Recombinant Photobacterium profundum UDP-N-acetylenolpyruvoylglucosamine reductase (murB)

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

Photobacterium profundum is a deep-sea Gammaproteobacterium belonging to the family Vibrionaceae. It possesses several characteristics that make it valuable for microbiological research, particularly for understanding adaptations to extreme environments. P. profundum is a gram-negative rod with two circular chromosomes that can grow at temperatures ranging from 0°C to 25°C and pressures from 0.1 MPa to 70 MPa, depending on the strain .

P. profundum has become a model organism for studying piezophilic (pressure-loving) and psychrophilic (cold-loving) adaptations. The most well-studied strain, SS9, demonstrates optimal growth at 15°C and 28 MPa, classifying it as both a psychrophile and a piezophile . Other characterized strains include 3TCK (isolated from San Diego Bay, optimal growth at 9°C and 0.1 MPa) and DSJ4 (isolated from the Ryukyu Trench at 5110m depth, optimal growth at 10°C and 10 MPa) .

The bacterium's ability to thrive under high-pressure conditions makes it an excellent model for studying how proteins and cellular processes adapt to extreme environments, providing insights into the molecular basis of pressure adaptation that may have applications in biotechnology and astrobiology.

What is the function of UDP-N-acetylenolpyruvoylglucosamine reductase (murB) in bacterial cell wall synthesis?

UDP-N-acetylenolpyruvoylglucosamine reductase (murB) catalyzes a crucial step in peptidoglycan biosynthesis, which is essential for bacterial cell wall formation. Specifically, murB reduces UDP-N-acetylenolpyruvoylglucosamine to UDP-N-acetylmuramate using NADPH as a cofactor. This reaction represents the second committed step in the cytoplasmic phase of peptidoglycan synthesis.

The peptidoglycan layer provides structural integrity to bacterial cells, protecting them from osmotic pressure and determining cell shape. In gram-negative bacteria like P. profundum, the peptidoglycan layer is thinner but still essential for survival. Inhibition of murB activity can lead to cell wall defects and bacterial death, making it a potential target for antibacterial agents.

In the context of P. profundum, murB function may be particularly interesting due to the bacterium's adaptation to high-pressure environments, which could necessitate modifications to cell wall structure and biosynthetic enzymes to maintain cellular integrity under these extreme conditions.

Why study recombinant murB from Photobacterium profundum specifically?

Studying recombinant murB from Photobacterium profundum offers several unique research opportunities:

• Pressure adaptation insights: As a piezophile, P. profundum's enzymes, including murB, may possess structural adaptations allowing function under high hydrostatic pressure. These adaptations might include modifications in protein flexibility, hydration, and ionic interactions that contribute to pressure resistance.

• Cold adaptation mechanisms: The psychrophilic nature of P. profundum suggests its enzymes may have evolved features enabling efficient catalysis at low temperatures, potentially including reduced activation energy barriers and increased active site flexibility.

• Comparative enzymology: Comparing murB from P. profundum with homologs from mesophilic bacteria can reveal how evolution has shaped this enzyme to function in extreme environments.

• Evolutionary biology: Understanding the molecular adaptations in murB contributes to our knowledge of how life adapts to extreme environments and the evolutionary mechanisms involved.

• Biotechnological applications: Pressure-adapted enzymes may have applications in industrial processes requiring biochemical reactions under non-standard conditions.

Additionally, cell wall biosynthesis under pressure represents a fundamental aspect of bacterial physiology in deep-sea environments, making murB a key enzyme for understanding how these organisms maintain cellular integrity in their natural habitat.

What expression systems are suitable for producing recombinant P. profundum murB?

When selecting an expression system for recombinant P. profundum murB, researchers should consider several factors to optimize protein yield, solubility, and activity:

Bacterial Expression Systems:

Expression Conditions Table:

Cell-free expression systems may be particularly valuable for studying pressure-adapted enzymes, as they allow protein synthesis under controlled pressure conditions that mimic the native environment of P. profundum.

Based on previous work with marine bacterial proteins, fusion tags such as MBP (maltose-binding protein) often improve the solubility of recombinant proteins from extremophiles and should be considered if solubility issues arise.

How do pressure and temperature affect murB activity in P. profundum?

