Recombinant Gloeobacter violaceus Phosphoenolpyruvate carboxylase (ppc), partial

What is Gloeobacter violaceus and why is it significant in evolutionary research?

Gloeobacter violaceus is a primitive cyanobacterium with unique cellular characteristics that distinguish it from all other known cyanobacteria. Most notably, G. violaceus completely lacks thylakoid membranes, forcing its photosynthetic machinery to operate within the cytoplasmic membrane instead . This distinctive organization limits its metabolism and growth rate compared to other cyanobacteria .

Phylogenetic analysis based on 16S ribosomal RNA places G. violaceus at the earliest branch of the cyanobacterial tree, indicating its position as one of the most primitive among living cyanobacteria . This basal position makes G. violaceus a key model organism in evolutionary studies of photosynthetic life and oxygenic photosynthesis . The genome of G. violaceus strain PCC 7421 has been fully sequenced, revealing many unique features compared to other cyanobacteria .

What is Phosphoenolpyruvate carboxylase (PEPC) and what role does it play in cyanobacteria?

Phosphoenolpyruvate carboxylase (PEPC) is the second major carbon-fixing enzyme in photoautotrophic organisms after RuBisCO . PEPC catalyzes the addition of bicarbonate to phosphoenolpyruvate (PEP) to form oxaloacetate, a reaction that plays several critical roles in cyanobacterial metabolism:

• It replenishes the TCA cycle by producing oxaloacetate

• It is required for the synthesis of amino acids of the glutamate and aspartate family

• Together with malate dehydrogenase and malic enzyme, it forms a metabolic shunt for synthesizing pyruvate from PEP

In this metabolic shunt process, CO₂ is first fixed and later released again, making PEPC vital for carbon flux regulation in the cell. Due to its central position in metabolism, understanding PEPC regulation is crucial for comprehending cellular carbon management .

What expression systems are suitable for producing recombinant G. violaceus PEPC?

Recombinant G. violaceus PEPC can be produced in multiple expression systems, each with distinct advantages:

According to product information, recombinant G. violaceus PEPC is commercially available from multiple expression sources including E. coli, yeast, baculovirus-infected cells, and mammalian cells . The choice depends on research requirements, with E. coli being the most common for basic studies.

What purification strategies yield high-purity recombinant G. violaceus PEPC?

Effective purification of recombinant G. violaceus PEPC typically involves multiple chromatographic steps:

• Affinity Chromatography: The initial capture step usually employs affinity tags such as His-tag or Avi-tag biotinylation. For instance, in vivo biotinylation using AviTag-BirA technology can create a highly specific binding site for streptavidin resins .

• Ion Exchange Chromatography: This secondary purification step separates proteins based on charge differences and helps remove contaminants that co-purify during affinity chromatography.

• Size Exclusion Chromatography: A final polishing step that separates proteins based on size and shape, useful for ensuring the proper quaternary structure of PEPC, which typically exists as a homotetramer.

Commercial preparations typically achieve >85% purity as assessed by SDS-PAGE . For functional studies, it's critical to verify that the purified enzyme retains catalytic activity.

How should recombinant G. violaceus PEPC be stored to maintain stability and activity?

Proper storage is critical for maintaining the structural integrity and enzymatic activity of recombinant G. violaceus PEPC:

• Short-term storage: Working aliquots can be kept at 4°C for up to one week

• Long-term storage: Add glycerol to 5-50% final concentration and store at -20°C/-80°C

• Format considerations: Lyophilized forms have longer shelf life (12 months) compared to liquid forms (6 months)

• Handling precautions: Avoid repeated freeze-thaw cycles as they can denature the protein and reduce activity

When reconstituting lyophilized protein, it's recommended to briefly centrifuge the vial before opening and use deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL .

What methods can be used to measure the enzymatic activity of recombinant G. violaceus PEPC?

