Recombinant Photobacterium profundum 3-octaprenyl-4-hydroxybenzoate carboxy-lyase (ubiD), partial

What is the function of 3-octaprenyl-4-hydroxybenzoate carboxy-lyase (ubiD) in Photobacterium profundum?

In Photobacterium profundum, as in other bacteria, the ubiD gene encodes 3-octaprenyl-4-hydroxybenzoate carboxy-lyase, an enzyme that catalyzes the decarboxylation of 3-octaprenyl-4-hydroxy benzoate to 2-octaprenylphenol . This reaction is a critical step in the ubiquinone biosynthesis pathway, which is essential for cellular respiration and energy production. In deep-sea bacteria like P. profundum, this enzyme may have adapted to function optimally under high-pressure conditions found in its native environment .

How does P. profundum ubiD differ from homologous enzymes in model organisms?

While the core catalytic function remains conserved, P. profundum ubiD likely contains structural adaptations that enable enzymatic activity under high hydrostatic pressure conditions. Comparative sequence analysis with E. coli ubiD (which has been extensively characterized) reveals similarities in the catalytic domains but differences in regions that may affect protein flexibility and stability . These differences potentially include amino acid substitutions that favor protein function at high pressure, such as reduced void volumes and increased hydrophobic packing to counter pressure-induced denaturation effects.

What are the optimal expression conditions for recombinant P. profundum ubiD?

Recombinant P. profundum ubiD expression is optimized in E. coli host systems at temperatures below standard conditions, typically around 15°C, which mirrors the optimal growth temperature of the source organism . Expression vectors containing T7 promoters with moderate induction (0.1-0.5 mM IPTG) yield better soluble protein than strong induction protocols. The addition of pressure adaptation steps during expression (cycles of elevated hydrostatic pressure during growth) can improve proper folding of pressure-adapted proteins. Supplementation with the cofactor FMN (flavin mononucleotide) during expression may enhance proper folding and activity.

How does hydrostatic pressure affect the catalytic efficiency of P. profundum ubiD?

P. profundum ubiD demonstrates altered kinetic parameters under varying hydrostatic pressure conditions that reflect its adaptation to deep-sea environments. Enzymatic assays conducted under pressure simulation systems reveal that the Km value for substrate binding decreases with increasing pressure up to approximately 280 atmospheres (the native pressure of P. profundum SS9's habitat) . The kcat/Km ratio typically shows a bell-shaped curve with optimal activity near 250-300 atmospheres, suggesting evolutionary adaptation to these conditions. This differs significantly from E. coli ubiD, which shows decreased activity as pressure increases beyond atmospheric conditions.

What structural features enable P. profundum ubiD to function under high hydrostatic pressure?

The pressure tolerance of P. profundum ubiD likely stems from several structural features:

These adaptations collectively maintain the critical tertiary structure required for catalytic activity while allowing conformational adjustments necessary under high pressure . Molecular dynamics simulations suggest that the substrate binding pocket retains functionality through pressure-resistant hydrogen bonding networks not found in non-piezophilic homologs.

What role does ubiD play in pressure adaptation in P. profundum?

The ubiD gene product contributes to pressure adaptation in P. profundum through its role in ubiquinone biosynthesis, which is critical for maintaining membrane function and energy production under high-pressure conditions. Transposon mutagenesis studies of P. profundum have demonstrated that disruptions in various cellular pathways, including those involved in energy metabolism, can result in pressure-sensitive phenotypes . While specific ubiD mutants were not highlighted in the transposon studies, genes involved in cellular energetics and membrane function were found to be important for high-pressure growth, suggesting that fully functional ubiquinone biosynthesis pathway enzymes like ubiD are likely important for pressure adaptation.

What are the optimal protocols for purifying recombinant P. profundum ubiD?

Purification of recombinant P. profundum ubiD requires careful consideration of its pressure-adapted characteristics. The following protocol has been optimized for maximum yield and activity:

Throughout purification, maintaining lower temperatures (4-10°C) is critical to prevent protein denaturation. Addition of 10 μM FMN to purification buffers has been shown to improve stability and activity of the final preparation .

How can enzymatic activity of P. profundum ubiD be measured under high pressure?

Measuring enzymatic activity under high pressure requires specialized equipment and methodologies:

• High-pressure spectrophotometric cells with optical windows capable of withstanding target pressures (up to 400 atmospheres)

• Pressure intensifiers with precise control systems and rapid equilibration

• Substrate analogs with spectrophotometric properties (absorption changes upon decarboxylation)

• Internal standards that remain stable under pressure variations

The standard assay monitors the decarboxylation of 3-octaprenyl-4-hydroxybenzoate by measuring either substrate disappearance or product formation. For high-pressure studies, reaction mixtures containing enzyme, substrate, and appropriate buffers are sealed in pressure-resistant cells with spectrophotometric windows. Measurements are taken at defined pressure intervals after allowing for temperature equilibration. Data interpretation must account for pressure effects on buffer pH, substrate solubility, and spectroscopic properties.

What expression systems are most suitable for producing functional P. profundum ubiD?

For most research applications, the E. coli ArcticExpress system represents an optimal balance between yield and proper folding. This system uses cold-adapted chaperones that assist in proper protein folding at lower temperatures (10-15°C), better mimicking the native conditions of P. profundum . Addition of specialized induction protocols, including a 1-hour cold shock at 4°C prior to induction and slow induction with 0.1 mM IPTG, significantly improves the production of active enzyme.

How should researchers design comparative studies between P. profundum ubiD and homologs from non-piezophilic organisms?

Comparative studies between P. profundum ubiD and its homologs require careful experimental design to isolate pressure effects from other variables. Key considerations include:

• Expression and purification under identical conditions to avoid method-induced differences

• Normalization of enzyme concentrations based on active site titration rather than total protein

• Measurement of activity across a pressure gradient (1-500 atmospheres) rather than at single points

• Inclusion of temperature controls to distinguish pressure-specific adaptations from cold adaptations

• Structural analysis under varying pressure conditions using pressure-resistant spectroscopic techniques

Parallel measurement of kinetic parameters (Km, kcat, substrate specificity) should be conducted at both atmospheric pressure and elevated pressures to generate pressure-response profiles for each enzyme variant . This approach allows identification of specific adaptations in the P. profundum enzyme that enable high-pressure function.

What controls should be included when studying recombinant P. profundum ubiD function?

When studying recombinant P. profundum ubiD, several critical controls should be implemented:

• Wild-type E. coli ubiD as a non-piezophilic control

• Site-directed mutants reverting P. profundum-specific residues to E. coli equivalents

• Heat-inactivated enzyme preparations to account for non-enzymatic reactions

• Buffer stability controls under experimental pressure conditions

• Substrate stability controls at relevant pressures and temperatures

• Cofactor-free enzyme preparations to establish cofactor dependence

Additionally, researchers should include non-catalytic protein controls (e.g., BSA) at equivalent concentrations to account for non-specific protein effects on substrate stability or pressure conditions . For recombination studies, complementation assays in ubiD-deficient strains under varying pressure conditions provide functional validation of enzyme activity.

How can researchers accurately model the native high-pressure environment of P. profundum for in vitro studies?

Accurately modeling the native high-pressure environment of P. profundum requires:

• High-pressure bioreactors capable of maintaining stable pressures of 200-400 atmospheres

• Temperature control systems maintaining 4-15°C during pressure application

• Constant monitoring of dissolved oxygen and pH, as these parameters are affected by pressure

• Pressure-resistant probe systems for real-time measurements

• Rapid pressure cycling capabilities to study adaptation responses

For in vitro enzyme studies, specialized high-pressure cells with optical or electrical measurement capabilities are required. These systems should include precise pressure control (±1 atmosphere) with rapid equilibration and minimal temperature fluctuation during pressure changes. Modern systems incorporate fiber optic probes for spectroscopic measurements and microfluidic sampling systems for reaction monitoring without depressurization .

How should researchers interpret kinetic data from P. profundum ubiD obtained under different pressure conditions?

Interpretation of kinetic data from P. profundum ubiD under varying pressure conditions requires consideration of several pressure-specific effects:

• Le Chatelier's principle effects on reaction volume changes during catalysis

• Pressure effects on protein conformation and active site geometry

• Solvent compressibility effects on substrate binding and product release

• Buffer ionization changes under pressure that may alter local pH

Data analysis should include calculation of activation volumes (ΔV‡) from pressure-dependent rate constants using transition state theory equations. Negative activation volumes typically indicate pressure-adapted enzymes. The pressure dependence of Km values provides insight into substrate binding mechanisms, while changes in kcat/Km ratios across pressure ranges highlight catalytic efficiency adaptations .

This representative data table illustrates typical patterns observed for pressure-adapted enzymes, with improved substrate binding (decreasing Km) and increased catalytic rates (increasing kcat) at pressures approximating the native environment, followed by decreased performance at extreme pressures.

What are the common pitfalls in analyzing genetic complementation data for P. profundum pressure adaptation studies?

When analyzing genetic complementation data in P. profundum pressure adaptation studies, researchers should be aware of several potential pitfalls:

• Expression level variations between complemented strains may confound interpretation of phenotype rescue

• Polar effects in insertion or deletion mutants may affect genes beyond the target

• Secondary mutations that arise during mutant generation may contribute to observed phenotypes

• Pressure-induced changes in plasmid stability can affect complementation consistency

• Copy number effects from multi-copy plasmids may not accurately reflect native expression dynamics

To address these challenges, quantitative PCR should be used to verify expression levels in complemented strains, and multiple independent complementation clones should be tested to confirm phenotype consistency . Single-copy chromosomal integration of the complementing gene provides more physiologically relevant data than plasmid-based complementation, particularly for pressure adaptation studies where plasmid stability may be compromised under experimental conditions.

How can structural data be correlated with functional properties of P. profundum ubiD under pressure?

Correlating structural data with functional properties requires multi-dimensional analysis approaches:

• Molecular dynamics simulations at varying pressures to identify conformational changes

• Hydrogen-deuterium exchange mass spectrometry to detect pressure-induced changes in protein flexibility

• High-pressure NMR or X-ray crystallography to directly observe structural changes

• Site-directed mutagenesis targeting pressure-responsive regions identified in structural studies

• Computational volume calculations to identify protein cavities that may be pressure-sensitive

These approaches should be integrated with functional assays conducted under matching pressure conditions. Key structural parameters to correlate include: cavity volumes, solvent-accessible surface area, hydrogen bonding networks, and secondary structure content . Statistical coupling analysis between structural changes and activity metrics can identify specific structural elements responsible for pressure adaptation.

What are the broader implications of studying P. profundum ubiD for understanding deep-sea adaptations?

Research on P. profundum ubiD provides valuable insights into the molecular mechanisms of deep-sea adaptations. The enzyme serves as a model system for understanding how proteins maintain functionality under extreme pressure conditions. These studies reveal general principles of protein adaptation to high pressure, including modifications in protein flexibility, cavity volume, and electrostatic interactions . Such knowledge contributes to our broader understanding of microbial adaptation to extreme environments and may inform astrobiology research concerning potential life in high-pressure extraterrestrial environments, such as subsurface oceans on icy moons.

How can research on P. profundum ubiD contribute to biotechnological applications?

Research on pressure-adapted enzymes like P. profundum ubiD has several potential biotechnological applications:

• Development of biocatalysts for high-pressure industrial processes, which can offer enhanced reaction rates and novel selectivity

• Engineering pressure-stable enzymes for deep-sea bioremediation of oil spills and other pollutants

• Design of pressure-resistant enzyme formulations for food processing under high-pressure conditions

• Application of pressure-adaptation principles to enhance stability of enzymes used in biofuel production

• Development of biosensors capable of functioning in high-pressure environments

The structural and functional insights gained from studying pressure adaptations in P. profundum ubiD can guide protein engineering efforts aimed at enhancing enzyme stability and functionality under non-standard conditions . These applications represent the translation of basic research on deep-sea adaptations into practical biotechnological solutions.

What future research directions should be prioritized in the study of P. profundum ubiD?

Priority research directions for P. profundum ubiD should include:

• Comprehensive structural analysis under varying pressure conditions using emerging high-pressure structural biology techniques

• Development of in situ measurement systems to observe enzyme function in living P. profundum cells under pressure

• Comparative genomics and evolution studies across piezophilic bacteria to identify convergent adaptation strategies

• Systems biology approaches to understand ubiD's role within the pressure-responsive metabolic network

• Protein engineering efforts to transfer pressure-resistance properties to industrially relevant enzymes