pykF E.Coli

What is pykF and what does it encode in E. coli?

pykF is a gene in Escherichia coli that encodes Pyruvate Kinase I (PykF), one of two pyruvate kinases present in E. coli K-12. This enzyme catalyzes the formation of pyruvate in the final step of glycolysis, a reaction that is irreversible under physiological conditions. The reaction is critical for controlling metabolic flux in the second part of glycolysis . PykF represents approximately 80% of the total pyruvate kinase activity within the cell during growth, making it the dominant isozyme compared to PykA (Pyruvate Kinase II) .

What is the amino acid sequence of the PykF protein?

The PykF protein in E. coli K-12 is a full-length protein of 470 amino acids. The complete sequence is:

MGSSHHHHHHSSGLVPRGSHM GSHM KKTKIVCTIGP KTESEEMLAK MLDAGMNVMR LNFSHGDYAE HGQRIQNLRN VMSKTGKTAA ILLDTKGPEI RTMKLEGGND VSLKAGQTFT FTTDKSVIGN SEMVAVTYEG FTTDLSVGNT VLVDDGLIGM EVTAIEGNKV ICKVLNNGDL GENKGVNLPG VSIALPALA EKDKQDLIFG CEQGVDFVAA SFIRKRSDVI EIREHGGENIHIISIKIENQ EGLNNFDEIL EASDGIMVAR GDLGVEIPVE EVIFAQKMMI EKCIRARKVV ITATQMLDSM IKNPRPTRAE AGDVANAIL DGTDAVMLSG ESAKGKYPLE AVSIMATICE RTDRVMNSRL EFNNDNRKLR ITEAVCRGAV ETAEKLDAPL IVVATQGGKS ARAVRKYFPD ATILALTTNE KTAHQLVLSK GVVPQLVKEI TSTDDFYRLG KELALQSGLA HKGDVVVMVS GALVPSGTT NTASVHVL

How does PykF interact with other metabolic enzymes?

PykF interacts with other glycolytic enzymes such as triosephosphate isomerase and phosphoglycerate kinase, ensuring efficient substrate flow and energy generation . These protein-protein interactions likely form metabolic complexes or "metabolons" that enhance reaction rates through substrate channeling, which allows for more efficient transfer of metabolic intermediates between enzymes in the pathway.

What happens to E. coli metabolism when pykF is knocked out?

When pykF is deleted in E. coli, significant metabolic rearrangements occur:

• Flux through phosphoenolpyruvate carboxylase and malic enzyme is up-regulated

• Acetate formation is significantly reduced

• Flux through the phosphofructose kinase pathway is reduced

• Flux through the oxidative pentose phosphate (PP) pathway increases

These changes are accompanied by corresponding alterations in enzyme activities and increases in the concentrations of key metabolites including phosphoenolpyruvate, glucose-6-phosphate, and 6-phosphogluconate .

How does a pykF knockout affect growth in different culture conditions?

The effects of pykF deletion on E. coli growth vary depending on culture conditions:

• In batch culture with glucose as a carbon source, growth may be moderately affected

• In continuous culture, enzyme activities of the oxidative PP and Entner-Doudoroff pathways increase as the dilution rate increases for the pykF mutant

• When using ribose as a non-phosphotransferase system-transporting carbon source, growth is significantly impaired, especially when pykA is also disrupted

For a comprehensive understanding of these metabolic changes, it is important to integrate information on intracellular metabolic flux distribution, enzyme activities, and intracellular metabolite concentrations .

What techniques are most effective for analyzing metabolic flux changes in pykF knockout strains?

Metabolic flux analysis based on 13C-labeling experiments provides the most comprehensive view of metabolic changes in pykF mutants. This typically involves:

• Culturing cells with 13C-labeled carbon sources

• Measuring intracellular isotope distribution using complementary techniques:

  • Two-dimensional nuclear magnetic resonance (2D NMR)

  • Gas chromatography-mass spectrometry (GC-MS)

• Measuring activities of related enzymes (16 enzymes were measured in one study)

• Monitoring concentrations of key intracellular metabolites

This integrated approach allows researchers to quantitatively map how carbon flux is redistributed when pykF is deleted, providing insights into the metabolic adaptations that occur in response to this genetic change.

What methods can be used to conditionally silence the pykF gene?

A promising technique for conditional silencing of pykF involves anti-terminated convergent transcription:

• λPL is convergently integrated into the chromosome downstream of pykF, face-to-face with its native promoter

• In the presence of λ cIts857, efficient temperature-sensitive silencing of pykF can be achieved

• The 5′-terminus of the PL-originated antisense RNA (asRNA), consisting of the rrnG-AT sequence, converts elongation complexes of RNA polymerase to a form resistant to Rho-dependent transcription termination

This approach leads to silencing through transcriptional interference due to collisions between converging RNA polymerases, although cis-antisense RNA effects may also play a role .

