Recombinant Photobacterium profundum Glycerol-3-phosphate dehydrogenase [NAD (P)+] (gpsA)

What is the biological role of Glycerol-3-phosphate dehydrogenase (gpsA) in Photobacterium profundum?

Glycerol-3-phosphate dehydrogenase (gpsA) in P. profundum plays a critical role in glycerol metabolism, particularly in the conversion of dihydroxyacetone phosphate to glycerol-3-phosphate (G3P). This enzyme is essential for maintaining G3P homeostasis, which appears to be important for bacterial growth and adaptation to different environmental conditions. Similar to what has been observed in Pseudomonas aeruginosa, proper G3P homeostasis likely influences growth rates, stress responses, and potentially adaptation to the deep-sea environment characteristic of P. profundum . In P. profundum SS9, a deep-sea piezophilic ("pressure-loving") bacterium belonging to the Vibrionaceae family, metabolic adaptation to high pressure environments may involve regulation of glycerol metabolism pathways .

What are the optimal conditions for expressing recombinant P. profundum gpsA in E. coli or other expression systems?

For optimal expression of recombinant P. profundum gpsA, researchers should consider the following methodological approach:

• Vector selection: Choose an expression vector with an appropriate promoter system that allows for controlled induction (such as pET vectors with T7 promoter)

• Host strain optimization: Select an E. coli strain optimized for recombinant protein expression (BL21(DE3), Rosetta, or Arctic Express for cold-adapted proteins)

• Temperature considerations: Given that P. profundum is a psychrotolerant organism capable of growth at temperatures from 0°C to 25°C , lower expression temperatures (15-20°C) may improve proper folding

• Media composition: Include osmolytes or salt in the growth medium, as P. profundum has a requirement for salt

• Induction parameters: Use lower IPTG concentrations (0.1-0.5 mM) and longer induction times at reduced temperatures

A methodological table for optimization:

How does pressure affect the kinetic properties and stability of P. profundum gpsA compared to homologous enzymes from non-piezophilic organisms?

P. profundum SS9 is a moderately piezophilic bacterium capable of growth at pressures from 0.1 MPa to 70 MPa . This adaptation to high-pressure environments likely influences the structural and kinetic properties of its enzymes, including gpsA. When studying P. profundum gpsA, researchers should consider:

• Pressure-dependent activity assays: Measure enzyme activity across a range of hydrostatic pressures (0.1-70 MPa) using specialized high-pressure equipment to determine optimal pressure for activity

• Comparative kinetics: Compare kinetic parameters (Km, Vmax, kcat) of P. profundum gpsA with homologs from non-piezophilic organisms at different pressures

• Structural stability analysis: Evaluate protein stability using circular dichroism, differential scanning calorimetry, or intrinsic fluorescence under varying pressure conditions

• Molecular dynamics simulations: Use computational approaches to predict structural changes and substrate binding under high pressure

Research indicates that adaptations to high-pressure environments typically involve modifications to maintain flexibility and function of proteins under compression. Based on studies of other piezophilic organisms, P. profundum gpsA may exhibit specific amino acid substitutions or structural features that confer pressure resistance without compromising catalytic efficiency .

What are the structural features of P. profundum gpsA that might contribute to its function under high-pressure conditions?

While specific structural information about P. profundum gpsA is not provided in the search results, structural adaptations likely include:

To investigate these features, researchers could employ X-ray crystallography, cryo-electron microscopy, or SAXS (small-angle X-ray scattering) to determine the structure under different pressure conditions. Site-directed mutagenesis of key residues followed by activity and stability testing could identify crucial amino acids for pressure adaptation.

How does G3P homeostasis contribute to P. profundum adaptation to deep-sea environments?

