AroK is the shikimate kinase (EC 2.7.1.71) of Pseudomonas putida KT2440. It catalyzes the fifth step of the shikimate pathway, the ATP-dependent phosphorylation of the 3-hydroxyl group of shikimate to form shikimate 3-phosphate (3-phosphoshikimate), with release of ADP and a proton. The enzyme requires a Mg(2+) cofactor (one ion bound per subunit) and adopts the P-loop (Walker A) NTPase fold characteristic of the shikimate kinase family, with a glycine-rich nucleotide-binding loop near the N-terminus. As a cytoplasmic monomer, AroK provides shikimate 3-phosphate as the substrate for the downstream EPSP synthase (AroA) and ultimately chorismate, the branch-point precursor for aromatic amino acids (Phe, Tyr, Trp), folate, ubiquinone, and other aromatic metabolites. In bacteria, shikimate kinase activity can be encoded by two isoenzymes, AroK (shikimate kinase I) and AroL (shikimate kinase II); this step is a recognized flux-control point in shikimate-pathway metabolic engineering.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000287 magnesium ion binding | IEA GO_REF:0000104 | ACCEPT | Summary: Shikimate kinase requires a divalent Mg(2+) cofactor for catalysis; UniProt records binding of one Mg(2+) ion per subunit, coordinated near the ATP-binding P-loop. This is a well-supported, family-conserved molecular function. Reason: Magnesium binding is intrinsic to shikimate kinase catalysis and is supported by the HAMAP rule and conserved metal-binding residue; consistent with the enzymology of the shikimate kinase family. |
| GO:0004765 shikimate kinase activity | IEA GO_REF:0000120 | ACCEPT | Summary: This is the core molecular function of AroK, supported by the EC 2.7.1.71 assignment, the RHEA:13121 catalytic-activity mapping, HAMAP rule MF_00109, and strong family/domain evidence (Pfam SKI, shikimate kinase signature). Reason: Directly represents the defining enzymatic activity of the gene product and is the central core function. |
| GO:0005524 ATP binding | IEA GO_REF:0000104 | ACCEPT | Summary: AroK is an ATP-dependent kinase that uses ATP as the phosphoryl donor; it contains a P-loop/Walker A motif (residues 11-16) and additional ATP-contacting residues. ATP binding is a required, well-supported molecular function. Reason: ATP binding is an obligatory part of the shikimate kinase reaction and is supported by the conserved P-loop nucleotide-binding fold. |
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | ACCEPT | Summary: AroK is a soluble cytoplasmic enzyme acting in central metabolism, consistent with UniProt subcellular location and the general biology of bacterial shikimate-pathway enzymes. Reason: Correct localization for a cytoplasmic biosynthetic enzyme; consistent with the cytosol annotation below. |
| GO:0005829 cytosol | IEA GO_REF:0000118 | ACCEPT | Summary: TreeGrafter assigns the cytosol component, the more specific child of cytoplasm, consistent with a soluble bacterial shikimate kinase. Reason: Accurate and slightly more specific localization than GO:0005737; both are appropriate for this enzyme. |
| GO:0009423 chorismate biosynthetic process | IEA GO_REF:0000120 | ACCEPT | Summary: AroK catalyzes step 5 of 7 in chorismate biosynthesis (the shikimate pathway), converting shikimate to shikimate 3-phosphate en route to chorismate. This is the precise biological process for the gene. Reason: Directly and specifically captures the pathway role of AroK; UniPathway UPA00053/UER00088 corroborates the chorismate-biosynthesis placement. |
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Download this section (compressed HTML)Q: Does P. putida KT2440 encode a second shikimate kinase isoenzyme (AroL/shikimate kinase II), and what is the relative contribution of AroK versus any paralog to in vivo shikimate kinase flux?
Experiment: Determine steady-state kinetic parameters (Km/kcat for shikimate and ATP, Mg(2+) dependence) of recombinant KT2440 AroK to confirm substrate specificity and rule out promiscuous quinate kinase activity.
Experiment: Assess essentiality/fitness of an aroK deletion in KT2440 on minimal medium with and without aromatic amino acid supplementation to test for aromatic auxotrophy.
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