The effects of pressure and temperature on murB activity in P. profundum represent a fascinating area of research that bridges enzymology and extreme environment adaptation. Based on studies of other P. profundum proteins, we can infer several likely effects:

Pressure Effects:

Pressure typically alters enzyme kinetics through several mechanisms:

• Modification of reaction volume changes (ΔV‡)

• Alteration of protein conformational equilibria

• Changes in hydration around the active site

• Effects on substrate binding affinity

For P. profundum murB specifically, researchers would need to conduct activity assays under varying pressure conditions using specialized high-pressure equipment. The enzyme from strain SS9 would likely show optimal activity around 28 MPa, corresponding to its native depth environment, while enzymes from strains adapted to different pressures (like 3TCK and DSJ4) would display different pressure optima .

Temperature Effects:

As a psychrophilic organism, P. profundum's murB would be expected to show:

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

• Lower activation energy

• Greater structural flexibility, particularly around the active site

• Potentially lower thermal stability as a trade-off for cold activity

Experimental Approach Table:

Previous studies have shown that in P. profundum SS9, several stress response genes (htpG, dnaK, dnaJ, and groEL) are upregulated in response to atmospheric pressure , suggesting the involvement of chaperones in maintaining proper protein folding and function. Similar mechanisms might protect murB functionality when the organism experiences pressure stress.

What structural adaptations might be present in murB from piezophilic bacteria?

Structural adaptations in murB from piezophilic bacteria like P. profundum would likely reflect general principles observed in pressure-adapted proteins, while also incorporating specific modifications related to its catalytic function:

General Piezophilic Adaptations:

• Increased internal hydration: Piezophilic enzymes often contain more internal water molecules and polar residues that maintain hydration networks, counteracting the water-excluding effects of high pressure.

• Reduced void volumes: High pressure compresses protein structure, so piezophilic proteins typically have fewer and smaller internal cavities than their mesophilic counterparts.

• Flexible active sites: Enhanced flexibility in the active site region allows catalysis to proceed despite the compressing effects of high pressure.

• Altered surface charge distribution: Often involves increased acidic residue content on the protein surface, influencing hydration and protein-solvent interactions.

• Modified oligomeric interfaces: If murB functions as an oligomer, pressure adaptation would likely involve strengthened subunit interactions to prevent pressure-induced dissociation.

murB-Specific Adaptations:

For the specific case of murB, which binds UDP-linked substrates and uses NADPH as a cofactor, structural adaptations might include:

• Modified substrate binding pocket: Potentially larger or more flexible to accommodate changes in substrate conformation under pressure.

• Adapted cofactor binding site: Modified interactions with NADPH that remain optimal under pressure conditions.

• Pressure-resistant catalytic mechanism: Alterations in residues involved in catalysis to maintain efficient reaction coordination under pressure.

Structural Investigation Methods:

A comparative analysis between murB structures from P. profundum strains adapted to different pressures (SS9 vs. 3TCK) could provide particularly valuable insights into pressure adaptation mechanisms.

How can researchers optimize the purification protocol for recombinant P. profundum murB?

Purifying recombinant murB from P. profundum requires careful consideration of the enzyme's psychrophilic and piezophilic nature. The following protocol considerations are designed to maintain enzyme stability and activity throughout the purification process:

Recommended Purification Strategy:

• Low-temperature processing: All purification steps should be performed at 4-8°C to preserve the likely thermolabile nature of this psychrophilic enzyme.

• Buffer optimization:

  • Include osmolytes like glycine betaine that are accumulated by deep-sea bacteria

  • Maintain physiologically relevant salt concentrations (P. profundum has a salt requirement )

  • Consider testing buffers with enhanced pressure stability (Good's buffers with minimal pressure-dependent pKa shifts)

• Affinity purification approach:

  • His-tag purification with careful optimization of imidazole concentrations to minimize structural perturbation

  • Alternative tags like MBP or GST may improve solubility and can be considered for a tandem purification approach

• Pressure considerations:

  • If available, consider using high-pressure equipment during some purification steps to maintain native conformation

  • At minimum, avoid repeated cycles of pressurization/depressurization that might denature the protein

Optimized Purification Protocol Table:

Quality Control Assessments:

• SDS-PAGE and Western blotting to confirm purity and identity

• Activity assays under various conditions, including:

  • Standard atmospheric pressure (0.1 MPa)

  • Native pressure (28 MPa for SS9 strain)

  • Range of temperatures (0-25°C)

• Thermal shift assays to determine stability profiles

• Dynamic light scattering to assess aggregation state

When designing a purification strategy, researchers should also consider that P. profundum has stress response genes (htpG, dnaK, dnaJ, and groEL) that are upregulated in response to atmospheric pressure , suggesting that protein folding and stability may be particularly sensitive to pressure changes.

