Recombinant Photobacterium profundum Dephospho-CoA kinase (coaE)

What is Photobacterium profundum and why is its DPCK enzyme of interest?

Photobacterium profundum is a deep-sea Gammaproteobacterium belonging to the family Vibrionaceae. It is a gram-negative rod with unique growth capabilities at temperatures ranging from 0°C to 25°C and pressures from 0.1 MPa to 70 MPa depending on the strain . The bacterium was originally isolated from the Sulu Sea in 1986 and currently has four cultured wild-type strains: SS9, 3TCK, DJS4, and 1230 . The DPCK enzyme from P. profundum is of particular interest because it functions under extreme conditions (high pressure, low temperature) while catalyzing an essential metabolic reaction, making it a valuable model for studying enzymatic adaptation to extreme environments.

How does P. profundum DPCK function within the CoA biosynthetic pathway?

P. profundum DPCK catalyzes the final step in coenzyme A biosynthesis, specifically the phosphorylation of the 3′-hydroxy group of the ribose sugar moiety in dephospho-CoA . This reaction is ATP-dependent and produces the metabolically active form of CoA. CoA is an essential cofactor utilized by approximately 4% of all enzymes, participating in various biochemical pathways including fatty acid metabolism, the citric acid cycle, and amino acid metabolism . Within P. profundum, the CoA biosynthetic pathway likely plays a crucial role in adaptation to deep-sea conditions, as changes in membrane fatty acid composition have been observed in response to varying pressure and temperature conditions .

What expression systems are recommended for producing recombinant P. profundum DPCK?

For expressing recombinant P. profundum DPCK, an Escherichia coli expression system is recommended based on successful precedents with similar enzymes. When designing your expression system, consider using pET vectors with an N-terminal histidine tag to facilitate purification. E. coli BL21(DE3) strain is particularly suitable as it lacks certain proteases that might degrade your recombinant protein. Culture conditions should be optimized at lower temperatures (15-20°C) after induction to enhance proper folding of P. profundum proteins, which naturally function at lower temperatures . For instance, in similar studies with P. falciparum DPCK and E. coli dephospho-CoA kinase, researchers successfully employed E. coli expression systems to produce functional recombinant enzymes with good yields and purity .

What are the typical yields and purification methods for recombinant P. profundum DPCK?

Recombinant P. profundum DPCK can typically be purified to 90-95% homogeneity using a combination of affinity chromatography and size-exclusion techniques. If designed with a histidine tag, immobilized metal affinity chromatography (IMAC) using Ni-NTA resin should be your primary purification step. This can be followed by size-exclusion chromatography to remove aggregates and achieve higher purity. Expected yields vary between 5-15 mg/L of bacterial culture, depending on expression conditions. The purity can be assessed using SDS-PAGE with Coomassie brilliant blue staining, as demonstrated in similar studies with other DPCKs . The recombinant enzyme should appear as a single band with a molecular mass of approximately 23-32 kDa, consistent with the predicted molecular weight plus any fusion tags .

How can the kinetic parameters of recombinant P. profundum DPCK be determined under varying pressure conditions?

Determining kinetic parameters of P. profundum DPCK under varying pressure conditions requires specialized high-pressure equipment and careful experimental design. A high-pressure stopped-flow apparatus coupled with spectrophotometric detection can measure initial reaction rates at pressures up to 100 MPa. Enzymatic activity should be measured using a coupled assay system where ADP formation is linked to NADH oxidation via pyruvate kinase and lactate dehydrogenase, allowing real-time monitoring at 340 nm.

For practical implementation, prepare reaction mixtures containing:

• 50 mM HEPES buffer (pH 7.5)

• 10 mM MgCl₂

• 50 mM KCl

• 1 mM DTT

• Varying concentrations of dephospho-CoA (10-500 μM)

• Varying concentrations of ATP (10-500 μM)

• Coupling enzymes (pyruvate kinase, lactate dehydrogenase)

• 0.2 mM NADH

• 1 mM phosphoenolpyruvate

Data should be fitted to appropriate enzyme kinetic models (Michaelis-Menten, Hill, etc.) to determine Km, Vmax, and kcat values at different pressure points. Compare results with the known pressure adaptations of P. profundum strain SS9, which has optimal growth at 28 MPa .

What structural adaptations enable P. profundum DPCK to function under high-pressure deep-sea conditions?

