Recombinant Photobacterium profundum UDP-N-acetylmuramate--L-alanine ligase (murC)

What is UDP-N-acetylmuramate--L-alanine ligase (MurC) and what role does it play in P. profundum cell wall biosynthesis?

UDP-N-acetylmuramate--L-alanine ligase (MurC) is an essential enzyme in bacterial peptidoglycan biosynthesis, catalyzing the addition of L-alanine to UDP-N-acetylmuramic acid (UDP-MurNAc). This represents the first step in peptide stem formation of peptidoglycan, requiring ATP and Mg²⁺ as cofactors. The peptidoglycan layer is crucial for maintaining cellular integrity against osmotic pressure and determining cell shape.

In P. profundum, MurC belongs to the Mur ligase family that sequentially adds amino acids to build the peptidoglycan structure. The reaction follows the general mechanism:

UDP-MurNAc + L-alanine + ATP → UDP-MurNAc-L-alanine + ADP + Pi

This pathway is particularly significant since Mur ligases are attractive antibacterial targets due to their essential role in peptidoglycan biosynthesis . The enzyme's structure and function may have adaptations specific to P. profundum's deep-sea habitat, potentially including pressure-adaptive features in deep-sea strains.

How do the different strains of P. profundum vary in their growth conditions, and how might this affect MurC expression and activity?

P. profundum has multiple strains adapted to different ocean depths with remarkable physiological differences in pressure response. These adaptations likely affect MurC expression and function:

These strain-specific adaptations likely influence MurC in several ways:

• Pressure adaptation: MurC from piezophilic strains (SS9, DSJ4) may have structural modifications that maintain functionality under high pressure conditions . These adaptations could include altered amino acid compositions that allow the enzyme to maintain proper folding and flexibility at depths where pressure would typically inhibit protein function.

• Temperature sensitivity: Each strain grows at different optimal temperatures, suggesting their MurC enzymes may have different temperature optima for activity . Cold-adapted enzymes typically display higher catalytic efficiency at lower temperatures compared to mesophilic counterparts.

• Gene expression regulation: Under suboptimal pressure conditions, P. profundum SS9 upregulates chaperones and DNA repair enzymes , suggesting MurC expression might also be regulated differently in response to environmental stressors.

• Evolutionary adaptation: The existence of phylogenetically cohesive "bathytypes" suggests that genetic modifications for depth-specific adaptations can evolve rapidly , potentially affecting MurC structure and function across strains.

What expression systems are optimal for producing recombinant P. profundum MurC?

For successful recombinant production of P. profundum MurC, several expression systems should be considered:

• E. coli expression systems:

  • BL21(DE3): Most commonly used, suitable for T7 promoter-driven expression

  • Arctic Express: Contains cold-adapted chaperonins (Cpn10 and Cpn60), beneficial for expressing psychrophilic proteins like those from P. profundum

  • C41/C43(DE3): Engineered strains that overcome toxicity issues with membrane-associated proteins

  • Rosetta: Supplies rare codons that might be present in P. profundum genes

• Expression parameters optimization:

  • Temperature: Lower induction temperatures (10-15°C) often yield higher amounts of soluble protein for enzymes from psychrophilic organisms like P. profundum

  • IPTG concentration: Lower concentrations (0.1-0.5 mM) typically reduce inclusion body formation

  • Media composition: Rich media with osmolytes may improve yield for marine bacterial proteins

  • Induction duration: Extended induction times (16-24 hours) at lower temperatures often increase yield

• Fusion tags for enhanced solubility and stability:

  • SUMO tag: Enhances solubility and can be precisely removed by SUMO protease

  • MBP (maltose-binding protein): Highly soluble carrier that can aid in purification

  • Thioredoxin: Promotes proper disulfide bond formation

  • His6 tag: Enables purification by metal affinity chromatography

A comparison of expression conditions for optimal MurC production might yield:

When designing an expression system, consider that P. profundum strains grow at different optimal temperatures—SS9 at 15°C and 3TCK at 9°C —suggesting recombinant expression may benefit from similarly low temperatures to ensure proper folding.

