| Claim/Aspect | P. putida-specific evidence (with citation id) | Ortholog/Inference evidence (with citation id) | Notes/Implications |
|---|---|---|---|
| Gene IDs / identity | In *P. putida* KT2440, the central-metabolism map labels two pyruvate kinase genes: **PykA (PP_1362)** and **PykF (PP_4301)**; a transcriptomics table also annotates **PP1362** as pyruvate kinase, matching UniProt Q88N54 / **pykA** (pqac-00000004, pqac-00000002) | — | Confirms the requested target is the **PP_1362 / pykA** gene product, distinct from the second isozyme **pykF / PP_4301**. |
| Pathway position | The KT2440 pathway map places PykA/PykF at the **phosphoenolpyruvate → pyruvate** step in lower central carbon metabolism, feeding pyruvate toward acetyl-CoA/TCA metabolism (pqac-00000004, pqac-00000014) | In *Pseudomonas* pyruvate kinase studies, PykA/PykF are described as the enzymes catalyzing the terminal glycolytic/ED-linked pyruvate kinase step (pqac-00000008) | Supports annotation of PykA as a cytosolic central-carbon enzyme connecting EDEMP/ED metabolism to pyruvate supply. |
| Catalyzed reaction / EC | Direct reaction wording was not recovered from the KT2440-specific texts examined; however PP_1362 is explicitly annotated as pyruvate kinase in pathway/expression resources (pqac-00000002, pqac-00000004) | Pyruvate kinase is **ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40**, catalyzing **phosphoenolpyruvate + ADP ↔ pyruvate + ATP** (pqac-00000008) | Reaction/EC assignment is strong at the family level and consistent with the UniProt entry, but direct KT2440 biochemical validation was not located in the retrieved corpus. |
| Allosteric regulation | No KT2440-specific allosteric effector data for PP_1362 were located in the retrieved sources (pqac-00000004) | In *P. aeruginosa* PykA, activity is strongly activated by **glucose-6-phosphate (G6P)** and also by **F6P, G3P, and reductive PPP intermediates**; G6P increases apparent catalytic efficiency about **3-fold** (pqac-00000003, pqac-00000005, pqac-00000006, pqac-00000008) | Suggests likely metabolite-level control of carbon flux at the PEP→pyruvate node in pseudomonads, but this remains inference for KT2440 unless directly tested. |
| Cofactors / ions | No KT2440-specific cofactor measurements were found in the retrieved texts (pqac-00000004) | Closely related PykA contains an **active-site Mg2+** and pyruvate kinases generally require divalent cations; *P. aeruginosa* PykA was reported as **K+-independent**, with added monovalent cations decreasing activity (pqac-00000006, pqac-00000007) | For KT2440 PykA, **Mg2+ dependence** is plausible by homology; **K+ independence** is a reasonable but unverified inference. |
| Oligomeric state | No KT2440-specific oligomerization data were recovered (pqac-00000004) | *P. aeruginosa* PykA is a **tetramer** in solution/structure (about **200 kDa**) (pqac-00000003, pqac-00000006) | Tetrameric organization is typical for bacterial pyruvate kinases and likely applies to KT2440 PykA, but direct demonstration is lacking here. |
| Kinetics / substrate behavior | No KT2440-specific kinetic constants were found in the retrieved literature set (pqac-00000004) | Orthologous PykA showed **KM(ADP) = 0.07 mM**, **S0.5(PEP) = 0.67 mM**, **Hill coefficient 2.14**, and regulator-dependent conversion from sigmoidal to hyperbolic PEP behavior (pqac-00000006, pqac-00000007) | Indicates cooperative control at the PEP branchpoint is plausible for pseudomonad PykA enzymes. |
| Physiological / pathway context in *P. putida* | KT2440 central metabolism emphasizes the **EDEMP/ED** architecture rather than a classical complete EMP pathway; pyruvate kinase occupies a key lower-pathway step in this context (pqac-00000004, pqac-00000014) | In pseudomonads relying heavily on ED-linked metabolism, pyruvate kinase is described as a major lower-pathway pacemaker/regulatory point (pqac-00000005, pqac-00000008) | This explains why pyruvate kinase is attractive for flux redirection in KT2440 engineering. |
| Engineering application: ΔpykF locus used for insertions | In muconate engineering, overexpression cassettes (**gpmI, maeB, rpiA, aroK, aroB**) were inserted at the **ΔpykF locus**; the locus diagram shows the **PP_4300–PP_4302 / ΔpykF** region used as a genomic landing pad (pqac-00000010, pqac-00000013, pqac-00000015) | — | Demonstrates direct practical use of a pyruvate-kinase locus in KT2440 strain construction, even when pyruvate kinase was not itself the final performance bottleneck. |
| Engineering application: conserve PEP for shikimate / muconate | In a model-guided aromatics strategy, knockout sets in KT2440 included **pykA, pykF, and ppc** to conserve **PEP** for shikimate-pathway product formation; however expected yield gains could be offset by alternative flux through the **EDEMP cycle** (pqac-00000016) | — | Important expert insight: pyruvate kinase deletions can be rational, but network plasticity may blunt the benefit unless companion bottlenecks are addressed. |
| Quantitative production outcomes linked to pyruvate-kinase engineering context | The 2022 muconate study achieved **33.7 g L−1** muconate at **0.18 g L−1 h−1** and **46% molar yield (92% of maximum theoretical yield)** in a rationally engineered KT2440 strain; overexpression constructs were installed at **ΔpykF** (pqac-00000011, pqac-00000015) | A related aromatics engineering analysis reported baseline yield values such as **6.4% ± 0.18% (mol/mol)** for one target and discussed pyruvate-kinase deletion logic in cMCS-guided designs (pqac-00000016) | Shows that pyruvate-kinase loci and PEP-partitioning logic are relevant to real KT2440 bioproduction, especially for shikimate-derived products. |


*Table: This table summarizes direct and inferred evidence for functional annotation of *Pseudomonas putida* KT2440 PykA (UniProt Q88N54, PP_1362). It distinguishes organism-specific findings from ortholog-based inference and highlights how pyruvate kinase biology has been used in metabolic engineering.*