Recombinant Photobacterium profundum Lysine--tRNA ligase (lysS), partial

What is the basic structure and function of Photobacterium profundum lysS?

Photobacterium profundum lysS encodes a lysine-tRNA ligase (LysRS) that belongs to the class-II aminoacyl-tRNA synthetase family. The protein consists of 502 amino acids with a molecular weight of approximately 57.1 kDa . Like other class-II aminoacyl-tRNA synthetases, it likely has two primary domains: a smaller N-terminal domain that binds the tRNA anticodon and a larger C-terminal domain with the catalytic activity characteristic of class II synthetases . The enzyme catalyzes the attachment of lysine to its cognate tRNA, which is an essential step in protein synthesis.

How does recombinant expression of P. profundum lysS differ from other bacterial lysS systems?

While E. coli possesses two isoforms of lysyl-tRNA synthetase (LysS and LysU), with LysU being expressed under stress conditions like heat shock , P. profundum SS9 appears to have adapted its protein expression systems to function optimally under high-pressure conditions. When expressing P. profundum lysS recombinantly, researchers should consider that this protein evolved to function in a high-pressure environment (approximately 28 MPa), which may affect its stability and activity when expressed in standard laboratory conditions .

What expression systems are recommended for producing recombinant P. profundum lysS?

Based on similar work with E. coli lysyl-tRNA synthetase, an effective approach would be to use a tac promoter fusion system for overproduction . The protein can be expressed in E. coli, as demonstrated by similar experiments with other P. profundum proteins . When designing expression constructs, it's advisable to include affinity tags for purification while ensuring they don't interfere with the protein's structure or function. Codon optimization might also be necessary when expressing in heterologous hosts due to potential codon usage bias differences between P. profundum and the expression host.

How does high hydrostatic pressure affect the structure-function relationship of P. profundum lysS?

While specific data on P. profundum lysS's pressure adaptation is limited in the provided search results, insights can be drawn from studies on other proteins from this organism. Cytochrome P450 from P. profundum SS9 shows pressure-induced transitions characterized by volume changes (ΔV) of around -100 to -200 mL/mol and a P1/2 of 300-800 bar, which is close to P. profundum's natural habitat pressure . For lysS, pressure adaptation might involve:

• Altered protein conformational dynamics that maintain activity at high pressure

• Modified substrate binding pocket architecture

• Changes in protein hydration and water accessibility to active sites

• Adaptations in protein-protein interactions under pressure

Research methodologies should include:

• Comparative activity assays at different pressures (0.1-80 MPa)

• Pressure perturbation spectroscopy to analyze conformational changes

• Molecular dynamics simulations under various pressure conditions

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

What methodological approaches are recommended for investigating potential synthetic lethality between lysS and other genes in P. profundum SS9?

Based on the synthetic lethality observed between fabD and pfaA in P. profundum SS9 , similar investigations with lysS could reveal important pathway interactions. A recommended methodological framework would include:

• Construction of conditional mutants: Create strains with inducible expression of lysS to avoid lethality during strain construction.

• Genome-wide screening: Employ transposon mutagenesis or CRISPR interference screens in the conditional lysS background to identify synthetic lethal or sick interactions.

• Complementation studies: Express heterologous lysS variants to test for functional conservation and pathway complementation.

How can researchers distinguish between the effects of temperature and pressure when working with recombinant P. profundum lysS?

P. profundum SS9 is both piezophilic (pressure-loving) and psychrophilic (cold-loving), requiring careful experimental design to distinguish these effects:

• Orthogonal experimental design: Establish a matrix of conditions varying both temperature (4-30°C) and pressure (0.1-60 MPa) independently.

• Comparative analysis with mesophilic homologs: Express lysS from non-piezophilic organisms alongside P. profundum lysS and compare their activities under various pressure/temperature combinations.

• Thermal stability vs. pressure stability analysis: Employ differential scanning calorimetry at various pressures to generate a comprehensive stability landscape.

• Kinetic parameter determination: Measure enzyme kinetics (kcat, KM) across the temperature/pressure matrix to identify parameters most affected by each variable.