What purification strategies yield the highest purity and activity for recombinant P. profundum MurC?

Purifying recombinant P. profundum MurC requires a strategic approach to maintain both purity and enzymatic activity:

• Initial capture steps:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged proteins

    • Ni-NTA or Co-NTA resins with gradient elution (20-250 mM imidazole)

    • Consider using EDTA-free protease inhibitors to prevent metal chelation

  • Affinity chromatography for fusion proteins

    • Amylose resin for MBP-tagged proteins

    • Glutathione-Sepharose for GST-tagged proteins

  • Ion exchange chromatography based on theoretical pI

    • Calculate pI from amino acid sequence to determine appropriate resin

• Intermediate purification:

  • Tag removal (if necessary)

    • TEV, PreScission, or SUMO protease cleavage

    • Reverse IMAC to remove cleaved tag

  • Ammonium sulfate fractionation

    • Useful for concentrating protein and removing contaminants

    • Determine optimal precipitation percentage through pilot experiments

• Polishing steps:

  • Size exclusion chromatography

    • Superdex 200 or similar for final purification

    • Provides information on oligomeric state

  • Hydroxyapatite chromatography

    • Useful for removing nucleic acid contaminants that may co-purify

• Considerations specific to P. profundum MurC:

  • Cold purification (4-10°C) throughout the process, essential for psychrophilic enzymes

  • Addition of substrate analogs or product for stabilization during purification

  • Inclusion of reducing agents (DTT or TCEP) to maintain cysteine residues

  • Consider adding glycerol (10-20%) to prevent cold denaturation

A representative purification table showing yields and purities:

For piezophilic P. profundum strains like SS9, consider that proteins may have evolved specific structural features for high-pressure environments . These adaptations may affect protein stability during purification at atmospheric pressure, potentially requiring additional stabilizing agents.

What assays can be used to measure recombinant P. profundum MurC activity?

Several assays can be employed to measure P. profundum MurC activity, each with specific advantages:

• Coupled enzyme assays:

  • ATP consumption coupled to NADH oxidation via pyruvate kinase and lactate dehydrogenase

  • Monitors ADP formation by following NADH decrease at 340 nm

  • Advantages: Continuous measurement, high sensitivity

  • Limitations: Coupling enzymes may be affected by experimental conditions (temperature, pressure)

• Direct product detection:

  • HPLC separation of UDP-MurNAc-L-alanine from UDP-MurNAc

  • LC-MS identification and quantification

  • Advantages: Direct measurement, high specificity

  • Limitations: Requires specialized equipment, not continuous

• Inorganic phosphate release assays:

  • Colorimetric detection using malachite green

  • Advantages: Simple, high sensitivity, compatible with high-throughput screening

  • Limitations: Endpoint assay, potential interference from buffers containing phosphate

• Radioactive assays:

  • Using 14C-labeled L-alanine or 32P-ATP

  • Quantification by scintillation counting after product separation

  • Advantages: Extremely high sensitivity, direct measurement

  • Limitations: Handling radioactive materials, waste disposal challenges

For studying MurC activity in piezophilic strains, specialized high-pressure equipment would be necessary to perform assays under native conditions. Activity comparisons between strains should use consistent methods, as seen in studies with E. coli MurC where cellular activity was measured by substrate accumulation and product decrease .

When developing assays, studies with E. coli MurC have shown that substrate levels can be monitored over time to confirm enzyme inhibition, with experiments showing increases in substrate levels and decreases in product levels when MurC is inhibited .

How does hydrostatic pressure affect MurC activity in different P. profundum strains?

