Recombinant Thermoplasma volcanium Phosphoenolpyruvate carboxykinase [GTP] (pckG), partial

What is the biochemical function of Thermoplasma volcanium phosphoenolpyruvate carboxykinase?

Phosphoenolpyruvate carboxykinase (PCK) catalyzes the nucleotide-dependent, reversible decarboxylation of oxaloacetate to yield phosphoenolpyruvate and CO₂. This enzyme plays a critical role in the interconversion between C₃ and C₄ metabolites in metabolic pathways . In archaeal organisms like Thermoplasma volcanium, PCK is particularly important for gluconeogenesis, providing phosphoenolpyruvate from oxaloacetate as the first step in this pathway . Similar to the characterized archaeal PCK from Thermococcus kodakaraensis, T. volcanium PCK likely shows preference for phosphoenolpyruvate formation from oxaloacetate rather than the reverse reaction .

How does T. volcanium PCK compare structurally to other archaeal PCKs?

While specific structural data for T. volcanium PCK is limited in the provided search results, archaeal PCKs typically share 30-35% sequence identity with GTP-dependent PCKs from mammals and bacteria . Based on comparative analysis with Thermococcus kodakaraensis PCK, T. volcanium PCK likely contains several conserved catalytically important regions found in all known PCKs, but with a unique GTP-binding region compared to other GTP-dependent PCKs . Phylogenetically, archaeal PCKs from Thermoplasma, Pyrococcus, and Sulfolobus form a distinct branch separate from classical PCK enzymes .

What expression patterns does PCK show in T. volcanium under different growth conditions?

While specific expression data for PCK in T. volcanium is not directly provided, research on archaeal PCK expression in T. kodakaraensis reveals that transcription and activity levels are higher under gluconeogenic conditions compared to glycolytic conditions . By extension, T. volcanium PCK expression likely increases when the organism is grown on substrates that require gluconeogenesis, such as amino acids or pyruvate, and decreases in the presence of carbohydrates . This expression pattern would be consistent with PCK's primary role in gluconeogenesis in archaea.

What are effective strategies for heterologous expression and purification of recombinant T. volcanium PCK?

For heterologous expression of thermophilic archaeal proteins like T. volcanium PCK, E. coli expression systems with heat-stable vectors are recommended. Based on protocols used for other T. volcanium proteins, the following methodological approach is suggested:

• Vector selection: Use a pET-based expression system with a 6×His-tag for easy purification.

• Expression conditions: Transform into E. coli BL21(DE3) or Rosetta strains, induce with IPTG (0.1-0.5 mM) at lower temperatures (20-30°C) to enhance proper folding.

• Purification protocol:

  • Initial heat treatment (60°C for 20 min) to denature host proteins

  • Ammonium sulfate fractionation (40-60%)

  • Ion exchange chromatography (DEAE or Q-Sepharose)

  • Affinity chromatography (Ni-NTA)

  • Size exclusion chromatography for final purification

For optimization of recombinant T. volcanium PCK activity, consider testing different buffer systems (HEPES, Tris-HCl, or phosphate) at pH 6.5-7.5 with various concentrations of stabilizing agents (glycerol 10-20%, NaCl 100-300 mM).

How can researchers design experiments to characterize the kinetic properties of T. volcanium PCK?

A comprehensive kinetic characterization requires analyzing both forward and reverse reactions under various conditions. The following experimental design is recommended:

• Substrate concentration ranges:

  • For forward reaction: Oxaloacetate (0.05-10 mM), GTP (0.01-5 mM)

  • For reverse reaction: Phosphoenolpyruvate (0.1-20 mM), GDP (0.01-5 mM), HCO₃⁻ (1-50 mM)

• Reaction conditions to test:

  • Temperature range: 30-80°C

  • pH range: 5.0-8.0

  • Divalent cations: Mg²⁺, Mn²⁺, Ca²⁺, Co²⁺ (0.5-10 mM)

  • Metal chelators: EDTA, EGTA (to determine metal dependence)

• Assay methods:

  • Coupled spectrophotometric assays tracking NADH oxidation

  • Direct monitoring of oxaloacetate consumption at 280 nm

  • Fixed-time point assays with HPLC quantification

• Inhibition studies: Test potential physiological regulators such as pyruvate, 2-oxoglutarate, ATP, and ADP

Based on studies of other archaeal PCKs, researchers should expect a Km value for oxaloacetate in the millimolar range (approximately 2-3 mM) and potential weak inhibition by pyruvate and 2-oxoglutarate .

What experimental approaches can reveal structure-function relationships in T. volcanium PCK?