What biochemical assays are suitable for characterizing the activity of recombinant murB under varying pressure conditions?

Characterizing recombinant P. profundum murB activity under varying pressure conditions requires specialized techniques that can measure enzyme kinetics while maintaining controlled pressure environments. The following assays and approaches are particularly suitable:

Spectrophotometric Assays Under Pressure:

• NADPH consumption monitoring: The murB reaction consumes NADPH, which can be monitored by the decrease in absorbance at 340 nm. High-pressure optical cells with sapphire windows connected to a spectrophotometer allow real-time measurement of this activity under pressure.

• Coupled enzyme assays: These can be adapted for high-pressure work by ensuring all coupling enzymes remain active under the experimental pressure range.

• Stopped-flow analysis: High-pressure stopped-flow equipment enables measurement of rapid kinetics under pressure, allowing determination of individual rate constants.

Pressure-Jump Techniques:

Pressure-jump experiments allow researchers to rapidly change pressure during enzyme catalysis, providing insights into conformational changes and their rates during the catalytic cycle. These experiments are particularly valuable for understanding how pressure affects different steps in the catalytic mechanism.

Activity Measurement Parameters Table:

Data Analysis Approaches:

• Linear free energy relationships: Plotting ln(k) vs pressure allows determination of activation volumes.

• Pressure-temperature phase diagrams: These reveal the combined effects of these parameters on enzyme activity and stability.

• Michaelis-Menten analysis under pressure: Determination of how pressure affects both substrate binding (Km) and catalytic rate (kcat).

Given that P. profundum strains show different pressure optima (strain SS9: 28 MPa; strain 3TCK: 0.1 MPa; strain DSJ4: 10 MPa) , comparative analysis of murB from these strains would provide valuable insights into pressure adaptation mechanisms of this enzyme.

How can site-directed mutagenesis be used to investigate pressure-adaptive features of P. profundum murB?

Site-directed mutagenesis represents a powerful approach for investigating the molecular basis of pressure adaptation in P. profundum murB. By systematically altering specific amino acids hypothesized to contribute to pressure adaptation, researchers can experimentally validate structural features critical for function under high-pressure conditions.

Strategic Approaches for Mutagenesis Studies:

• Comparative sequence analysis-guided mutagenesis:

  • Align murB sequences from P. profundum strains adapted to different pressures (SS9, 3TCK, DSJ4)

  • Identify residues that differ between high-pressure and low-pressure adapted strains

  • Perform reciprocal mutations (converting high-pressure to low-pressure residues and vice versa)

• Structural feature targeting:

  • Target residues in cavities (volume-change sensitive regions)

  • Modify surface-exposed charged residues that may influence hydration

  • Alter flexibility-conferring regions (glycine residues, loop regions)

  • Modify substrate binding pocket residues

• Catalytic mechanism investigation:

  • Mutate residues involved in NADPH binding

  • Target residues participating in substrate recognition

  • Modify catalytic residues to assess pressure effects on different steps of the reaction

Mutagenesis Experimental Design Table:

Experimental Validation Approaches:

• Activity assays under pressure: Compare wild-type and mutant enzymes across pressure ranges (0.1-70 MPa) to determine changes in pressure optima.

• Stability measurements: Assess how mutations affect pressure and thermal stability using techniques like differential scanning calorimetry under pressure.

• Structural analysis: When possible, obtain structures of wild-type and key mutants to directly visualize the structural consequences of mutations.

• Computational approaches: Molecular dynamics simulations of wild-type and mutant proteins under various pressure conditions can provide mechanistic insights.

This systematic mutagenesis approach would help identify specific amino acids and structural features responsible for murB's adaptation to high pressure in P. profundum, contributing to our broader understanding of protein adaptation to extreme environments.

What potential applications exist for recombinant P. profundum murB in biotechnology?

Recombinant P. profundum murB offers several promising applications in biotechnology, leveraging its unique adaptations to extreme conditions:

Enzyme Engineering and Biocatalysis:

• Pressure-resistant biocatalysts: The pressure-adapted properties of P. profundum murB could serve as a template for engineering other enzymes to function under high-pressure industrial processes, which can enhance reaction rates and selectivity for certain chemical transformations.

• Cold-active catalysts: As a psychrophilic enzyme, murB could inspire the development of energy-efficient biocatalytic processes that operate at lower temperatures, reducing energy costs in industrial applications.

• Structure-based design: Understanding the structural basis of pressure adaptation in murB provides principles that can be applied to enhance the pressure resistance of other industrially relevant enzymes.

