Recombinant Photobacterium profundum Tyrosine--tRNA ligase (tyrS)

What is Photobacterium profundum TyrS and how does it differ from other bacterial tyrosyl-tRNA synthetases?

Photobacterium profundum tyrosine--tRNA ligase (TyrS) is an aminoacyl-tRNA synthetase that catalyzes the attachment of tyrosine to tRNA^Tyr in a two-step reaction: first activating tyrosine with ATP to form Tyr-AMP, then transferring the tyrosine to the acceptor end of tRNA^Tyr .

P. profundum TyrS differs from other bacterial TyrS enzymes primarily in its adaptation to high-pressure environments. P. profundum SS9, isolated from the Sulu Sea at depths of approximately 2.5 km, grows optimally at 28 MPa and 15°C . These extreme conditions have likely influenced the structural and functional properties of its TyrS enzyme, particularly in terms of pressure stability and substrate binding efficiency under high hydrostatic pressure.

How does the genomic context of the tyrS gene in P. profundum relate to its pressure adaptation?

The P. profundum SS9 genome consists of two chromosomes and an 80 kb plasmid . The genomic context of the tyrS gene reflects the organism's adaptation to varying pressure conditions. Like other pressure-responsive genes in P. profundum, tyrS likely has pressure-sensitive regulatory elements in its promoter region that control expression levels under different pressure conditions.

Comparative genomic analyses have revealed that P. profundum contains 15 rRNAs, the largest number reported for any bacterium, with significant variation among these operons . This genomic adaptation allows rapid response to pressure changes. Similar regulatory mechanisms may influence tyrS expression, allowing for appropriate levels of protein synthesis under varying pressure conditions.

What are the optimal expression systems and conditions for producing functional recombinant P. profundum TyrS?

For functional expression of recombinant P. profundum TyrS, the following methodological approach is recommended:

Expression System Selection:

• E. coli BL21(DE3) strains are suitable for initial expression attempts, but cold-adapted strains such as ArcticExpress may improve folding of this psychrophilic enzyme

• Consider using the pET system with an N-terminal His-tag for purification purposes

Culture Conditions:

• Growth temperature: 15-17°C (matching P. profundum's natural environment)

• Media: Marine broth supplemented with glucose (28 g/L 2216 medium with 20 mM glucose and 100 mM HEPES, pH 7.5)

• Induction: Lower IPTG concentrations (0.1-0.5 mM) with extended expression times (18-24 hours)

To verify proper expression, monitor growth at OD600 and confirm protein production via SDS-PAGE and Western blot analysis using anti-His antibodies if a His-tag was incorporated.

What challenges arise during purification of recombinant P. profundum TyrS and how can they be addressed?

Purification of recombinant P. profundum TyrS presents several challenges related to its psychrophilic and piezophilic origin:

Common Challenges and Solutions:

A typical purification protocol would involve:

• Cell lysis under gentle conditions (French press or sonication with cooling)

• IMAC (immobilized metal affinity chromatography) using Ni-NTA resin

• Size-exclusion chromatography for final polishing

• Storage in buffer containing 20 mM Tris-HCl (pH 8.0), 20% glycerol, 0.1 M NaCl, and 1 mM DTT

How does hydrostatic pressure affect the catalytic activity and specificity of P. profundum TyrS?

P. profundum TyrS exhibits pressure-dependent alterations in catalytic activity and specificity:

Pressure Effects on Enzymatic Parameters:

The enzyme's adaptation to high pressure likely involves structural modifications that optimize substrate binding and catalytic efficiency under deep-sea conditions. Research methodology for studying these effects includes:

• High-pressure enzyme assays using specialized equipment such as the high-pressure diamond anvil cell or high-pressure stopped-flow apparatus

• Monitoring aminoacylation activity by measuring AMP or PPi production under various pressure conditions

• Comparing reaction rates and substrate affinities at different pressures

These pressure-dependent changes may contribute to maintaining precise protein synthesis under the extreme conditions of the deep sea.

What is the temperature dependence profile of recombinant P. profundum TyrS and how does it compare to TyrS enzymes from mesophilic organisms?

As a psychrophilic enzyme from an organism that grows optimally at 15°C, P. profundum TyrS exhibits a distinct temperature-dependent activity profile compared to mesophilic homologs:

Temperature Profile Comparison:

To investigate temperature dependence:

• Measure aminoacylation activity across a temperature range (0-40°C)

• Generate Arrhenius plots to determine activation energies

• Conduct thermal denaturation studies using circular dichroism spectroscopy or differential scanning calorimetry

The low-temperature activity of P. profundum TyrS likely results from increased flexibility in catalytic domains, which enhances catalysis at low temperatures but reduces thermal stability compared to mesophilic counterparts.