G3P homeostasis likely plays a critical role in P. profundum adaptation to deep-sea environments through multiple mechanisms. Drawing parallels from studies in other bacteria like P. aeruginosa, where G3P homeostasis has been shown to be important for growth and virulence factor production , we can hypothesize that:

• Membrane composition modification: G3P is a precursor for phospholipid biosynthesis, and membrane composition is critical for adaptation to high pressure environments

• Energy metabolism regulation: G3P serves as an important metabolic intermediate linking glycolysis and oxidative phosphorylation

• Osmoregulation: G3P may function as a compatible solute under high-pressure, low-temperature conditions

• Stress response: Similar to what has been observed in P. aeruginosa, proper G3P concentration may influence oxidative stress tolerance

Research approaches to investigate this question should include:

• Creation of gpsA mutants in P. profundum with subsequent growth and metabolomic analysis at different pressures

• Transcriptomic profiling of wild-type and gpsA mutants under varying pressure conditions

• Lipidomic analysis to determine changes in membrane composition in response to pressure and gpsA mutation

Is P. profundum gpsA regulated by ToxR, and how might this relate to pressure-responsive gene expression?

Based on the search results, ToxR in P. profundum SS9 has been identified as necessary for pressure-responsive gene expression . To determine if gpsA is regulated by ToxR:

• Comparative transcriptomics: RNA sequencing of wild-type and toxR mutant strains at different pressures could reveal if gpsA expression is ToxR-dependent

• RNA arbitrarily primed PCR (RAP-PCR): This technique has successfully identified ToxR-regulated genes in P. profundum SS9

• Promoter analysis: Identification of potential ToxR binding sites in the gpsA promoter region through bioinformatic analysis and DNA-binding assays

• Reporter gene assays: Construction of gpsA promoter-reporter fusions to monitor expression in wild-type and toxR mutant backgrounds

Previous studies have identified seven ToxR-activated transcripts and one ToxR-repressed transcript in P. profundum SS9, with the regulated genes falling into categories related to membrane structure alteration and starvation response . If gpsA is indeed regulated by ToxR, this would suggest a coordinated response linking glycerol metabolism to membrane composition adaptation under different pressure conditions.

What are the most effective purification strategies for obtaining active recombinant P. profundum gpsA?

For successful purification of active recombinant P. profundum gpsA, a methodical approach should include:

• Affinity tag selection: His6-tag is commonly used, but consider alternative tags (MBP, GST) if solubility issues arise

• Cold purification: Conduct all purification steps at 4-10°C to maintain stability of this psychrotolerant enzyme

• Buffer optimization: Include:

  • Osmolytes (glycerol 5-10%, NaCl 100-300mM)

  • Reducing agents (DTT or β-mercaptoethanol) to protect cysteine residues

  • pH optimization (typically pH 7.0-8.0)

  • Stabilizing cofactors (NAD+ or NADP+)

• Chromatographic steps:

  • IMAC (Immobilized Metal Affinity Chromatography) as initial capture step

  • Ion exchange chromatography for intermediate purification

  • Size exclusion chromatography as polishing step

• Activity preservation: Add glycerol (20-30%) to storage buffer and store at -80°C in small aliquots

A suggested purification protocol based on the properties of P. profundum:

What assay methods are most suitable for measuring P. profundum gpsA activity under varying pressure conditions?

To accurately measure P. profundum gpsA activity under varying pressure conditions, researchers should consider the following methodological approaches:

• Spectrophotometric assays under pressure:

  • Use specialized high-pressure optical cells connected to a spectrophotometer

  • Monitor NAD(P)H formation/consumption at 340 nm

  • Calculate activity by determining the rate of absorbance change

• Stopped-flow techniques for rapid kinetics:

  • Conduct reactions under pressure, then rapidly decompress for measurement

  • Useful for determining initial reaction rates

• High-pressure enzyme reactors:

  • Conduct reactions in pressure vessels, removing samples at defined intervals

  • Analyze product formation using HPLC or coupled enzyme assays

• Fluorescence-based assays:

  • Utilize fluorescent NAD(P)H or fluorescently-labeled substrates

  • Can be more sensitive than absorbance measurements

• Nuclear Magnetic Resonance (NMR) spectroscopy:

  • Use high-pressure NMR tubes to monitor reactions in real-time

  • Provides detailed information about reaction intermediates

Standardized assay conditions for meaningful comparisons:

How should researchers interpret discrepancies between in vitro and in vivo findings when studying P. profundum gpsA function?

