AroD is the type I 3-dehydroquinate dehydratase (3-dehydroquinase, EC 4.2.1.10) of Escherichia coli, the only 3-dehydroquinase of this organism. It catalyses the third step of the shikimate (chorismate) pathway, the reversible dehydration of 3-dehydroquinate to 3-dehydroshikimate, which introduces the first double bond of the future aromatic ring. Catalysis proceeds by a syn elimination through a covalent Schiff base (imine) formed between the substrate keto group and Lys-170, with His-143 acting as the general base and also assisting Schiff base formation and hydrolysis; the K170A and H143A mutants lose about 10^6-fold activity while still binding ligand. The enzyme is a soluble cytosolic homodimer of 27.5 kDa subunits with a TIM-barrel (class I aldolase) fold, Km for 3-dehydroquinate of about 17-18 uM and kcat of about 140 s-1. Through chorismate it supplies the precursor of the aromatic amino acids, folate, ubiquinone, menaquinone and the enterobactin siderophore.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003855 3-dehydroquinate dehydratase activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBA (GO_REF:0000033) from ancestral node PTN000472499 of the type I 3-dehydroquinase family PTHR43699; E. coli AroD is itself one of the experimentally characterized members seeding the node, and its own activity is shown directly. Reason: The IBD places 3-dehydroquinate dehydratase activity at the base of the type I DHQase clade, which AroD sits inside, and no loss is recorded on its lineage (no IRD/IKR reported by paint-losses). AroD appearing in the WITH/FROM is the expected marker of its own experimental grounding (PMID:2950851, PMID:7592767). This is the defining activity of the protein. Supporting Evidence: file:ECOLI/aroD/aroD-uniprot.txt Catalyzes the cis-dehydration of 3-dehydroquinate (DHQ) and introduces the first double bond of the aromatic ring to yield 3-dehydroshikimate. file:ECOLI/aroD/aroD-uniprot.txt SIMILARITY: Belongs to the type-I 3-dehydroquinase family. |
| GO:0003855 3-dehydroquinate dehydratase activity | IDA PMID:2950851 Purification and characterization of 3-dehydroquinase from E... | ACCEPT | Summary: Chaudhuri et al. (1986) purified E. coli 3-dehydroquinase to homogeneity and determined its catalytic properties (Km about 18 uM for 3-dehydroquinate). Reason: Direct assay of the purified E. coli enzyme; the activity matches the GO definition (3-dehydroquinate = 3-dehydroshikimate + H2O). Core function. Supporting Evidence: PMID:2950851 Homogeneous enzyme with specific activity 163 units/mg of protein was obtained in 19% overall yield. PMID:2950851 The catalytic properties of the enzyme have been determined and shown to be very similar to those of the biosynthetic 3-dehydroquinase component of the arom multifunctional enzyme of Neurospora crassa. |
| GO:0003855 3-dehydroquinate dehydratase activity | IDA PMID:7592767 Mutagenesis of active site residues in type I dehydroquinase... | ACCEPT | Summary: Leech et al. (1995) characterized wild-type and active-site mutant enzymes, showing that Lys-170 (Schiff base) and His-143 (general base) are essential for the conversion of 3-dehydroquinate to 3-dehydroshikimate. Reason: Direct kinetic and mutational characterization of the E. coli enzyme, including trapping of the product 3-dehydroshikimate as a Schiff base on the H143A mutant. Core function. Supporting Evidence: PMID:7592767 When the highly conserved, Schiff base-forming lysine residue was mutated (K170A) the resulting enzyme showed a approximately 10(6)-fold reduction in catalytic activity, but was still able to bind both substrate and product PMID:7592767 the H143A mutant is isolated from overexpressing cells with a significant proportion of the enzyme monomers covalently bound to the product, 3-dehydroshikimate, via a Schiff base linkage. |