What structural features of P. profundum TyrS contribute to its pressure adaptation, and how can they be investigated?

Several structural adaptations likely contribute to the pressure tolerance of P. profundum TyrS:

Pressure-Adaptive Features:

• Increased flexibility in catalytic domains

• Reduced number of ion pairs and hydrogen bonds

• Larger solvent-accessible cavities

• Modified hydrophobic core packing

These adaptations can be investigated through:

• High-resolution structural analysis:

  • X-ray crystallography under various pressure conditions

  • NMR spectroscopy for solution structure determination

  • Cryo-electron microscopy for visualizing conformational states

• Computational approaches:

  • Molecular dynamics simulations under varying pressure conditions

  • Analysis of volume changes during catalysis

  • Computational prediction of pressure effects on protein stability

• Mutational analysis:

  • Site-directed mutagenesis of residues predicted to be involved in pressure adaptation

  • Creation of chimeric enzymes with domains from mesophilic TyrS

Research has shown that pressure-adapted proteins often display increased flexibility at atmospheric pressure, which may appear counterintuitive but actually represents a pre-adaptation to maintain sufficient rigidity under the compressing effects of high hydrostatic pressure .

How does the amino acid specificity of P. profundum TyrS compare with other bacterial TyrRS enzymes, particularly regarding mischarging with phenylalanine?

Amino acid specificity is a critical aspect of tRNA synthetase function, with implications for translational fidelity. For P. profundum TyrS:

Comparative Specificity Analysis:

The specificity ratio represents how much better the enzyme discriminates its cognate amino acid (tyrosine) over the near-cognate amino acid (phenylalanine). Deep-sea organisms may have evolved higher specificity due to:

• Pressure effects on amino acid recognition

• The critical need for translational accuracy under extreme conditions

• Possible differences in the amino acid binding pocket architecture

To investigate this experimentally:

• Conduct aminoacylation assays with both tyrosine and phenylalanine

• Perform steady-state kinetic analysis to determine kcat and Km values for both amino acids

• Analyze the amino acid binding pocket through structural studies and molecular modeling

• Create mutations at key residues responsible for amino acid discrimination

Understanding these specificity differences has implications for protein synthesis fidelity under extreme conditions and may reveal novel mechanisms of amino acid recognition.

How can recombinant P. profundum TyrS be used to study pressure adaptation mechanisms in protein synthesis?

Recombinant P. profundum TyrS serves as an excellent model system for investigating pressure adaptation in protein synthesis through several methodological approaches:

• In vitro translation systems:

  • Reconstitute protein synthesis using P. profundum components under varying pressures

  • Compare translation efficiency and fidelity with mesophilic systems

  • Assess the role of TyrS in maintaining translational accuracy under pressure

• Chimeric enzyme studies:

  • Create hybrids between P. profundum TyrS and mesophilic TyrS enzymes

  • Identify domains responsible for pressure adaptation

  • Test functionality under varying pressure conditions

• Systems biology approaches:

  • Integrate proteomic and transcriptomic data on pressure response

  • Compare expression patterns of TyrS with other components of the translation machinery

  • Develop models of translation efficiency under pressure

Recent proteomic studies of P. profundum grown at atmospheric versus high pressure (28 MPa) have revealed differential expression of multiple translation components, including ribosomal proteins . TyrS likely participates in this coordinated pressure response of the translation machinery, making it a valuable probe for studying how protein synthesis adapts to extreme conditions.

What are the methodological considerations for measuring TyrS activity under high-pressure conditions?

Measuring enzymatic activity under high pressure presents unique challenges. For P. profundum TyrS activity assays under pressure:

Experimental Approaches:

• High-pressure stopped-flow spectroscopy:

  • Allows rapid mixing and real-time measurement

  • Can monitor ATP hydrolysis or pyrophosphate release

  • Requires specialized equipment rated for high pressure

• High-pressure cell systems:

  • Custom pressure chambers compatible with spectrophotometric measurements

  • Can maintain constant temperature and pressure during assays

  • Examples include diamond anvil cells adapted for biochemical assays

• Radioactive assays:

  • Pre-mix enzyme with substrates (including radiolabeled tyrosine)

  • Expose to pressure in specialized equipment

  • Quench reaction and analyze aminoacylated tRNA by acid precipitation

Key Methodological Considerations:

For accurate measurements, control experiments should include well-characterized enzymes with known pressure responses to validate the experimental setup.