When encountering discrepancies between in vitro and in vivo findings related to P. profundum gpsA function, researchers should systematically evaluate:

• Environmental context differences:

  • In vitro assays often lack the complex physiological environment found in cells

  • P. profundum's natural deep-sea environment includes high pressure, low temperature, and specific ionic conditions that may be difficult to fully replicate in vitro

• Methodological considerations:

  • Evaluate whether experimental conditions (buffer composition, pH, temperature, pressure) adequately mimic cellular conditions

  • Consider if enzyme modifications during purification (tags, truncations) affect function

• Biological complexity factors:

  • Cellular regulatory mechanisms may modify enzyme activity in vivo

  • Potential involvement of ToxR regulation pathway in modulating gpsA expression under different conditions

  • Interaction with other proteins or metabolites in vivo not present in purified system

• Data interpretation framework:

  • Develop integrated models that account for both in vitro and in vivo observations

  • Use systems biology approaches to contextualize enzymatic data within metabolic networks

Resolution strategies include:

• Refining in vitro conditions to better mimic in vivo environment

• Developing cell-free extract assays as intermediate between purified enzymes and whole cells

• Using genetic approaches (point mutations rather than gene deletions) for more nuanced in vivo studies

• Employing metabolomics to track G3P levels and related metabolites in vivo

What are common technical challenges in working with P. profundum gpsA and how can they be overcome?

When working with P. profundum gpsA, researchers may encounter several technical challenges:

• Protein solubility issues:

  • Challenge: Recombinant expression may lead to inclusion body formation

  • Solution: Lower induction temperature (15-18°C), use solubility-enhancing tags (MBP, SUMO), include osmolytes in buffer

• Enzyme stability concerns:

  • Challenge: Loss of activity during purification or storage

  • Solution: Maintain cofactors (NAD(P)+) in all buffers, include stabilizing agents (glycerol, trehalose), avoid freeze-thaw cycles

• Assay interference:

  • Challenge: Spectrophotometric assays at 340nm may have background interference

  • Solution: Carefully design controls, consider alternative assay methods (fluorescence-based, HPLC)

• Pressure equipment limitations:

  • Challenge: Specialized high-pressure equipment may be limited or costly

  • Solution: Collaborate with geophysics or food science departments that routinely use high-pressure systems, develop simplified pressure application methods

• Expression host compatibility:

  • Challenge: E. coli may not properly fold psychrophilic/piezophilic proteins

  • Solution: Consider cold-adapted expression hosts or cell-free systems

Troubleshooting table for common issues:

How can researchers effectively compare P. profundum gpsA with homologs from other organisms to elucidate pressure-adaptation mechanisms?

To systematically compare P. profundum gpsA with homologs from other organisms for understanding pressure-adaptation mechanisms, researchers should employ a multi-faceted approach:

• Phylogenetic analysis:

  • Construct robust phylogenetic trees of gpsA homologs from diverse bacteria

  • Classify organisms by habitat (deep-sea, shallow marine, terrestrial)

  • Identify instances of convergent evolution in pressure adaptation

• Comparative biochemistry:

  • Measure enzymatic parameters at various pressures for multiple homologs:

    • Km, Vmax, kcat at pressures from 0.1 to 100 MPa

    • Activation volumes (ΔV‡) calculated from pressure effects on kinetics

    • Pressure stability profiles (P50 - pressure at which 50% activity remains)

• Structural comparison:

  • Analyze amino acid composition differences (especially charged vs. hydrophobic residues)

  • Compare volume and compressibility of enzymes

  • Examine differences in secondary structure elements and flexibility

• Molecular dynamics simulations:

  • Model behavior of different homologs under pressure

  • Identify regions with differential responses to pressure

  • Calculate volume fluctuations and compressibility

• Experimental validation:

  • Create chimeric enzymes by domain swapping between homologs

  • Use site-directed mutagenesis to introduce "pressure-adaptive" features into non-piezophilic homologs

  • Test predictions from computational analysis with experimental measurements

A comprehensive comparative framework might include:

How might understanding P. profundum gpsA contribute to biotechnological applications of pressure-adapted enzymes?

