aroA (PP_1770) of Pseudomonas putida KT2440 is a 746-residue bifunctional cytoplasmic enzyme of aromatic amino acid biosynthesis. Its EPSP synthase module (3-phosphoshikimate 1-carboxyvinyltransferase, EC 2.5.1.19; 5-enolpyruvylshikimate-3-phosphate synthase) catalyzes the penultimate step of the shikimate pathway, transferring the enolpyruvyl moiety of phosphoenolpyruvate to the 5-hydroxyl of shikimate-3-phosphate to yield 5-enolpyruvylshikimate-3-phosphate (EPSP) plus inorganic phosphate; EPSP is then converted to chorismate, the branch-point precursor of phenylalanine, tyrosine, tryptophan, folate, ubiquinone, and other aromatic metabolites. In addition to the canonical EPSP synthase domain (Pfam EPSP_synthase; COG0128; TIGR01356 aroA), the protein carries an N-terminal prephenate/arogenate dehydrogenase (TyrA) module (Pfam PDH_N/PDH_C; COG0287) with a NAD(P)-binding Rossmann fold. UniProt annotates a second catalytic activity for this module, prephenate dehydrogenase (prephenate + NAD+ -> 4-hydroxyphenylpyruvate + CO2 + NADH, EC 1.3.1.12), placing it in the tyrosine-biosynthetic conversion of prephenate to 4-hydroxyphenylpyruvate. The protein is thus a fused EPSP-synthase / prephenate-dehydrogenase enzyme contributing to both chorismate formation and downstream L-tyrosine biosynthesis. The shikimate pathway is absent in animals, making EPSP synthase the molecular target of the herbicide glyphosate, a competitive inhibitor at the PEP site.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: Root-level catalytic activity term; uninformative given the specific enzymatic activities annotated below. Reason: GO:0003824 is the top-level molecular-function catalytic term and conveys no specific information. The protein has well-supported specific activities (EPSP synthase, EC 2.5.1.19; prephenate dehydrogenase, EC 1.3.1.12) that should be used instead. |
| GO:0003866 3-phosphoshikimate 1-carboxyvinyltransferase activity | IEA GO_REF:0000120 | ACCEPT | Summary: EPSP synthase activity (EC 2.5.1.19); the canonical, core molecular function of aroA. Reason: Directly supported by sequence/domain evidence: the EPSP synthase Pfam domain (PF00275), HAMAP rule MF_00210, COG0128, NCBIfam TIGR01356 (aroA), conserved PEP and shikimate-3-phosphate binding residues, and mapping to Rhea:21256 / EC 2.5.1.19. This is the defining function of aroA. |
| GO:0004665 prephenate dehydrogenase (NADP+) activity | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: NADP+-dependent prephenate dehydrogenase activity inferred from the fused TyrA domain. The cofactor specificity (NADP+ vs NAD+) is not experimentally established for this protein. Reason: The protein carries a genuine N-terminal prephenate/arogenate dehydrogenase (TyrA) module (Pfam PDH_N/PDH_C; COG0287), so prephenate dehydrogenase activity is a plausible second function. However, UniProt's curated CATALYTIC ACTIVITY block lists only the NAD+ route (EC 1.3.1.12), and the NADP+ specificity here is purely an InterPro electronic inference (IPR003099) with no cofactor evidence. Retain as a non-core, lower-confidence activity rather than a core function. |
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | ACCEPT | Summary: Cytoplasmic localization, consistent with a soluble shikimate-pathway metabolic enzyme. Reason: EPSP synthase is a soluble cytosolic enzyme of central aromatic amino acid biosynthesis; cytoplasmic localization is supported by UniProt-SubCell (SL-0086) and HAMAP rule MF_00210, with no signal/transmembrane features. |
| GO:0006571 L-tyrosine biosynthetic process | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: Tyrosine biosynthesis; supported via the fused prephenate dehydrogenase (TyrA) domain that converts prephenate to 4-hydroxyphenylpyruvate. Reason: The TyrA (prephenate dehydrogenase) module places this protein in the tyrosine-specific branch (prephenate -> 4-hydroxyphenylpyruvate, UniPathway step 1/1 of the NAD+ route). This is a real but secondary process relative to the core EPSP synthase / chorismate-biosynthesis role, hence non-core. |
| GO:0008652 amino acid biosynthetic process | IEA GO_REF:0000104 | MARK AS OVER ANNOTATED | Summary: General amino acid biosynthetic process; correct but non-specific given the more precise aromatic/chorismate terms. Reason: True but high-level. The more specific processes (chorismate biosynthetic process, aromatic amino acid biosynthetic process, L-tyrosine biosynthetic process) capture the role precisely, making this generic parent redundant. |
