Recombinant Photobacterium profundum Probable 2- (5''-triphosphoribosyl)-3'-dephosphocoenzyme-A synthase (citG)

What is Photobacterium profundum and why is it significant for enzyme research?

Photobacterium profundum is a psychrotolerant and moderately piezophilic bacterium first isolated from an amphipod homogenate enrichment from the Sulu Sea. This microorganism is capable of growth at temperatures ranging from below 2°C to above 20°C (with an optimal temperature of 15°C) and at pressures from 0.1 MPa to nearly 90 MPa (with an optimal pressure of 28 MPa) . These characteristics make P. profundum an excellent model organism for studying adaptations to deep-sea environments, particularly how enzymes function under high pressure and low temperature conditions.

The significance of P. profundum in enzyme research lies in its remarkable adaptability to extreme conditions. Analysis of the P. profundum SS9 genome and transcriptome has revealed a high degree of metabolic diversity and redundancy, which likely contributes to its environmental adaptations . By studying enzymes like citG from P. profundum, researchers can gain insights into pressure and temperature adaptations at the molecular level, potentially revealing novel catalytic mechanisms that function efficiently under extreme conditions.

What is the function of citG in Photobacterium profundum?

The citG gene in P. profundum encodes a Probable 2-(5''-triphosphoribosyl)-3'-dephosphocoenzyme-A synthase, which is involved in coenzyme A biosynthesis. This enzyme catalyzes a key step in the conversion of intermediates in the coenzyme A biosynthetic pathway. The enzyme's function must be understood within the context of P. profundum's adaptations to deep-sea environments.

In piezophilic organisms like P. profundum, metabolic enzymes often exhibit structural and functional adaptations that allow them to maintain activity under high pressure. For citG specifically, these adaptations might include altered substrate binding affinities, modified active site architectures, or pressure-resistant structural features that enable efficient catalysis even at depths where pressure exceeds 28 MPa. The experimental characterization of this enzyme requires specialized techniques that can simulate the native high-pressure environment of P. profundum.

What are the optimal conditions for heterologous expression of P. profundum citG?

For heterologous expression of P. profundum citG, researchers should consider the following methodological approach:

• Expression System Selection: While E. coli is commonly used for recombinant protein expression, the psychrophilic nature of P. profundum proteins may require lower induction temperatures. Based on protocols used for similar deep-sea bacterial enzymes, expression in E. coli BL21(DE3) strains at reduced temperatures (15-18°C) after IPTG induction can improve the solubility and proper folding of psychrophilic enzymes.

• Growth Medium Optimization: E. coli strains can be cultured in Luria-Bertani medium at 37°C until reaching mid-log phase (OD600 of 0.5-0.7), followed by temperature reduction to 15-18°C prior to induction . For certain experiments, antibiotic selection with kanamycin (200 μg/ml) or streptomycin (150 μg/ml) may be required, as demonstrated with P. profundum cultures .

• Induction Parameters: Inducer concentration (typically 0.1-0.5 mM IPTG) and post-induction incubation time (often extended to 16-24 hours for cold-adapted enzymes) should be optimized through small-scale expression trials to maximize yield of properly folded protein.

• Codon Optimization: Consider codon optimization of the citG gene to match the codon usage bias of the expression host, which can significantly improve expression levels of heterologous proteins.

For pressure-adapted enzymes like those from P. profundum, standard expression systems may not replicate the native folding environment. Researchers might need to explore specialized expression systems or post-expression treatments that simulate high-pressure conditions to ensure proper protein folding and activity.

What purification strategy is most effective for recombinant citG from P. profundum?

A multi-step purification strategy is recommended for recombinant citG from P. profundum:

• Initial Capture: Affinity chromatography using an N-terminal or C-terminal His-tag is often effective for initial capture. Purification can be performed under cold conditions (4-10°C) to preserve the activity of this psychrophilic enzyme.

• Intermediate Purification: Ion exchange chromatography (typically using a Q-Sepharose column) can separate the target protein from similarly sized contaminants.

• Polishing Step: Size exclusion chromatography using a Superdex 200 column equilibrated with a buffer containing stabilizing agents (such as glycerol or specific divalent cations) can achieve high purity.

• Buffer Optimization: Throughout the purification process, maintain buffer conditions that mimic aspects of the deep-sea environment—slightly elevated salt concentrations (300-500 mM NaCl) and the inclusion of pressure-stabilizing osmolytes may help maintain the native conformation of the enzyme.

• Quality Control: Assess purity using SDS-PAGE and confirm identity through Western blotting or mass spectrometry. Evaluate the oligomeric state through native PAGE or analytical size exclusion chromatography.

Researchers should be aware that enzymes from piezophilic organisms might exhibit altered behavior during standard purification procedures performed at atmospheric pressure. In some cases, incorporating a high-pressure treatment step or performing activity assays under pressure may be necessary to fully restore or evaluate the enzyme's native activity.

How can the enzymatic activity of recombinant citG be assessed under conditions mimicking the deep-sea environment?

Assessing the enzymatic activity of recombinant citG under deep-sea-like conditions requires specialized methodologies:

• High-Pressure Reaction Vessels: Custom-designed stainless steel pressure vessels, similar to those used for P. profundum growth studies (as described in the literature for pressures up to 45 MPa) , can be adapted for enzyme assays. These systems allow researchers to control both pressure and temperature simultaneously.

• Spectrophotometric Coupled Assays: For measuring citG activity, develop coupled enzyme assays that link the reaction to a spectrophotometrically detectable product. These assays can be modified to function in pressure-resistant cuvettes or microplate formats that can be inserted into high-pressure chambers.

• Temperature Control: Maintain assay conditions at temperatures relevant to the deep sea (typically 4-15°C) to properly evaluate the enzyme's cold-adapted properties. P. profundum exhibits optimal growth at 15°C, suggesting its enzymes may be similarly optimized .

• Pressure Gradient Analysis: Perform activity measurements across a range of pressures (0.1 MPa to 90 MPa) to determine the pressure optimum and compare it to the organism's growth optimum of approximately 28 MPa .

• Substrate Concentration Optimization: Determine Michaelis-Menten kinetic parameters (Km, Vmax, kcat) at different pressures to assess how hydrostatic pressure affects substrate binding and catalytic efficiency.

It's important to note that pressure affects the pH of buffer systems (typically decreasing pH with increasing pressure), so researchers should use buffers with minimal pressure sensitivity or account for these changes when interpreting results. Additionally, the impact of pressure on any coupling enzymes used in the assay must be considered to ensure they don't become rate-limiting under experimental conditions.

What analytical techniques are recommended for determining substrate specificity of P. profundum citG?

For determining the substrate specificity of P. profundum citG, a comprehensive analytical approach is recommended:

• High-Performance Liquid Chromatography (HPLC): Develop an HPLC method with appropriate columns (typically reverse-phase C18) to separate and quantify the reaction products. For coenzyme A-related metabolites, specialized columns designed for nucleotide detection may be more suitable.

• Mass Spectrometry Coupling: LC-MS/MS analysis can provide definitive identification of reaction products and intermediates, allowing for the detection of even minor conversion products that might indicate secondary substrate preferences.

• Isothermal Titration Calorimetry (ITC): This technique can be used to directly measure binding affinities for different potential substrates, providing thermodynamic parameters of binding even in cases where catalytic conversion is minimal.

• Substrate Library Screening: Test a panel of structurally related compounds as potential substrates, including both natural substrates and synthetic analogs. This approach can reveal the structural determinants of substrate recognition.

• Competitive Inhibition Studies: Assess how different substrate analogs compete with the natural substrate, providing insights into binding site preferences and substrate specificity determinants.

When working with enzymes from extremophiles like P. profundum, it's crucial to perform these analyses under conditions that reflect the enzyme's native environment. For example, substrate binding studies should be conducted at various pressures to determine if substrate specificity changes under deep-sea conditions. This is particularly relevant as pressure can alter enzyme conformations and binding pocket geometries, potentially shifting substrate preferences.

What structural features of P. profundum citG contribute to its pressure adaptation?

The structural features of P. profundum citG that likely contribute to its pressure adaptation include:

• Increased Flexibility: Proteins from piezophilic organisms often exhibit higher structural flexibility, particularly in loop regions and around the active site, which helps maintain catalytic activity under compression. This flexibility is typically achieved through a higher ratio of glycine residues and fewer proline residues in key positions.

• Modified Hydrophobic Core: Pressure-adapted enzymes frequently display a reorganized hydrophobic core with fewer and/or smaller hydrophobic amino acids, reducing the volume change associated with protein folding under pressure.

• Increased Surface Hydration: Enhanced hydration of the protein surface through strategic positioning of charged and polar residues can counteract the dehydration effects of high pressure.

• Salt Bridge Networks: Extensive networks of salt bridges and electrostatic interactions can provide structural stability under high pressure conditions while maintaining necessary flexibility for catalysis.

• Reduced Void Volumes: Pressure-adapted proteins typically have fewer and smaller internal cavities, minimizing compressibility under high pressure.

To identify these features in P. profundum citG, researchers should conduct comparative structural analyses with homologous enzymes from non-piezophilic organisms. Advanced structural biology techniques such as X-ray crystallography, cryo-electron microscopy, or NMR spectroscopy under varying pressure conditions can reveal pressure-induced conformational changes. Molecular dynamics simulations at different pressures can also provide insights into the dynamic behavior of the enzyme's structure and how it responds to high-pressure environments.

What protein engineering strategies can enhance the stability and activity of recombinant citG?

Protein engineering strategies to enhance the stability and activity of recombinant citG include:

• Directed Evolution Approaches: Implement error-prone PCR or DNA shuffling techniques similar to those used for other industrial enzymes, such as the transaminase engineered for sitagliptin manufacture . This approach allows for the exploration of sequence space without requiring detailed structural knowledge.

• Structure-Guided Rational Design: Based on structural analysis or homology modeling, introduce specific mutations that could enhance stability while maintaining catalytic activity. Focus on:

  • Introducing additional salt bridges for thermostability

  • Optimizing surface charge distribution

  • Rigidifying flexible regions away from the active site

  • Modifying substrate binding residues to improve affinity

• Semi-Rational Approaches: Combine computational design with experimental screening by creating focused libraries of variants targeting specific regions predicted to influence stability or activity.

• Substrate Walking Strategy: Similar to the approach described for transaminase engineering , gradually modify the enzyme to accept the desired substrate through incremental changes, using intermediate substrates as stepping stones.

• Computational Enzyme Design: Utilize advanced computational tools to redesign the active site or substrate binding pocket for enhanced catalytic efficiency or altered substrate specificity.

The interplay between computational design and protein engineering approaches has proven successful in developing enzymes with improved properties. For example, the transaminase enzyme for sitagliptin manufacture was developed through substrate walking, modeling, and directed evolution, resulting in a biocatalyst with practical application in manufacturing settings .

How can sequence similarity tools help in studying P. profundum citG?

Sequence similarity tools provide valuable insights for studying P. profundum citG through the following methodological approaches:

• Homolog Identification: Tools like BLAST can identify homologous proteins from diverse organisms. A similarity analysis approach similar to that illustrated in search result can identify the closest homologs to citG. For example, a BLAST search might reveal sequence similarities to enzymes from other deep-sea or psychrophilic organisms with sequence identities in the range of 30-40%, which is common for functionally related enzymes across different species.

• Multiple Sequence Alignment (MSA): Tools like Clustal Omega, available in databases such as LICEDB , can align citG with homologous sequences to identify:

  • Conserved catalytic residues

  • Regions of variability that might confer specific adaptations

  • Insertions or deletions unique to piezophilic homologs

• Phylogenetic Analysis: Constructing phylogenetic trees based on sequence alignments can reveal the evolutionary relationships between citG variants from different environmental niches, potentially correlating sequence features with adaptation to specific conditions.

• Structure Prediction: Sequence similarity can inform homology modeling when crystal structures are unavailable, helping predict the three-dimensional structure of citG based on solved structures of homologous proteins.

• Functional Annotation Transfer: Experimental data from well-characterized homologs can guide hypothesis generation about citG function, substrate specificity, and reaction mechanism.

Table 1 illustrates a hypothetical sequence similarity analysis output for P. profundum citG, similar to the BLAST results format shown in search result :

This type of analysis reveals the degree of conservation across different organisms and helps identify species with potentially similar enzymatic properties or adaptations.

What can comparative studies with mesophilic homologs reveal about citG adaptation to deep-sea conditions?

Comparative studies between P. profundum citG and its mesophilic homologs can reveal several important aspects of adaptation to deep-sea conditions:

• Amino Acid Composition Analysis: Quantitative comparison of amino acid frequencies between piezophilic and mesophilic homologs can identify:

  • Increased glycine content in flexible regions of piezophilic enzymes

  • Reduced hydrophobic core volume through substitution of large hydrophobic residues with smaller ones

  • Enhanced surface hydration through strategic positioning of charged and polar residues

• Thermal Stability Profiles: Comparing thermal denaturation curves of citG with mesophilic homologs can reveal:

  • Lower thermal stability in the piezophilic enzyme (common in cold-adapted proteins)

  • Pressure-dependent shifts in thermal stability

  • Different unfolding pathways under varying pressure conditions

• Kinetic Parameter Comparison: Analyzing enzyme kinetics across pressure gradients can demonstrate:

  • How Km and kcat values respond to pressure changes in piezophilic versus mesophilic enzymes

  • Pressure-dependent changes in substrate specificity or catalytic mechanism

  • Adaptations that maintain catalytic efficiency under high pressure

• Structural Flexibility Assessment: Techniques such as hydrogen-deuterium exchange mass spectrometry or molecular dynamics simulations can reveal:

  • Regions of enhanced flexibility in the piezophilic enzyme

  • Pressure-responsive elements that undergo conformational changes

  • Differences in protein dynamics across varying pressure conditions

• Volume Change Determination: Measuring the activation volume (ΔV‡) and volume change of unfolding (ΔVunfolding) for both piezophilic and mesophilic variants can quantify adaptations that minimize volume changes under pressure.

These comparative approaches provide mechanistic insights into how citG has evolved to function optimally in the deep-sea environment, potentially revealing generalizable principles of protein adaptation to high-pressure conditions that could inform biotechnological applications.

How can recombinant P. profundum citG be utilized in biotechnological applications?

Recombinant P. profundum citG offers several promising biotechnological applications based on its unique adaptations to deep-sea conditions:

• Low-Temperature Biocatalysis: The cold-adapted properties of citG make it potentially valuable for biotransformations that benefit from low-temperature processing, such as:

  • Synthesis of thermolabile compounds

  • Reactions where reduced temperature minimizes undesired side reactions

  • Processes requiring reduced energy input for cooling

• High-Pressure Enzymatic Processes: The pressure adaptation of citG could be exploited for:

  • High-pressure biocatalytic processes where elevated pressure enhances reaction rates or selectivity

  • Enzymatic reactions in supercritical CO2 or other pressurized solvent systems

  • Biocatalysis under conditions that suppress microbial contamination

• Enzyme Engineering Platform: The pressure-adapted structural features of citG could serve as a scaffold for engineering other enzymes to function under non-conventional conditions, similar to how directed evolution approaches have been applied to other industrial enzymes .

• Coenzyme A Derivative Synthesis: As a 2-(5''-triphosphoribosyl)-3'-dephosphocoenzyme-A synthase, engineered variants of citG could potentially catalyze the synthesis of valuable coenzyme A derivatives or analogs used in metabolic studies, drug development, or as enzyme cofactors.

• Biosensors for High-Pressure Environments: Exploiting the pressure-responsive properties of citG could lead to the development of biosensors for monitoring deep-sea conditions or industrial high-pressure processes.

To effectively apply P. profundum citG in these contexts, researchers would need to optimize expression and purification protocols, engineer improved variants through directed evolution or rational design, and develop specialized reaction systems capable of maintaining controlled pressure conditions. The successful implementation would draw on approaches similar to those used in developing the biocatalytic synthesis of sitagliptin, where an enzyme with marginal initial activity was engineered to create a commercially viable biocatalytic process .

What are the challenges in studying conformational changes of citG under high pressure conditions?

Studying conformational changes of citG under high pressure presents several methodological challenges that require specialized techniques:

• High-Pressure Spectroscopic Methods: Adapting standard spectroscopic techniques for high-pressure environments requires:

  • Specialized pressure cells with optical windows for circular dichroism (CD) and fluorescence measurements

  • Pressure-resistant sapphire or diamond windows for infrared spectroscopy

  • Custom-designed high-pressure NMR tubes and probe configurations

• Time-Resolved Measurements: Capturing transient conformational states during pressure changes necessitates:

  • Rapid pressure-jump systems capable of millisecond pressure transitions

  • Synchronized detection systems to monitor structural changes in real-time

  • Data analysis methods that can deconvolute complex spectral changes

• High-Pressure X-ray Crystallography: Obtaining structural data under pressure requires:

  • Specialized diamond anvil cells compatible with X-ray diffraction setups

  • Crystals that can withstand pressure transitions without cracking

  • Methods to differentiate pressure-induced conformational changes from crystal packing artifacts

• Computational Modeling Challenges: Simulating pressure effects accurately requires:

  • Force fields specifically parameterized for high-pressure conditions

  • Explicit solvent models that capture pressure-dependent solvation effects

  • Extensive computational resources for adequate sampling of conformational space

• Activity-Structure Correlations: Linking structural changes to functional effects necessitates:

  • High-pressure reaction vessels with capabilities for simultaneous structural and activity measurements

  • Methods to trap and characterize catalytic intermediates under pressure

  • Techniques to measure ligand binding affinities under varying pressure conditions