How can molecular evolution studies with P. profundum TyrS advance our understanding of pressure adaptation in enzymes?

Molecular evolution studies with P. profundum TyrS can provide significant insights into pressure adaptation mechanisms through:

• Ancestral sequence reconstruction:

  • Infer ancestral sequences of TyrS enzymes

  • Resurrect these ancestral proteins through recombinant expression

  • Test their pressure resistance to trace the evolutionary path to piezophily

• Directed evolution approaches:

  • Apply pressure as a selection force on TyrS libraries

  • Screen for variants with enhanced or altered pressure responses

  • Identify key residues involved in pressure adaptation

• Comparative genomics and phylogenetics:

  • Analyze TyrS sequences across bacterial species from different depth zones

  • Identify convergent adaptations to pressure in independent lineages

  • Correlate sequence features with depth distribution

This research could identify evolutionary principles governing enzyme adaptation to extreme conditions. Studies of P. profundum have already revealed that some genes show evidence of horizontal gene transfer, potentially facilitating rapid adaptation to the deep-sea environment . Similar mechanisms may have influenced TyrS evolution.

What are the common challenges in aminoacylation assays with recombinant P. profundum TyrS and how can they be overcome?

Aminoacylation assays with P. profundum TyrS present several technical challenges:

Common Issues and Solutions:

Recommended Assay Protocol Modifications:

• Pre-incubate reaction components at 10-15°C

• Include 10-20% glycerol and 1 mM DTT in reaction buffers

• Use freshly prepared enzyme preparations whenever possible

• Consider radioactive assays ([³H]-tyrosine or [¹⁴C]-tyrosine) for highest sensitivity

• Include appropriate negative controls (reactions without enzyme, without tRNA, or with heat-denatured enzyme)

How does P. profundum culture under different pressure conditions affect the expression and properties of native TyrS?

P. profundum cultivation under varying pressure conditions significantly impacts its proteome, including TyrS expression and characteristics:

Pressure Effects on Native TyrS:

To study these effects:

• Culture P. profundum in pressure vessels at different pressures (0.1 MPa, 28 MPa, 45 MPa)

• Use the established cultivation method: marine broth supplemented with glucose and HEPES buffer (pH 7.5) at 15-17°C

• Harvest cells and extract proteins under conditions that preserve native state

• Analyze TyrS expression by Western blotting, enzyme activity assays, and mass spectrometry

Previous proteomic studies have shown that P. profundum differentially expresses many proteins involved in translation under varying pressure conditions . The ToxR/ToxS pressure-sensing system likely plays a role in regulating pressure-responsive genes, potentially including tyrS .

How can structural information from P. profundum TyrS contribute to the design of pressure-stable enzymes for biotechnological applications?

Structural insights from P. profundum TyrS can guide rational design of pressure-stable enzymes through:

Structure-Based Design Strategies:

• Identification of pressure-adaptive motifs:

  • Analyze structural elements that maintain function under pressure

  • Map these elements onto industrial enzymes to enhance pressure stability

• Rational engineering approaches:

  • Modify surface charge distribution based on P. profundum TyrS patterns

  • Redesign cavity volumes and flexibility in target enzymes

  • Introduce specific salt bridges or hydrophobic interactions identified in TyrS

• Computational prediction methods:

  • Develop algorithms based on P. profundum TyrS structure-function relationships

  • Create predictive models for pressure effects on protein stability

  • Simulate pressure effects on engineered variants before experimental testing

These approaches could lead to enzymes with enhanced stability for high-pressure biotechnological processes such as high-pressure biocatalysis, deep-sea bioremediation, and pressure-assisted food processing.

What insights can comparative studies between P. profundum TyrS and methanotrophic bacterial TyrS provide about enzymatic adaptations to extreme environments?

Comparative studies between P. profundum TyrS (adapted to high pressure and low temperature) and methanotrophic bacterial TyrS (adapted to utilize methane) can reveal convergent and divergent evolutionary strategies for extreme environment adaptation:

Comparative Analysis Framework:

Research approaches include:

• Comparative genomics and phylogenetic analysis

• Parallel structural studies of both enzyme types

• Swapping domains between the two enzymes to create functional chimeras

• Testing both enzymes under combinations of extreme conditions (pressure, temperature, pH)

This research could identify general principles of enzyme adaptation to extreme environments and reveal whether similar structural solutions have evolved independently in different extreme habitats.