| GO:0008977 prephenate dehydrogenase (NAD+) activity | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: NAD+-dependent prephenate dehydrogenase activity from the fused TyrA domain; the second catalytic function of this bifunctional protein. Reason: Supported by the prephenate/arogenate dehydrogenase domain (residues ~14-302; Pfam PDH_N/PDH_C; COG0287) and by UniProt's curated CATALYTIC ACTIVITY block citing the NAD+ reaction (Rhea:13869, EC 1.3.1.12) and the L-tyrosine biosynthesis (NAD+ route) pathway. This is the better-supported of the two prephenate dehydrogenase cofactor variants but remains the secondary (non-core) function relative to EPSP synthase. |
| GO:0009073 aromatic amino acid biosynthetic process | IEA GO_REF:0000120 | ACCEPT | Summary: Aromatic amino acid biosynthesis; accurate at the family level for an EPSP-synthase / chorismate-pathway enzyme also feeding tyrosine biosynthesis. Reason: Both functional modules act within aromatic amino acid biosynthesis: EPSP synthase produces the chorismate precursor common to Phe/Tyr/Trp, and the TyrA domain feeds the tyrosine branch. The term is appropriately specific. |
| GO:0009423 chorismate biosynthetic process | IEA GO_REF:0000120 | ACCEPT | Summary: Chorismate biosynthesis; the core biological process of the EPSP synthase activity (penultimate shikimate-pathway step). Reason: EPSP synthase catalyzes step 6/7 of chorismate biosynthesis from D-erythrose-4-phosphate and PEP (UniPathway UPA00053/UER00089). This is the most precise and well-supported biological-process term for the core function. Consistent with KT2440-specific metabolic-engineering evidence that tuning aroA expression contributes to flux through the shikimate pathway toward chorismate-derived products (see aroA-deep-research-falcon.md, citing PMID:41029715). |
| GO:0016491 oxidoreductase activity | IEA GO_REF:0000104 | MARK AS OVER ANNOTATED | Summary: Generic oxidoreductase parent term covering the prephenate dehydrogenase activity. Reason: Redundant high-level parent of the specific prephenate dehydrogenase activities (GO:0008977 / GO:0004665) already annotated. Provides no additional information beyond the specific terms. |
| GO:0016628 oxidoreductase activity, acting on the CH-CH group of donors, NAD or NADP as acceptor | IEA GO_REF:0000117 | MARK AS OVER ANNOTATED | Summary: Intermediate oxidoreductase-class parent term for the prephenate dehydrogenase activity. Reason: A grouping parent of the specific prephenate dehydrogenase (NAD+/NADP+) activities. The leaf terms GO:0008977 / GO:0004665 are retained, so this mid-level class is redundant over-annotation. |
| GO:0016765 transferase activity, transferring alkyl or aryl (other than methyl) groups | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: Generic enolpyruvyl/alkyl transferase parent term for the EPSP synthase activity. Reason: High-level parent of the specific EPSP synthase activity (GO:0003866, enolpyruvyl transferase) which is retained. Redundant given the leaf term. |
| GO:0070403 NAD+ binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: NAD+ cofactor binding by the Rossmann-fold prephenate dehydrogenase (TyrA) domain. Reason: Consistent with the NAD(P)-binding Rossmann fold of the fused TyrA domain (InterPro IPR046826) and with the NAD+-dependent prephenate dehydrogenase activity. A supporting cofactor-binding term for the secondary activity, so non-core rather than a primary function. |
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Download this section (compressed HTML)Q: Is the prephenate dehydrogenase (TyrA) module of P. putida AroA catalytically active in vivo, and does it prefer NAD+ or NADP+ as cofactor?
Q: Does the AroA-TyrA domain fusion form a substrate channel or otherwise functionally couple chorismate biosynthesis with the tyrosine branch in P. putida?
Experiment: Heterologously express and purify Q88M05 and assay both EPSP synthase (S3P + PEP) and prephenate dehydrogenase (prephenate + NAD+/NADP+) activities to confirm bifunctionality and determine cofactor preference.
Experiment: Construct an aroA deletion/complementation in P. putida KT2440 and test for aromatic amino acid (and specifically tyrosine) auxotrophy to establish in vivo requirement of each catalytic module.
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