Pharmaceutical Research:

• Antibiotic development: As murB is essential for bacterial cell wall synthesis but absent in humans, insights from P. profundum murB could contribute to the development of novel antibiotics targeting this enzyme in pathogenic bacteria, potentially including pressure-stable antibiotics for deep-wound infections.

• High-pressure protein crystallography: The study of pressure-adapted enzymes like P. profundum murB advances methodologies for high-pressure protein crystallography, which has applications in understanding protein conformational states relevant to drug binding.

Environmental Biotechnology:

• Bioremediation under extreme conditions: Understanding pressure adaptation mechanisms could help develop microorganisms or enzymes capable of degrading pollutants in deep-sea environments.

• Biosensors for deep-sea applications: The pressure-sensing mechanisms identified through murB research could be applied to develop biosensors for deep-sea monitoring.

Technological Applications Table:

The study of P. profundum murB thus contributes both to our fundamental understanding of protein adaptation to extreme environments and provides valuable principles that can be applied across multiple biotechnological fields.

How does the kinetic behavior of P. profundum murB differ from mesophilic homologs?

The kinetic behavior of P. profundum murB likely exhibits distinctive characteristics when compared to mesophilic homologs, reflecting adaptations to both cold temperatures and high pressure. These differences would be expected to manifest in several key kinetic parameters:

Comparative Kinetic Parameters:

• Catalytic efficiency (kcat/Km): P. profundum murB would likely show higher catalytic efficiency at low temperatures compared to mesophilic homologs, a hallmark adaptation of psychrophilic enzymes that compensates for reduced reaction rates in cold environments.

• Temperature dependence: The activation energy (Ea) for P. profundum murB would be expected to be lower than for mesophilic homologs, resulting in less pronounced temperature dependence of activity.

• Pressure effects on kinetics: Unlike mesophilic enzymes that typically show reduced activity under pressure, P. profundum murB would maintain activity or even show enhanced catalysis under moderate pressure conditions, particularly for strain SS9 which grows optimally at 28 MPa .

• Substrate affinity: Psychrophilic enzymes often display higher Km values (lower affinity) at low temperatures compared to mesophilic counterparts, a trade-off that contributes to maintaining high kcat values.

Expected Kinetic Behavior Table:

Mechanistic Implications:

The kinetic differences likely reflect specific structural adaptations in P. profundum murB:

• Active site flexibility: Increased flexibility around the active site would facilitate catalysis at low temperatures and under pressure, contributing to higher kcat values.

• Substrate binding dynamics: Altered binding pocket dynamics would affect substrate recognition and product release, potentially manifesting as changes in Km values.

• Conformational landscape: The enzyme's energy landscape would be shifted to maintain a proper balance between stability and flexibility under cold, high-pressure conditions.

• Solvation effects: Different interactions with water molecules around the active site could contribute to unique pressure and temperature dependencies of catalytic parameters.

Understanding these kinetic differences not only provides insights into how murB has adapted to function in the deep-sea environment but also offers principles that could be applied to engineer enzymes for biotechnological applications under non-standard conditions.

What are the major challenges in expressing and purifying active recombinant P. profundum murB?

Researchers working with recombinant P. profundum murB face several significant challenges due to the enzyme's origin from a piezophilic and psychrophilic organism. Understanding these challenges and implementing appropriate solutions is crucial for successful expression and purification of the active enzyme:

Expression Challenges:

• Codon usage bias: P. profundum's genome has different codon preferences compared to common expression hosts like E. coli, potentially leading to translation inefficiency or premature termination.

• Protein folding at non-native pressure: When expressed at atmospheric pressure, the protein may not fold correctly if its native folding pathway is pressure-dependent.

• Temperature sensitivity: As a psychrophilic enzyme, expression at standard temperatures (37°C) may lead to misfolding, aggregation, or inclusion body formation.

• Post-translational modifications: Any required modifications in the native organism may be absent in the expression host.

Purification Challenges:

• Stability during purification: The enzyme may be unstable when removed from its native high-pressure environment or when exposed to temperatures above its normal range.

• Maintaining activity: Loss of activity during purification is particularly concerning for enzymes adapted to extreme conditions.

• Aggregation propensity: Extremophilic proteins often show increased aggregation when handled under standard laboratory conditions.

• Buffer incompatibility: Standard purification buffers may not be optimal for maintaining the structure of a pressure-adapted enzyme.

