Recombinant Photobacterium profundum 3-methyl-2-oxobutanoate hydroxymethyltransferase (panB)

Basic Research Questions

• What is Photobacterium profundum 3-methyl-2-oxobutanoate hydroxymethyltransferase (panB) and what reaction does it catalyze?

PanB from Photobacterium profundum catalyzes the reversible reaction in which a hydroxymethyl group from 5,10-methylenetetrahydrofolate is transferred onto alpha-ketoisovalerate to form ketopantoate . This represents the first committed step in the biosynthesis of pantothenate (vitamin B5), which serves as a precursor for coenzyme A and acyl carrier protein cofactor . The enzyme belongs to the PanB family and in P. profundum SS9, the protein consists of 264 amino acids with a molecular mass of approximately 28.6 kDa .

The reaction mechanism likely involves coordination of the substrate by a divalent metal ion (typically Mg²⁺), which orients the C3 carbon for deprotonation, similar to what has been observed in the E. coli homolog . This transferase activity is crucial for microbial survival, as many bacteria cannot acquire pantothenate from their environment and must synthesize it de novo.

• Why is Photobacterium profundum used as a source for studying panB?

Photobacterium profundum presents several unique characteristics that make it particularly valuable for studying panB:

• P. profundum is a piezopsychrophilic (pressure-loving and cold-loving) bacterium that grows optimally at 28 MPa and 15°C, providing insights into enzyme adaptation to extreme environments .

• It can grow under a wide pressure range from atmospheric pressure (0.1 MPa) up to 90 MPa, making it an excellent model organism for studying pressure adaptation mechanisms .

• Its ability to grow at atmospheric pressure allows for easier genetic manipulation and culture compared to other piezophiles, facilitating molecular biology studies .

• P. profundum SS9's genome has been fully sequenced, consisting of two chromosomes and an 80 kb plasmid, providing a comprehensive genetic context for studying its metabolic enzymes .

• The organism shows differential protein expression under varying pressure conditions, including key metabolic enzymes, suggesting unique adaptations that might extend to panB .

These characteristics make P. profundum panB an excellent model for understanding how enzymes adapt to function under high pressure and low temperature conditions, with potential implications for biotechnological applications.

• How does the structure and function of P. profundum panB compare to homologs from other organisms?

While the specific structure of P. profundum panB has not been fully characterized in the provided research, comparative analysis with the E. coli homolog reveals valuable insights:

The E. coli ketopantoate hydroxymethyltransferase (KPHMT) has been crystallized at 1.9 Å resolution, showing that it adopts a (βα)₈ barrel fold and belongs to the phosphoenolpyruvate/pyruvate superfamily . The active site contains ketopantoate bidentately coordinated to Mg²⁺, and similar binding is likely for the substrate, alpha-ketoisovalerate, orienting the C3 for deprotonation .

Based on our understanding of piezophilic proteins, P. profundum panB likely exhibits structural adaptations that include:

These structural adaptations would allow P. profundum panB to maintain catalytic activity under the high pressure conditions of its native deep-sea environment, while potentially sacrificing stability at atmospheric pressure and higher temperatures.

• What expression systems are optimal for recombinant production of P. profundum panB?

For efficient recombinant production of P. profundum panB, several expression systems can be employed with specific considerations for this piezopsychrophilic enzyme:

E. coli-based expression systems:

• BL21(DE3) strain with pET vectors (e.g., pET15b) for T7 RNA polymerase-driven expression

• Addition of an N-terminal 6xHis-tag for purification via immobilized metal affinity chromatography

• Co-expression with cold-adapted chaperones may improve folding

Optimized expression protocol:

• Clone the P. profundum panB gene into pET15b vector with an N-terminal His-tag

• Transform into E. coli BL21(DE3)

• Grow cultures at 37°C until OD₆₀₀ reaches 0.6-0.8

• Shift temperature to 15-18°C before induction with 0.1-0.5 mM IPTG

• Continue expression at 15-18°C for 16-20 hours

• Harvest cells by centrifugation at 4°C

This approach has been successfully applied to other proteins from P. profundum and similar cold-adapted organisms, with lower induction temperatures helping to ensure proper folding of psychrophilic proteins .

For challenging cases where E. coli expression yields poor results, alternative systems to consider include:

• Cold-adapted expression hosts like Pseudoalteromonas haloplanktis

• Yeast systems such as Pichia pastoris for proteins requiring eukaryotic folding machinery

• Cell-free protein synthesis systems that allow precise control of reaction conditions

• What purification strategies yield the highest activity for recombinant P. profundum panB?

A multi-step purification strategy is recommended to obtain high-activity recombinant P. profundum panB:

Purification workflow:

• Cell lysis:

  • Resuspend cells in buffer containing 50 mM HEPES pH 7.5, 300 mM NaCl, 10% glycerol, 1 mM DTT, 5 mM MgCl₂

  • Include protease inhibitors (e.g., PMSF or commercial cocktail)

  • Lyse cells by sonication or French press at 4°C

• Immobilized Metal Affinity Chromatography (IMAC):

  • Load clarified lysate onto a Ni-NTA or HisTrap column

  • Wash with buffer containing 20-50 mM imidazole

  • Elute with buffer containing 250-300 mM imidazole

  • Immediately add 5 mM MgCl₂ to the eluted fractions to stabilize the enzyme

• Size Exclusion Chromatography:

  • Apply concentrated protein to a Superdex 200 column

  • Elute with buffer containing 25 mM HEPES pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 1 mM DTT, 10% glycerol

• Optional tag removal:

  • If the His-tag affects activity, consider removing it using an appropriate protease (e.g., TEV protease)

  • Re-purify by passing through IMAC to remove the cleaved tag

Critical considerations for maintaining activity:

• Keep all buffers and equipment at 4°C throughout purification

• Include Mg²⁺ in all buffers as it is likely required for structural integrity and activity

• Add glycerol (10-20%) to prevent cold denaturation and improve stability

• Monitor activity after each purification step to track recovery and specific activity

This purification approach has been successful for related enzymes and should yield highly pure and active P. profundum panB suitable for biochemical and structural studies.

Intermediate Research Questions

• How do pressure and temperature affect the activity and stability of P. profundum panB?

As an enzyme from a piezopsychrophilic organism, P. profundum panB likely exhibits specific responses to pressure and temperature variations:

Pressure effects:
Based on studies of P. profundum proteomics under different pressure conditions , we can predict that panB may show:

• Higher catalytic efficiency at elevated pressures (around 28 MPa), corresponding to the organism's optimal growth pressure

• Structural stabilization under pressure, potentially through reduction of protein volume

• Pressure-dependent changes in substrate binding affinity

Temperature effects:
As a psychrophilic enzyme, P. profundum panB likely exhibits:

• Optimal activity at low temperatures (around 15°C), corresponding to the organism's growth temperature

• Reduced thermal stability compared to mesophilic homologs

• Higher catalytic activity at low temperatures compared to mesophilic equivalents

Expected activity profile under various conditions:

To experimentally determine these effects, one would need to:

• Express and purify recombinant P. profundum panB

• Measure enzymatic activity under varying pressure conditions using specialized high-pressure equipment similar to that described for P. profundum culture

• Analyze thermal stability through methods like differential scanning fluorimetry at different pressures

• Compare kinetic parameters (Km, kcat) across a matrix of temperature and pressure conditions

• What cofactors and reaction conditions are required for optimal activity of P. profundum panB?

Based on the information available for ketopantoate hydroxymethyltransferase enzymes and the specific characteristics of P. profundum:

Essential cofactors:

• Magnesium ions (Mg²⁺):

  • Critical for catalytic activity, likely coordinating the substrate in the active site as observed in the E. coli homolog

  • Optimal concentration typically 1-5 mM

  • May be partially substitutable with Mn²⁺ with reduced activity

• 5,10-methylenetetrahydrofolate:

  • Serves as the hydroxymethyl group donor in the reaction

  • Must be present in stoichiometric or excess amounts

Optimal reaction conditions:

Protocol for activity assay:

• Prepare reaction buffer: 50 mM HEPES pH 7.8, 150 mM NaCl, 5 mM MgCl₂, 2 mM DTT

• Add enzyme (1-10 μg)

• Add α-ketoisovalerate (1-5 mM)

• Initiate reaction by adding 5,10-methylenetetrahydrofolate (0.5-1 mM)

• Incubate at 15°C under appropriate pressure conditions

• Monitor reaction progress by:

  • HPLC analysis of ketopantoate formation

  • Spectrophotometric monitoring of 5,10-methylenetetrahydrofolate consumption

  • Coupled enzyme assay with the next enzyme in the pantothenate biosynthesis pathway

• How can the enzymatic activity of P. profundum panB be accurately measured in vitro?

Several methodological approaches can be employed to measure P. profundum panB activity with high accuracy:

Direct activity measurement methods:

• HPLC-based product detection:

  • Separate reaction components using reverse-phase HPLC

  • Quantify ketopantoate formation using UV detection at ~210-220 nm

  • Include appropriate standards for calibration

  • This method offers high specificity and sensitivity

• Spectrophotometric monitoring:

  • Follow the decrease in absorbance of 5,10-methylenetetrahydrofolate at ~290-300 nm

  • Requires correction for potential protein absorbance changes

  • Provides continuous real-time monitoring capability

• Coupled enzyme assay:

  • Link panB reaction to ketopantoate reductase (panE) activity

  • Monitor NADPH oxidation at 340 nm

  • Calculate panB activity based on the rate of NADPH consumption

  • This approach amplifies the signal for improved sensitivity

Detailed protocol for HPLC-based assay:

• Prepare reaction mixture containing:

  • 50 mM HEPES buffer, pH 7.5

  • 5 mM MgCl₂

  • 1 mM DTT

  • 0.1-1.0 mM 5,10-methylenetetrahydrofolate

  • 1-5 mM α-ketoisovalerate

  • 0.1-1 μM purified panB enzyme

• Incubate at 15°C (optionally under pressure)

• Stop reaction at various time points by heat inactivation or TCA precipitation

• Analyze samples by HPLC using:

  • C18 reverse-phase column

  • Mobile phase: 5-10% methanol in phosphate buffer, pH 6.5

  • Flow rate: 0.8-1.0 ml/min

  • UV detection at 210-220 nm

• Quantify ketopantoate using a standard curve

• Calculate initial reaction rates from the linear portion of the product formation curve

For kinetic characterization, vary substrate concentrations and analyze data using appropriate enzyme kinetics software to determine Km, Vmax, and kcat values.

• What unique adaptations in the P. profundum panB sequence contribute to its pressure tolerance?

While specific sequence information for pressure adaptation in P. profundum panB is not explicitly detailed in the search results, we can infer likely adaptations based on proteomic studies of P. profundum under different pressure conditions and general principles of protein adaptation to high pressure:

Potential sequence adaptations in P. profundum panB:

• Amino acid composition differences:

  • Likely reduced content of bulky hydrophobic residues (Phe, Trp, Leu)

  • Increased proportion of small residues (Ala, Gly) in core regions

  • Higher content of charged residues (Asp, Glu) on protein surface

  • Reduced number of proline residues in loop regions to enhance flexibility

• Key structural regions likely to contain adaptations:

  • Active site architecture: Modifications to maintain catalytic geometry under pressure

  • Subunit interfaces: If oligomeric, adaptations to maintain proper assembly

  • Loop regions: Increased flexibility to accommodate pressure effects

  • Hydrophobic core: Modifications to reduce volume changes under pressure

• Comparative analysis approach:
  To identify specific pressure adaptations, one would:

  • Align P. profundum panB sequence with homologs from non-piezophilic organisms

  • Identify conserved catalytic residues that remain unchanged

  • Locate positions with consistent substitutions unique to piezophilic lineages

  • Perform computational analysis of volume, flexibility, and charge distribution

• Experimental validation:

  • Generate site-directed mutants reversing putative pressure adaptations

  • Measure activity and stability under varying pressure conditions

  • Perform structural studies under pressure to observe conformational changes

Proteomic studies of P. profundum have shown that proteins involved in key metabolic pathways are differentially expressed under high pressure conditions , suggesting that adaptations to maintain essential functions like pantothenate biosynthesis under pressure would be evolutionarily favored.

The role of panB in P. profundum's metabolic adaptation to high pressure environment can be understood by examining the broader context of cellular responses to pressure:

Metabolic significance:

• PanB catalyzes the first committed step in pantothenate biosynthesis, which is essential for producing coenzyme A (CoA)

• CoA is critical for central carbon metabolism, including the TCA cycle and fatty acid metabolism

• Maintaining these pathways under high pressure is essential for cell survival

Integration with pressure-responsive pathways:
Proteomic studies of P. profundum have revealed that:

• Proteins involved in the glycolysis/gluconeogenesis pathway are up-regulated at high pressure (28 MPa)

• Conversely, several proteins involved in oxidative phosphorylation are up-regulated at atmospheric pressure (0.1 MPa)

• These differential expressions suggest a metabolic remodeling under pressure that would require maintained production of pantothenate and CoA

Specific adaptations in metabolic enzymes:

• The expression of some proteins involved in nutrient transport or assimilation appears to be directly regulated by pressure

• Different hydrostatic pressures represent distinct ecosystems with particular nutrient limitations and abundances

• PanB may be adapted to function optimally within this altered metabolic landscape

Genetic context considerations:

• The genomic organization of panB and related genes in P. profundum might reveal co-regulation with other pressure-responsive genes

• While not directly mentioned in the search results for P. profundum, the genomic context of biosynthetic genes can provide insights into their regulation under environmental stress

Experimental approach to investigate metabolic integration:

• Compare panB expression and activity levels under different pressure conditions

• Measure intracellular CoA levels at varying pressures

• Perform metabolic flux analysis using isotope-labeled precursors under different pressure regimes

• Create panB knockout or knockdown strains and assess pressure tolerance

This integrated understanding places panB within the broader context of P. profundum's remarkable adaptation to the deep-sea environment.