| Annotation element | Current best-supported statement for *Pseudomonas putida* KT2440 enolase (Q88MF9; **eno**; PP_1612) | Key evidence/citations |
|---|---|---|
| Target identity | The target matches the canonical bacterial **enolase** annotated in UniProt as **EC 4.2.1.11** and encoded in the KT2440 genome context as **eno / PP_1612**; available KT2440 literature discusses enolase as a central-carbon enzyme in this organism, consistent with the UniProt family/domain assignment. | (pqac-00000005, pqac-00000012) |
| Enzyme name / EC | Enolase (2-phospho-D-glycerate hydro-lyase; 2-phosphoglycerate dehydratase), **EC 4.2.1.11**. | (pqac-00000012, pqac-00000013) |
| Reaction | Enolase catalyzes the reversible conversion of **2-phosphoglycerate (2-PG) to phosphoenolpyruvate (PEP)**; this is the standard penultimate glycolytic step. | (pqac-00000013, pqac-00000014) |
| Substrates / products | Best-supported substrate/product pair is **2-phosphoglycerate ⇄ phosphoenolpyruvate**; PEP depletion under fluoride stress in KT2440 is consistent with impaired flux through this reaction. | (pqac-00000012, pqac-00000013) |
| Cofactor dependence | Enolase activity is metal-dependent; fluoride toxicity is discussed as likely involving sequestration/interference with **Mg2+/Mn2+**-dependent catalytic function, consistent with known enolase chemistry. | (pqac-00000012, pqac-00000006) |
| Pathway role | In KT2440, enolase functions in **central carbon metabolism** downstream of upper glycolytic sugar-phosphate pools and upstream of pyruvate-generating steps; this fits the organism’s glucose-processing architecture centered on the **ED/EDEMP network** while retaining the EMP enolase step to generate PEP. | (pqac-00000005, pqac-00000012) |
| Organism-specific metabolic context | KT2440 is a stress-tolerant metabolic chassis whose core metabolism is organized to favor redox generation and flexible carbon processing; enolase sits within this highly engineered/engineerable central metabolic backbone. | (pqac-00000000, pqac-00000005) |
| Cellular localization | No direct KT2440 localization evidence was retrieved here; the best-supported annotation is therefore **cytosolic central-metabolism enzyme**, with no organism-specific evidence in this evidence set for surface exposure in *P. putida* KT2440. | (pqac-00000012, pqac-00000013) |
| Essentiality | No direct experimental essentiality evidence for **PP_1612/eno** in KT2440 was retrieved in the available context; enolase should therefore be described as **likely important for glycolytic flux, but essentiality unresolved in this evidence set**. | (pqac-00000005, pqac-00000012) |
| Regulation / stress link: fluoride inhibition | Fluoride is a strong mechanistic link for KT2440 enolase annotation: authoritative recent synthesis cites **Ki ~80 µM** for fluoride inhibition of enolase and KT2440 metabolomics showing **upper-glycolysis metabolite accumulation with depletion of PEP and downstream/TCA intermediates**, consistent with enolase inhibition. | (pqac-00000013, pqac-00000014) |
| Organism-specific fluoride phenotype | In KT2440, NaF triggers broad stress and central-metabolism remodeling; metabolomics reported **PEP depletion over time** with accumulation of upstream sugar phosphates (e.g., G6P, S7P, F6P, R5P), supporting impaired lower glycolytic throughput at or near enolase. | (pqac-00000012, pqac-00000009) |
| Omics/proteomics evidence | Enolase was detected in KT2440 proteomic work under carbon/phosphorus limitation during mcl-PHA studies, supporting expression of the enzyme under relevant industrial/physiological conditions even though that study was not a dedicated functional dissection of **eno**. | (pqac-00000001) |
| Moonlighting evidence | Enolase has broad **bacterial moonlighting** literature (especially surface plasminogen binding in pathogens), but **no direct evidence was retrieved for moonlighting of KT2440 enolase**; such functions should not be transferred to this strain without organism-specific data. | (pqac-00000013, pqac-00000014) |
| Applications / real-world relevance | Because KT2440 is a major **synthetic biology and metabolic-engineering chassis**, enolase matters as a core node in carbon partitioning, stress physiology, and productivity phenotypes relevant to bioproduction, lignin valorization, and central-metabolism rewiring. | (pqac-00000000, pqac-00000005) |
| PHA / industrial biotechnology relevance | KT2440 is widely used for **mcl-PHA production** and other biotechnological processes; enolase is relevant indirectly as part of the glycolytic/central-carbon supply network that supports growth, redox balance, and polymer/product formation in this chassis. | (pqac-00000001, pqac-00000000) |


*Table: This table condenses the strongest organism-relevant functional annotation points for *Pseudomonas putida* KT2440 enolase (Q88MF9/PP_1612). It highlights verified enzyme identity, reaction chemistry, pathway context, fluoride inhibition evidence, and why the enzyme matters in KT2440 biotechnology.*