What purification strategy is recommended for obtaining high-purity recombinant P. profundum lysS?

Based on successful purification of E. coli lysyl-tRNA synthetase and other similar proteins, a multi-step purification strategy is recommended:

• Initial capture: Affinity chromatography using either:

  • Nickel-NTA (if His-tagged)

  • Glutathione sepharose (if GST-tagged)

• Intermediate purification: Ion exchange chromatography

  • Given the theoretical pI of P. profundum lysS, use anion exchange at pH 8.0

• Polishing step: Size exclusion chromatography to ensure homogeneity and remove aggregates

• Quality control assessments:

  • SDS-PAGE: Should show a single band at approximately 57.1 kDa

  • Western blot: Using antibodies against the tag or specific to lysS

  • Activity assay: Measuring aminoacylation of tRNA^Lys

  • Mass spectrometry: For identity confirmation

What are the critical parameters for assessing the activity of recombinant P. profundum lysS?

A comprehensive activity assessment should include:

• Aminoacylation assay conditions optimization:

  Parameter	Range to test	Notes
  pH	6.5-8.5	Test in 0.5 increments
  Temperature	4-30°C	Include 15°C (growth optimum for SS9)
  Pressure	0.1-60 MPa	Include 28 MPa (natural habitat)
  Salt concentration	100-500 mM NaCl	SS9 is moderately halophilic
  Mg²⁺ concentration	1-20 mM	Critical for ATP binding
  ATP concentration	0.1-5 mM	Substrate for reaction
  Lysine concentration	0.1-5 mM	Substrate for reaction
  tRNA^Lys concentration	0.1-10 μM	Substrate for reaction

• Kinetic parameters determination:

  • KM and kcat for all three substrates (ATP, lysine, tRNA^Lys)

  • Product inhibition studies

  • pH rate profiles to determine optimal conditions

• Pressure effects characterization:

  • Activity vs. pressure plots

  • Determination of pressure optima

  • Calculation of activation volumes

How can researchers verify that recombinant P. profundum lysS is properly folded and active?

Multiple complementary approaches should be employed:

• Circular dichroism spectroscopy: To assess secondary structure content and thermal stability

• Intrinsic fluorescence measurements: To evaluate tertiary structure integrity

• Size exclusion chromatography with multi-angle light scattering (SEC-MALS): To determine oligomeric state and homogeneity

• Activity assays under various conditions:

  • Standard assay measuring aminoacylation activity

  • ATP-PPi exchange assay to assess amino acid activation

  • Thermal shift assays in the presence and absence of substrates

• Comparative analysis with mesophilic homologs: Benchmark against well-characterized E. coli LysS/LysU

• Complementation testing: Ability to complement E. coli lysS/lysU mutants

What approaches are recommended to investigate the structural basis of pressure adaptation in P. profundum lysS?

Given the known crystal structure of E. coli LysU at 2.8 Å resolution , several approaches can be applied:

• Homology modeling and molecular dynamics simulations:

  • Generate a structural model of P. profundum lysS based on E. coli LysU

  • Perform comparative MD simulations at various pressures

• X-ray crystallography under pressure:

  • Crystallize P. profundum lysS

  • Collect diffraction data at various pressures using specialized equipment

• High-pressure NMR studies:

  • Investigate structural changes and dynamics under pressure

  • Identify regions with altered flexibility or conformation

• Hydrogen-deuterium exchange mass spectrometry:

  • Compare exchange rates at different pressures

  • Identify pressure-sensitive regions of the protein

• Mutational analysis targeting unique residues:

  • Identify residues unique to P. profundum lysS compared to mesophilic homologs

  • Create point mutations and assess pressure sensitivity

How does P. profundum lysS compare to other bacterial lysyl-tRNA synthetases in terms of substrate specificity?

Lysyl-tRNA synthetases demonstrate important differences in substrate recognition that affect their function:

• Lysine analog recognition:

  • LysRS1 and LysRS2 show significant differences in their potential to bind lysine analogs with backbone replacements

  • S-(2-aminoethyl)-L-cysteine is a poor substrate for LysRS1 but can be utilized by LysRS2

  • P. profundum lysS likely belongs to the LysRS2 family based on its sequence, suggesting it may have similar substrate specificities to E. coli LysS/LysU

• Comparative substrate specificity analysis:

  Enzyme source	Relative activity with lysine	S-(2-aminoethyl)-L-cysteine utilization	Inhibition by S-(2-aminoethyl)-L-cysteine
  E. coli LysS	100%	Yes	Strong
  E. coli LysU	Variable (stress-induced)	Yes	Strong
  LysRS1 family	100%	Poor	200-fold less effective than for LysRS2
  P. profundum lysS	To be determined	To be determined	To be determined

• tRNA recognition elements: Comparative analysis of tRNA recognition by various LysRS enzymes would reveal whether P. profundum lysS has evolved specialized features for recognizing tRNA^Lys under high-pressure conditions.

What is known about the evolution of lysyl-tRNA synthetases in piezophilic organisms compared to mesophilic bacteria?

While the search results don't directly address the evolution of lysyl-tRNA synthetases in piezophilic organisms, we can infer potential evolutionary patterns:

• Studies on other P. profundum proteins show pressure adaptations, such as in cytochrome P450, which exhibits constrained water access to the active site under high pressure .

• Homologous proteins from piezophilic organisms often show amino acid substitutions that favor protein function under high pressure. For instance, a single amino acid substitution was attributed to pressure adaptation in 3-isopropylmalate dehydrogenase from an extremely piezophilic bacterium .

• Comparative genomic analysis between P. profundum and related mesophilic species would likely reveal selective pressure on genes involved in protein synthesis, including lysyl-tRNA synthetase.

• The distribution of LysRS1 and LysRS2 in different organisms has been linked to their differential sensitivity to noncanonical amino acids , suggesting that environmental factors can drive the selection of specific aminoacyl-tRNA synthetase variants.

How might recombinant P. profundum lysS be utilized in biotechnological applications?

Several potential applications emerge from understanding this pressure-adapted enzyme:

• Pressure-stable cell-free protein synthesis systems:

  • Incorporation of P. profundum lysS into cell-free systems could enhance protein production under high-pressure conditions

  • This could enable the synthesis of pressure-sensitive proteins that are difficult to produce in conventional systems

• Bioorthogonal amino acid incorporation:

  • If P. profundum lysS shows altered specificity compared to conventional LysRS enzymes, it might be engineered for incorporation of noncanonical amino acids

  • This could expand the genetic code for novel protein engineering applications

• Antibiotic development targeting LysRS:

  • Understanding the structural differences between P. profundum lysS and human lysyl-tRNA synthetase could inform the development of selective inhibitors

  • This approach has already shown promise for M. tuberculosis LysRS

• Model system for studying pressure adaptation:

  • P. profundum lysS could serve as a model protein for investigating the molecular basis of pressure adaptation

  • Insights could be applied to engineering pressure-stable variants of industrial enzymes

What genetic engineering approaches could be used to study the role of lysS in P. profundum SS9 adaptation to high pressure?

Based on the genetic approaches used to study other genes in P. profundum SS9 , several strategies could be employed:

• Conditional mutant construction:

  • If lysS is essential, create strains with inducible or temperature-sensitive alleles

  • This would allow for controlled depletion studies

• Domain swapping experiments:

  • Create chimeric proteins by swapping domains between P. profundum lysS and mesophilic homologs

  • Identify which domains confer pressure adaptation

• Site-directed mutagenesis:

  • Target residues unique to P. profundum lysS

  • Create point mutations to assess their contribution to pressure adaptation

• Heterologous complementation tests:

  • Express P. profundum lysS in E. coli or other mesophilic bacteria

  • Test whether it confers any pressure-related phenotypes

• Global gene expression studies:

  • Analyze transcriptome changes in response to lysS depletion under various pressure conditions

  • Identify genes that are co-regulated with lysS or respond to lysS perturbation

• Suppressor mutation analysis:

  • Similar to the approach used for fabD/pfaA , identify suppressor mutations that rescue lysS defects

  • This could reveal functional interactions with other cellular pathways