To investigate structure-function relationships, consider the following multi-faceted approach:

• Site-directed mutagenesis:

  • Target conserved residues in the active site based on sequence alignments with other characterized PCKs

  • Focus on unique residues in the GTP-binding domain that differentiate archaeal PCKs

  • Create chimeric constructs with domains from other archaeal PCKs

• Structural studies:

  • X-ray crystallography of the enzyme with various ligands (substrates, products, inhibitors)

  • Circular dichroism spectroscopy to monitor thermal stability and secondary structure

  • Small-angle X-ray scattering (SAXS) for solution-state structural information

• Computational approaches:

  • Homology modeling based on existing PCK structures

  • Molecular dynamics simulations under different temperature conditions

  • Docking studies with substrates and potential inhibitors

• Domain analysis:

  • Limited proteolysis to identify stable domains

  • Expression of individual domains to assess their functionality

This multi-method approach will help elucidate how T. volcanium PCK's unique structural features contribute to its thermostability and catalytic efficiency.

How does environmental stress affect T. volcanium PCK expression and activity?

Based on stress response studies in T. volcanium, design experiments to investigate PCK regulation under various stressors:

• Experimental design for stress studies:

  • Heat stress: Culture exposure to temperatures from 55°C to 70°C

  • pH stress: Adjustment of culture medium to pH 1.0-4.0

  • Oxidative stress: Treatment with H₂O₂ (0.01-0.05 mM)

  • Nutrient limitation: Carbon or nitrogen starvation

• Expression analysis methods:

  • Quantitative RT-PCR for transcript levels

  • Western blotting for protein levels

  • Enzyme activity assays under each stress condition

• Time-course experiments:

  • Short-term response (15-120 minutes after stress)

  • Long-term adaptation (2-24 hours after stress)

What are the differences in catalytic mechanisms between archaeal and bacterial/eukaryotic PCKs?

To investigate catalytic mechanism differences, researchers should focus on:

• Nucleotide specificity:

  • Determine whether T. volcanium PCK is strictly GTP-dependent or can use other nucleotides

  • Compare kinetic parameters with GTP vs. ATP as phosphoryl donors

  • Analyze structural elements determining nucleotide specificity

• Metal ion requirements:

  • Characterize the dual cation-binding sites observed in archaeal PCKs

  • Determine the specific roles of Mn²⁺ and Mg²⁺ in catalysis

  • Investigate whether metal preferences change at different temperatures

• Reaction intermediates:

  • Use rapid kinetics techniques to identify and characterize reaction intermediates

  • Employ isotope exchange studies to elucidate the reaction mechanism

  • Determine the rate-limiting step in both forward and reverse reactions

• Evolutionary divergence analysis:

  • Perform comparative analyses with PCKs from all three domains of life

  • Focus on conserved vs. divergent catalytic residues

  • Investigate adaptive features related to thermophilic lifestyle

Based on studies of T. kodakaraensis PCK, archaeal PCKs appear to have a unique GTP-binding region compared to other GTP-dependent PCKs, suggesting potential mechanistic differences in nucleotide recognition and utilization .

How can genome-wide expression analysis inform our understanding of T. volcanium PCK's metabolic role?

Microarray or RNA-Seq approaches can provide comprehensive insights into PCK's metabolic context:

• Experimental design for transcriptome analysis:

  • Compare expression profiles under gluconeogenic vs. glycolytic conditions

  • Analyze co-expression patterns with other metabolic genes

  • Use gene knockout/knockdown approaches to investigate regulatory networks

• Data analysis approaches:

  • Pathway enrichment analysis to identify co-regulated metabolic modules

  • Comparative transcriptomics across multiple archaeal species

  • Integration with metabolomics data for metabolic flux analysis

• Validation experiments:

  • Confirm key findings with qRT-PCR of selected genes

  • Use Western blotting to verify protein-level changes

  • Perform enzyme activity assays to correlate transcript levels with function

When designing these experiments, researchers should consider that in T. volcanium, similar to other archaea, transcription of gluconeogenic enzymes is likely higher under gluconeogenic conditions (growth on pyruvate or amino acids) and lower under glycolytic conditions (growth on carbohydrates) . Comprehensive microarray analysis can reveal the broader metabolic context and potential moonlighting functions of PCK.

What are the best approaches for measuring T. volcanium PCK activity in crude extracts?

Measuring PCK activity in crude extracts presents specific challenges due to interfering enzymes. Consider these methodological approaches:

• Sample preparation:

  • Use gentle cell lysis methods (sonication in 50 mM HEPES, pH 7.0, with 5 mM MgCl₂, 1 mM DTT)

  • Heat treatment (55-60°C) to denature most mesophilic proteins while preserving thermophilic PCK

  • Prepare cell-free extracts by centrifugation (20,000 × g, 30 min, 4°C)

• Activity assays:

  • Direct assay: Monitor oxaloacetate consumption at 280 nm

  • Coupled assay: Link PCK reaction to malate dehydrogenase and monitor NADH oxidation

  • Radiometric assay: Use ¹⁴C-labeled substrates for increased sensitivity

• Controls and validations:

  • Measure activity without GTP to determine background reactions

  • Include specific PCK inhibitors to confirm specificity

  • Perform activity assays at different temperatures (30-70°C) to distinguish thermophilic PCK from mesophilic interfering enzymes

Researchers should be aware that in extracts of T. volcanium grown on pyruvate or amino acids, PCK activity is expected to be significantly higher than in cells grown on carbohydrates , which should be considered when designing experiments and interpreting results.