How does acetylation affect PykF function?

Acetylation is an important post-translational modification that affects PykF function:

• Multiple lysine residues in PykF can be acetylated, including K13, K19, K52, K59, K68, K145, K317, K319, K340, K368, and K382

• The deacetylation of different sites has varying effects on acetylation level:

  • Deacetylation of K52, K68, K317, and K382 significantly decreases acetylation level

  • Deacetylation of other sites shows no significant difference in acetylation level

• PykF activity generally decreases with increased acetylation level

• Acetylation occurs in a time- and acetyl phosphate (AcP) dose-dependent manner

What deacetylation mechanisms regulate PykF activity?

The deacetylase CobB can deacetylate PykF, representing an important regulatory mechanism. The site-specificity of acetylation and deacetylation depends on:

• Surface accessibility of the target lysine residue

• Reactivity of the lysine residue

• Three-dimensional microenvironment surrounding the target lysine

These factors determine which lysine residues are most likely to be acetylated or deacetylated under specific conditions, providing a mechanism for fine-tuning PykF activity in response to metabolic changes .

What are the functional differences between pykF and pykA in E. coli?

The two pyruvate kinase genes in E. coli show important functional differences:

How do pykF and pykA mutations affect virulence and fitness during infection?

The effects of pyruvate kinase mutations on E. coli virulence reveal complex interactions:

• Loss of pykF results in a defect in bladder and kidney colonization during urinary tract infection

• Loss of pykA actually provides a fitness advantage during infection

• Interestingly, a pykA pykF double mutant performs similarly to wild-type

• This suggests that the presence of Pyk II (encoded by pykA) rather than the absence of Pyk I itself is responsible for the fitness defect in pykF mutants

• These findings indicate that E. coli suppresses latent enzymes (like Pyk II) to survive in the host urinary tract

How can pykF mutations be leveraged for metabolic engineering?

The metabolic changes induced by pykF mutations can be exploited for various biotechnological applications:

• Reduced acetate formation in pykF mutants can improve the production of recombinant proteins sensitive to acidic conditions

• Increased flux through the pentose phosphate pathway can enhance the supply of NADPH for biosynthetic reactions

• Elevated levels of phosphoenolpyruvate (PEP) can be advantageous for PEP-dependent biosynthetic pathways

• The conditional silencing of pykF through anti-terminated convergent transcription provides a tunable system for metabolic engineering applications

These properties make pykF an attractive target for rational strain design in metabolic engineering projects.

What insights do pykF studies provide about metabolic flexibility in bacteria?

Research on pykF contributes to our understanding of bacterial metabolic flexibility:

• The presence of dual pyruvate kinase isozymes (pykF and pykA) provides E. coli with metabolic redundancy and flexibility

• The observation that pykF suppresses potentially detrimental effects of pykA suggests complex regulatory relationships between isozymes

• The metabolic rearrangements that occur in pykF mutants demonstrate the remarkable adaptability of bacterial central metabolism

• The different fitness effects of pykF and pykA mutations during infection highlight how metabolic flexibility contributes to pathogenesis

These insights have broader implications for understanding bacterial adaptation to diverse environments.

What are the most accurate methods for measuring PykF activity?

Several complementary approaches can be used to measure PykF activity:

• Enzymatic assays: Coupling the production of pyruvate to lactate dehydrogenase activity, which can be monitored spectrophotometrically by measuring NADH oxidation

• Western blotting: To quantify PykF protein levels using specific antibodies

• RT-PCR: For measuring pykF mRNA levels, which can indicate expression levels

• In vivo flux analysis: Using 13C-labeled substrates to trace carbon flow through the pyruvate kinase reaction in living cells

For the most comprehensive analysis, researchers should combine these approaches to correlate enzyme activity with protein and mRNA levels.

How can researchers distinguish between the activities of PykF and PykA?

Distinguishing between the activities of the two pyruvate kinase isozymes requires specialized approaches:

• Genetic approach: Using single knockouts (pykF or pykA) to measure the contribution of each isozyme

• Biochemical approach: Exploiting differences in allosteric regulation, substrate affinity, or optimal reaction conditions

• Expression analysis: Measuring the expression levels of each isozyme under different growth conditions

• Protein purification: Isolating each isozyme for in vitro characterization

These approaches can provide insights into the relative contributions and specific roles of PykF and PykA in different metabolic contexts.