The effect of hydrostatic pressure on MurC activity varies significantly between P. profundum strains based on their evolutionary adaptations:

• Enzymatic activity under pressure:

  • SS9 (piezophile) MurC likely maintains or increases activity up to 28 MPa (optimal growth pressure)

  • 3TCK (non-piezophile) MurC activity probably decreases with increasing pressure above 0.1 MPa

  • These differences reflect strain-specific adaptations to different ocean depths

• Kinetic parameter shifts:

  • Km values may decrease under pressure for piezophilic MurC, indicating increased substrate affinity

  • kcat values might be maintained or enhanced under pressure for piezophilic enzymes

  • Volume changes during catalysis (ΔV‡) are typically smaller for pressure-adapted enzymes

• Experimental approaches to measure pressure effects:

  • High-pressure bioreactors with sampling capabilities

  • Pressure perturbation calorimetry

  • Custom high-pressure spectroscopic cells for continuous monitoring

A hypothetical dataset comparing MurC relative activity across pressures might show:

Growth experiments with P. profundum strains should be performed in pressure vessels with no gas space, as described in previous studies . Equipment used for high-pressure cultivation includes heat-sealable plastic bulbs containing media that are placed in pressure vessels .

The pressure adaptation of SS9 is reflected in its optimal growth at 28 MPa, while strain 3TCK grows optimally at 0.1 MPa . These growth optima likely correlate with the pressure optima for their respective MurC enzymes due to evolutionary adaptation of the entire cellular machinery.

What structural adaptations does P. profundum MurC have for functioning under high pressure?

P. profundum MurC from piezophilic strains likely exhibits several structural adaptations that enable function in high-pressure environments:

• Protein compressibility adaptations:

  • Reduced void volumes within the protein structure

  • Optimization of internal packing to minimize compressibility

  • Strategic placement of water-filled cavities to buffer pressure effects

• Active site modifications:

  • More flexible active site to maintain catalytic efficiency under pressure

  • Reduced ΔV of reaction to minimize pressure inhibition

  • Altered charge distribution to stabilize transition states under pressure

• Surface characteristics:

  • Increased number of surface-exposed hydrophilic residues

  • Modified surface charge distribution

  • Optimized salt bridge networks that are less affected by pressure

• Amino acid composition shifts:

  • Increased glycine content for enhanced backbone flexibility

  • Reduced proline content to avoid structural rigidity

  • More hydrophobic interactions that are strengthened by pressure

Comparative analysis of amino acid composition between piezophilic and non-piezophilic MurC might show patterns similar to those observed in other P. profundum proteins:

Studies on P. profundum have shown that deep-sea strains upregulate stress response genes like htpG, dnaK, dnaJ, and groEL in response to atmospheric pressure , suggesting MurC might also need chaperone assistance when not at optimal pressure.

How can site-directed mutagenesis be used to study the catalytic mechanism of P. profundum MurC?

Site-directed mutagenesis provides a powerful approach to probe the catalytic mechanism of P. profundum MurC:

• Key residues to target:

  • Catalytic residues: Those directly involved in bond making/breaking

  • Substrate binding residues: Those that interact with UDP-MurNAc, L-alanine, or ATP

  • Metal-coordinating residues: Those that coordinate Mg²⁺

  • Conformational change mediators: Residues at domain interfaces or hinge regions

• Types of mutations to consider:

  • Conservative mutations: Substituting with similar amino acids to assess specific contributions

  • Alanine scanning: Systematic replacement with alanine to identify essential residues

  • Charge inversions: Changing charge to assess electrostatic contributions

  • Cysteine substitutions: For subsequent chemical modification studies

• Analytical approaches:

  • Steady-state kinetics to determine changes in Km and kcat

  • Pre-steady-state kinetics to identify rate-limiting steps

  • Isothermal titration calorimetry to measure binding thermodynamics

  • Structural studies of mutant proteins

• Pressure-related investigations:

  • Comparing mutations' effects at different pressures

  • Identifying pressure-sensing residues

  • Engineering pressure-tolerant variants of pressure-sensitive MurC

To implement these studies, genetic tools developed for P. profundum can be utilized, such as tri-parental conjugations using helper E. coli strain pRK2073 . Plasmid construction techniques as described for other P. profundum genes can be adapted for MurC mutagenesis.

Example experimental design for site-directed mutagenesis:

*Note: These residue numbers are hypothetical and would need to be identified from sequence alignments or structural models of P. profundum MurC.

How can inhibitor studies of P. profundum MurC inform antibacterial drug development?

Inhibitor studies of P. profundum MurC can provide valuable insights for antibacterial drug development:

• Identification of novel inhibitory scaffolds:

  • High-throughput screening of chemical libraries

  • Fragment-based drug discovery approaches

  • Natural product screening

  • Virtual screening using homology models

• Structure-activity relationship (SAR) studies:

  • Systematic modification of lead compounds

  • Determination of pharmacophore requirements

  • Optimization of potency, selectivity, and physicochemical properties

• Comparison with MurC from pathogenic bacteria:

  • Cross-species activity profiling

  • Identification of conserved binding sites

  • Use of P. profundum MurC as a model for hard-to-express MurC enzymes

• Inhibition mechanism studies:

  • Determination of inhibition modality (competitive, uncompetitive, noncompetitive)

  • Time-dependent inhibition analysis

  • Structure-based investigations of binding modes

Examples from previous MurC inhibitor studies show that pyrazolopyrimidine compounds (like compound A) can potently inhibit MurC from various bacterial species, including E. coli and P. aeruginosa . Cellular studies with E. coli tolC mutants have demonstrated that MurC inhibition results in accumulation of the UDP-MurNAc substrate and decreased levels of the UDP-MurNAc-L-alanine product .

Experiments have shown that overexpression of MurC in E. coli tolC mutant leads to a ≥16-fold shift in MIC for compound A, confirming that inhibition of MurC expression results in growth suppression . This validates MurC as an antibacterial target.

What controls should be included when studying pressure effects on P. profundum MurC activity?

• Enzyme source controls:

  • Purified MurC from multiple P. profundum strains (piezophilic SS9 and non-piezophilic 3TCK)

  • MurC from non-piezophilic reference organisms (e.g., E. coli)

  • Site-directed mutants with altered pressure sensitivity

• Experimental condition controls:

  • Temperature control (±0.1°C) during pressure application

  • pH stability verification (some buffers have pressure-dependent pKa)

  • Substrate stability under pressure

  • Equipment calibration under pressure

• Activity measurement controls:

  • Pressure effects on any coupling enzymes or detection reagents

  • Reversibility of pressure effects

  • Time-course measurements to ensure linearity

  • Multiple pressure application/release cycles

• Data analysis controls:

  • Normalization to ambient pressure activity

  • Statistical analysis of pressure dependence

  • Comparison to theoretical models of pressure effects

Protocol considerations for high-pressure enzyme assays:

High-pressure experiments with P. profundum have been conducted using pressure vessels and heat-sealable plastic bulbs containing media with no gas space . Similar approaches could be adapted for enzyme assays.

How might recombinant P. profundum MurC be used as a model for studying extremozymes?

Recombinant P. profundum MurC offers a valuable model system for studying extremozymes (enzymes from extreme environments):

• Pressure adaptation studies:

  • MurC from piezophilic strains (SS9, DSJ4) versus non-piezophilic strains (3TCK) provides natural variants adapted to different pressure environments

  • Comparison reveals molecular basis of pressure adaptation

  • Insights can be applied to other pressure-sensitive enzymes or processes

• Cold adaptation mechanisms:

  • P. profundum strains grow at low temperatures (9-15°C)

  • MurC likely exhibits cold-adaptation features

  • Understanding these adaptations can inform the engineering of cold-active enzymes for biotechnology

• Molecular evolution studies:

  • Different bathytypes of P. profundum represent evolutionary adaptations to specific depths

  • MurC sequence and structural comparisons can reveal selection pressures

  • Genome plasticity between bathytypes highlights adaptation mechanisms

• Enzyme engineering applications:

  • Identifying pressure-resistant structural elements

  • Creating chimeric enzymes with enhanced pressure tolerance

  • Developing enzymes for industrial processes under non-standard conditions

P. profundum's genome shows evidence of rapid adaptation to depth-specific environmental stresses, including temperature, pressure, and nutrient availability . This makes its enzymes, including MurC, excellent models for studying how proteins adapt to extreme conditions.

The fact that different strains of P. profundum have been isolated from environments ranging from shallow waters (3TCK from San Diego Bay) to deep sea (SS9 from Sulu Sea) provides a natural experimental system with enzymes adapted to different pressures but still within the same species, minimizing other evolutionary variables.