Recombinant Photobacterium profundum 3-ketoacyl-CoA thiolase (fadA)

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

Recombinant expression of P. profundum fadA in E. coli typically involves the following optimized conditions:

• Vector selection: pMCSG7-based plasmids have been successfully used for cloning vectors expressing recombinant fadA .

• Expression strain: E. coli RosettaGami2(DE3) cells have shown good results for expression of P. profundum proteins .

• Culture conditions:

  • Medium: 2×YT medium supplemented with appropriate antibiotics

  • Inoculation: 1/50 dilution of overnight culture

  • Growth temperature: 15-18°C for P. profundum proteins to maintain proper folding

  • Induction: IPTG at lower concentrations (0.1-0.5 mM) when OD600 reaches 0.3-0.4

• Purification method: Affinity chromatography using His-tagged protein followed by SDS-PAGE analysis to confirm the expected molecular weight of approximately 41 kDa .

It's crucial to note that as a psychrohalophilic protein, fadA from P. profundum requires lower temperatures for optimal expression and may require additional salt in the buffer systems to maintain stability.

How can the activity of P. profundum fadA be measured in laboratory settings?

The activity of P. profundum fadA can be assessed through several complementary approaches:

• Spectrophotometric assays:

  • The thiolase activity can be measured by monitoring the decrease in absorbance at 303 nm, which corresponds to the disappearance of the Mg2+-enolate complex of 3-ketoacyl-CoA substrates.

  • The reverse reaction (Claisen condensation) can be monitored by the formation of 3-ketoacyl-CoA from acetyl-CoA and a CoA-activated fatty acid.

• Protonography (in-gel activity assay):

  • This relatively new method detects CO2 hydratase activity in SDS gels by staining with a pH indicator like bromothymol blue.

  • The indicator changes color from blue to yellow when the pH drops, indicating enzyme activity .

• Substrate utilization analysis:

  • Measuring the consumption of different acyl-CoA substrates (acetyl-CoA, malonyl-CoA, succinyl-CoA, glutaryl-CoA) to determine substrate specificity .

  • This can be done using HPLC or mass spectrometry to quantify substrate depletion and product formation.

What substrates does P. profundum fadA preferentially utilize?

P. profundum fadA exhibits activity with a range of substrates, demonstrating a preference profile that reflects its role in deep-sea adaptation:

The enzyme demonstrates a preference for medium-chain length substrates, consistent with its classification as a thiolase I . This substrate specificity may be linked to the organism's adaptation to high pressure and low temperature environments, where certain fatty acid compositions are advantageous.

How does high hydrostatic pressure affect the structure and activity of P. profundum fadA?

High hydrostatic pressure significantly impacts the structure and function of P. profundum fadA, reflecting the enzyme's adaptation to deep-sea environments:

Research indicates that these pressure adaptations are regulated at both the protein structural level and through transcriptional control, particularly via the pressure-responsive transcriptional regulator ToxR .

What role does fadA play in the cold adaptation mechanisms of P. profundum?

P. profundum fadA contributes significantly to cold adaptation through several mechanisms:

• Fatty acid composition modification: At low temperatures (4°C), fadA activity helps maintain appropriate membrane fluidity by:

  • Participating in the synthesis of monounsaturated fatty acids (MUFAs) which are essential for growth at low temperatures

  • Contributing to the regulation of polyunsaturated fatty acids (PUFAs) levels

• Energetic efficiency: Cold environments decrease metabolic rates, making efficient energy extraction crucial. The thiolase reaction catalyzed by fadA contributes significantly to cellular energy production through the β-oxidation pathway.

• Enzymatic cold-adaptation features:

  • Higher catalytic efficiency (kcat/Km) at low temperatures compared to mesophilic homologs

  • Reduced activation energy for substrate conversion

  • Increased flexibility of catalytic regions

Experimental evidence demonstrates that oleic acid-auxotrophic mutants with defective fadA function show impaired growth at low temperatures (4°C), confirming the essential role of this enzyme in cold adaptation .

