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 |
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Download this section (compressed HTML)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
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