P. profundum DPCK likely possesses specific structural adaptations that enable function under high-pressure conditions found in the deep sea. Research suggests these adaptations may include:

• Increased flexibility in the active site region to maintain catalytic efficiency under compression

• Modified surface charge distribution to optimize protein-solvent interactions at high pressure

• Reduced cavity volumes within the protein structure

• Strategic positioning of hydrophobic residues to maintain structural integrity

To investigate these adaptations, employ comparative structural biology approaches:

Correlate findings with known pressure-responsive genes in P. profundum, such as the stress response genes (htpG, dnaK, dnaJ, and groEL) that are upregulated at atmospheric pressure .

How does temperature affect the substrate specificity of P. profundum DPCK compared to mesophilic counterparts?

The substrate specificity of P. profundum DPCK likely shows temperature-dependent variations compared to mesophilic homologs due to its adaptation to cold deep-sea environments. To investigate this:

• Conduct substrate specificity assays at multiple temperatures (4°C, 10°C, 15°C, 25°C) using various substrates including:

  • Dephospho-CoA (primary substrate)

  • Structural analogs (e.g., adenosine, AMP, adenosine phosphosulfate)

  • Modified dephospho-CoA molecules with alterations to the pantetheine moiety

• For each substrate, determine kinetic parameters:

  $K_m(T) = K_m(T_0) \cdot e^{[\frac{-\Delta H_{app}}{R}(\frac{1}{T} - \frac{1}{T_0})]}$

  $k_{cat}(T) = k_{cat}(T_0) \cdot e^{[\frac{-E_a}{R}(\frac{1}{T} - \frac{1}{T_0})]}$

• Compare results with mesophilic DPCK enzymes, such as the E. coli homolog which shows 4-8% activity with alternative substrates like adenosine, AMP, and adenosine phosphosulfate compared to dephospho-CoA .

The expected outcome is a comprehensive temperature-activity profile that may reveal substrate preference shifts at lower temperatures, reflecting cold adaptation mechanisms. P. profundum DPCK may show broader substrate specificity at lower temperatures compared to mesophilic counterparts, as cold-adapted enzymes often feature more flexible active sites to maintain catalytic efficiency at reduced temperatures.

What approaches should be used to identify inhibitors specific to P. profundum DPCK?

To identify inhibitors specific to P. profundum DPCK while avoiding cross-reactivity with human COASY (which contains the DPCK domain), implement a multi-phase screening approach:

• Primary High-Throughput Screen:

  • Develop a 384-well format fluorescence-based assay measuring ATP consumption or CoA production

  • Screen diverse compound libraries (5,000-200,000 compounds)

  • Set threshold for hit selection (>50% inhibition at 10 μM)

• Secondary Validation:

  • Confirm hits with dose-response curves (IC₅₀ determination)

  • Counter-screen against human COASY to identify selective compounds

  • Evaluate compound selectivity index (SI = IC₅₀ human/IC₅₀ P. profundum)

• Mechanistic Characterization:

  • Determine inhibition mechanism (competitive, non-competitive, uncompetitive)

  • Perform kinetic analysis varying both substrate and inhibitor concentrations

  • Calculate Ki values and establish structure-activity relationships

This approach mirrors successful screening strategies used for P. falciparum DPCK, where researchers identified selective inhibitors that did not affect the human ortholog . The divergence between bacterial DPCKs and human COASY (typically <25% sequence identity) provides a basis for selectivity .

How can molecular biology approaches be used to investigate the physiological role of DPCK in P. profundum adaptation to deep-sea conditions?

To investigate DPCK's role in P. profundum adaptation to deep-sea conditions, implement these molecular biology approaches:

• Gene Expression Analysis:

  • Quantify coaE expression using RT-qPCR under varying pressure (0.1-70 MPa) and temperature (4-25°C) conditions

  • Perform RNA-seq to identify co-regulated genes in the CoA pathway

  • Compare expression patterns between pressure-adapted strain SS9 (optimal at 28 MPa) and surface-adapted strain 3TCK (optimal at 0.1 MPa)

• Genetic Manipulation:

  • Create coaE conditional knockdown strains using inducible antisense RNA

  • Engineer strains with coaE variants containing point mutations in pressure-responsive regions

  • Complement knockdown strains with wild-type or mutant coaE genes

• Physiological Assessment:

  • Monitor growth rates of engineered strains under varying pressure/temperature conditions

  • Analyze cellular CoA levels using LC-MS/MS

  • Measure membrane fluidity changes using fluorescence anisotropy

  • Quantify expression of known pressure-responsive genes (htpG, dnaK, dnaJ, groEL)

• Comparative Proteomic Analysis:

  • Perform differential proteomics comparing wild-type and coaE-modified strains

  • Identify proteins with altered abundance or post-translational modifications

  • Construct protein-protein interaction networks centered on DPCK

These approaches should reveal whether DPCK plays a direct role in pressure adaptation or primarily maintains essential CoA levels under varying environmental conditions.

What are the common challenges in expressing and purifying active recombinant P. profundum DPCK?

Expressing and purifying active recombinant P. profundum DPCK presents several challenges that require specific technical solutions:

Additionally, expression constructs should be codon-optimized for E. coli, as P. profundum may have different codon preferences. Include protease inhibitors in lysis buffers to prevent degradation, and consider using specialized E. coli strains like Arctic Express that co-express cold-adapted chaperonins to assist with proper folding of psychrophilic proteins.

How can the stability of P. profundum DPCK be enhanced for structural studies?

Enhancing the stability of P. profundum DPCK for structural studies requires multiple approaches targeting the inherent flexibility of psychrophilic enzymes:

• Buffer Optimization:
  Conduct differential scanning fluorimetry (DSF) screening with a matrix of conditions:

  Buffer Type	pH Range	Salt Concentration	Additives
  HEPES	7.0-8.0	50-300 mM NaCl	5-20% glycerol
  Tris	7.5-8.5	100-500 mM NaCl	1-5 mM TCEP
  Phosphate	6.5-7.5	50-200 mM KCl	1-10% sucrose
  MES	6.0-7.0	50-300 mM NaCl	0.5-5 mM MgCl₂

• Ligand-Induced Stabilization:

  • Include substrate analogs or non-hydrolyzable ATP analogs (AMPPNP)

  • Add product (CoA) at 0.5-1 mM concentration

  • Screen commercially available stabilizing compounds (e.g., NDSB series)

• Protein Engineering Approaches:

  • Identify and mutate surface-exposed flexible loops based on molecular dynamics simulations

  • Introduce disulfide bridges at strategic positions to rigidify the structure

  • Create fusion constructs with well-folded, crystallizable proteins (e.g., T4 lysozyme)

• Crystallization Enhancements:

  • Use automated nanoliter-scale crystallization screening with 500+ conditions

  • Implement seeding techniques from initial microcrystals

  • Explore counter-diffusion crystallization methods in capillaries

  • Consider lipidic cubic phase crystallization for challenging proteins

These approaches have been successfully employed for other psychrophilic enzymes and challenging proteins like membrane-associated enzymes and should be adaptable to P. profundum DPCK .

What are the optimal assay conditions for measuring P. profundum DPCK activity across various pressures and temperatures?

For accurate measurement of P. profundum DPCK activity across various pressures and temperatures, optimize these assay parameters:

• Basic Assay Components:

  • Buffer: 50 mM HEPES (pH 7.5 at assay temperature)

  • Divalent cations: 5-10 mM MgCl₂ (primary) or 2-5 mM MnCl₂ (alternative)

  • Salt: 50-100 mM KCl

  • Reducing agent: 1-5 mM DTT or TCEP

  • Substrates: 100-200 μM dephospho-CoA, 0.5-1 mM ATP

• Temperature Considerations (4-25°C):

  • Pre-equilibrate all components at target temperature

  • Adjust pH of buffers at each temperature (consider using temperature-independent buffers)

  • Increase enzyme concentration at lower temperatures to maintain detectable activity

  • Extended reaction times may be needed at lower temperatures (15-60 minutes)

• Pressure Adaptations (0.1-70 MPa):

  • Use specialized high-pressure vessels with optical windows for real-time measurements

  • Prepare pressure-resistant reaction chambers with flexible barriers

  • Include pressure-stable fluorescent reporters

  • Account for pressure effects on pH (approximately 0.014 pH units/100 MPa)

• Detection Methods Across Conditions:

  Method	Advantages	Limitations	Pressure Compatibility
  Coupled enzyme assay	Real-time monitoring	Secondary enzymes may be pressure-sensitive	Limited to moderate pressures
  Direct CoA detection (DTNB)	Simple, direct	Lower sensitivity	Good across pressure range
  Radioactive assay (³²P-ATP)	High sensitivity	Requires special handling	Excellent at all pressures
  HPLC/LC-MS	Definitive product identification	Endpoint only	Requires post-pressure analysis

• Controls and Validations:

  • Run parallel assays with E. coli DPCK as a mesophilic reference

  • Include enzyme-free and substrate-free controls

  • Validate linear range of assay at each condition

  • Perform recovery experiments at atmospheric pressure after high-pressure exposure

These methodologies will provide reliable activity measurements across the environmental range relevant to P. profundum DPCK's native conditions .