| GO:0003855 3-dehydroquinate dehydratase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Combined automated IEA (GO_REF:0000120) from InterPro type I DHQase signatures (IPR001381, active-site IPR018508), the EC 4.2.1.10/RHEA:21096 mapping and the HAMAP MF_00214 UniRule. Reason: Electronic routes agree with the direct experimental evidence; AroD carries the type I DHQase signatures and the active-site lysine motif. Supporting Evidence: file:ECOLI/aroD/aroD-uniprot.txt InterPro; IPR018508; 3-dehydroquinate_DH_AS. file:ECOLI/aroD/aroD-uniprot.txt HAMAP; MF_00214; AroD; 1. |
| GO:0005829 cytosol | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBA placing type I DHQase activity in the cytosol, seeded by the E. coli AroD cytosol IDA annotations. Reason: AroD is a soluble enzyme without signal peptide or transmembrane segment, found in the cytosolic fraction of E. coli; the chorismate pathway is cytosolic in bacteria. The node placement is sound for this bacterial clade. Supporting Evidence: PMID:18304323 we identified 1103 proteins from the cytosolic fraction of the Escherichia coli strain MC4100. |
| GO:0005829 cytosol | IDA PMID:15911532 Localization, annotation, and comparison of the Escherichia ... | ACCEPT | Summary: Lopez-Campistrous et al. (2005) assigned E. coli K-12 proteins to cytosol, periplasm, inner and outer membrane by biochemical fractionation and 2D-gel/MS; the EcoCyc curators annotated AroD to cytosol from this dataset. Reason: The gene-level assignment is in the paper's data tables, which are not cached (abstract only); the fractionation design supports a cytosol call and it agrees with the biology of a soluble biosynthetic enzyme. Deferring to the curator. Supporting Evidence: PMID:15911532 2,160 were annotated and assigned to the cytosol, periplasm, inner membrane, and outer membrane by biochemical fractionation followed by two-dimensional gel electrophoresis and tandem mass spectrometry. |
| GO:0005829 cytosol | IDA PMID:18304323 Protein abundance profiling of the Escherichia coli cytosol. | ACCEPT | Summary: Ishihama et al. (2008) identified 1103 proteins in the cytosolic fraction of E. coli MC4100 by LC-MS/MS; AroD is annotated to cytosol from this dataset. Reason: Cytosolic-fraction proteomics; aroD is not named in the cached main text (the protein list is supplementary), but the assignment is consistent with the soluble enzyme and the other cytosol rows. Supporting Evidence: PMID:18304323 we identified 1103 proteins from the cytosolic fraction of the Escherichia coli strain MC4100. |
| GO:0009423 chorismate biosynthetic process | IEA GO_REF:0000120 | ACCEPT | Summary: Combined automated IEA (GO_REF:0000120) from the HAMAP MF_00214 UniRule and UniPathway UPA00053 (chorismate biosynthesis, step 3/7). Reason: AroD catalyses step 3 of the seven-step shikimate pathway from erythrose 4-phosphate and phosphoenolpyruvate to chorismate and is the only 3-dehydroquinase of E. coli K-12, so it performs a step of this process. This is the correct process term for the enzyme's activity. Supporting Evidence: file:ECOLI/aroD/aroD-uniprot.txt Involved in the third step of the chorismate pathway, which leads to the biosynthesis of aromatic amino acids (AroAA). file:ECOLI/aroD/aroD-notes.md returned only P05194 (aroD), so AroD is the only annotated 3-dehydroquinase of E. coli K-12 |
| GO:0042803 protein homodimerization activity | IDA PMID:2950851 Purification and characterization of 3-dehydroquinase from E... | KEEP AS NON CORE | Summary: Gel permeation chromatography of the purified enzyme gave a native Mr of 52,000-58,000 against a 29,000 subunit, indicating a dimer. Reason: The homodimer is directly supported, but quaternary structure is a structural property of the enzyme rather than its function; the core activity is the dehydratase. Supporting Evidence: PMID:2950851 The native Mr, estimated by gel permeation chromatography on Sephacryl S-200 (superfine) and on TSK G3000SW, was in the range 52,000-58,000, indicating that the enzyme is dimeric. |