Several assay systems can be employed to measure PEPC activity:

• Spectrophotometric Coupled Enzyme Assay: This is the most common method, where PEPC activity is coupled to the oxidation of NADH by malate dehydrogenase. The decrease in NADH absorbance at 340 nm directly correlates with PEPC activity.

• Direct Measurement of Oxaloacetate Formation: Using colorimetric or HPLC-based methods to detect the product.

• Radiometric Assay: Incorporation of ¹⁴C-labeled bicarbonate into acid-stable products.

For G. violaceus PCC 7421 RuBisCO (another carbon-fixing enzyme), researchers have used a spectrophotometric estimation in a coupled enzyme assay system, which revealed a carboxylation activity of 5 nMol of phosphoglycerate min⁻¹ mg⁻¹ of protein . Similar methodological approaches could be adapted for PEPC activity measurements.

How do PEPC activity levels in G. violaceus compare to other cyanobacteria?

While specific comparative data for PEPC activity across multiple cyanobacterial species is limited in the provided search results, some inferences can be made from studies on other carbon-fixing enzymes in G. violaceus.

For RuBisCO, G. violaceus PCC 7421 showed relatively low carboxylation activity (5 nMol of phosphoglycerate min⁻¹ mg⁻¹ of protein), which aligns with the organism's slow growth rate . Similar patterns might be expected for PEPC activity, reflecting the primitive nature and slower metabolism of G. violaceus compared to more derived cyanobacterial lineages.

A four-fold enhancement in RuBisCO activity was observed through in vitro complementation of RbcL with RbcS in the presence of RbcX from G. violaceus . This suggests that protein-protein interactions may significantly influence carbon-fixing enzyme activity in this organism.

What factors influence G. violaceus PEPC regulation in experimental settings?

Based on research with cyanobacterial PEPCs, several factors likely influence G. violaceus PEPC activity:

When designing experiments with recombinant G. violaceus PEPC, these factors should be systematically controlled to ensure reproducible results. The regulatory patterns observed in other cyanobacterial PEPCs suggest that G. violaceus PEPC activity is likely integrated with the cell's broader carbon/nitrogen sensing mechanisms .

How can G. violaceus PEPC contribute to understanding the evolution of carbon fixation pathways?

As a member of the earliest diverging lineage of cyanobacteria, G. violaceus offers unique insights into the evolution of carbon fixation mechanisms:

• Evolutionary Reconstruction: Comparing G. violaceus PEPC with those from more derived cyanobacteria and other photosynthetic organisms can help reconstruct the evolutionary history of carbon fixation pathways.

• Ancestral Feature Preservation: G. violaceus may retain ancestral features that have been modified or lost in more derived lineages. For example, G. violaceus lacks thylakoid membranes, reflecting a cellular organization that may represent an earlier evolutionary stage .

• Unique Operon Organization: Similar to how the photosystem II psbADC operon in G. violaceus represents the first example of a transcribed gene cluster containing core photosystem subunits , the genomic context of the PEPC gene may provide insights into the co-evolution of carbon fixation enzymes.

• Comparative Genomics: Recent pangenomic analysis revealed that G. violaceus contains unique gene clusters not shared with other cyanobacteria , potentially including novel regulatory elements for carbon fixation enzymes.

Can G. violaceus PEPC be integrated with artificial photosynthetic systems?

Recent research has demonstrated the potential for integrating components from primitive photosynthetic organisms into engineered systems:

• Light-Driven Proton Pumping: G. violaceus rhodopsin (GR) has been successfully expressed in Ralstonia eutropha and shown to function as a light-driven proton pump . Similar approaches could be applied to coupling PEPC activity with light-harvesting systems.

• Carbon Fixation Engineering: The unique properties of G. violaceus enzymes may provide advantages for engineered carbon fixation pathways. For example, research has shown that GR-expressing Ralstonia eutropha cells can be engineered for photo-electrosynthetic CO₂ fixation .

• Synthetic Biology Applications: By combining G. violaceus PEPC with other carbon-fixing enzymes in artificial pathways, researchers might develop more efficient systems for converting CO₂ into valuable compounds or biomass.

