron acceptor"
# ================== CYTOPLASM (NEW) ==================
- term:
id: GO:0005737
label: cytoplasm
evidence_type: IEA
original_reference_id: GO_REF:0000120
review:
summary: >-
NadX is a soluble enzyme with no predicted transmembrane segments or signal
peptides, consistent with cytoplasmic localization. As a central metabolic
enzyme in NAD biosynthesis, cytoplasmic localization is expected for this
archaeal protein.
action: NEW
reason: >-
Cytoplasmic localization is consistent with the enzyme's soluble nature and
its role in the cytoplasmic de novo NAD biosynthesis pathway. No transmembrane
domains are predicted.
supported_by:
- reference_id: PMID:16731057
supporting_text: "a homodimeric protein with a molecular mass of about 48 kDa"
references:
- id: PMID:12496312
title: "Aspartate dehydrogenase, a novel enzyme identified from structural and functional studies of TM1643"
findings:
- statement: >-
TM1643 from Thermotoga maritima is an NAD/NADP-dependent L-aspartate
dehydrogenase that produces iminoaspartate, representing an alternative
to aspartate oxidase (NadB) for the first step of NAD biosynthesis.
supporting_text: "two different enzymes, an oxidase and a dehydrogenase, may have evolved to catalyze the first step of NAD biosynthesis in prokaryotes. TM1643 establishes a new class of amino acid dehydrogenases"
- statement: >-
The crystal structure reveals an N-terminal Rossmann fold domain with bound
NAD+ and a C-terminal catalytic domain.
supporting_text: "The structure reveals the presence of an N-terminal Rossmann fold domain with a bound NAD(+) cofactor and a C-terminal alpha+beta domain"
- statement: >-
TM1643 has no aspartate oxidase activity, confirming it is a dehydrogenase
and not an oxidase.
supporting_text: "The enzymatic characterization of TM1643 revealed that it possesses NAD or NADP-dependent dehydrogenase activity toward l-aspartate but no aspartate oxidase activity"
- id: PMID:16731057
title: "The first archaeal L-aspartate dehydrogenase from the hyperthermophile Archaeoglobus fulgidus: gene cloning and enzymological characterization."
findings:
- statement: >-
The A. fulgidus L-aspartate dehydrogenase is a homodimeric protein (~48 kDa)
that is highly thermostable with optimum temperature of ~80 degrees C.
supporting_text: "a homodimeric protein with a molecular mass of about 48 kDa...The enzyme specifically utilized L-aspartate as the electron donor, while either NAD or NADP could serve as the electron acceptor"
- statement: >-
Homologs of L-aspartate dehydrogenase are found in many methanogenic archaea
but not in Thermococcales or Sulfolobales.
supporting_text: "Within the archaeal domain, homologues of this enzyme occurred in many Methanogenic species, but not in Thermococcales or Sulfolobales species"
- id: PMID:17651440
title: "Crystal structure of archaeal highly thermostable L-aspartate dehydrogenase/NAD/citrate ternary complex"
findings:
- statement: >-
The A. fulgidus L-aspDH crystal structure at 1.9 A resolution shows a dimeric
enzyme with two domains per subunit separated by a deep active-site cleft.
supporting_text: "each subunit consists of two domains separated by a deep cleft containing an active site"
- statement: >-
Substrate binding induces a large conformational change with movement of two
loops, and thermostability is achieved through inter- and intrasubunit ion pairs.
supporting_text: "A. fulgidus L-aspDH assumes a closed conformation and that a large movement of the two loops takes place during substrate binding"
- id: PMID:8688087
title: "Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii"
findings:
- statement: >-
The complete genome sequence of M. jannaschii, which includes the MJ0915
locus (nadX).
supporting_text: "Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii"
- id: GO_REF:0000002
title: Gene Ontology annotation through association of InterPro records with GO terms
findings: []
- id: GO_REF:0000104
title: Electronic Gene Ontology annotations created by transferring manual GO annotations
between related proteins based on shared sequence features
findings: []
- id: GO_REF:0000120
title: Combined Automated Annotation using Multiple IEA Methods
findings: []
core_functions:
- description: >-
NadX catalyzes the NAD(P)-dependent oxidative deamination of L-aspartate to
iminoaspartate as the first committed step of de novo NAD+ biosynthesis from
L-aspartate. This is an alternative to the FAD-dependent aspartate oxidase
(NadB) found in many bacteria. The iminoaspartate product is channeled to
quinolinate synthase (NadA) for quinolinate biosynthesis.
molecular_function:
id: GO:0033735
label: L-aspartate dehydrogenase [NAD(P)+] activity
directly_involved_in:
- id: GO:0034628
label: "'de novo' NAD+ biosynthetic process from L-aspartate"
locations:
- id: GO:0005737
label: cytoplasm
supported_by:
- reference_id: PMID:12496312
supporting_text: "two different enzymes, an oxidase and a dehydrogenase, may have evolved to catalyze the first step of NAD biosynthesis in prokaryotes"
- reference_id: PMID:16731057
supporting_text: "Characterization revealed the enzyme to be a highly thermostable L-aspartate dehydrogenase"
suggested_questions:
- question: >-
Has L-aspartate dehydrogenase activity been directly demonstrated for the
M. jannaschii NadX protein (Q58325), or is all experimental evidence from
homologs (T. maritima TM1643, A. fulgidus)?
experts:
- "Toshihisa Ohshima"
- "Liang Tong"
- question: >-
Is there experimental evidence for physical interaction between NadX and
NadA (quinolinate synthase) in M. jannaschii to channel the unstable
iminoaspartate intermediate?
suggested_experiments:
- hypothesis: >-
The M. jannaschii NadX (Q58325) has L-aspartate dehydrogenase activity
with similar kinetic parameters to the characterized A. fulgidus homolog.
description: >-
Express and purify recombinant M. jannaschii NadX. Perform kinetic
characterization measuring NAD- and NADP-dependent oxidation of L-aspartate
by monitoring NADH/NADPH production spectrophotometrically at 340 nm.
Determine Km values for L-aspartate, NAD, and NADP.
experiment_type: enzyme kinetics
- hypothesis: >-
NadX physically interacts with NadA to channel the unstable iminoaspartate
intermediate in M. jannaschii.
description: >-
Perform co-purification or co-immunoprecipitation experiments with tagged
NadX and NadA from M. jannaschii. Use cross-linking mass spectrometry to
identify interaction surfaces. Test whether coupled NadX-NadA reactions
show enhanced quinolinate production compared to sequential reactions.
experiment_type: protein-protein interaction