| GO:0046279 3,4-dihydroxybenzoate biosynthetic process | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: PAINT IBA from PTN000472499 asserting 3,4-dihydroxybenzoate (protocatechuate) biosynthesis, with E. coli AroD (P05194) and Salmonella Typhimurium AroD (P58687) in the WITH/FROM. Reason: The term means formation of 3,4-dihydroxybenzoate. DHQase makes 3-dehydroshikimate, which becomes protocatechuate only through a separate 3-dehydroshikimate dehydratase (EC 4.2.1.118, AroZ/QuiC/AsbF); E. coli K-12 encodes none, so in this organism AroD's product goes to chorismate, not protocatechuate. The seed annotations measured 3-dehydroquinate dehydration, not 3,4-dihydroxybenzoate formation. The term is defensible only for type I DHQases in organisms with an AroZ route, so propagating it across the clade over-annotates. Kept consistent with the IDA and IEA rows on the same term. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: LINEAGE OR TAXON MISMATCH Sources checked: PANTHER:PTN000472499 SOURCE WEAK OR INFERRED The IBD on GO:0046279 is seeded by experimental rows on E. coli AroD (P05194) and Salmonella Typhimurium AroD (P58687); the E. coli papers measured 3-dehydroquinate dehydration only (the Salmonella source was not inspected), and protocatechuate formation needs a 3-dehydroshikimate dehydratase that E. coli K-12 lacks. Supporting Evidence: file:ECOLI/aroD/aroD-notes.md returned no entries, i.e. no E. coli K-12 protein carries EC 4.2.1.118 file:ECOLI/aroD/aroD-notes.md E. coli K-12 therefore has no known route from 3-dehydroshikimate to protocatechuate |
| GO:0046279 3,4-dihydroxybenzoate biosynthetic process | IDA PMID:13198937 Aromatic biosynthesis. XII. Conversion of 5-dehydroquinic ac... | MARK AS OVER ANNOTATED | Summary: Mitsuhashi and Davis (1954) described the E. coli 5-dehydroquinase converting 5-dehydroquinic acid to 5-dehydroshikimic acid. Only the citation is cached; the title names 5-dehydroshikimate, not 3,4-dihydroxybenzoate, as the product. Reason: The assay is of 3-dehydroquinate dehydration (supporting GO:0003855), not of protocatechuate formation. E. coli K-12 lacks the 3-dehydroshikimate dehydratase that would make 3,4-dihydroxybenzoate, so the process is not one the organism carries out. Not REMOVE because the enzyme does supply the immediate protocatechuate precursor where such a dehydratase is present (for example, in engineered strains), so the claim is not wholly baseless. Supporting Evidence: file:ECOLI/aroD/aroD-notes.md E. coli K-12 therefore has no known route from 3-dehydroshikimate to protocatechuate file:ECOLI/aroD/aroD-uniprot.txt Catalyzes the cis-dehydration of 3-dehydroquinate (DHQ) and introduces the first double bond of the aromatic ring to yield 3-dehydroshikimate. |
| GO:0046279 3,4-dihydroxybenzoate biosynthetic process | IDA PMID:7592767 Mutagenesis of active site residues in type I dehydroquinase... | MARK AS OVER ANNOTATED | Summary: Leech et al. (1995) is an active-site mutagenesis study whose measured product is 3-dehydroshikimate; it does not report 3,4-dihydroxybenzoate. Reason: The paper supports the dehydratase activity (GO:0003855), already annotated from the same reference. Formation of 3,4-dihydroxybenzoate requires a further enzyme that E. coli K-12 does not have, so the process term over-reaches for this gene. Supporting Evidence: PMID:7592767 the H143A mutant is isolated from overexpressing cells with a significant proportion of the enzyme monomers covalently bound to the product, 3-dehydroshikimate, via a Schiff base linkage. file:ECOLI/aroD/aroD-notes.md returned no entries, i.e. no E. coli K-12 protein carries EC 4.2.1.118 |
| GO:0046279 3,4-dihydroxybenzoate biosynthetic process | IEA GO_REF:0000117 | MARK AS OVER ANNOTATED | Summary: ARBA machine-learning rule (GO_REF:0000117) assigning 3,4-dihydroxybenzoate biosynthesis, presumably learned from the existing DHQase annotations to this term. Reason: Same claim as the IDA and IBA rows and the same objection; the ARBA rule repeats the existing annotations rather than adding independent evidence. Kept consistent with those rows. Supporting Evidence: file:ECOLI/aroD/aroD-notes.md E. coli K-12 therefore has no known route from 3-dehydroshikimate to protocatechuate |
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