• Environmental Considerations: The adaptation of G. violaceus to rock-dwelling environments suggests its enzymes may have unique properties suitable for deployment in harsh conditions or non-traditional settings.

What methodological challenges exist when studying protein-protein interactions involving G. violaceus PEPC?

Investigating protein-protein interactions with G. violaceus PEPC presents several challenges:

• Membrane Association: In G. violaceus, the photosynthetic machinery operates within the cytoplasmic membrane . This membrane association may affect PEPC interactions with other proteins and complicate in vitro reconstitution efforts.

• Complex Formation Dynamics: Studies of Synechocystis PEPC showed that complex formation with regulatory proteins like P₁₁ is highly dependent on metabolite concentrations . Similar dynamic interactions may exist for G. violaceus PEPC, requiring careful control of experimental conditions.

• Structural Considerations: The primitive nature of G. violaceus may be reflected in unique structural features of its PEPC that affect interaction interfaces. Techniques like hydrogen-deuterium exchange mass spectrometry or cross-linking coupled with mass spectrometry can help map these interfaces.

• Heterologous Expression Issues: When studying interactions with other G. violaceus proteins, co-expression in compatible systems may be necessary, potentially requiring optimization of codon usage and expression conditions for multiple proteins simultaneously.

How should researchers design experiments to compare G. violaceus PEPC with PEPCs from other organisms?

When designing comparative studies:

• Standardized Assay Conditions: Use identical buffer compositions, substrate concentrations, and detection methods across all enzyme variants to ensure valid comparisons.

• Parallel Expression and Purification: Express and purify all enzymes using the same system and protocol to minimize variation from production methods.

• Kinetic Parameter Determination: Systematically measure and compare kinetic parameters (Km, Vmax, kcat/Km) for substrates (PEP, bicarbonate) and response to regulators under identical conditions.

• Structural Comparison: Employ techniques like circular dichroism, thermal shift assays, and limited proteolysis to compare structural features and stability.

• Evolutionary Context: Interpret differences in light of phylogenetic relationships, considering that G. violaceus represents one of the earliest diverging cyanobacterial lineages .

What controls are essential when performing functional studies with recombinant G. violaceus PEPC?

Essential controls include:

• Enzyme-Free Control: Reaction mixture without PEPC to establish baseline activity and rule out non-enzymatic reactions.

• Heat-Inactivated Enzyme: PEPC that has been heat-denatured to confirm that measured activity is due to the active enzyme rather than contaminants.

• Substrate Dependence: Reactions with and without PEP or bicarbonate to confirm substrate specificity.

• Known PEPC Standards: Include well-characterized PEPC from organisms like E. coli or Synechocystis as positive controls and reference points for activity comparisons.

• Tag-Only Controls: When using tagged proteins, include controls with the tag alone to ensure observed effects are not due to the tag.

• Buffer Components: Systematically test the effects of buffer components, particularly divalent cations and potential regulatory molecules.

How can genetic approaches complement biochemical studies of G. violaceus PEPC?

Integrating genetic and biochemical approaches can provide comprehensive understanding:

• Heterologous Complementation: Express G. violaceus PEPC in PEPC-deficient mutants of other organisms to assess functional conservation and unique properties.

• Site-Directed Mutagenesis: Create specific mutations in conserved or unique residues to determine their roles in catalysis, regulation, or protein-protein interactions.

• Domain Swapping: Generate chimeric enzymes with domains from G. violaceus PEPC and other PEPCs to identify regions responsible for specific functional properties.

• Regulatory Element Analysis: Investigate the native promoter and regulatory elements of the G. violaceus PEPC gene to understand its transcriptional control.

• Genomic Context Examination: Analyze genes adjacent to PEPC in the G. violaceus genome to identify potential functional partners or co-regulated components of carbon fixation pathways.

How do recent findings about G. violaceus photosystems relate to understanding its carbon fixation mechanisms?

Recent research has revealed several important connections:

• Unique Operon Organization: The identification of the psbADC operon in G. violaceus, encoding three of the five reaction center core subunits (D1, D2, and CP43), represents the first example of a transcribed gene cluster containing these core photosystem components in any oxygenic phototroph . This unique genomic arrangement suggests that carbon fixation genes might also have distinctive regulatory patterns in this organism.

• Evolutionary Implications: G. violaceus has five copies of the photosystem II psbA gene encoding the D1 reaction center protein subunit, widely distributed throughout its 4.6 Mbp genome . This gene organization pattern differs markedly from other cyanobacteria and provides context for understanding how other metabolic pathways, including those involving PEPC, may be organized and regulated.

• Light-Driven Processes: Studies have shown that G. violaceus expresses rhodopsin (GR) that functions as a light-driven proton pump . This alternative mechanism for generating proton gradients may interact with carbon fixation pathways, potentially affecting PEPC regulation through changes in cellular pH or energetics.

What advances in structural biology techniques could enhance our understanding of G. violaceus PEPC?

Recent advances in structural biology offer new opportunities:

• Cryo-Electron Microscopy: The resolution revolution in cryo-EM now enables structural determination of large enzymes like PEPC without crystallization, potentially revealing conformational states difficult to capture by X-ray crystallography.

• Integrative Structural Biology: Combining multiple techniques (X-ray crystallography, NMR, SAXS, mass spectrometry) can provide comprehensive structural models of PEPC in different functional states.

• Time-Resolved Structural Methods: Emerging techniques like time-resolved X-ray crystallography and cryo-EM can capture transient conformational states during the catalytic cycle.

• In-Cell Structural Biology: Methods to study protein structures in their native cellular environment could reveal how G. violaceus PEPC functions within the context of the cytoplasmic membrane, where the organism's photosynthetic machinery resides .

• Computational Approaches: Advanced molecular dynamics simulations can model PEPC conformational changes and interactions with substrates, regulators, and other proteins based on structural data.

How might the ecological context of G. violaceus inform research on its PEPC?

The unique ecological niche of G. violaceus provides important context:

• Rock-Dwelling Lifestyle: G. violaceus has been identified as a common rock-dwelling cyanobacterium , suggesting its enzymes may be adapted to this challenging environment.

• Primitive Freshwater Origin: Evidence suggests a freshwater origin for cyanobacteria, with G. violaceus representing an early diverging lineage . This ecological context might explain certain biochemical properties of its enzymes.

• Slow Growth Adaptation: The slow growth rate of G. violaceus correlates with its relatively low RuBisCO activity . Similar adaptations might be observed in PEPC, reflecting a metabolic strategy optimized for its ecological niche.

• Experimental Design Implications: When studying G. violaceus PEPC, researchers should consider experimental conditions that reflect the organism's natural habitat, including appropriate pH, temperature ranges, and ion concentrations that might influence enzyme activity and regulation.

What are common pitfalls when working with recombinant G. violaceus PEPC and how can they be addressed?

Researchers should be aware of several common challenges:

How can researchers validate the authenticity and quality of recombinant G. violaceus PEPC preparations?

Multiple validation approaches should be employed:

• Mass Spectrometry Analysis: Confirm protein identity through peptide mass fingerprinting or intact mass determination.

• Activity Assays: Verify enzymatic function using established PEPC activity assays.

• Circular Dichroism: Assess secondary structure content to confirm proper folding.

• Size Exclusion Chromatography: Confirm the proper oligomeric state (typically tetrameric for PEPC).

• Thermal Shift Assays: Evaluate protein stability under different buffer conditions.

• SDS-PAGE Analysis: Assess purity and integrity, looking for a single band at the expected molecular weight (typically >85% purity by SDS-PAGE for research-grade preparations) .

• Western Blotting: Use specific antibodies to confirm identity and detect potential degradation products.