How can researchers optimize experimental design for thermostability studies of T. volcanium PCK?

For robust thermostability characterization of T. volcanium PCK, implement the following experimental design:

• Thermal inactivation kinetics:

  • Preincubate purified enzyme at temperatures ranging from 50-90°C

  • Remove aliquots at specified time intervals (0-120 min)

  • Measure residual activity under standard conditions

  • Calculate half-life values at each temperature

• Differential scanning calorimetry (DSC):

  • Determine melting temperature (Tm) under various buffer conditions

  • Analyze the effects of substrates, cofactors, and metals on thermal stability

  • Compare thermal unfolding profiles with mesophilic PCK homologs

• Circular dichroism spectroscopy:

  • Monitor secondary structure changes during thermal denaturation

  • Perform thermal melts with gradual temperature increases (25-95°C)

  • Assess reversibility of thermal denaturation by cooling and reheating

• Statistical design for optimization:

  • Use response surface methodology to optimize multiple parameters simultaneously

  • Apply Design of Experiments (DoE) approaches to efficiently explore stability conditions

  • Analyze interaction effects between pH, salt concentration, and temperature

When designing these experiments, researchers should consider using specialized software packages for DoE like those mentioned in search result , which can help efficiently explore the multidimensional parameter space affecting enzyme thermostability.

What are the challenges in interpreting kinetic data for thermophilic enzymes like T. volcanium PCK?

Interpreting kinetic data for thermophilic enzymes presents unique challenges that researchers should address:

• Temperature-dependent kinetic parameters:

  • Measure kinetic parameters across a wide temperature range (30-80°C)

  • Construct Arrhenius plots to determine activation energies

  • Consider that optimal activity temperature may differ from physiological temperature

• Buffer considerations:

  • Account for temperature-dependent changes in buffer pH (e.g., Tris buffers)

  • Use buffers with minimal temperature-dependent pKa shifts for consistent conditions

  • Consider testing multiple buffer systems to identify potential buffer-specific effects

• Data analysis approaches:

  • Apply temperature correction factors to compare data across temperatures

  • Use non-linear regression for complex kinetic models beyond Michaelis-Menten

  • Consider global fitting approaches for integrated dataset analysis

• Common interpretational pitfalls:

  • Misattributing stability effects to catalytic effects

  • Overlooking the impact of protein dynamics on catalysis at different temperatures

  • Neglecting substrate/cofactor stability at elevated temperatures

Temperature-dependent conformational changes may significantly impact kinetic parameters, making linear extrapolations from standard temperature conditions inappropriate for thermophilic enzymes like T. volcanium PCK.

How can recombinant T. volcanium PCK be used in metabolic engineering applications?

The thermostable nature of T. volcanium PCK makes it potentially valuable for metabolic engineering applications:

• Potential applications:

  • Engineering thermotolerant microorganisms for elevated-temperature bioprocesses

  • Creating synthetic metabolic pathways for carbon fixation or C3-C4 interconversion

  • Developing cell-free systems for biocatalysis at elevated temperatures

• Research approaches:

  • Heterologous expression in industrial microorganisms (E. coli, yeast, thermophiles)

  • Enzyme engineering to optimize activity, stability, or substrate specificity

  • Integration with other thermostable enzymes for multi-step conversions

• Performance metrics to evaluate:

  • Catalytic efficiency (kcat/Km) at different temperatures

  • Long-term operational stability under process conditions

  • Compatibility with industrial substrates and co-solvents

• Proof-of-concept experiments:

  • In vitro reconstitution of metabolic pathways incorporating T. volcanium PCK

  • Small-scale bioreactor tests with engineered strains

  • Comparison with mesophilic PCK variants under standard and elevated-temperature conditions

Researchers should note that PCK's preference for the gluconeogenic direction (conversion of oxaloacetate to phosphoenolpyruvate) makes it particularly suitable for pathways requiring PEP generation .

What are the considerations for using T. volcanium PCK in structural biology research?

T. volcanium PCK represents an excellent target for structural studies of thermophilic enzymes:

• Crystallization strategies:

  • Screen wide ranges of pH (5.0-9.0) and precipitant concentrations

  • Include GTP, GDP, oxaloacetate, or phosphoenolpyruvate as co-crystallization ligands

  • Consider crystallization at elevated temperatures (20-37°C) to maintain native conformation

  • Use surface entropy reduction approaches if initial crystallization attempts fail

• NMR studies:

  • Prepare isotopically labeled protein (¹⁵N, ¹³C, ²H)

  • Optimize buffer conditions for long-term stability at room temperature

  • Consider TROSY-based experiments for better resolution

• Cryo-EM approaches:

  • Evaluate whether PCK forms oligomers or can be engineered to form larger assemblies

  • Optimize grid preparation conditions to prevent preferential orientation

  • Consider GraFix method to stabilize protein complexes

• Structure-guided investigations:

  • Design mutagenesis experiments based on structural information

  • Investigate thermostability determinants through comparative structural analysis

  • Explore substrate channeling or protein-protein interaction interfaces

When planning structural biology experiments, researchers should consider that archaeal PCKs like T. volcanium PCK have unique features compared to bacterial and eukaryotic counterparts, including a distinctive GTP-binding region , which may require specific optimization of experimental conditions.

How should researchers approach experimental design for studying the effects of post-translational modifications on T. volcanium PCK?

To investigate potential post-translational modifications (PTMs) of T. volcanium PCK:

• PTM identification strategies:

  • Mass spectrometry-based proteomic analysis of native PCK

  • Comparison between recombinant and native enzyme properties

  • Western blotting with PTM-specific antibodies (phosphorylation, acetylation)

  • Chemical labeling approaches for specific modifications

• Functional impact assessment:

  • Site-directed mutagenesis of identified or predicted PTM sites

  • Activity assays under varying conditions that might affect PTM status

  • Stability and folding analysis of modified vs. unmodified forms

• Experimental design considerations:

  • Growth condition variations to potentially alter PTM patterns

  • Time-course analysis to capture dynamic modification changes

  • Control experiments with dephosphorylation or deacetylation treatments

• Data analysis approaches:

  • Quantitative comparison of enzyme kinetics before and after PTM manipulation

  • Structural modeling to predict PTM effects on protein conformation

  • Statistical analysis to ensure reproducibility and significance of observed differences

While specific information about PTMs in T. volcanium PCK is not provided in the search results, research on other archaeal proteins suggests that phosphorylation and acetylation may play regulatory roles, particularly under stress conditions .

What statistical approaches are most appropriate for analyzing T. volcanium PCK kinetic data?

Robust statistical analysis of enzyme kinetic data requires careful consideration:

• Experimental design for statistical robustness:

  • Use Design of Experiments (DoE) approaches to efficiently explore parameter space

  • Perform sufficient technical and biological replicates (minimum n=3)

  • Include appropriate positive and negative controls in each experimental set

• Data analysis methods:

  • Non-linear regression for fitting enzyme kinetic models

  • Residual analysis to validate model assumptions

  • Bootstrap or jackknife methods for parameter uncertainty estimation

  • ANOVA or mixed-effects models for comparing conditions

• Software tools and packages:

  • R packages specialized for enzyme kinetics analysis

  • GraphPad Prism or similar for visualization and statistical testing

  • Python with scipy.optimize for custom model fitting

• Addressing common statistical challenges:

  • Dealing with substrate inhibition or activation kinetics

  • Accounting for temperature effects on equilibrium constants

  • Managing datasets with missing values or outliers

Researchers should consider applying specialized statistical approaches from the Design of Experiments field, as outlined in search result , particularly when optimizing multiple experimental parameters simultaneously.

How can researchers resolve contradictory experimental results when characterizing T. volcanium PCK?

When faced with contradictory results in enzyme characterization:

• Systematic troubleshooting approach:

  • Review experimental conditions for subtle differences (buffer components, sample preparation)

  • Check enzyme purity and integrity (SDS-PAGE, mass spectrometry)

  • Verify assay specificity and sensitivity

  • Consider batch-to-batch variation in enzyme preparations

• Cross-validation strategies:

  • Use multiple independent assay methods to measure the same parameter

  • Employ different expression systems to produce the recombinant enzyme

  • Compare results across different laboratories if possible

  • Validate key findings using native enzyme from T. volcanium

• Resolution framework:

  • Develop testable hypotheses to explain disparities

  • Design critical experiments that can differentiate between competing explanations

  • Consider whether contradictions reflect genuine biological complexity rather than technical issues

  • Use statistical meta-analysis approaches to integrate conflicting datasets

• Documentation and reporting:

  • Maintain detailed records of all experimental conditions

  • Report seemingly contradictory results in publications with possible explanations

  • Consider whether protein heterogeneity (isoforms, post-translational modifications) might explain variations

When analyzing contradictory results, researchers should consider whether differences in experimental temperature, pH, or ionic conditions might particularly affect thermophilic enzymes like T. volcanium PCK, which may display distinct behavior under different conditions.