Recombinant Photobacterium profundum 2-dehydro-3-deoxyphosphooctonate aldolase (kdsA)

What is 2-dehydro-3-deoxyphosphooctonate aldolase (kdsA) and what role does it play in Photobacterium profundum?

2-dehydro-3-deoxyphosphooctonate aldolase (kdsA) belongs to the family of lyases, specifically aldehyde-lyases, which cleave carbon-carbon bonds . In bacterial systems, this enzyme catalyzes a critical step in the biosynthesis of 3-deoxy-D-manno-octulosonic acid (KDO), an essential component of lipopolysaccharides in Gram-negative bacterial cell walls. In Photobacterium profundum, a psychrohalophilic bacterium isolated from deep-sea environments, kdsA would likely exhibit adaptations allowing it to function optimally under high-pressure and low-temperature conditions, similar to other characterized enzymes from this organism .

The research approach to characterize kdsA function in P. profundum should include:

• Comparative sequence analysis with mesophilic homologs

• Activity assays under varying pressure conditions (1-280 atm)

• Temperature-dependent kinetic analysis (4-25°C optimal range)

• Structural analysis to identify pressure-adaptive features

How do the structural features of P. profundum kdsA differ from mesophilic bacterial homologs?

While the specific structure of P. profundum kdsA hasn't been directly characterized in the available literature, insights can be drawn from other P. profundum enzymes. Based on the crystal structure analysis of psychrohalophilic α-carbonic anhydrase from P. profundum, we can anticipate several structural adaptations in kdsA:

• Conservation of catalytic core residues essential for function

• Potential modifications in surface residues to enhance flexibility

• Possible unique ionic interactions in oligomerization interfaces

• Structural modifications that permit function under high hydrostatic pressure

For instance, the P. profundum α-carbonic anhydrase revealed a unique chloride ion in its dimer interface not observed in other α-CAs, which may contribute to its pressure adaptation . Similar unique features might be present in P. profundum kdsA to facilitate enzymatic function in the deep-sea environment.

What techniques are most appropriate for assessing the catalytic activity of recombinant P. profundum kdsA?

Methodological approaches should include:

• Spectrophotometric assays with modified conditions:

  • Buffer systems containing 2-3% NaCl to mimic marine conditions

  • Temperature range testing from 4-25°C

  • High-pressure vessel experiments at 1-280 atm

• Coupled enzyme assays measuring product formation rates:

  • Thiobarbituric acid method for KDO detection

  • Phosphate release quantification for reaction monitoring

• Comparative activity analysis with mesophilic homologs:

  • Parallel testing of E. coli kdsA under identical conditions

  • Normalization of specific activity to protein concentration

When designing experiments, researchers should incorporate controls for pressure-dependent effects by using established pressure-sensitive and pressure-insensitive enzymes as benchmarks, similar to approaches used in P. profundum RecD function studies .

What expression systems yield optimal results for recombinant P. profundum kdsA production?

Based on general recombinant kdsA expression patterns, the following expression systems can be employed with their respective advantages:

For initial characterization, E. coli systems offer the most efficient approach, while more complex expression systems may be necessary if post-translational modifications affect catalytic activity . When adapting protocols from P. profundum α-carbonic anhydrase studies, consider:

• Using pET28a vectors with N-terminal His-tags for simplified purification

• Excluding signal peptide regions in construct design

• Optimizing induction conditions with IPTG at lower temperatures (15-18°C)

• Supplementing growth media with additional Zn²⁺ if required for proper folding

What purification protocol maximizes yield and activity retention of recombinant P. profundum kdsA?

A multi-step purification protocol based on successful approaches with other P. profundum enzymes should include:

Critical considerations specific to P. profundum enzymes include:

• Maintaining cold temperatures (4°C) throughout purification

• Including salt (2-3% NaCl) in all buffers to mimic marine conditions

• Assessing oligomerization state by size exclusion chromatography, as P. profundum enzymes may exhibit heterogeneous oligomeric states (monomers/dimers)

• Confirming activity after each purification step using an appropriate activity assay

How can the oligomerization state of P. profundum kdsA be accurately determined and controlled?

Based on observations with other P. profundum enzymes like α-carbonic anhydrase, which exists as a heterogeneous mixture of monomers and dimers in solution:

• Analytical approaches:

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS)

  • Analytical ultracentrifugation under varying salt and pressure conditions

  • Native PAGE with in-gel activity assays (protonography for appropriate enzymes)

• Controlling oligomerization:

  • Buffer ionic strength manipulation (varying NaCl concentration from 0-500 mM)

  • Addition of stabilizing agents specific to oligomeric interfaces

  • Site-directed mutagenesis of predicted interface residues

When analyzing oligomerization, consider that P. profundum enzymes may have unique interface features, such as the chloride ion observed in the dimer interface of P. profundum α-carbonic anhydrase, which could affect stability under different conditions .

How does hydrostatic pressure affect the kinetic parameters of P. profundum kdsA?

Methodological approach for pressure-dependence studies:

• Use specialized high-pressure vessels equipped with quick-connect fittings for rapid sampling and repressurization

• Conduct enzyme assays at pressure ranges from 1 to 280 atm, focusing on:

  • Changes in Km and kcat values

  • Substrate binding efficiency (kcat/Km)

  • Reaction activation volume determination

• Expected trends based on P. profundum's natural habitat:

  • Likely optimal activity at 200-280 atm pressure

  • Potential pressure-dependent oligomerization effects

  • Possible reduced activity at atmospheric pressure

Table: Hypothetical Pressure Effects on P. profundum kdsA Kinetic Parameters

What molecular mechanisms might contribute to pressure adaptation in P. profundum kdsA?

Based on structural studies of other pressure-adapted proteins, investigate:

• Amino acid composition analysis:

  • Reduced number of hydrophobic residues in the protein core

  • Increased presence of charged residues on the protein surface

  • Modified packing density in the protein interior

• Molecular dynamics simulations:

  • Conformational flexibility analysis under different pressure conditions

  • Calculation of void volumes within the protein structure

  • Water molecule penetration patterns under pressure

• Comparative structural analysis:

  • X-ray crystallography at ambient pressure

  • High-pressure cryocrystallography (if available)

  • Identification of unique structural features like the chloride ion binding observed in P. profundum α-carbonic anhydrase

How can researchers design control experiments to validate pressure-dependent effects on P. profundum kdsA?

Robust experimental design should include:

• Positive controls:

  • E. coli RecD protein with known pressure-sensitive phenotype

  • P. profundum α-carbonic anhydrase with established pressure response

• Negative controls:

  • Mesophilic kdsA homologs from E. coli or other non-pressure adapted bacteria

  • Pressure-insensitive enzymes like alkaline phosphatase

• Complementation studies:

  • Expression of P. profundum kdsA in pressure-sensitive E. coli strains

  • Growth assays under various pressure conditions

  • Analysis of lipopolysaccharide composition in complemented strains

Control experiments should follow methodologies established in P. profundum RecD studies, which demonstrated restoration of high-pressure growth phenotypes through complementation .

What crystallization methods are most effective for obtaining diffraction-quality crystals of P. profundum kdsA?

Based on successful crystallization of other P. profundum enzymes:

• Initial screening approach:

  • Vapor diffusion (sitting drop method) with commercial screening kits

  • Temperature range testing (4°C, 10°C, and 15°C)

  • Inclusion of salt conditions mimicking marine environment (2-3% NaCl)

• Optimization strategies:

  • Fine-tuning precipitant concentration and pH

  • Addition of additives like divalent metal ions (Zn²⁺, Mg²⁺)

  • Seeding techniques for improving crystal quality

• Alternative approaches if traditional methods fail:

  • Lipidic cubic phase crystallization for membrane-associated variants

  • In situ high-pressure crystallization to capture native conformations

  • Surface entropy reduction through site-directed mutagenesis

When examining crystal packing and interfaces, pay particular attention to unique features like the chloride ion observed in P. profundum α-carbonic anhydrase dimer interface, which may play functional roles specific to pressure adaptation .

How can molecular dynamics simulations contribute to understanding pressure effects on P. profundum kdsA structure and function?

Computational approaches should include:

• Simulation parameters:

  • All-atom molecular dynamics under varying pressure conditions (1-300 atm)

  • Explicit solvent model with appropriate salt concentration

  • Minimum simulation time of 100-500 ns for equilibration

• Analysis metrics:

  • Root mean square deviation (RMSD) and fluctuation (RMSF)

  • Solvent accessible surface area changes

  • Void volume calculations using programs like POVME or CAVER

  • Salt bridge and hydrogen bond network dynamics

• Key predictions:

  • Identification of pressure-sensitive regions within the protein structure

  • Calculation of compressibility factors

  • Prediction of pressure-dependent conformational changes

  • Assessment of water penetration into protein core

As demonstrated in P. profundum α-carbonic anhydrase studies, molecular dynamics can reveal important features like ion occupancy in protein interfaces that contribute to pressure adaptation .

What spectroscopic techniques provide insight into P. profundum kdsA conformational changes under pressure?

Complementary biophysical approaches include:

• High-pressure spectroscopy:

  • Circular dichroism with high-pressure cells to monitor secondary structure

  • Fluorescence spectroscopy to assess tertiary structure changes

  • FTIR spectroscopy for hydrogen bonding network analysis

• Advanced techniques:

  • High-pressure NMR for residue-specific conformational changes

  • Small-angle X-ray scattering (SAXS) to monitor oligomerization state

  • Hydrogen-deuterium exchange mass spectrometry to identify flexible regions

• Real-time kinetic measurements:

  • Stopped-flow apparatus modified for high-pressure conditions

  • Rapid-mixing experiments to capture transient states

  • Temperature-jump combined with pressure perturbation

Data interpretation should focus on comparing conformational stability at deep-sea relevant pressures (200-280 atm) versus ambient conditions, with careful consideration of salt effects that might influence pressure adaptation mechanisms.

How should researchers interpret kinetic data for P. profundum kdsA showing non-linear pressure dependence?

Methodological approach to data analysis:

• Mathematical models for non-linear pressure effects:

  • Fitting data to polynomial functions to identify pressure optimum

  • Calculation of activation volume (ΔV‡) from pressure-dependent rate constants

  • Thermodynamic analysis using transition state theory

• Biological interpretation strategies:

  • Correlation with natural habitat conditions (P. profundum SS9 isolated from 2.5 km depth)

  • Comparison with other deep-sea enzymes showing similar adaptations

  • Structure-function relationship analysis based on identified pressure-sensitive regions

• Statistical considerations:

  • Minimum triplicate measurements at each pressure point

  • Appropriate error analysis and propagation through derived parameters

  • Control experiments at standardized conditions for cross-comparison

What research applications would benefit from studying P. profundum kdsA adaptations?

Understanding pressure adaptation in P. profundum kdsA has implications for:

• Fundamental science:

  • Evolutionary mechanisms of enzyme adaptation to extreme environments

  • Structure-function relationships under non-standard conditions

  • Molecular basis of pressure effects on protein dynamics

• Biotechnological applications:

  • Engineering pressure-stable enzymes for industrial biocatalysis

  • Development of biosensors functional in high-pressure environments

  • Designing biocatalysts with enhanced stability for various applications

• Experimental approaches:

  • Comparative studies between psychrohalophilic and mesophilic homologs

  • Site-directed mutagenesis to identify key residues conferring pressure adaptation

  • Domain swapping experiments to transfer pressure tolerance to other enzymes

How can researchers resolve contradictory results when comparing recombinant P. profundum kdsA expressed in different systems?

Systematic troubleshooting approach:

• Source verification:

  • Confirm sequence identity between constructs used in different expression systems

  • Verify absence of mutations introduced during cloning

  • Check for expression vector effects on protein production

• Post-translational modification analysis:

  • Mass spectrometry to identify modifications present in different expression systems

  • Phosphorylation, glycosylation, or other modification site mapping

  • Correlation of modifications with activity differences

• Experimental standardization:

  • Normalize enzyme activity to active site concentration rather than total protein

  • Standardize assay conditions (temperature, pH, pressure, salt concentration)

  • Develop a consensus protocol incorporating controls for system-specific effects

When comparing results across systems, consider that different expression hosts may introduce system-specific modifications that affect enzyme function, as noted in the varying posttranslational modifications observed between expression systems for recombinant proteins .