Understanding the molecular basis of pressure adaptation in P. profundum gpsA could enable several biotechnological applications:

• Biocatalysis under high-pressure conditions:

  • Development of pressure-stable enzymatic processes for industrial applications

  • Enhanced reactions where high pressure improves selectivity or yield

  • Design of biocatalysts for deep-sea natural product synthesis

• Protein engineering principles:

  • Identification of structural motifs that confer pressure stability

  • Application of these principles to enhance stability of industrially relevant enzymes

  • Creation of design rules for developing pressure-resistant proteins

• Environmental biotechnology:

  • Development of biosensors for deep-sea environments

  • Engineered microorganisms for bioremediation in high-pressure environments

  • Tools for studying deep-sea microbial communities

• Fundamental research tools:

  • Pressure as a perturbation tool to study protein dynamics and function

  • Pressure-stable enzymes as model systems for biophysical studies

  • New insights into protein-solvent interactions under extreme conditions

The study of P. profundum gpsA may reveal molecular adaptations that could be transferred to other enzymes, potentially enabling industrial processes at high pressures where reaction rates or selectivity may be enhanced . The mechanisms elucidated might also inform our understanding of how life adapts to extreme environments, contributing to both fundamental knowledge and applied biotechnology.

What research gaps remain in our understanding of glycerol metabolism in piezophilic bacteria like P. profundum?

Despite advances in understanding P. profundum biology, significant research gaps remain regarding glycerol metabolism in piezophilic bacteria:

• Regulatory networks:

  • How pressure signals are sensed and transduced to regulate metabolic genes

  • Comprehensive characterization of ToxR regulon and its role in glycerol metabolism

  • Integration of pressure, temperature, and nutrient signals in metabolic regulation

• Metabolic flux distribution:

  • How carbon flux through glycerol pathways changes under different pressures

  • Integration of glycerol metabolism with central carbon metabolism under pressure

  • Energetic consequences of pressure on metabolic efficiency

• Comparative metabolism:

  • Differences in glycerol metabolism between piezophilic, piezotolerant, and piezosensitive organisms

  • Evolutionary convergence/divergence in metabolic adaptations to pressure

  • Role of horizontal gene transfer in acquisition of pressure-adapted metabolic genes

• Systems-level understanding:

  • How G3P homeostasis affects global cellular function under pressure

  • Metabolomic profiles across pressure gradients

  • Integration of transcriptomic, proteomic, and metabolomic data

Future research directions should include:

• Development of genetic systems for P. profundum to enable more sophisticated in vivo studies

• Application of high-throughput approaches to study pressure effects on metabolism

• Integration of experimental data with computational modeling to predict metabolic adaptations

How can researchers effectively design experiments to study the role of P. profundum gpsA in pressure adaptation?

To effectively investigate the role of P. profundum gpsA in pressure adaptation, researchers should design experiments that systematically address multiple levels of biological organization:

• Genetic approaches:

  • Create precise gpsA mutants (point mutations, regulated expression)

  • Complement mutations with wild-type or modified gpsA variants

  • Use RNA arbitrarily primed PCR (RAP-PCR) to identify genes co-regulated with gpsA under pressure

• Physiological characterization:

  • Growth curves at various pressures (0.1-70 MPa) and temperatures (0-25°C)

  • Membrane composition analysis under different pressure regimes

  • Stress response characterization (oxidative, osmotic stress tolerance)

• Biochemical analysis:

  • Enzyme activity assays across pressure range

  • Protein stability measurements under pressure

  • Interaction studies with potential protein partners

• Systems biology approaches:

  • Transcriptomics comparing wild-type and gpsA mutants at different pressures

  • Metabolomics focusing on glycerol-related metabolites

  • Flux analysis using isotope labeling

Experimental design considerations should include: