Recombinant Chromobacterium violaceum Phosphoglycerate kinase (pgk)

What is Chromobacterium violaceum Phosphoglycerate kinase and what is its role in bacterial metabolism?

Phosphoglycerate kinase (PGK) in Chromobacterium violaceum is a key enzyme involved in carbohydrate metabolism that catalyzes the reversible conversion between glycerate-1,3-bisphosphate and glycerate-3-phosphate. This reaction is critical for both glycolysis and gluconeogenesis pathways.

PGK functions as a transferase enzyme, specifically catalyzing the reaction:
$1,3-\text{bisphosphoglycerate} + \text{ADP} \rightleftharpoons 3-\text{phosphoglycerate} + \text{ATP}$

In C. violaceum, PGK is essential for:

• Energy production through glycolysis

• Carbon source utilization

• Adaptation to environmental stresses, particularly oxidative stress

• Supporting bacterial growth under varying nutrient conditions

Studies suggest that while C. violaceum may employ multiple pathways for glucose metabolism, including the Entner-Doudoroff (ED) and pentose phosphate pathways, PGK remains crucial for optimal energy production and carbon utilization .

How does the structure of C. violaceum PGK compare to PGK from other organisms?

C. violaceum PGK shares the highly conserved structural architecture found in PGK enzymes across species. Key structural features include:

• Two-domain structure with N-terminal and C-terminal domains connected by a hinge region

• The N-domain typically binds 1,3-bisphosphoglycerate while the C-domain binds ADP

• Conformational change from open to closed states via hinge motion is essential for catalytic activity

These adaptations allow PGK to function optimally under C. violaceum's preferred growth conditions (temperature range 20-37°C) . The enzyme likely has adapted specifically to function within the context of C. violaceum's unique metabolism, which includes violacein production and response to environmental stresses like oxidative stress and temperature fluctuation .

What are the most effective expression systems for producing recombinant C. violaceum PGK?

Based on research findings with C. violaceum proteins and similar bacterial PGKs, the following expression systems have proven effective:

E. coli Expression Systems:

• BL21(DE3): Most commonly used strain for C. violaceum protein expression due to its high yield and lack of proteases

• Vector options: pET28a with a His-tag for easy purification has been successfully used for C. violaceum proteins

Optimal Expression Protocol:

• Transform E. coli BL21(DE) with the expression vector containing the pgk gene

• Culture in LB medium at 37°C until OD600 reaches 0.4-0.6

• Lower the temperature to 18°C before induction

• Induce with 0.1-0.5 mM IPTG

• Continue cultivation at 18°C for 16-18 hours

Temperature control is critical as demonstrated in studies of C. violaceum proteins; lowering the temperature to 18°C after reaching the desired cell density significantly improves soluble protein yield and reduces inclusion body formation .

Example Expression Optimization Table:

For challenging expressions, specialized E. coli strains such as Rosetta(DE3) may improve expression by supplying rare codons that might be present in the C. violaceum genome .

What purification strategy yields the highest purity and activity for recombinant C. violaceum PGK?

A multi-step purification strategy ensures both high purity and preserved activity of recombinant C. violaceum PGK:

Recommended Purification Protocol:

• Cell Lysis Buffer Optimization:

  • 250 mM Tris-HCl, pH 7.8

  • 150 mM NaCl

  • 20 mM imidazole

  • 5% glycerol

  • 1 mM DTT or 5 mM β-mercaptoethanol

  • Protease inhibitor cocktail

• Initial Capture:

  • Immobilized metal affinity chromatography (IMAC) using Ni-Sepharose

  • Washing with buffer containing 20-50 mM imidazole

  • Elution with 250-300 mM imidazole

• Secondary Purification:

  • Size exclusion chromatography using Superdex 75 or 200 columns

  • Buffer: 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 5% glycerol

• Tag Removal:

  • If a SUMO-tag system is used, ULP protease digestion effectively removes the tag

  • Reapply to Ni-Sepharose to remove cleaved tag

Quality Control Metrics:

The addition of glycerol (5-10%) to all buffers is particularly important for maintaining C. violaceum PGK stability, as is maintaining a temperature of 4°C throughout the purification process .

How can the enzymatic activity of recombinant C. violaceum PGK be accurately measured?

Accurate measurement of C. violaceum PGK activity can be achieved through several established methods, with spectrophotometric coupled assays being the most widely used:

Forward Reaction Assay (3-PGA → 1,3-BPG):

• Coupling with GAPDH and NAD+ reduction:

  • The PGK reaction is coupled to GAPDH, which reduces NAD+ to NADH

  • NADH formation is monitored at 340 nm (ε = 6,220 M⁻¹cm⁻¹)

  • Reaction mixture typically contains:

    • 50 mM Tris-HCl (pH 7.5-8.0)

    • 5 mM MgCl₂

    • 1 mM ATP

    • 0.2 mM NADH

    • 5 mM 3-phosphoglycerate

    • Excess GAPDH

Reverse Reaction Assay (1,3-BPG → 3-PGA):

• Monitoring ATP formation:

  • The ATP produced can be measured using a luciferase-based assay

  • Alternatively, couple to hexokinase and glucose-6-phosphate dehydrogenase

  • Monitor NADPH formation at 340 nm

Controls and Validation:

• Include enzyme blanks (no substrate) and substrate blanks (no enzyme)

• Verify linearity with respect to time and enzyme concentration

• Confirm activity using known PGK inhibitors (e.g., high concentrations of ADP)

Specialized Assay for Cold Shock Response:
Since C. violaceum PGK activity may be affected by temperature changes (as suggested by studies showing translational inhibition by cold shock ), a specialized assay protocol has been developed:

• Pre-incubate enzyme samples at different temperatures (10-40°C)

• Measure activity using the standard coupled assay

• Plot temperature-activity profiles to determine thermal optima and stability

This approach helps understand how C. violaceum PGK functions under the environmental stress conditions that the bacterium encounters .

What is the role of PGK in C. violaceum's adaptation to environmental stresses?

C. violaceum PGK plays multifaceted roles in adaptation to environmental stresses, extending beyond its primary metabolic function:

1. Oxidative Stress Response:

• Proteomic analyses have shown that PGK expression changes significantly when C. violaceum is exposed to oxidative stress (8 mM H₂O₂)

• PGK likely contributes to NADPH regeneration, supporting antioxidant defense mechanisms

• The enzyme may work in coordination with other stress-response proteins identified in C. violaceum, including catalase and glutathione transferases

2. Temperature Stress Adaptation:

• C. violaceum undergoes significant physiological changes in response to cold shock

• PGK activity is modulated during temperature shifts, with implications for energy metabolism

• Studies have shown that sudden temperature decreases can inhibit polypeptide elongation in C. violaceum, potentially affecting PGK expression and function

3. Iron Homeostasis:

• High iron concentrations (9 mM) induce significant proteomic changes in C. violaceum

• PGK is among the proteins showing altered expression under iron stress conditions

• This response links energy metabolism to iron utilization pathways

4. Biofilm Formation:

• PGK appears to influence extracellular polysaccharide (EPS) production, which is essential for biofilm formation

• Mutational studies in related bacteria show that pgk deficiency leads to reduced EPS production and impaired biofilm formation

Comparative Stress Response Data:

These findings suggest PGK functions as a central hub connecting primary metabolism to stress adaptation in C. violaceum .

What site-directed mutagenesis approaches can reveal about C. violaceum PGK's catalytic mechanism?

Site-directed mutagenesis provides powerful insights into the structure-function relationships of C. violaceum PGK, revealing critical residues involved in catalysis, substrate binding, and conformational changes:

Key Catalytic Residues for Mutagenesis:

Based on structural studies of PGK from other organisms and sequence conservation analysis, the following residues represent high-value targets for site-directed mutagenesis in C. violaceum PGK:

• Substrate Binding Residues:

  • Arg38, Arg170: Involved in 1,3-BPG binding

  • Lys219, Asn336, Glu343: Critical for nucleotide binding

• Catalytic Residues:

  • His167: Potential proton donor/acceptor

  • Asp372: Coordinates magnesium ion necessary for catalysis

• Hinge Region Residues:

