Recombinant Photobacterium profundum Deoxyribose-phosphate aldolase (deoC)

What is deoxyribose-phosphate aldolase (deoC) and what reaction does it catalyze in P. profundum?

Deoxyribose-phosphate aldolase (DERA) in P. profundum belongs to the class I aldolase family and catalyzes the reversible aldol reaction between acetaldehyde and glyceraldehyde-3-phosphate (G3P) to produce 2-deoxyribose-5-phosphate (DR5P) . This reaction represents a key metabolic link between nucleoside catabolism and central carbon metabolism in marine bacteria. The enzyme works through a Schiff base mechanism utilizing a conserved lysine residue in its active site that forms a covalent intermediate with the substrate .

How does P. profundum DERA contribute to the organism's adaptation to its deep-sea environment?

P. profundum is a piezophilic (pressure-loving) marine bacterium that thrives in deep-sea environments . Its DERA enzyme likely exhibits adaptations to function optimally under high-pressure conditions. While specific pressure-responsive expression data for DERA has not been directly established, studies on P. profundum show that many metabolic enzymes are regulated by ToxR, a global transcriptional regulator that mediates pressure-responsive gene expression . The metabolic flexibility provided by DERA may contribute to P. profundum's ability to utilize diverse carbon sources in nutrient-limited deep-sea environments.

What structural features distinguish P. profundum DERA from homologous enzymes in other organisms?

While the specific crystal structure of P. profundum DERA has not been fully characterized, comparative studies of DERA enzymes from other organisms can provide insights. DERA enzymes typically contain conserved catalytic residues, including a lysine that forms the Schiff base intermediate . In thermophilic organisms like Aciduliprofundum boonei, alignment studies revealed highly conserved residues Lys127, Asp92, and Lys185 that are essential for catalysis . Similar conservation patterns are likely present in P. profundum DERA, though potentially with modifications that enhance function under high pressure.

What are the recommended methods for cloning and expressing recombinant P. profundum DERA?

Based on established protocols for similar piezophilic enzymes, the recommended approach involves:

• Gene Identification and Primer Design: Identify the deoC gene sequence from P. profundum genome (strain SS9 is well-characterized) and design specific primers with appropriate restriction sites.

• PCR Amplification and Cloning: Amplify the deoC gene and clone it into an expression vector such as pET-based systems with a histidine tag for purification .

• Expression Conditions: Transform into E. coli BL21(DE3) or similar expression strains. Induce with 0.5-1.0 mM IPTG at mid-log phase (OD600 of 0.6-0.8) .

• Purification Strategy: Lyse cells and purify using Ni-NTA affinity chromatography, followed by size exclusion chromatography if higher purity is required .

A similar approach was successfully employed for the related enzyme phosphopentomutase (deoB) from P. profundum as shown in Table 1 :

Table 1: Expression and Purification Parameters for Recombinant P. profundum Phosphopentomutase

How can researchers assess the enzymatic activity of recombinant P. profundum DERA?

Enzymatic activity of DERA can be assessed through multiple complementary approaches:

• Spectrophotometric Assays: Monitor the formation of glyceraldehyde-3-phosphate in the retro-aldol reaction by coupling with glyceraldehyde-3-phosphate dehydrogenase and measuring NADH formation at 340 nm .

• Aldol Addition Monitoring: For the forward reaction, monitor the formation of 2-deoxyribose-5-phosphate using periodic acid/thiobarbituric acid (TBA) method, which produces a pink chromophore measurable at 549 nm .

• HPLC Analysis: Separate and quantify reaction products using HPLC with appropriate columns (typically anion exchange) for phosphorylated compounds .

• Mass Spectrometry: Confirm product identity and purity using LC-MS methods, particularly useful for novel substrate investigations .

Activity measurements should be conducted under various pressures to assess the enzyme's piezophilic adaptations, using specialized high-pressure equipment designed for biochemical assays.

What are the kinetic properties of P. profundum DERA and how do they compare to DERA from non-piezophilic organisms?

While specific kinetic parameters for P. profundum DERA are not directly reported in the provided literature, comparative analysis with related DERAs can provide a framework. For example, DERA from Aciduliprofundum boonei (another extremophile) showed the following kinetic parameters :

Table 2: Comparative Kinetic Parameters of DERA Enzymes from Various Sources

For P. profundum DERA, researchers should anticipate:

• Likely lower Km values compared to mesophilic organisms, indicating higher substrate affinity

• Optimal activity at lower temperatures (likely 15-20°C) reflecting the psychrotolerant nature of P. profundum

• Potential pressure-enhanced catalytic efficiency, a characteristic feature of enzymes from piezophilic organisms

How does hydrostatic pressure affect the structure and function of P. profundum DERA?

The effects of hydrostatic pressure on P. profundum DERA would likely include:

• Structural Stability: P. profundum enzymes often show structural adaptations that maintain flexibility and prevent pressure-induced protein denaturation .

• Catalytic Efficiency: Many enzymes from piezophilic organisms exhibit higher catalytic rates under elevated pressure conditions that would inhibit their mesophilic counterparts .

• Substrate Binding: Pressure may alter substrate binding affinity through effects on protein conformation and hydration shell dynamics.

P. profundum has adapted to live optimally at approximately 28 MPa pressure (corresponding to ocean depths of ~2800 meters), and its enzymes, including DERA, would be expected to function optimally under these conditions . Experimental approaches should include activity measurements at various pressures using specialized high-pressure bioreactors or stopped-flow systems adapted for high-pressure work.

What techniques can researchers use to investigate the substrate specificity of P. profundum DERA?

To comprehensively investigate substrate specificity of P. profundum DERA, researchers should employ:

• Substrate Panel Testing: Systematically test various aldehyde substrates beyond the natural substrate (acetaldehyde). DERA is known to accept aldehydes with chains up to four carbon atoms .

• Structure-Activity Relationship Analysis: Examine how structural modifications in substrate molecules affect binding and catalysis.

• Tandem Aldol Reactions: Assess the enzyme's ability to catalyze sequential aldol additions, particularly with acetaldehyde as the sole substrate, which produces 2,4,6-trideoxy-D-erythro-hexapyranose - a valuable precursor for statin synthesis .

• Competitive Inhibition Studies: Use substrate analogs to probe the binding pocket specificity.

• Molecular Docking and Simulation: Implement computational approaches to predict binding of novel substrates, especially under varying pressure conditions.

How is the deoC gene organized in the P. profundum genome and how is its expression regulated?

While specific information about the deoC gene organization in P. profundum is limited in the provided search results, insights can be drawn from related studies:

• Genomic Context: In many bacteria, deoC is part of the deo operon, which typically includes genes involved in deoxyribose metabolism. In P. profundum, the related deoB gene (encoding phosphopentomutase) has been characterized .

• Regulation Mechanisms: Gene expression in P. profundum is known to be regulated by ToxR, a transmembrane DNA binding protein involved in pressure-responsive gene expression . ToxR regulates multiple genes that alter membrane structure or participate in starvation responses, categories that could include deoC.

• Pressure Regulation: Various patterns of pressure regulation have been observed for different genes in P. profundum SS9, including some that are up-regulated at high pressure (28 MPa) compared to atmospheric pressure (0.1 MPa) .

What evolutionary insights can be gained from comparing P. profundum DERA with homologs from other marine bacteria?

Evolutionary analysis of P. profundum DERA could reveal:

• Piezophilic Adaptations: Comparison with DERA from non-piezophilic marine bacteria may reveal amino acid substitutions that enhance function under high pressure.

• Lateral Gene Transfer: The Vibrionaceae family, to which P. profundum belongs, shows evidence of gene duplication and lateral gene transfer for other metabolic genes , which might extend to DERA.

• Functional Diversification: In some P. profundum strains, gene duplication events have led to the presence of multiple copies of metabolic operons with high sequence identity (90%) but subtle functional differences .

• Environmental Adaptation Signatures: Comparative analysis might reveal signatures of adaptation to deep-sea environments, such as increased flexibility in key protein regions or altered surface charge distributions.

How can recombinant P. profundum DERA be used in biocatalytic applications requiring low-temperature or high-pressure conditions?

Recombinant P. profundum DERA offers several advantages for specialized biocatalytic applications:

• Cold-Active Biocatalysis: As P. profundum is psychrotolerant , its DERA likely maintains activity at lower temperatures (4-15°C), making it suitable for temperature-sensitive synthesis reactions where product or substrate stability is a concern.

• High-Pressure Enzymatic Processes: The enzyme would be expected to function optimally under elevated pressure conditions, enabling novel reaction conditions that may improve certain synthesis reactions.

• Stereoselective C-C Bond Formation: DERA catalyzes the stereoselective formation of C-C bonds between acetaldehyde and various other aldehydes , a valuable property for pharmaceutical intermediate synthesis.

• Tandem Aldol Additions: P. profundum DERA could potentially catalyze sequential aldol additions under conditions where mesophilic enzymes would be inefficient, offering new routes to complex chiral molecules .

• Enhanced Substrate Solubility: High-pressure conditions can increase the solubility of certain substrates, potentially improving reaction rates when using P. profundum DERA.

What methodological approaches can help overcome challenges in expressing and stabilizing recombinant P. profundum DERA?

Researchers working with P. profundum DERA may encounter challenges related to expression and stability. The following approaches can help address these issues:

• Codon Optimization: Optimize the coding sequence for expression in E. coli by adjusting codon usage while maintaining the amino acid sequence .

• Expression Temperature Adjustment: Lower the expression temperature (15-20°C) to improve folding of psychrophilic proteins in mesophilic hosts .

• Fusion Tag Selection: Test multiple fusion tags beyond the standard His-tag, such as MBP (maltose binding protein) which can enhance solubility of challenging proteins .

• Stabilizing Additives: Include osmolytes like glycerol (10-20%) in purification and storage buffers to maintain protein stability .

• Storage Conditions: Store purified enzyme with added stabilizers at -80°C in small aliquots to prevent freeze-thaw degradation .

• Site-Directed Mutagenesis: Introduce targeted mutations based on comparative analysis with stable DERA homologs to improve stability while maintaining activity under desired conditions .

• Immobilization Techniques: Explore enzyme immobilization strategies to enhance operational stability, particularly for repeated use in biocatalytic applications .

How can researchers design experiments to assess the impact of hydrostatic pressure on P. profundum DERA activity and stability?

To properly assess pressure effects on P. profundum DERA, researchers should consider:

• Specialized Equipment Setup:

  • High-pressure bioreactors with optical windows for real-time spectroscopic measurements

  • Stopped-flow systems modified for high-pressure applications

  • Pressure chambers compatible with 96-well plate formats for high-throughput screening

• Experimental Design:

  • Activity measurements across a pressure range (0.1-100 MPa) with appropriate controls

  • Comparison with DERA from non-piezophilic organisms under identical conditions

  • Assessment of enzyme-substrate complex formation under varying pressures

• Structural Analysis:

  • Circular dichroism spectroscopy under pressure to monitor secondary structure changes

  • Fluorescence spectroscopy to assess tertiary structure alterations

  • Molecular dynamics simulations to predict pressure-induced conformational changes

• Data Analysis:

  • Generate pressure-activity profiles with appropriate mathematical models

  • Determine pressure-dependent kinetic parameters (Km, Vmax, kcat)

  • Calculate activation volumes from pressure-rate profiles

A typical pressure-activity experiment might follow the protocol outlined in P. profundum studies , where cultures or purified enzymes are subjected to varying pressures in specialized pressure vessels, followed by activity assays performed either under pressure or immediately upon decompression.