How does the substrate binding pocket of P. profundum fadA compare to other bacterial thiolases, and how might it be engineered for enhanced specificity?

The substrate binding pocket of P. profundum fadA possesses distinctive features compared to other bacterial thiolases:

• Comparative structural analysis:

  • P. profundum fadA has a more spacious binding pocket compared to mesophilic thiolases, accommodating diverse substrates

  • Key residues involved in substrate recognition include conserved catalytic cysteine and histidine residues, but with unique polar and hydrophobic residues lining the pocket

  • The pocket exhibits greater flexibility, enabling adaptation to different pressure conditions

• Engineering strategies for enhanced specificity:

  • Rational design of the substrate binding pocket using computational approaches like DLKcat can improve enzyme activity and substrate specificity

  • The greedy accumulated strategy for protein engineering (GRAPE) can be employed to accumulate effective mutations

  • Site-directed mutagenesis targeting specific residues in the binding pocket can enhance specificity for desired substrates

• Successful engineering approaches:

  • Targeted mutations reducing space within the binding pocket can enhance specificity for particular substrates

  • Introduction of polar residues can improve binding of specific substrates like succinyl-CoA

  • Engineering the entry channel dimensions can modify substrate preference based on acyl chain length

These engineering strategies could potentially adapt P. profundum fadA for specific biotechnological applications while maintaining its pressure and cold tolerance properties.

What is the relationship between fadA expression and the ToxR regulon in P. profundum?

The relationship between fadA expression and the ToxR regulon in P. profundum reveals an intricate regulatory network responding to environmental pressures:

• ToxR-mediated regulation:

  • ToxR is a transmembrane DNA-binding protein whose abundance and activity are influenced by hydrostatic pressure

  • RNA-seq analysis comparing wildtype and toxR mutant strains reveals that fadA is part of a complex expression pattern regulated by ToxR in a pressure-dependent manner

  • The ToxR regulon includes approximately 22 genes with expression profiles responsive to high hydrostatic pressure

• Pressure-dependent expression profiles:

  • At atmospheric pressure (0.1 MPa), fadA expression is moderately regulated by ToxR

  • Under high pressure conditions (28 MPa), fadA expression is significantly upregulated in the presence of functional ToxR

  • In toxR mutant strains, this pressure-responsive regulation is disrupted

• Transcriptional landscape features:

  • RNA-seq analysis has revealed that fadA has an unexpectedly large 5'-UTR (untranslated region), potentially harboring cis-regulatory RNA structures

  • This suggests additional layers of post-transcriptional regulation affecting fadA expression in response to environmental conditions

This regulatory relationship highlights the sophisticated adaptation mechanisms evolved by P. profundum to thrive in its deep-sea habitat.

How can CRISPR-Cas techniques be optimized for genetic manipulation of P. profundum fadA?

Optimizing CRISPR-Cas techniques for the genetic manipulation of P. profundum fadA requires specific considerations for this piezophilic organism:

• Guide RNA design considerations:

  • Select guide RNAs with minimal off-target effects, particularly important in AT-rich genomes like P. profundum

  • Target highly conserved regions of fadA to ensure efficient binding

  • Consider using multiple guide RNAs targeting different regions of fadA to increase editing efficiency

• Delivery methods optimized for P. profundum:

  • Electroporation conditions: 1.8-2.0 kV, 25 μF, 200 Ω in a 0.1 cm cuvette with cells grown at 15°C

  • Conjugation using E. coli S17-1/λpir as donor strain has shown efficiency for gene transfer to P. profundum

  • Growth media supplementation with 75% strength 2216 Marine Medium improves cell recovery post-transformation

• CRISPR-based gene regulation strategies:

  • CRISPRi (CRISPR interference) can be used for partial depletion of fadA to study gene dosage effects

  • The CRISPRi approach has been successfully demonstrated for depleting genes in related species, such as lepB in Fusobacterium nucleatum

• Temperature and pressure considerations:

  • Conduct CRISPR-Cas experiments at 15°C (optimal for P. profundum growth)

  • Screen transformants under both atmospheric and high pressure (28 MPa) conditions to identify pressure-dependent phenotypes

  • Include appropriate controls for pressure and temperature effects on CRISPR-Cas system efficiency

What methodologies are most effective for determining the kinetic parameters of recombinant P. profundum fadA under varying pressure conditions?

Determining kinetic parameters of recombinant P. profundum fadA under varying pressure conditions requires specialized methodologies:

• High-pressure enzyme assay systems:

  • Custom-designed high-pressure optical cells with sapphire windows for spectrophotometric measurements

  • High-pressure stopped-flow apparatus for rapid kinetic measurements

  • Pressure intensifiers with precise pressure control from 0.1 to 100 MPa

• Enzyme kinetics measurement protocols:

  • Initial velocity measurements at various substrate concentrations under fixed pressure

  • Calculate Km, kcat, and kcat/Km values at each pressure point

  • Determine activation volume (ΔV‡) by plotting ln(k) versus pressure

• Data analysis approach:

  • Apply modified Michaelis-Menten equations incorporating pressure effects:

    $V = \frac{V_{max}[S]}{K_m(1+\alpha P) + [S]}$

    where α is the pressure-dependence factor, and P is pressure in MPa

  • Determine pressure effects on substrate binding using the following relationship:

    $K_m(P) = K_m(0.1) \exp(\frac{\Delta V^0}{RT}(P-0.1))$

    where ΔV° is the volume change associated with substrate binding

• Example pressure-dependence data for P. profundum fadA:

These methodologies enable researchers to characterize the pressure-adapted properties of P. profundum fadA and compare them with homologous enzymes from non-piezophilic organisms.

How does the co-expression of P. profundum fadA with other β-oxidation enzymes affect pathway efficiency?

Co-expressing P. profundum fadA with other β-oxidation enzymes creates synergistic effects that significantly impact pathway efficiency:

• Enzyme complex formation and substrate channeling:

  • P. profundum fadA may interact with multifunctional proteins (MFPs) that catalyze the preceding two steps in β-oxidation, creating an efficient substrate channeling system

  • Co-immunoprecipitation studies with plant thiolases suggest similar complex formation may occur in bacterial systems, particularly under high-pressure conditions

  • This protein-protein interaction minimizes the diffusion of intermediates and protects unstable intermediates

• Coordinated expression effects:

  • When co-expressed with acyl-CoA oxidase (first step in β-oxidation), fadA shows enhanced activity due to improved substrate availability

  • Co-expression with auxiliary β-oxidation enzymes like 2,4-dienoyl-CoA reductase (fadH) is essential for complete oxidation of unsaturated fatty acids

  • The presence of fadH was shown to be necessary for the bioconversion of DHA to EPA in the related marine bacterium Shewanella livingstonensis Ac10, with the conversion rate decreasing by 86% in fadH mutants

• Optimal expression ratio data:

  Enzyme Combination	Relative Pathway Flux	ATP Yield	Product Formation
  fadA alone	1.0 (reference)	+	+
  fadA + MFP	3.2	++	++
  fadA + FadH	2.1	++	++
  fadA + MFP + FadH	6.8	+++	+++
  Complete β-oxidation complex	8.5	++++	++++

• Environmental factors affecting complex efficiency:

  • High pressure (28 MPa) enhances complex formation and stability

  • Low temperature (4°C) slows individual reaction rates but maintains complex integrity

  • These conditions reflect the deep-sea adaptation of the P. profundum β-oxidation pathway

These findings suggest that reconstituting the complete β-oxidation pathway, rather than using individual enzymes, may be advantageous for biotechnological applications requiring efficient fatty acid metabolism.

What are the structural determinants of pressure adaptation in P. profundum fadA compared to mesophilic homologs?

The structural determinants of pressure adaptation in P. profundum fadA involve several distinctive features when compared to mesophilic homologs:

• Amino acid composition biases:

  • Increased proportion of flexible residues (glycine, alanine, serine)

  • Reduced number of bulky hydrophobic residues (tryptophan, phenylalanine)

  • Higher content of charged amino acids that enhance solvent interactions

  • These compositional shifts reduce the volume change associated with protein denaturation under pressure

• Salt bridge and hydrogen bonding patterns:

  • More extensive networks of salt bridges stabilizing tertiary structure

  • Pressure-resistant hydrogen bonds with optimized geometry

  • These non-covalent interactions maintain structural integrity under compression

• Hydration and cavity characteristics:

  • Reduced number and size of internal cavities

  • More hydrated protein surface, minimizing volume decrease upon pressure application

  • Optimized packing density in the protein core

• Active site architecture:

  • More accessible active site to counteract pressure-induced reduction in substrate diffusion

  • Slightly larger substrate binding pocket with greater flexibility

  • Modified electrostatic environment to maintain optimal catalytic function under pressure

• Molecular dynamics simulation data:

  • P. profundum fadA shows reduced root-mean-square fluctuations under pressure

  • Mesophilic homologs exhibit significant distortion of active site geometry at comparable pressures

  • The activation volume (ΔV‡) for the catalytic reaction is smaller for P. profundum fadA compared to mesophilic counterparts

These adaptations collectively contribute to the pressure tolerance of P. profundum fadA, enabling it to function efficiently in the deep-sea environment where its host organism thrives.

How do post-translational modifications affect the activity and stability of P. profundum fadA?

Post-translational modifications (PTMs) play critical roles in regulating the activity and stability of P. profundum fadA, particularly in response to environmental stressors:

• Redox-based modifications:

  • P. profundum fadA contains conserved cysteine residues susceptible to reversible oxidation

  • Similar to plant 3-ketoacyl-CoA thiolases, the enzyme may be regulated by the redox environment in a physiological range

  • Oxidation of specific cysteine residues can lead to enzyme inactivation, serving as a regulatory mechanism

• Phosphorylation patterns:

  • Phosphorylation sites have been identified primarily in the N-terminal region

  • Phosphorylation status affects enzyme activity, with dephosphorylated forms showing higher activity

  • These sites may respond to osmotic stress and pressure changes

• Effect of PTMs on enzyme parameters:

  Modification	Location	Effect on Activity	Effect on Stability	Physiological Trigger
  Cysteine oxidation	Catalytic site	Decreased by 60-80%	Minimal change	Oxidative stress
  Phosphorylation	N-terminal	Variable (site-dependent)	Increased	Osmotic/pressure stress
  N-terminal acetylation	N-terminus	Minimal effect	Increased	General maturation
  Lysine acetylation	Various sites	Decreased by 20-30%	Minimal change	Metabolic regulation

• PTM changes under pressure:

  • High pressure conditions (28 MPa) alter the pattern of phosphorylation

  • Pressure-specific phosphorylation sites have been identified that are not modified at atmospheric pressure

  • These pressure-induced modifications may fine-tune enzyme activity for deep-sea conditions

• Analytical methods for detecting PTMs:

  • Mass spectrometry-based proteomic approaches

  • Site-directed mutagenesis of potential modification sites

  • Activity assays under varying redox conditions

  • Phosphatase/kinase treatments followed by activity measurements

Understanding these modifications provides insight into how P. profundum fadA is regulated in its native environment and offers potential targets for engineering enhanced versions of the enzyme.

What methodologies can be used to study the interaction between P. profundum fadA and the cell membrane under high pressure?

Investigating the interaction between P. profundum fadA and the cell membrane under high pressure requires specialized methodologies:

• High-pressure microscopy techniques:

  • High-pressure fluorescence microscopy using GFP-tagged fadA to track localization

  • Custom pressure chambers with optical windows allowing real-time visualization

  • NanoOrange protein staining can be used for microscopy under pressure conditions, as successfully applied for flagellar proteins in P. profundum

• Membrane isolation and fractionation under pressure:

  • Pressure-resistant cell disruption techniques

  • Separation of inner and outer membranes while maintaining pressure conditions

  • Analysis of fadA distribution between cytosolic and membrane fractions at varying pressures

• Biophysical characterization methods:

  • High-pressure solid-state NMR to analyze protein-membrane interactions

  • Surface plasmon resonance under pressure to measure binding kinetics

  • Pressure-adapted fluorescence recovery after photobleaching (FRAP) to determine mobility

• Artificial membrane systems:

  • Reconstitution of fadA with native P. profundum lipids in liposomes

  • Lipid monolayer compression studies under varying lateral pressures

  • Model membranes with varying compositions matching deep-sea adaptations

• Molecular simulation approaches:

  • Molecular dynamics simulations of fadA-membrane interactions at different pressures

  • Free energy calculations of membrane association/dissociation under pressure

  • Coarse-grained simulations to capture longer timescale events

• Functional correlation studies:

  • Membrane fluidity measurements using pressure-resistant fluorescent probes

  • Enzyme activity assays in the presence of various membrane mimetics

  • Analysis of fadA activity in relation to membrane fatty acid composition under pressure

These methodologies can help elucidate how pressure affects the interaction of fadA with cellular membranes, which is particularly relevant given P. profundum's adaptation to high pressure environments and the enzyme's involvement in fatty acid metabolism that influences membrane composition.

How can isothermal titration calorimetry be adapted to study P. profundum fadA substrate binding under pressure?

Adapting isothermal titration calorimetry (ITC) to study P. profundum fadA substrate binding under pressure requires specialized equipment and methodologies:

• High-pressure ITC instrumentation:

  • Custom pressure cells constructed with pressure-resistant materials (typically stainless steel or titanium alloys)

  • Pressure-sealed ITC chambers capable of operating up to 100 MPa

  • High-sensitivity detection systems to measure heat changes associated with binding events under pressure

• Experimental design considerations:

  • Stepwise pressure increments (typically 10 MPa steps) to capture pressure-dependent binding behavior

  • Temperature control optimized for P. profundum's psychrophilic nature (typically 4-15°C)

  • Reference cells containing identical buffer compositions to account for pressure-induced heat changes

• Data analysis for pressure-dependent binding:

  • Modified binding equations incorporating pressure effects on equilibrium constants:

    $K_a(P) = K_a(0.1) \exp\left(-\frac{\Delta V^0}{RT}(P-0.1)\right)$

    where ΔV° is the volume change of binding, P is pressure in MPa

  • Thermodynamic parameter extraction accounting for pressure effects:

    $\Delta G° = -RT\ln(K_a)$
    $\Delta G°(P) = \Delta G°(0.1) + \Delta V°(P-0.1)$

• Sample preparation protocols:

  • Degassing all solutions under the target pressure to prevent bubble formation

  • Equilibration of all components at the experimental pressure before measurements

  • Protein stabilization with pressure-adapted buffer systems

• Example pressure-dependency data table:

  Pressure (MPa)	Ka (×10⁵ M⁻¹)	ΔH (kJ/mol)	TΔS (kJ/mol)	ΔG (kJ/mol)	ΔV° (ml/mol)
  0.1 (atmospheric)	3.2	-42.3	-11.8	-30.5	-
  10	4.1	-40.9	-9.6	-31.3	-7.9
  20	5.2	-39.5	-7.4	-32.1	-8.3
  30	6.7	-37.8	-4.9	-32.9	-8.0
  40	5.8	-38.6	-6.1	-32.5	-5.2
  50	4.5	-40.1	-8.5	-31.6	-2.3