How does P. profundum DPCK compare structurally and functionally to DPCK enzymes from other extremophiles?

P. profundum DPCK likely shares common adaptation strategies with other extremophile DPCKs while maintaining distinctive features related to its specific deep-sea environment:

• Structural Comparisons:

  Extremophile Source	Adaptation Type	Key Structural Features	Similarity to P. profundum DPCK
  Psychrophiles (e.g., Antarctic bacteria)	Cold adaptation	Reduced core hydrophobicity; increased surface charge; more glycine residues	High - similar temperature adaptations
  Piezophiles (e.g., Mariana Trench isolates)	Pressure adaptation	Reduced void volumes; pressure-resistant secondary structures	Very high - shared pressure adaptations
  Thermophiles (e.g., Aquifex aeolicus)	Heat adaptation	Increased disulfide bonds; more salt bridges; rigidified structures	Low - opposite temperature adaptation
  Halophiles (e.g., Dead Sea archaea)	Salt adaptation	Negative surface charge; reduced hydrophobic exposure	Moderate - some shared osmotic stress responses

• Functional Differences:

  The kinetic parameters of P. profundum DPCK likely reflect combined adaptations to cold and pressure. Comparative enzymatic studies would be expected to show:

  • Lower kcat values compared to mesophilic/thermophilic counterparts at standard temperatures

  • Lower activation energy compared to mesophilic homologs

  • Maintained catalytic efficiency (kcat/Km) at low temperatures

  • Pressure-dependent activity profile with optimum at 20-30 MPa, similar to its optimal growth pressure of 28 MPa

  • Broader substrate specificity compared to non-extremophile homologs

• Evolutionary Implications:

  Sequence alignments and phylogenetic analyses would likely reveal that P. profundum DPCK shows highest homology to marine Vibrionaceae family members, consistent with its taxonomy . It may share conserved catalytic residues with other DPCKs like the E. coli enzyme while displaying distinctive amino acid compositions in flexible regions and surface areas that reflect adaptation to the deep-sea environment .

What insights can comparative genomics provide about the evolution of the coaE gene in P. profundum relative to other marine bacteria?

Comparative genomics analysis of the coaE gene in P. profundum and other marine bacteria can reveal evolutionary adaptations and selective pressures:

• Gene Context and Operon Structure:
  The genomic organization of coaE may differ between P. profundum and shallow-water relatives. In P. profundum, coaE might be part of a pressure-responsive operon or genomic island associated with deep-sea adaptation. Analysis should focus on:

  • Adjacent genes and potential co-regulation mechanisms

  • Presence of pressure-responsive promoter elements

  • Conservation of gene order among Photobacterium species from different depths

  • Potential horizontal gene transfer signatures

• Sequence Evolution Patterns:

  Calculating evolutionary rates (dN/dS ratios) for coaE across marine bacteria sampled from different depths can identify:

  • Sites under positive selection in deep-sea lineages

  • Conservation patterns of catalytic versus structural residues

  • Depth-correlated amino acid substitution patterns

  • Coevolution with other CoA pathway genes

• Functional Implications:

  Analysis	Methodology	Expected Outcome
  Amino acid composition analysis	Compare frequency of specific residues	Higher Gly, Ser content in P. profundum vs. shallow-water relatives
  Hydrophobicity profile	Plot hydrophobicity along sequence	Altered hydrophobic core in P. profundum DPCK
  Predicted flexibility analysis	B-factor prediction from sequence	Higher predicted flexibility in non-catalytic regions
  Domain architecture comparison	InterPro/Pfam analysis	Potential additional domains or modified linkers

• Adaptation Signals:

  Look for convergent evolution patterns between P. profundum and unrelated deep-sea bacteria, which would suggest independent adaptations to similar selective pressures. The coaE gene in P. profundum might show molecular signatures similar to those in piezophilic bacteria from different phylogenetic backgrounds, particularly in regions associated with pressure stability .

These analyses would contribute to understanding how essential metabolic enzymes adapt to extreme environments while maintaining their catalytic function.

How can structural insights from P. profundum DPCK inform the design of pressure-stable enzymes for biotechnology?

Structural insights from P. profundum DPCK can provide valuable design principles for engineering pressure-stable enzymes for various biotechnological applications:

• Key Structural Features to Transfer:

  • Optimized void volumes and packing density to prevent pressure-induced denaturation

  • Strategic distribution of hydrophobic residues to maintain core stability

  • Surface charge patterns that favor pressure resistance

  • Flexible active site architecture that accommodates pressure-induced conformational changes

• Practical Engineering Approaches:

  Strategy	Methodology	Expected Outcome
  Cavity-filling mutations	Identify and fill internal cavities with bulky hydrophobic residues	Increased pressure stability
  Surface charge optimization	Introduce charged residues in specific patterns based on P. profundum model	Enhanced solubility under pressure
  Disulfide engineering	Add strategic disulfide bridges in locations identified from P. profundum structure	Stabilized tertiary structure
  Active site flexibility	Engineer glycine residues around active site based on P. profundum patterns	Maintained activity under pressure

• Application-Specific Considerations:

  • High-pressure biocatalysis: Focus on pressure range 100-300 MPa

  • Deep-sea enzyme replacement therapy: Target 10-50 MPa stability

  • Pressure-cycling industrial processes: Engineer pressure reversibility

  • Food processing applications: Combine pressure and temperature stability

• Validation Approaches:

  • High-pressure enzyme activity assays to confirm functionality

  • Structural characterization under pressure using specialized equipment

  • Molecular dynamics simulations to predict behavior

  • Industrial-scale pilot testing in relevant applications

These principles derived from P. profundum DPCK could revolutionize enzyme engineering for high-pressure applications by providing nature-inspired solutions to pressure sensitivity problems .

What role might P. profundum DPCK play in understanding the adaptation of metabolic pathways to deep-sea environments?

P. profundum DPCK serves as an excellent model for understanding broader metabolic adaptation to deep-sea environments:

• CoA Metabolism as an Adaptation Indicator:

  • CoA-dependent processes (e.g., fatty acid metabolism) are critical for membrane composition adjustments required for pressure adaptation

  • Changes in DPCK activity directly affect available CoA pools, potentially serving as a regulatory point for pressure response

  • The rate-limiting nature of DPCK in the CoA biosynthetic pathway positions it as a potential metabolic control point

• Systems Biology Perspective:

  • Construct metabolic flux models incorporating P. profundum DPCK kinetic parameters at different pressures

  • Map pressure-dependent changes in CoA-utilizing pathways

  • Identify potential metabolic bottlenecks or rerouting under pressure

  • Compare with pressure-naive organisms to highlight adaptive differences

• Environmental Implications:

  • Use P. profundum DPCK as a marker for adaptation to specific deep-sea niches

  • Correlate enzyme properties with depth distribution of related bacteria

  • Understand biochemical constraints on deep-sea colonization

  • Model the energetic costs of maintaining metabolism at high pressure

• Future Research Directions:

  • Investigate pressure effects on entire CoA-dependent pathways rather than isolated enzymes

  • Develop high-pressure metabolomics approaches to track CoA-related metabolites in vivo

  • Create genetic tools to manipulate DPCK expression in deep-sea organisms

  • Explore potential pressure-sensing roles of DPCK through conformational changes

This research has broader implications for understanding the biochemical limits of life in extreme environments and could inform astrobiology studies on potential metabolic adaptations in high-pressure extraterrestrial environments .

How might research on P. profundum DPCK contribute to the development of novel antimicrobial strategies?

Research on P. profundum DPCK could significantly advance antimicrobial development through several avenues:

• Structural Basis for Selective Inhibition:
  The relatively low sequence identity between bacterial DPCKs and the human COASY (typically <25%) provides a foundation for developing selective inhibitors . P. profundum DPCK research could:

  • Identify unique structural features in bacterial DPCKs absent in human counterparts

  • Map binding pocket differences amenable to selective targeting

  • Reveal potential allosteric sites specific to bacterial enzymes

• Enzyme-Based Screening Platform:
  Using recombinant P. profundum DPCK as a model system could accelerate drug discovery:

  • Develop high-throughput screening assays based on P. profundum DPCK

  • Test compound libraries for inhibitory activity

  • Perform comparative screening against human COASY to identify selective hits

  • Validate hits against clinically relevant pathogens (e.g., Vibrio cholerae, which is related to Photobacterium)

• Applications to Pathogen-Specific Targeting:

  Pathogen	Relationship to P. profundum	Potential Application
  Vibrio cholerae	Close phylogenetic relative	Direct application of inhibitors
  Pseudomonas aeruginosa	Similar Gram-negative physiology	Structure-based design of modified inhibitors
  Plasmodium falciparum	Distinct DPCK with essential function	Comparative approach to improve selectivity
  Multi-drug resistant bacteria	Various	Novel target less susceptible to existing resistance mechanisms

• Advantages over Current Approaches:

  • Essential nature of DPCK makes it less susceptible to bypass mutations

  • Low sequence conservation with human enzyme enables selectivity

  • Targeting CoA biosynthesis affects multiple downstream pathways

  • Novel target with limited existing resistance mechanisms

This research direction parallels successful work on P. falciparum DPCK as an antimalarial target, where researchers identified selective inhibitors through high-throughput screening of diverse compound libraries . The structural and functional insights from P. profundum DPCK could extend this approach to a broader range of pathogens.

What specialized techniques are required to study the enzyme kinetics of P. profundum DPCK under deep-sea conditions?

Studying P. profundum DPCK kinetics under authentic deep-sea conditions requires specialized equipment and methodological adaptations:

• High-Pressure Enzyme Assay Systems:

  System Type	Pressure Range	Advantages	Limitations
  Stopped-flow with pressure cell	Up to 200 MPa	Real-time measurements; rapid mixing	Limited observation time; expensive
  High-pressure optical cell	Up to 100 MPa	Direct spectroscopic measurements	Static conditions only
  High-pressure bioreactor	Up to 70 MPa	Large volume; biological relevance	Endpoint measurements only
  Diamond anvil cell	Up to 1000 MPa	Extreme pressure range	Microscopic volume; specialized detectors needed

• Kinetic Parameter Determination Under Pressure:

  • Develop pressure-resistant fluorescent or colorimetric assays

  • Implement on-line monitoring systems with pressure-resistant optical windows

  • Use quench-flow techniques for time-resolved measurements

  • Account for pressure effects on pH, ionic strength, and substrate conformation

• Technical Adaptations for Low-Temperature/High-Pressure Studies:

  • Temperature-controlled pressure vessels

  • Pressure-stable cooling systems

  • Extended reaction times to compensate for slower reaction rates

  • Increased enzyme concentrations to maintain detectable signal

• Data Analysis Considerations:

  • Apply pressure-dependent kinetic models:

    $K_m(P) = K_m(P_0) \cdot e^{[\frac{\Delta V_m \cdot (P-P_0)}{RT}]}$

    $k_{cat}(P) = k_{cat}(P_0) \cdot e^{[\frac{-\Delta V^\ddagger \cdot (P-P_0)}{RT}]}$

    where ΔVm is the volume change associated with substrate binding and ΔV‡ is the activation volume

  • Calculate activation volumes from pressure-dependent kinetic data

  • Compare with volumetric properties of the native enzyme environment

These specialized techniques allow researchers to accurately measure how P. profundum DPCK functions under conditions mimicking its native deep-sea habitat, providing insights into natural pressure adaptation mechanisms .

What are the best approaches for analyzing the substrate specificity of P. profundum DPCK compared to mesophilic homologs?

To comprehensively analyze substrate specificity differences between P. profundum DPCK and mesophilic homologs, implement these methodological approaches:

• Comparative Substrate Panel Testing:
  Systematically evaluate a panel of substrates with both enzymes under standardized conditions:

  Substrate Category	Examples	Analytical Method
  Natural substrate	Dephospho-CoA	HPLC/LC-MS quantification of CoA formation
  Close analogs	Dephospho-acyl-CoAs (various chain lengths)	Coupled enzyme assay
  Structural variants	Dephospho-CoA with modified pantetheine	Radiometric assay with γ-³²P-ATP
  Distant analogs	Adenosine, AMP, APS	Malachite green phosphate detection
  Non-adenosine nucleotides	Dephospho-GMP, dephospho-UMP	LC-MS/MS

• Temperature-Dependent Substrate Profiling:

  • Conduct parallel specificity assays at 4°C, 15°C, and 25-37°C

  • Calculate temperature coefficients (Q₁₀) for each substrate

  • Determine activation energy (Ea) using Arrhenius plots

  • Compare thermal dependency patterns between P. profundum and mesophilic DPCKs

• Structural Basis for Specificity Differences:

  • Perform molecular docking simulations with various substrates

  • Identify key residues determining specificity through homology modeling

  • Use site-directed mutagenesis to convert specificity from one type to another

  • Validate with binding studies (isothermal titration calorimetry, surface plasmon resonance)

• High-Resolution Kinetic Analysis:

  • Determine complete kinetic profiles (Km, kcat, kcat/Km) for each substrate

  • Analyze product inhibition patterns

  • Investigate potential allosteric effects

  • Perform competition assays between substrates

This comprehensive approach will likely reveal that P. profundum DPCK has broader substrate tolerance at lower temperatures compared to mesophilic homologs, which may show 4-8% activity with alternative substrates like adenosine, AMP, and adenosine phosphosulfate compared to dephospho-CoA .

What bioinformatic tools are most appropriate for identifying and analyzing DPCK sequences from deep-sea metagenomes?

For identifying and analyzing DPCK sequences from deep-sea metagenomes, employ these specialized bioinformatic approaches:

• Metagenomic Sequence Mining:

  Tool	Application	Key Features for DPCK Analysis
  HMMER	Profile-based homology search	Create profiles from known piezophilic DPCKs including P. profundum
  MMseqs2	Fast sequence clustering and search	Efficient for large metagenomic datasets
  DIAMOND	Accelerated BLAST-like search	Process deep-sea metagenomes with billions of reads
  MetaGeneAnnotator	Gene prediction in metagenomes	Optimized for short reads and partial genes

• Specialized Analysis Pipelines:

  • Implement pressure-depth correlation analysis to identify depth-stratified DPCK variants

  • Develop codon usage analysis specific to deep-sea organisms

  • Create automated workflows for identifying pressure-adaptive signatures

  • Design phylogenetic pipelines with reference to known piezophile sequences

• Structural Prediction and Analysis:

  • Use AlphaFold2 or RoseTTAFold for accurate structure prediction of identified sequences

  • Implement cavity analysis algorithms to detect pressure adaptations

  • Apply molecular dynamics simulations at varying pressures

  • Develop automated analysis of hydrostatic pressure adaptation features

• Functional Prediction:

  • Map sequences onto metabolic networks using KEGG and MetaCyc

  • Predict substrate specificity using machine learning approaches

  • Analyze coevolution patterns with other CoA pathway enzymes

  • Identify potential horizontal gene transfer events in extreme environments

These approaches will efficiently identify novel DPCK variants from deep-sea environments and characterize their potential adaptations to high-pressure conditions, building on our understanding of P. profundum DPCK .

How can researchers address the challenge of low enzyme stability when working with recombinant P. profundum DPCK?

Addressing stability issues with recombinant P. profundum DPCK requires systematic troubleshooting:

• Expression Optimization:

  • Reduce expression temperature to 12-15°C

  • Extend induction time to 24-48 hours

  • Co-express with cold-adapted chaperones from Arctic Express system

  • Use auto-induction media for gentle, gradual protein production

• Purification Strategies:

  Issue	Solution	Implementation
  Rapid activity loss	Minimize purification steps	Design one-step purification protocol
  Aggregation	Add solubilizing agents	Include 5-10% glycerol, 0.1% Triton X-100, or 500 mM arginine
  Oxidation sensitivity	Maintain reducing environment	Use 5 mM DTT or TCEP; work under nitrogen atmosphere
  Metal ion loss	Supplement metal cofactors	Add 1-5 mM MgCl₂ to all buffers
  Proteolytic degradation	Add protease inhibitors	Use EDTA-free protease inhibitor cocktail plus 1 mM PMSF

• Storage Optimization:

  • Test multiple storage conditions in parallel:

    • Flash freezing in liquid nitrogen with 15-20% glycerol

    • Storage at 4°C with weekly buffer exchange

    • Lyophilization with stabilizing excipients

    • Addition of 50% glycerol and storage at -20°C

  • Add stabilizing ligands (ATP, dephospho-CoA) at sub-saturating concentrations

  • Consider storage as ammonium sulfate precipitate

• Activity Recovery Protocols:

  • Develop refolding protocols using step-wise dialysis

  • Test activity rescue with osmolytes (trimethylamine N-oxide, betaine)

  • Identify minimum buffer components necessary for stability

  • Determine optimal protein concentration range to prevent concentration-dependent aggregation

These approaches address the intrinsic instability often associated with cold-adapted enzymes from extreme environments, which typically sacrifice stability for activity at low temperatures .

What strategies help overcome challenges in crystallizing P. profundum DPCK for structural studies?

Crystallizing P. profundum DPCK presents unique challenges due to its deep-sea origin and likely inherent flexibility. Implement these specialized strategies:

• Pre-Crystallization Screening:

  • Deploy thermal shift assays (TSA/DSF) to identify stabilizing conditions

  • Use differential scanning calorimetry to quantify stability improvements

  • Perform size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) to ensure monodispersity

  • Apply limited proteolysis to identify and remove flexible regions

• Construct Optimization:

  Approach	Methodology	Rationale
  N/C-terminal truncations	Create series of truncated constructs	Remove disordered termini
  Surface entropy reduction	Replace surface Lys/Glu clusters with Ala	Reduce surface entropy to promote crystal contacts
  Fusion proteins	T4 lysozyme, MBP, or BRIL fusions	Provide rigid scaffold for crystallization
  Synthetic antibody fragments	Co-crystallization with Fab fragments	Stabilize flexible regions and provide crystal contacts

• Crystallization Condition Innovations:

  • Implement temperature cycling protocols (4°C to 18°C)

  • Test pressure-assisted crystallization in specialized chambers

  • Use counter-diffusion methods in capillaries

  • Explore lipidic cubic phase or bicelle crystallization

  • Test crystallization under moderate pressure (10-50 MPa)

• Additive Strategies:

  • Screen with substrate analogs and product (CoA)

  • Try non-hydrolyzable ATP analogs (AMPPNP, AMPPCP)

  • Test transition state mimics

  • Use silver bullets library (cocktails of small molecules)

  • Add osmolytes characteristic of deep-sea environments

• Alternative Structural Approaches:

  • Cryo-electron microscopy for single-particle analysis

  • Small-angle X-ray scattering (SAXS) for solution structure

  • Nuclear magnetic resonance (NMR) for dynamics studies

  • Hydrogen-deuterium exchange mass spectrometry for conformational analysis

These specialized approaches address the particular challenges of crystallizing proteins from extremophiles, which often have structural adaptations that make crystallization difficult .

How can researchers validate that recombinant P. profundum DPCK retains native-like properties and activities?

To ensure recombinant P. profundum DPCK maintains its native properties and activities, implement this comprehensive validation strategy:

• Biochemical Validation:

  Property	Validation Method	Expected Result
  Enzymatic activity	Coupled enzyme assay	Activity comparable to native enzyme
  Substrate specificity	Test panel of substrates	Similar preference pattern to native enzyme
  Optimal temperature	Activity profiling at 4-30°C	Optimum at 10-15°C
  Pressure response	High-pressure activity assays	Activity enhancement at 20-30 MPa
  Salt requirement	Activity assay with varying NaCl	Optimal activity at marine concentrations

• Structural Validation:

  • Circular dichroism (CD) spectroscopy to confirm secondary structure

  • Intrinsic fluorescence to assess tertiary structure

  • Dynamic light scattering (DLS) to confirm appropriate oligomeric state

  • Thermal denaturation to verify low melting temperature characteristic of psychrophiles

  • Limited proteolysis to compare digestion patterns with native enzyme

• Functional Comparisons:

  • Perform complementation studies in DPCK-deficient bacterial strains

  • Compare kinetic parameters with those of the native enzyme (if available)

  • Test stability under pressure cycling conditions

  • Verify cold adaptation properties like higher activity at low temperatures

  • Measure activation energy and compare with mesophilic homologs

• Advanced Biophysical Validation:

  • Hydrogen-deuterium exchange mass spectrometry to map flexibility regions

  • Pressure perturbation calorimetry to quantify volumetric properties

  • Molecular dynamics simulations under various pressure conditions

  • Nuclear magnetic resonance (NMR) to analyze protein dynamics

  • Analytical ultracentrifugation to determine pressure-dependent oligomerization