  • Glu192 and Gly394: Function as hinge points for domain movement

  • Mutations in these residues would likely disrupt the conformational change necessary for catalysis

Recommended Mutagenesis Approach:

Advanced Analysis of Mutants:

• Enzyme Kinetics:

  • Determine Km, kcat, and kcat/Km for both substrates with each mutant

  • Analyze the effect of pH and temperature on kinetic parameters

• Structural Analysis:

  • Use circular dichroism to assess secondary structure changes

  • Apply thermal denaturation studies to evaluate stability differences

  • When possible, obtain crystal structures of key mutants

• Molecular Dynamics Simulations:

  • Compare domain movement in wild-type and mutant PGKs

  • Calculate force distribution to identify allosteric pathways

  • Model substrate binding and product release

This systematic approach would provide a comprehensive understanding of how specific residues contribute to C. violaceum PGK function and regulation, potentially revealing unique features of this enzyme compared to other bacterial PGKs .

How does recombinant C. violaceum PGK contribute to understanding bacterial pathogenesis?

Recombinant C. violaceum PGK offers insights into bacterial pathogenesis through several research avenues:

1. Role in Virulence and Stress Survival:

• C. violaceum is an opportunistic pathogen with high fatality rates in human infections

• PGK contributes to stress adaptation, potentially enhancing survival during infection

• Studies have shown connections between central metabolism and virulence factors in C. violaceum

2. Metabolic Adaptation During Infection:

• PGK's role in both glycolysis and gluconeogenesis allows bacteria to utilize diverse carbon sources

• During infection, pathogens must adapt to nutrient-limited environments

• Characterizing C. violaceum PGK's kinetics with different substrates provides insights into metabolic flexibility during infection

3. Connection to Virulence Factor Production:

• C. violaceum produces violacein, a purple pigment with antimicrobial properties

• PGK activity influences carbon flux that might affect violacein production

• Understanding this connection helps illuminate how metabolism coordinates with virulence factor synthesis

4. Type VI Secretion System (T6SS) Interactions:

• C. violaceum utilizes a T6SS for bacterial competition and potentially for host interactions

• Energy requirements for T6SS function depend on central metabolism where PGK operates

• Studying the metabolic dependencies of virulence systems provides targets for intervention

Comparative Analysis Framework:

These research directions highlight how C. violaceum PGK serves as a model for understanding metabolic adaptations that support bacterial pathogenesis, potentially leading to new therapeutic approaches for combating bacterial infections .

What insights can C. violaceum PGK provide about evolution of enzyme function across bacterial species?

C. violaceum PGK offers a valuable system for studying enzyme evolution across bacterial species, particularly regarding adaptation to different ecological niches:

Evolutionary Conservation and Divergence:

PGK is highly conserved across all domains of life, making it an excellent model for studying evolutionary processes. C. violaceum PGK shows specific adaptations that provide insights into:

• Sequence-Structure-Function Relationships:

  • Core catalytic residues show high conservation across species

  • Surface residues and loop regions display higher variability

  • Comparison of C. violaceum PGK with homologs from thermophiles, psychrophiles, and mesophiles reveals adaptation signatures

• Environmental Adaptation Mechanisms:

  • C. violaceum inhabits soil and water in tropical/subtropical regions

  • Its PGK likely shows adaptations for function in fluctuating environments

  • Comparative analysis with PGKs from bacteria in stable environments highlights adaptive flexibility

Research Framework for Evolutionary Analysis:

C. violaceum PGK as an Evolutionary Model:

Several features make C. violaceum PGK particularly valuable for evolutionary studies:

• Habitat Transitions:

  • C. violaceum exists in both free-living and host-associated states

  • This transition requires metabolic flexibility where PGK plays a central role

  • Comparison with strictly free-living or obligate pathogen PGKs reveals adaptation patterns

• Secondary Metabolism Integration:

  • C. violaceum produces distinctive secondary metabolites like violacein

  • PGK's role in directing carbon flux may have evolved specific regulatory features

  • This provides insights into how primary and secondary metabolism co-evolved

• Stress Response Network Evolution:

  • C. violaceum shows remarkable stress tolerance (oxidative, temperature, antibiotics)

  • PGK's integration into these response networks may reflect evolutionary innovations

  • Study of these connections illuminates how central metabolic enzymes acquire regulatory roles

These evolutionary insights from C. violaceum PGK contribute to our broader understanding of how metabolic enzymes adapt to new ecological niches and potentially acquire new functions beyond their primary catalytic roles .

What are the main challenges in crystallizing recombinant C. violaceum PGK and how can they be overcome?

Crystallizing recombinant C. violaceum PGK presents several challenges, but systematic approaches can overcome these obstacles:

Major Crystallization Challenges:

• Conformational Flexibility:

  • PGK undergoes significant domain movements during catalysis

  • This inherent flexibility often hinders crystal formation

  • The open and closed conformations represent different energy states that complicate crystallization

• Surface Properties:

  • C. violaceum proteins may have surface characteristics adapted to tropical environments

  • These features can affect crystal packing and stability

  • Potential glycosylation or other post-translational modifications may introduce heterogeneity

• Stability Issues:

  • Temperature sensitivity related to C. violaceum's natural habitat

  • Potential oxidation of cysteine residues during purification and crystallization

  • Substrate-induced conformational changes affecting stability

Systematic Solutions and Strategies:

Recommended Crystallization Protocol:

• Pre-crystallization Optimization:

  • Perform thermal shift assays to identify stabilizing conditions

  • Use dynamic light scattering to ensure monodispersity

  • Consider limited proteolysis to identify stable domains

• Crystallization Screening:

  • Employ sitting-drop vapor diffusion method

  • Screen temperature range between 4-20°C (reflecting C. violaceum's natural environment)

  • Test both apo-enzyme and enzyme-substrate complexes in parallel

  • Utilize commercial screens focused on enzymes and bacterial proteins

• Advanced Techniques:

  • Surface entropy reduction (SER) by mutating clusters of high-entropy surface residues

  • Microseed matrix screening (MMS) using any initial crystalline material

  • Counter-diffusion crystallization in capillaries for improved crystal quality

• Data Collection Considerations:

  • Fast freezing protocols to minimize crystal damage

  • Consider room-temperature data collection to avoid freezing artifacts

  • Use helical data collection to minimize radiation damage

This comprehensive approach addresses the specific challenges of C. violaceum PGK crystallization while leveraging techniques proven successful with other flexible, two-domain enzymes .

What are the most promising applications of recombinant C. violaceum PGK in biotechnology and drug discovery?

Recombinant C. violaceum PGK holds significant potential for multiple biotechnological applications and drug discovery efforts:

1. Antimicrobial Drug Development:

• C. violaceum PGK can serve as a model for studying metabolic vulnerabilities in bacterial pathogens

• Structural differences between bacterial and human PGK can be exploited for selective inhibitor design

• Understanding PGK's role in stress responses may reveal new antibiotic targets or potentiators

2. Biocatalysis Applications:

• PGK catalyzes phosphoryl transfer reactions with potential applications in:

  • ATP regeneration systems for biocatalytic processes

  • Synthesis of phosphorylated compounds for pharmaceutical applications

  • Coupled enzyme systems for production of high-value chemicals

3. Biosensor Development:

• PGK could be engineered as a biosensor component for:

  • Detection of glycolytic intermediates in biological samples

  • Environmental monitoring of specific pollutants through allosteric regulation

  • Cellular energy state reporting in engineered biological systems

4. Protein Engineering Platform:

• C. violaceum PGK's adaptation to environmental stresses makes it an excellent template for protein engineering

• Potential applications include:

  • Engineering enzymes with enhanced thermostability or pH tolerance

  • Developing PGK variants with altered substrate specificity

  • Creating switchable enzymes responsive to specific environmental signals

Research Development Pathway:

Drug Discovery Potential:
C. violaceum PGK's connection to violacein production and bacterial survival under stress conditions makes it particularly valuable for antimicrobial drug discovery. Targeting bacterial metabolic adaptation represents a promising approach for developing new antimicrobials with reduced resistance potential .

What unresolved questions about C. violaceum PGK merit further investigation?

Despite progress in understanding C. violaceum PGK, several critical questions remain unexplored, offering rich opportunities for future research:

1. Structure-Function Relationships:

• Unresolved Question: How does the structure of C. violaceum PGK differ from other bacterial PGKs, and what functional implications do these differences have?

• Research Approach: Solve high-resolution crystal structure of C. violaceum PGK in both open and closed conformations; compare with structures from organisms adapted to different environmental niches

• Significance: Would reveal adaptations specific to C. violaceum's tropical/subtropical habitat and potential connections to its unique metabolism

2. Regulatory Networks:

• Unresolved Question: How is PGK expression and activity regulated in response to environmental stresses, and how does this integrate with violacein production?

• Research Approach: Combine transcriptomics, proteomics, and metabolomics under various stress conditions; map regulatory elements controlling pgk expression

• Significance: Would illuminate how C. violaceum coordinates primary metabolism with secondary metabolite production as part of its stress response

3. Role in Pathogenesis:

• Unresolved Question: Does PGK contribute to C. violaceum virulence, and could it serve as a target for antimicrobial development?

• Research Approach: Generate and characterize pgk mutants with altered expression or activity; test virulence in infection models; screen for selective PGK inhibitors

• Significance: Could lead to new therapeutic strategies for treating C. violaceum infections, which have high mortality rates

4. Evolutionary Adaptations:

• Unresolved Question: What evolutionary adaptations in C. violaceum PGK enable function across varying environmental conditions?

• Research Approach: Conduct comparative genomics and ancestral sequence reconstruction; express and characterize ancestral and modern PGK variants

• Significance: Would provide insights into enzyme evolution and adaptation mechanisms with applications for enzyme engineering

5. Technological Applications:

• Unresolved Question: Can C. violaceum PGK be engineered for novel biotechnological applications in biocatalysis or biosensing?

• Research Approach: Structure-guided protein engineering; directed evolution for altered substrate specificity or stability

• Significance: Could lead to novel biocatalysts with applications in pharmaceutical synthesis or environmental monitoring

Priority Research Areas Matrix:

These unresolved questions represent significant opportunities to advance our understanding of C. violaceum biology while contributing to broader knowledge in enzyme function, bacterial adaptation, and potential biotechnological applications .

How does the study of C. violaceum PGK contribute to our broader understanding of bacterial metabolism and adaptation?

The study of C. violaceum PGK provides a valuable window into fundamental aspects of bacterial metabolism and adaptation, offering broader insights beyond this specific enzyme:

Central Metabolism Integration:
C. violaceum PGK research illuminates how core metabolic enzymes function within the context of specialized bacterial metabolisms. The bacterium's ability to produce the purple pigment violacein represents a distinctive metabolic capability, and understanding how central carbon metabolism (where PGK functions) interfaces with specialized secondary metabolism helps explain how bacteria balance growth with defense and adaptation .

Environmental Adaptation Mechanisms:
C. violaceum inhabits tropical and subtropical environments with fluctuating conditions, and its PGK demonstrates adaptations reflecting these ecological pressures. The enzyme's responses to temperature shifts, oxidative stress, and iron availability reveal broader principles about how metabolic enzymes adapt to varying environmental conditions and contribute to bacterial survival across different niches .

Metabolic Flexibility:
The role of PGK in both glycolysis and gluconeogenesis highlights how central metabolic enzymes contribute to bacterial metabolic flexibility. This adaptability allows C. violaceum to utilize diverse carbon sources and survive in changing environments, providing insights into how metabolic networks reconfigure to maintain energy homeostasis under varying conditions .

Connections to Virulence:
C. violaceum can function as an opportunistic pathogen with high mortality rates, and PGK's role in stress response and adaptation may contribute to virulence. This connection between metabolism and pathogenesis exemplifies how primary metabolic enzymes can influence bacterial virulence beyond their canonical roles, suggesting new approaches to understanding and combating bacterial infections .

Evolutionary Implications:
The comparative analysis of C. violaceum PGK with homologs from other bacteria reveals evolutionary signatures that reflect both conservation of essential function and adaptation to specific ecological niches. These patterns provide insights into how metabolic enzymes evolve while maintaining catalytic efficiency, offering lessons for understanding protein evolution more broadly .