Solutions and Strategies Table:

Experimental Validation Approaches:

• Activity monitoring throughout purification: Track specific activity at each step to identify where activity loss occurs.

• Multiple construct design: Create various fusion constructs and truncations to identify the most stable and active form.

• Solubility screening: Test multiple expression conditions in parallel using fluorescent fusion reporters to rapidly identify conditions yielding soluble protein.

• Thermal shift assays: Use differential scanning fluorimetry to optimize buffer conditions for maximum stability.

By systematically addressing these challenges, researchers can successfully express and purify active recombinant P. profundum murB, enabling detailed biochemical and structural studies of this pressure-adapted enzyme.

How can researchers effectively study the structure-function relationship of murB under pressure conditions?

Investigating the structure-function relationship of P. profundum murB under pressure requires specialized techniques that can provide structural and functional data at elevated pressures. This represents a significant technical challenge but is essential for understanding how this enzyme has adapted to function in the deep sea.

Structural Analysis Under Pressure:

Functional Analysis Under Pressure:

• High-pressure stopped-flow kinetics:

  • Measures reaction rates under various pressures

  • Determines activation volumes for different steps in the catalytic cycle

  • Reveals pressure-dependent rate-limiting steps

• Pressure-perturbation spectroscopy:

  • Uses pressure jumps to trigger conformational changes

  • Monitors spectroscopic signals (fluorescence, absorbance) to track dynamics

Integrated Structure-Function Approaches Table:

Experimental Strategy:

• Begin with computational predictions (MD simulations) to identify regions likely to be pressure-sensitive

• Design targeted mutations based on these predictions

• Test wild-type and mutant enzymes using functional assays under pressure

• Obtain structural information through a combination of the techniques above

• Correlate structural changes with altered functional parameters

This integrated approach allows researchers to establish causal relationships between specific structural features and functional adaptations that enable murB to operate efficiently under the high-pressure conditions found in P. profundum's natural deep-sea habitat.

What are the most promising future research directions for P. profundum murB?

Research on P. profundum murB opens numerous avenues for future investigation that span from fundamental biochemistry to applied biotechnology. Several particularly promising directions include:

• Comparative genomics and evolution: Expanding the analysis to murB genes from other piezophilic bacteria would illuminate the evolutionary pathways leading to pressure adaptation. This could involve comparing murB sequences from various oceanic depths to identify convergent adaptations.

• Structural biology under extreme conditions: Developing improved methods for obtaining high-resolution structures under simultaneous high-pressure and low-temperature conditions would provide unprecedented insights into the native state of this enzyme.

• Systems biology of cell wall synthesis under pressure: Investigating how murB interacts with other enzymes in the peptidoglycan synthesis pathway under pressure would reveal adaptations at the pathway level rather than just the single enzyme level.

• Synthetic biology applications: Engineering pressure-adaptive elements from P. profundum murB into other proteins could create novel pressure-resistant enzymes for industrial applications.

• Astrobiology implications: Understanding how essential cellular processes adapt to extreme conditions provides insights relevant to the search for life in extreme environments, including potentially habitable zones on other celestial bodies.

The systematic study of P. profundum murB not only advances our understanding of life's adaptation to extreme environments but also provides valuable principles that can be applied across multiple scientific and technological fields, from enzyme engineering to deep-sea biotechnology.

How does understanding murB contribute to our broader knowledge of bacterial adaptation to extreme environments?

The study of UDP-N-acetylenolpyruvoylglucosamine reductase (murB) from Photobacterium profundum provides a valuable model system for understanding fundamental principles of bacterial adaptation to extreme environments. This enzyme's characteristics offer insights that extend beyond a single protein to broader biological adaptation mechanisms:

Fundamental Adaptation Principles:

• Molecular basis of pressure resistance: P. profundum murB exemplifies how essential enzymes adapt to maintain function under high hydrostatic pressure, revealing principles that likely apply across diverse pressure-adapted proteins.

• Dual adaptation strategies: As P. profundum is both piezophilic and psychrophilic, murB must balance adaptations to both pressure and cold, illustrating how proteins evolve under multiple selective constraints.

• Essential pathway conservation: Cell wall synthesis is non-negotiable for bacterial survival, making murB an excellent model for studying how critical pathways are preserved while adapting to extreme conditions.

• Evolutionary trajectories: Comparing murB across P. profundum strains adapted to different pressures (SS9: 28 MPa; 3TCK: 0.1 MPa; DSJ4: 10 MPa) reveals evolutionary pathways toward pressure adaptation.

Broader Implications Table: