Recombinant Photobacterium profundum Probable inorganic polyphosphate/ATP-NAD kinase (ppnK)

What is Photobacterium profundum and why is its ppnK enzyme significant to research?

Photobacterium profundum is a cosmopolitan marine bacterium capable of growth at low temperatures and high hydrostatic pressures. This organism has been isolated from various ocean depths, with different strains showing remarkable differences in their physiological responses to pressure . The ppnK enzyme (inorganic polyphosphate/ATP-NAD kinase) from P. profundum is particularly significant because it represents an important metabolic enzyme involved in NAD phosphorylation using either ATP or inorganic polyphosphate as phosphoryl donors.

The study of this enzyme provides insights into deep-sea adaptations and unique metabolic pathways that allow organisms to survive in extreme environments. The ability to use inorganic polyphosphate as a phosphoryl donor may represent a crucial adaptation to environments where ATP availability might be limited, such as in deep-sea conditions with high hydrostatic pressure.

How does the ppnK enzyme from P. profundum differ from similar enzymes in other organisms?

The ppnK enzyme from P. profundum belongs to a family of NAD kinases found across various organisms. While comprehensive comparative data specifically for P. profundum ppnK is limited in the provided search results, insights can be drawn from studies of similar enzymes. For instance, in Micrococcus flavus, another organism with inorganic polyphosphate/ATP-NAD kinase, alignment of the primary structure with other characterized NAD kinases revealed specific candidate amino acid residues, mainly charged ones, that would be related to inorganic polyphosphate utilization .

The distinct characteristics of ppnK enzymes across species can be generally summarized as follows:

Further detailed structural analysis would be required to fully characterize the specific adaptations in P. profundum ppnK that might relate to pressure adaptation.

What are the optimal conditions for expressing recombinant P. profundum ppnK in E. coli systems?

When expressing recombinant P. profundum ppnK in E. coli, researchers should consider several key parameters to maximize yield and maintain enzymatic activity:

• Expression Vector Selection: Based on available information, the gene encoding ppnK should be cloned into an expression vector with an appropriate promoter system. For recombinant expression, vectors containing T7 or tac promoters are commonly used .

• Host Strain Optimization: E. coli BL21(DE3) or similar strains designed for protein expression are recommended as they lack certain proteases that could degrade the recombinant protein .

• Induction Parameters: For IPTG-inducible systems, induction should typically be performed when cultures reach mid-log phase (OD600 ~0.6-0.8). For cold-adapted enzymes from deep-sea organisms like P. profundum, lower induction temperatures (15-20°C) may help maintain proper protein folding.

• Buffer Conditions: During purification, consider using buffers that mimic the native environment of P. profundum, potentially including salt concentrations that reflect marine conditions.

An effective experimental design would include control points to verify expression success, such as SDS-PAGE analysis of pre- and post-induction samples, and activity assays to confirm functional enzyme production.

How can researchers design experiments to accurately measure the dual substrate specificity of ppnK?

To accurately measure the dual substrate specificity of ppnK (its ability to use both ATP and inorganic polyphosphate), the following methodological approach is recommended:

• Enzyme Purification Protocol:

  • Express the recombinant enzyme as described above

  • Purify using affinity chromatography (His-tag systems are commonly employed)

  • Verify purity via SDS-PAGE and western blot

  • Perform buffer exchange to remove imidazole or other elution agents that might interfere with activity assays

• Activity Assay Design:

  • Prepare reaction mixtures containing the enzyme, NAD as substrate, and either ATP or inorganic polyphosphate as phosphoryl donors

  • Include appropriate controls: no enzyme, no phosphoryl donor, and heat-inactivated enzyme

  • Monitor NADP+ formation using spectrophotometric methods (absorption at 340 nm) or coupling with NADP+-dependent enzymes

• Comparative Kinetic Analysis:

  • Determine Km and Vmax values for both ATP and polyphosphate as substrates

  • Calculate catalytic efficiency (kcat/Km) for both substrates to quantify preference

  • Analyze the effect of different reaction conditions (pH, temperature, pressure) on substrate preference

Following the principles of sound experimental research design is critical in this process. This includes establishing clear hypotheses, using appropriate controls, ensuring reproducibility, and analyzing data with suitable statistical methods .

What structural features of P. profundum ppnK are responsible for its ability to use inorganic polyphosphate?

While the specific structural features of P. profundum ppnK have not been fully characterized in the provided search results, insights can be drawn from studies of similar enzymes, such as the one from Micrococcus flavus. Analysis of the primary structure alignment revealed several key features potentially associated with polyphosphate utilization:

• Charged Amino Acid Residues: Specific charged amino acids, likely positioned in the active site, play crucial roles in binding and utilizing inorganic polyphosphate . These charged residues create an electrostatic environment suitable for interaction with the negatively charged phosphate groups.

• Potential Binding Domains: Based on homology with other NAD kinases, the enzyme likely contains specialized domains for binding both NAD and polyphosphate substrates.

• Structural Homology: The enzyme shows homology with ATP synthase β chain, suggesting an evolutionary relationship between these phosphoryl transfer enzymes . This homology may provide clues about the mechanism of phosphoryl transfer.

To fully elucidate these structural features, researchers should consider:

• X-ray crystallography studies of the enzyme with bound substrates

• Site-directed mutagenesis of candidate residues to confirm their role

• Molecular dynamics simulations to understand the interaction between the enzyme and polyphosphate

How does hydrostatic pressure affect the structure-function relationship of ppnK from deep-sea versus shallow-water strains of P. profundum?

P. profundum strains isolated from different ocean depths show remarkable differences in their physiological responses to pressure . The deep-sea piezopsychrophilic strain SS9 and the shallow-water non-piezophilic strain 3TCK provide an excellent comparative model system to investigate pressure adaptation.

For ppnK specifically, the structural adaptations that might confer pressure resistance could include:

An experimental approach to investigate these differences would include:

• Comparative enzyme kinetics under varying pressure conditions using specialized high-pressure equipment

• Circular dichroism spectroscopy to monitor structural changes with pressure

• Hydrogen-deuterium exchange mass spectrometry to identify regions with altered flexibility

• Computational analysis of amino acid substitutions between deep-sea and shallow-water variants

Research on the genome sequence differences between these strains has provided clues regarding genetic features required for growth in the deep sea, and these differences range from variations in gene content to specific gene sequences under positive selection .

What evolutionary insights can be gained from comparing ppnK across different Photobacterium species and other marine bacteria?

Comparative analysis of ppnK across different Photobacterium species and other marine bacteria can provide valuable evolutionary insights:

• Phylogenetic Analysis: Constructing phylogenetic trees based on ppnK sequences can reveal evolutionary relationships and potential horizontal gene transfer events among marine bacteria.

• Selective Pressure Analysis: Calculating the ratio of non-synonymous to synonymous substitutions (dN/dS) across different lineages can identify regions under positive selection, potentially associated with environmental adaptation.

• Ecotype Diversification: P. profundum strains from different depths represent distinct ecotypes adapted to specific environmental conditions . Analysis of ppnK variation across these ecotypes can reveal how this enzyme contributed to niche adaptation.

• Functional Convergence: Similar adaptations might have evolved independently in unrelated deep-sea organisms, providing examples of convergent evolution at the molecular level.

The genome plasticity observed between Photobacterium strains suggests that comparative genomics approaches would be particularly valuable for understanding the evolution of metabolic capabilities including polyphosphate utilization.

How does the inorganic polyphosphate isolated from P. profundum compare to that from other bacterial sources in terms of chain length and biological function?

While the search results don't provide specific information about polyphosphate from P. profundum, insights from other organisms like M. flavus can guide research in this area. In M. flavus, inorganic polyphosphate isolated from cells was demonstrated to be used by the inorganic polyphosphate/ATP-NAD kinase as a phosphoryl donor, suggesting physiological significance rather than merely an evolutionary trait .

Key aspects for comparison across bacterial sources would include:

• Chain Length Distribution: Analysis of polyphosphate chain length using gel electrophoresis or HPLC methods.

• Cellular Localization: Determination of where polyphosphate accumulates within the cell (cytoplasmic, membrane-associated, or in specialized structures).

• Synthesis and Degradation Pathways: Identification and characterization of the enzymes involved in polyphosphate metabolism across species.

• Environmental Regulation: Analysis of how environmental factors (pressure, temperature, nutrient availability) affect polyphosphate accumulation and utilization.

A comprehensive analysis would require extraction and characterization of polyphosphate from P. profundum grown under various conditions, followed by comparative analysis with polyphosphate from other marine bacteria from different habitats.

How can ppnK be used as a model system to study enzyme adaptation to extreme environments?

The ppnK enzyme from P. profundum represents an excellent model system for studying enzymatic adaptation to extreme environments, particularly high-pressure deep-sea habitats:

• Comparative Genomics Approach: The availability of genome sequences from both deep-sea (SS9) and shallow-water (3TCK) strains of P. profundum provides a foundation for identifying genetic adaptations. Researchers can:

  • Compare ppnK sequences between strains

  • Analyze surrounding genomic regions for co-evolving genes

  • Identify regulatory elements that might influence expression under different pressure conditions

• Structure-Function Studies: Detailed structural analysis can reveal adaptations specific to pressure tolerance:

  • X-ray crystallography or cryo-EM studies under different pressure conditions

  • Molecular dynamics simulations to identify pressure-sensitive regions

  • Site-directed mutagenesis to convert between pressure-sensitive and pressure-resistant forms

• Experimental Evolution: Laboratory evolution experiments can provide insights into adaptive processes:

  • Subjecting shallow-water strains to gradually increasing pressure

  • Genomic and proteomic analysis of evolved strains

  • Reverse engineering identified mutations into the original strain to confirm their role

• Systems Biology Integration: Positioning ppnK within the broader metabolic network:

  • Metabolic flux analysis under different pressure conditions

  • Transcriptomic and proteomic studies to identify co-regulated pathways

  • In silico modeling of metabolic networks incorporating pressure effects

What are the methodological approaches for investigating the potential biotechnological applications of P. profundum ppnK?

The dual substrate specificity and potential pressure resistance of P. profundum ppnK make it an interesting candidate for biotechnological applications. Methodological approaches to investigate these applications include:

• Enzyme Engineering for Industrial Biocatalysis:

  • Directed evolution to enhance stability or activity

  • Rational design based on structural insights

  • Creation of chimeric enzymes combining beneficial features from different sources

• Development of Biosensors:

  • Coupling ppnK activity to detection systems for NAD/NADP+ ratios

  • Utilizing polyphosphate sensitivity for environmental monitoring applications

  • Engineering enzyme variants with altered substrate specificity for specific detection needs

• High-Pressure Biocatalysis Applications:

  • Testing ppnK activity in high-pressure industrial processes

  • Comparing performance metrics with conventional enzymes

  • Optimizing reaction conditions for industrial-scale applications

• Methodological Workflow for Application Development:

  • Initial characterization of enzymatic parameters

  • Stability testing under various conditions (temperature, pH, pressure)

  • Immobilization studies to enhance reusability

  • Scale-up experiments to evaluate industrial potential

  • Economic and lifecycle assessment of enzyme-based processes

These investigations should follow rigorous experimental research design principles, including appropriate controls, statistical validation, and systematic parameter optimization .

What are the common challenges in purifying active ppnK from recombinant expression systems and how can they be addressed?

Researchers working with recombinant P. profundum ppnK may encounter several technical challenges during protein purification:

• Protein Solubility Issues:

  • Challenge: Formation of inclusion bodies due to misfolding or overexpression

  • Solution: Lower induction temperature (15-20°C), reduce inducer concentration, use solubility-enhancing fusion tags (SUMO, MBP), or optimize codon usage for E. coli

• Maintaining Enzymatic Activity:

  • Challenge: Loss of activity during purification processes

  • Solution: Include stabilizing agents (glycerol, reducing agents), minimize freeze-thaw cycles, and use buffers that mimic the native environment of P. profundum

• Contaminant Phosphatases:

  • Challenge: Co-purification of phosphatases that may interfere with activity assays

  • Solution: Include phosphatase inhibitors during purification, apply additional purification steps, or use phosphatase-deficient expression hosts

• Methodological Approach to Troubleshooting:

  • Systematically vary expression conditions (temperature, time, media composition)

  • Test multiple purification strategies in parallel

  • Perform activity assays at each purification step to track activity loss

  • Consider on-column refolding techniques if inclusion bodies are unavoidable

How should researchers address data inconsistencies when comparing ppnK activity with ATP versus polyphosphate substrates?

When comparing ppnK activity with different phosphoryl donors (ATP versus polyphosphate), researchers may encounter data inconsistencies that require careful methodological consideration:

• Substrate Quality and Standardization:

  • Challenge: Variability in polyphosphate chain length and purity

  • Solution: Characterize polyphosphate preparations (average chain length, polydispersity), use defined fractions when possible, and maintain consistent preparation methods

• Assay Condition Optimization:

  • Challenge: Different optimal conditions for ATP versus polyphosphate utilization

  • Solution: Perform detailed pH and ionic strength profiling for each substrate, establish substrate-specific reaction conditions, and always include appropriate controls

• Data Analysis and Interpretation:

  • Challenge: Complex kinetics with polyphosphate due to heterogeneity

  • Solution: Apply appropriate kinetic models, consider multiple parameters beyond just Vmax and Km, and use global fitting approaches when appropriate

• Experimental Design Considerations:

  • Conduct parallel assays with both substrates under identical conditions

  • Include internal standards to normalize between experiments

  • Perform statistical analysis to determine significance of observed differences

  • Consider the impact of experimental design choices on outcomes

• Validation Approaches:

  • Use complementary assay methods to verify results

  • Test with engineered enzyme variants to confirm mechanistic hypotheses

  • Compare results with enzymes from related organisms as reference points