Recombinant Gloeobacter violaceus Phosphoglycerate kinase (pgk)

How does heterologous expression of Gloeobacter violaceus PGK typically work?

Heterologous expression of G. violaceus PGK typically utilizes E. coli as the host organism, following standard recombinant DNA technology protocols. The process begins with PCR amplification of the PGK gene from G. violaceus genomic DNA using specific primers that incorporate appropriate restriction sites. The amplified gene is then ligated into an expression vector, commonly one with a His-tag system for subsequent purification.

Based on protocols used for similar G. violaceus proteins, transformation into an E. coli expression strain (such as BL21(DE3)pLysS) would be performed, followed by induction with IPTG (isopropyl β-d-thiogalactopyranoside) at optimal temperatures between 18-37°C . Expression conditions must be carefully optimized, as G. violaceus proteins may require lower induction temperatures (around 18°C) to ensure proper folding and solubility . After cell lysis, the recombinant protein can be purified using affinity chromatography, typically with immobilized metal affinity chromatography (IMAC) if a His-tag was employed .

What storage conditions are recommended for recombinant G. violaceus PGK?

Recombinant G. violaceus PGK should be stored at -20°C to -80°C upon receipt. For multiple uses, aliquoting the protein is crucial to avoid repeated freeze-thaw cycles that could compromise enzyme activity. Storage buffer composition can significantly impact stability; a typical buffer might contain 50 mM HEPES-NaOH (pH 7.5), 150 mM NaCl, and 7.5 mM 2-mercaptoethanol or other reducing agents to protect thiol groups . Addition of glycerol (10-20%) can help prevent freeze damage during storage. For long-term storage, -80°C is preferred, while working aliquots may be kept at -20°C. Enzymatic activity should be periodically verified, especially after prolonged storage periods.

What spectrophotometric assays are suitable for measuring G. violaceus PGK activity?

G. violaceus PGK activity can be measured using coupled enzyme assay systems similar to those employed for other recombinant proteins from this organism. A suitable approach would parallel the spectrophotometric estimation method used for G. violaceus RuBisCO, which measured carboxylation activity in nMol of phosphoglycerate min⁻¹ mg⁻¹ of protein .

For PGK specifically, a coupled assay system typically includes:

• Forward reaction measurement:

  • Coupling with glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

  • Monitoring NADH oxidation at 340 nm

  • Reaction mixture containing 3-phosphoglycerate, ATP, NADH, and GAPDH

• Reverse reaction measurement:

  • Using 1,3-bisphosphoglycerate and ADP as substrates

  • Monitoring ATP formation via coupling with hexokinase and glucose-6-phosphate dehydrogenase

  • Following NADP⁺ reduction at 340 nm

Reaction conditions should be optimized for temperature (likely 25-30°C given G. violaceus's growth preferences) and pH (7.0-7.5 range). The slowness of G. violaceus metabolism suggests its enzymes might have lower specific activities compared to those from faster-growing organisms .

How can protein-protein interactions of G. violaceus PGK be studied in vitro?

In vitro investigation of G. violaceus PGK protein-protein interactions can employ several complementary techniques:

• Pull-down assays: Using purified His-tagged recombinant PGK as bait, followed by co-immunoprecipitation and mass spectrometry to identify binding partners from G. violaceus cell lysate.

• Surface Plasmon Resonance (SPR): For quantitative binding kinetics measurement between PGK and potential interaction partners, especially those involved in metabolic channeling within glycolysis.

• Isothermal Titration Calorimetry (ITC): To determine thermodynamic parameters of binding interactions.

• In vitro reconstitution experiments: Similar to the complementation approach used with G. violaceus RuBisCO, where RbcL with RbcS in presence of RbcX facilitated partial reconstitution and enhanced specific activity . This approach would involve mixing purified PGK with potential interaction partners and measuring resulting changes in enzymatic activity.

• Crosslinking studies: Using chemical crosslinkers followed by proteomic analysis to capture transient interactions.

When interpreting results, researchers should consider G. violaceus's unique cellular architecture (lacking thylakoid membranes), which may result in different spatial organization of metabolic enzymes compared to other cyanobacteria .

How does G. violaceus PGK activity correlate with the organism's slow growth rate?

The relationship between G. violaceus PGK activity and the organism's characteristically slow growth rate represents an intriguing research question. Evidence from G. violaceus RuBisCO studies indicates a carboxylation activity of only 5 nMol of phosphoglycerate min⁻¹ mg⁻¹ of protein, which researchers noted was "in coherence with the organism's slow growth rate" .

A comprehensive investigation of this correlation would require:

• Comparative kinetic analysis: Measuring and comparing the kinetic parameters (Km, kcat, kcat/Km) of recombinant G. violaceus PGK with those from faster-growing cyanobacteria.

• Metabolic flux analysis: Tracing carbon flow through the glycolytic pathway using isotope-labeled substrates to identify potential rate-limiting steps.

• Growth rate manipulation experiments: Examining changes in PGK expression and activity under conditions that alter growth rates.

• Heterologous complementation: Expressing G. violaceus PGK in PGK-deficient strains of faster-growing cyanobacteria to assess growth impacts.

What structural features distinguish G. violaceus PGK from homologs in other cyanobacteria?

Structural analysis of G. violaceus PGK compared to homologs in other cyanobacteria would likely reveal adaptations related to its unique evolutionary position and cellular architecture. While specific structural data for G. violaceus PGK is limited in the provided search results, a comprehensive structural investigation would include:

• Sequence analysis: Multiple sequence alignment comparing G. violaceus PGK with homologs from diverse cyanobacteria to identify conserved domains and unique residues.

• Homology modeling: Construction of a 3D structural model based on crystallographic data from related PGK enzymes.

• Domain organization analysis: Examination of the two-domain structure typical of PGK (N-terminal 3-PG binding domain and C-terminal nucleotide binding domain) for G. violaceus-specific features.

• Active site comparison: Identification of potential differences in catalytic residues or substrate binding pockets that might explain kinetic properties.

• Molecular dynamics simulations: Investigation of domain movement and hinge bending that are critical for PGK catalytic function.

G. violaceus's status as an early-diverging cyanobacterium suggests its PGK might retain more ancestral features compared to homologs from more recently evolved cyanobacteria, potentially offering insights into the evolution of this essential glycolytic enzyme.

How does the absence of thylakoid membranes in G. violaceus affect the organization and function of metabolic enzymes including PGK?

G. violaceus is unique among cyanobacteria in lacking thylakoid membranes , which has profound implications for its cellular organization and metabolic function. This architectural distinction likely impacts PGK function through several mechanisms:

• Spatial organization: Without thylakoid membranes, photosynthetic and respiratory components reside in the cytoplasmic membrane, potentially altering the spatial relationship between energy generation and glycolytic enzymes including PGK.

• Metabolic channeling: The altered subcellular organization may affect substrate channeling between PGK and other glycolytic enzymes or between glycolysis and photosynthetic metabolism.

• Regulatory mechanisms: The absence of thylakoid membranes might necessitate unique regulatory mechanisms for coordinating glycolytic activity with photosynthesis.

• Redox environment: The different arrangement of electron transport components could create a distinct redox environment affecting PGK activity.

Research approaches to investigate these effects would include:

• Immunolocalization studies to determine PGK distribution within G. violaceus cells

• Comparative proteomic analysis of membrane-associated versus soluble fractions

• Investigation of potential protein-protein interactions between PGK and photosynthetic components

• Examination of PGK activity under varying light conditions that affect photosynthetic electron transport

Understanding these relationships could provide insights into how primitive cyanobacteria integrated glycolysis with emerging photosynthetic mechanisms .

What strategies can enhance the expression and purification yield of recombinant G. violaceus PGK?

Optimizing expression and purification of recombinant G. violaceus PGK requires addressing several challenges specific to this primitive cyanobacterial enzyme. Based on protocols used for other G. violaceus proteins, the following strategies can enhance yields:

• Codon optimization: Adapting the G. violaceus PGK gene sequence to the codon usage bias of E. coli can significantly improve expression levels.

• Expression vector selection: Testing different vectors with varying promoter strengths and fusion tag options (His₆, GST, MBP) to identify optimal combination for soluble expression.

• Expression conditions optimization table:

• Co-expression with chaperones: Including molecular chaperones (GroEL/ES, DnaK/DnaJ/GrpE) to assist proper folding.

• Purification optimization: Using a combination of immobilized metal affinity chromatography and size exclusion chromatography, with buffers containing stabilizing agents (glycerol, reducing agents) appropriate for a glycolytic enzyme .

• On-column refolding: For protein recovered from inclusion bodies, implementing gradual removal of denaturants during affinity chromatography.

These approaches should be systematically tested and monitored via SDS-PAGE and activity assays to determine optimal conditions for functional G. violaceus PGK production.

What novel approaches could be employed to study G. violaceus PGK in its native cellular context?

Investigating G. violaceus PGK within its native cellular environment presents unique challenges due to the organism's slow growth rate and distinct cellular architecture. Innovative methodological approaches include:

• CRISPR-Cas genome editing: Development of CRISPR-based tools optimized for G. violaceus to create reporter fusions or conditional knockdowns of the PGK gene.

• Single-cell metabolomics: Application of mass spectrometry techniques to analyze metabolite profiles in individual G. violaceus cells, correlating glycolytic intermediate levels with PGK activity.

• In situ cryo-electron tomography: Visualization of the spatial organization of glycolytic enzymes within the unique thylakoid-less cellular architecture of G. violaceus.

• Activity-based protein profiling: Using activity-based probes specific for PGK to monitor enzyme activity directly within living G. violaceus cells.

• Metabolic sensors: Development of FRET-based biosensors to monitor PGK substrates or products in real-time within living cells.

• Comparative systems biology approach: Integration of transcriptomic, proteomic, and metabolomic data across different growth conditions to model PGK's role in G. violaceus metabolism.

• Native protein complex isolation: Gentle cell disruption methods followed by blue native PAGE or sucrose gradient ultracentrifugation to isolate and identify native complexes containing PGK.

These approaches would provide unprecedented insights into how this glycolytic enzyme functions within the context of one of the most primitive cyanobacteria, potentially revealing fundamental principles about the evolution of central carbon metabolism in photosynthetic organisms .

How does the kinetic efficiency of G. violaceus PGK compare to homologs from other cyanobacteria and photosynthetic organisms?

A comprehensive kinetic comparison between G. violaceus PGK and homologs from other photosynthetic organisms would reveal important evolutionary adaptations. While specific kinetic data for G. violaceus PGK is limited in the provided search results, a comparative analysis should include:

• Enzyme kinetic parameters comparison table:

• Substrate specificity analysis: Comparing the ability of G. violaceus PGK to utilize different phosphorylated substrates against enzymes from evolutionary diverse organisms.

• Temperature and pH profiles: Examining optima and stability ranges across different source organisms.

• Allosteric regulation: Investigating differences in response to regulatory metabolites.

G. violaceus's position as an early-diverging cyanobacterium suggests its PGK might display kinetic properties resembling a more ancestral form of the enzyme. Evidence from G. violaceus RuBisCO showing lower carboxylation activity consistent with slow growth suggests its PGK might similarly exhibit lower catalytic efficiency compared to homologs from more recently evolved photosynthetic organisms.

What evolutionary insights can be gained from studying G. violaceus PGK?

G. violaceus occupies a unique evolutionary position as one of the earliest-diverging cyanobacteria, lacking thylakoid membranes and possessing other primitive characteristics . Study of its PGK offers valuable evolutionary insights:

• Ancestral traits identification: Comparison of G. violaceus PGK sequence, structure and function with homologs from diverse organisms can reveal conserved ancestral features predating the evolution of thylakoid membranes.

• Evolutionary rate analysis: Examination of substitution rates in different domains of PGK across cyanobacterial lineages can identify regions under different selective pressures.

• Domain architecture evolution: Investigation of how the two-domain structure characteristic of PGK may have evolved from the early cyanobacterial ancestor represented by G. violaceus.

• Horizontal gene transfer assessment: Analysis of G. violaceus PGK sequence for signatures of potential horizontal gene transfer events that might have influenced early cyanobacterial metabolism.

• Co-evolution with other metabolic enzymes: Study of evolutionary rate correlations between PGK and other glycolytic enzymes in the G. violaceus lineage.

• Adaptation to ecological niche: Correlation of unique PGK features with G. violaceus's specialized habitat and growth characteristics.

This research could provide insights into the early evolution of glycolysis in photosynthetic organisms and help reconstruct the metabolic capabilities of the last common ancestor of cyanobacteria, offering a window into ancient metabolic systems predating the evolution of modern photosynthetic apparatus .

How is G. violaceus PGK activity coordinated with the unique photosynthetic apparatus of this organism?

G. violaceus possesses a unique photosynthetic apparatus that lacks thylakoid membranes, with photosynthetic components instead embedded in the cytoplasmic membrane . This distinctive arrangement raises important questions about coordination between glycolysis (involving PGK) and photosynthesis:

• Metabolic channeling mechanisms: Investigation of potential direct physical associations between PGK and components of the photosynthetic electron transport chain located in the cytoplasmic membrane.

• Redox regulation: Analysis of how the redox state generated by photosynthetic electron transport might regulate PGK activity through post-translational modifications or allosteric effects.

• Transcriptional co-regulation: Examination of whether PGK gene expression correlates with expression patterns of photosynthetic genes under varying light conditions, similar to the differential expression observed with the psbA gene family in G. violaceus .

• Energy partitioning: Study of how ATP generated through PGK activity in glycolysis is balanced with ATP production from photophosphorylation in the primitive photosynthetic apparatus.

• Carbon flux coordination: Investigation of the relationship between carbon fixation via the Calvin-Benson cycle (which produces glycolytic intermediates) and carbon flow through the PGK-catalyzed step of glycolysis.

This research would provide insights into how primitive cyanobacteria integrated glycolysis with early photosynthetic mechanisms, potentially revealing fundamental principles about the evolution of metabolic coordination in photosynthetic organisms .

What role might G. violaceus PGK play in stress response mechanisms?

G. violaceus demonstrates specific gene expression changes in response to environmental stresses, as evidenced by the differential regulation of its psbA gene family under high irradiance and UVB stress conditions . The potential role of PGK in stress response mechanisms represents an important research question:

• Light stress response: Investigation of whether PGK expression or activity changes under photoinhibitory high irradiance conditions, potentially coordinating with the observed strong induction of psbAIII under such conditions .

• UVB stress adaptation: Analysis of PGK involvement in metabolic adjustments during UVB exposure, which causes limited recovery in G. violaceus .

• Osmotic stress response: Examination of potential connections between PGK activity and osmolyte metabolism in G. violaceus, similar to how other cyanobacteria adjust their metabolism under osmotic stress.

• Oxidative damage protection: Investigation of whether PGK might play a role in NADPH generation for antioxidant systems during oxidative stress.

• Temperature stress adaptation: Study of temperature-dependent changes in PGK kinetics and expression as part of the organism's temperature stress response.

Research methods should include transcriptomic and proteomic analysis under various stress conditions, measurement of PGK activity and post-translational modifications following stress exposure, and phenotypic analysis of PGK-modified strains under stress conditions. This research would enhance understanding of how this primitive cyanobacterium coordinates central metabolism with stress response mechanisms .

What potential biotechnological applications could arise from studying G. violaceus PGK?

Investigation of G. violaceus PGK offers several promising biotechnological applications:

• Enzyme evolution studies: G. violaceus PGK represents an ancestral form of this glycolytic enzyme, providing a valuable starting point for directed evolution experiments aimed at understanding enzyme functional adaptation.

• Biosensor development: PGK-based biosensors could be developed for monitoring glycolytic intermediates or ATP levels in various biotechnological processes.

• Biocatalysis applications: The potentially unique properties of G. violaceus PGK might be exploited for stereospecific phosphoryl transfer reactions in biocatalytic processes.

• Cold-adapted enzyme engineering: If G. violaceus PGK shows adaptation to lower temperatures (consistent with the organism's growth conditions), it could serve as a template for engineering cold-active variants for industrial applications requiring low-temperature processes.

• Metabolic engineering platform: Understanding G. violaceus's primitive but functional glycolytic pathway could inform metabolic engineering strategies for minimized synthetic pathways in bioproduction hosts.

• Protein stability engineering: Insights from structural studies of G. violaceus PGK could guide engineering efforts to enhance stability of glycolytic enzymes for industrial applications.

These applications would build upon fundamental understanding of G. violaceus PGK structure-function relationships and its role in the unique metabolism of this early-diverging cyanobacterium .

How can in vitro reconstitution experiments with G. violaceus PGK inform synthetic biology approaches?

In vitro reconstitution experiments with G. violaceus PGK can provide valuable insights for synthetic biology approaches, similar to how reconstitution of G. violaceus RuBisCO components (RbcL with RbcS in presence of RbcX) revealed a four-fold enhancement in specific activity . Key research directions include:

• Minimal glycolytic module design: Reconstitution of G. violaceus PGK with other glycolytic enzymes to determine the minimal set required for efficient carbon flux through designed pathways.

• Enzyme scaffolding optimization: Testing different artificial scaffolds for co-localizing G. violaceus PGK with functionally related enzymes to enhance metabolic flux through synthetic pathways.

• Cross-species compatibility assessment: Systematic evaluation of G. violaceus PGK functionality when integrated with glycolytic enzymes from diverse organisms to inform hybrid pathway design.

• Cofactor regeneration systems: Development of reconstituted systems coupling G. violaceus PGK with other ATP-generating or ATP-consuming enzymes for efficient cofactor cycling in synthetic applications.

• Allosteric regulation engineering: In vitro testing of designed regulatory mechanisms for controlling G. violaceus PGK activity in response to specific molecular signals.

• Proteoliposome encapsulation: Incorporation of G. violaceus PGK into artificial vesicular systems as models for minimal synthetic cells.

The relatively simple cellular architecture of G. violaceus, lacking internal membrane systems like thylakoids , makes its enzymes potentially valuable components for minimal synthetic systems where reduced complexity is advantageous .

What are the most promising research frontiers for understanding G. violaceus PGK in the context of early photosynthetic evolution?

The study of G. violaceus PGK presents several exciting research frontiers that could illuminate early photosynthetic evolution:

• Ancestral sequence reconstruction: Computational resurrection and laboratory characterization of inferred ancestral forms of PGK from the last common ancestor of cyanobacteria, using G. violaceus PGK as a key reference point.

• Comparative structural biology: High-resolution structural analysis of G. violaceus PGK compared with homologs from diverse photosynthetic lineages to identify structural adaptations associated with the evolution of more complex photosynthetic systems.

• Metabolic network modeling: Computational reconstruction of the primitive glycolytic pathway in early cyanobacteria, with PGK as a central component, to understand evolutionary constraints and adaptive possibilities.

• Horizontal gene transfer investigation: Analysis of G. violaceus PGK sequence for evidence of ancient horizontal gene transfer events that might have influenced early cyanobacterial metabolism.

• Functional complementation studies: Testing whether G. violaceus PGK can functionally replace homologs in more recently evolved cyanobacteria, algae, or plants, and characterizing any fitness effects.

• Evolutionary rate analysis: Investigation of selection pressures acting on different domains of PGK across cyanobacterial evolution to identify functionally critical regions.

These approaches could reveal fundamental insights into how central carbon metabolism co-evolved with photosynthetic mechanisms during the early diversification of cyanobacteria, using G. violaceus as a valuable representative of primitive photosynthetic metabolism .

What interdisciplinary approaches might yield new insights into G. violaceus PGK function and evolution?

Advancing our understanding of G. violaceus PGK would benefit from innovative interdisciplinary approaches:

• Astrobiology connections: Analysis of G. violaceus PGK as a model for primitive Earth enzymes to inform astrobiological research on potential extraterrestrial biochemistry.

• Synthetic biology integration: Design of minimal artificial cells incorporating G. violaceus PGK and related enzymes to test hypotheses about early metabolic systems.

• Computational chemistry approaches: Quantum mechanical calculations to understand the electronic structure of the G. violaceus PGK active site and transition states during catalysis.

• Systems biology modeling: Integration of -omics data to model the behavior of G. violaceus PGK within the context of the organism's complete metabolic network.

• Paleobiochemistry perspectives: Correlation of G. violaceus PGK properties with reconstructed ancient Earth conditions to understand enzyme adaptation to early Earth environments.

• Evolutionary developmental biology frameworks: Application of evo-devo concepts to understand how metabolic networks involving PGK have evolved in complexity.

• Biophysics approaches: Advanced single-molecule techniques to characterize the conformational dynamics of G. violaceus PGK during catalysis.

• Geomicrobiology context: Study of G. violaceus PGK function under conditions mimicking ancient microbial mats to understand its role in early photosynthetic ecosystems.

These interdisciplinary approaches would place G. violaceus PGK research within broader scientific contexts, potentially yielding insights that extend beyond biochemistry into our understanding of early life evolution on Earth .