Recombinant Photobacterium profundum Peptide methionine sulfoxide reductase MsrA (msrA)

What is Photobacterium profundum MsrA and what is its primary function?

Photobacterium profundum MsrA is an enzyme that catalyzes the reduction of methionine sulfoxide residues back to methionine in proteins. Its primary function is to repair oxidative damage to proteins by reducing oxidized methionine residues, which is essential for maintaining protein functionality during oxidative stress. Similar to MsrA from other organisms, P. profundum MsrA can reduce both free oxidized-methionine and protein-contained oxidized methionine, with comparable specific activities reported for both Met(O) and N-Ac-Met(O) substrates (approximately 32-38 nmol/mg per min) . This repair mechanism is particularly important in deep-sea environments where P. profundum naturally inhabits, as these environments can present unique oxidative challenges.

How does P. profundum differ from other bacterial species in terms of environmental adaptation?

P. profundum SS9 is a Gram-negative bacterium originally isolated from the Sulu Sea. Unlike mesophilic bacteria, it is psychrotolerant and moderately piezophilic, capable of growth at temperatures from <2°C to >20°C (optimal temperature 15°C) and pressures from 0.1 MPa to nearly 90 MPa (optimal pressure 28 MPa) . Its ability to grow at atmospheric pressure allows for easier genetic manipulation and culture, making it an excellent model organism for studying piezophily. P. profundum possesses two chromosomes and an 80 kb plasmid, with greater genomic complexity than many other bacterial species, including 15 rRNAs (the largest reported in any bacterium), which may reflect its ability to rapidly respond to changes in pressure .

Why is MsrA considered part of the minimal gene set in bacteria?

MsrA is considered part of the minimal gene set because it performs an essential function in protecting proteins against oxidative damage across diverse organisms. Studies in various species, including E. chrysanthemi, have demonstrated that msrA is a virulence determinant . In yeast, deletion of the msrA gene results in increased sensitivity to oxidative stress and higher levels of free and protein-bound methionine sulfoxide under various oxidative stresses . This conservation across species highlights its fundamental importance in cellular function and survival, particularly under stress conditions. The enzyme's ability to repair oxidative damage in vivo is especially significant if methionine residues serve as antioxidants, as proposed by some researchers .

What expression systems are most effective for producing recombinant P. profundum MsrA?

Based on methodologies used for similar proteins, E. coli BL21 expression systems are generally effective for producing recombinant MsrA. In comparable studies with other MsrA proteins, researchers have successfully used pET expression vectors with histidine tags for efficient purification. For example, in work with E. chrysanthemi MsrA, cells containing the expression vector pTMS10 were grown in the presence of the inducer IPTG to overproduce MsrA-(His)6 protein, yielding a protein of approximately 27 kDa . Expression conditions should be optimized at temperatures below 37°C (typically 16-20°C) to ensure proper folding, particularly given P. profundum's native growth at 15°C .

What are the optimal conditions for purifying recombinant P. profundum MsrA while maintaining enzymatic activity?

For optimal purification of recombinant P. profundum MsrA while preserving enzymatic activity:

• Use nickel affinity chromatography for His-tagged protein (as demonstrated for other MsrA proteins)

• Maintain buffer pH between 7.0-7.5 (e.g., 100 mM HEPES buffer, pH 7.5)

• Include reducing agents (typically 1-5 mM DTT or β-mercaptoethanol) in all purification buffers to protect the catalytic cysteine residues from oxidation

• Conduct purification at 4°C to minimize protein degradation

• Consider including 10% glycerol in storage buffers to maintain stability

• Verify protein purity by SDS-PAGE and confirm identity through partial Edman degradation to determine N-terminal amino acid sequence

After purification, activity assays using Met(O) and N-Ac-Met(O) as substrates and thioredoxin as a reductant can verify that the enzyme maintains catalytic function .

How can I design an effective enzymatic assay to measure the activity of recombinant P. profundum MsrA?

An effective enzymatic assay for P. profundum MsrA activity should measure the reduction of methionine sulfoxide to methionine. Based on established protocols for MsrA enzymes:

Standard reaction mixture:

• 50 mM sodium phosphate buffer (pH 7.5)

• 50 mM NaCl

• 20 mM DTT or an alternative thioredoxin system (E. coli thioredoxin + thioredoxin reductase + NADPH)

• Substrate: Either free L-Met(O) (typically 200 μM) or N-acetyl methionine sulfoxide (N-Ac-Met(O)) at 200 μM

Measurement methods:

• HPLC-based assay: Measure the conversion of Met(O) to Met by reversed-phase HPLC

• Spectrophotometric assay: If using the thioredoxin system, monitor NADPH oxidation at 340 nm

• Coupled enzyme assay: Measure the reduction of the artificial substrate dabsyl-Met(O) to dabsyl-Met by HPLC with detection at 436 nm

Activity is typically expressed as nmol/mg per min, with expected specific activities around 32-38 nmol/mg per min for both Met(O) and N-Ac-Met(O) substrates, similar to E. coli MsrA .

How does P. profundum MsrA structure compare to MsrA from mesophilic bacteria?

While the search results don't provide direct structural information about P. profundum MsrA specifically, we can infer likely structural adaptations based on other pressure-adapted proteins in this organism:

Detailed structural studies using X-ray crystallography or cryo-EM would be necessary to confirm these predictions.

What molecular mechanisms enable P. profundum MsrA to function optimally under high-pressure conditions?

The molecular mechanisms enabling P. profundum MsrA to function optimally under high pressure likely include:

• Pressure-resistant protein folding: Modifications to the protein structure that resist pressure-induced denaturation or maintain proper folding under pressure. This is particularly important as high pressure can favor protein unfolding when there are large void volumes within the structure.

• Altered enzyme kinetics: Pressure affects reaction rates by influencing transition state volumes. P. profundum MsrA likely has evolved an active site that minimizes positive activation volumes or favors negative activation volumes under pressure.

• Membrane association adaptations: If P. profundum MsrA associates with membranes, it may have specific adaptations to function with pressure-adapted membranes, which tend to have different lipid compositions.

• Pressure-responsive regulation: The expression of P. profundum MsrA may be regulated by pressure-sensing systems. Proteomic studies of P. profundum have identified numerous proteins that are differentially expressed at different pressures , and regulatory systems including ToxR/ToxS have been implicated in pressure sensing .

• Coordinated antioxidant systems: MsrA likely works in concert with other antioxidant systems that are also pressure-adapted, forming a comprehensive defense against oxidative damage at high pressure.

How do substrate specificity and catalytic efficiency of P. profundum MsrA compare with MsrA from other organisms?

While specific kinetic parameters for P. profundum MsrA are not provided in the search results, we can make comparisons based on data from other organisms:

Substrate specificity:

• Like other MsrA enzymes, P. profundum MsrA likely has specificity for the S-epimer of methionine sulfoxide

• It can likely reduce both free Met(O) and protein-bound Met(O), as shown for other MsrA enzymes

• The enzyme would be expected to show similar activity toward N-Ac-Met(O), which is a model for peptide-bound Met(O)

Catalytic parameters:

• Based on data from E. chrysanthemi MsrA, specific activities for both Met(O) and N-Ac-Met(O) were approximately 38 nmol/mg per min and 32 nmol/mg per min, respectively

• P. profundum MsrA might show enhanced catalytic efficiency at higher pressures compared to mesophilic counterparts

• The enzyme likely utilizes a similar thioredoxin-dependent reduction system as observed in other organisms

Evolutionary adaptations:

• Some organisms have multiple MsrA copies (e.g., two MsrA proteins plus a fused MsrA-MsrB protein in cold-adapted organisms like D. psychrophila)

• P. profundum may have evolved specific residue substitutions that optimize activity at high pressure and low temperature

A comprehensive enzymological study comparing kinetic parameters (kcat, Km, kcat/Km) at different pressures would be needed to fully characterize these differences.

How does P. profundum MsrA contribute to the organism's adaptation to high hydrostatic pressure?

P. profundum MsrA likely contributes to high pressure adaptation through several mechanisms:

• Protection against pressure-induced oxidative stress: High pressure can increase the production of reactive oxygen species (ROS), leading to increased oxidation of methionine residues in proteins. MsrA's ability to repair these oxidative damages helps maintain protein function under pressure stress.

• Preservation of protein functionality: Pressure can affect protein conformation and function. By repairing oxidized methionine residues, MsrA helps maintain the proper structure and function of proteins essential for survival under high pressure.

• Integration with pressure-specific metabolic changes: Proteomic studies show that P. profundum undergoes significant metabolic shifts at high pressure, including up-regulation of glycolysis/gluconeogenesis pathway proteins and down-regulation of oxidative phosphorylation pathway proteins . MsrA may play a role in protecting these differentially expressed proteins from oxidative damage.

• Potential linkage to pressure-sensing systems: P. profundum uses transmembrane proteins like ToxR and ToxS for pressure sensing . MsrA may protect these sensor proteins or be regulated by them as part of the pressure response network.

• Supporting ribosomal function: High pressure affects ribosomal assembly, and P. profundum shows up-regulation of ribosomal proteins at high pressure . MsrA could protect ribosomal proteins from oxidative damage, supporting protein synthesis under pressure.

What evidence exists for pressure-dependent regulation of msrA gene expression in P. profundum?

While the search results don't specifically mention pressure-dependent regulation of msrA in P. profundum, several pieces of evidence suggest potential regulation patterns:

• Differential protein expression under pressure: Proteomic studies have identified numerous proteins in P. profundum that are differentially expressed at atmospheric pressure (0.1 MPa) versus high pressure (28 MPa) .

• Pressure-sensing systems: P. profundum utilizes the ToxR/ToxS transmembrane proteins as pressure sensors that regulate gene expression in response to pressure changes .

• Anti-correlation between transcriptome and proteome: Studies have shown that there can be anti-correlation between transcript levels and protein levels for some genes in P. profundum under different pressure conditions, suggesting complex post-transcriptional regulation mechanisms, possibly including antisense RNA inhibition of translation .

• Stress response regulation: Proteins associated with cellular stress responses (like DnaK, DnaJ, and GroEL) show complex regulation patterns under different pressure conditions, with some differences between transcript and protein levels .

To definitively determine pressure-dependent regulation of msrA, specific studies examining msrA transcript and protein levels at different pressures would be necessary, potentially using RT-PCR or quantitative proteomics approaches similar to those employed for other genes in P. profundum .

How does oxidative stress at different pressures affect MsrA activity and expression in P. profundum?

The relationship between oxidative stress, pressure, and MsrA activity/expression in P. profundum likely involves:

• Pressure-induced oxidative stress: High pressure environments may generate different patterns of oxidative stress compared to atmospheric pressure. Proteomic studies show that proteins involved in responding to reactive oxygen species are differentially expressed at different pressures in P. profundum .

• Metabolic shifts affecting ROS production: P. profundum shows up-regulation of glycolysis/gluconeogenesis pathway proteins at high pressure and up-regulation of oxidative phosphorylation pathway proteins at atmospheric pressure . These metabolic shifts likely result in different patterns of ROS production, potentially necessitating different levels of MsrA activity.

• Integrated stress responses: Studies in other organisms show that MsrA is part of an integrated response to oxidative stress. In yeast, msrA mutants showed increased sensitivity to oxidative stress and higher levels of free and protein-bound methionine sulfoxide under various oxidative stresses .

• Potential cold-adaptation link: P. profundum is both pressure-adapted and cold-adapted (optimal growth at 15°C) . Some cold-adapted organisms have multiple MsrA copies, suggesting a potentially enhanced role in cold environments . This might interact with pressure adaptation mechanisms.

• Regulatory connections: Regulatory proteins including MarR family regulators (involved in response to antibiotics and oxidative stresses in E. coli) are differentially expressed between pressure conditions in P. profundum , suggesting complex regulation of stress responses that might include MsrA.

Experimental approaches comparing MsrA activity and expression under controlled oxidative stress conditions at different pressures would be needed to fully characterize these relationships.

How does P. profundum MsrA compare to MsrA from other marine or piezophilic bacteria?

While the search results don't provide direct comparisons of MsrA across piezophilic bacteria, we can infer likely differences based on general adaptations observed in P. profundum and other marine bacteria:

• Evolutionary adaptations in piezophiles: P. profundum MsrA likely shares adaptations with other piezophilic bacteria compared to shallow-water relatives, potentially including:

  • Structural modifications that maintain function under pressure

  • Regulatory mechanisms that respond to pressure changes

  • Integration with pressure-specific metabolic pathways

• Photobacterium species variations: Other Photobacterium species (such as P. phosphoreum, P. iliopiscarium, and P. carnosum) inhabit different environments ranging from marine settings to modified atmosphere packaged meats . MsrA in these species likely shows adaptations specific to their respective ecological niches, with P. profundum's enzyme uniquely adapted to the deep sea.

• Redundancy patterns: Deep-sea adaptations in P. profundum include a high degree of metabolic diversity and redundancy . Some organisms have multiple MsrA copies (e.g., cold-adapted organisms) , and P. profundum may have similar redundancy in its methionine sulfoxide reduction systems.

• Integration with pressure-specific systems: P. profundum has pressure-specific adaptations in many cellular systems including flagellar motility, with separate flagellar systems for swimming and swarming under high-pressure conditions . MsrA may show integration with these pressure-specific cellular functions.

Comparative genomic and biochemical studies would be needed to fully characterize these differences across piezophilic and marine bacteria.

What can comparative genomics tell us about the evolution of msrA genes in piezophilic bacteria?

Comparative genomics could reveal several important aspects of msrA evolution in piezophilic bacteria:

• Gene duplication and specialization: Some organisms have multiple MsrA copies that may have specialized functions. For example, some bacteria have two MsrA copies, with one being present in all related genomes and another found specifically in certain cold-adapted species . Genomic analysis could reveal whether P. profundum has single or multiple msrA genes and their evolutionary relationships.

• Horizontal gene transfer versus vertical inheritance: Comparative genomics could determine whether msrA genes in piezophilic bacteria were acquired through horizontal gene transfer from other deep-sea organisms or evolved from ancestral genes through gradual adaptation.

• Sequence adaptations: Analysis of amino acid compositions and substitution patterns in MsrA from piezophilic versus non-piezophilic bacteria could reveal specific adaptations for high-pressure environments.

• Regulatory element evolution: Examining the promoter regions and regulatory elements of msrA genes could reveal how expression regulation evolved in response to high pressure.

• Genomic context: The P. profundum genome consists of two chromosomes and an 80 kb plasmid . Determining which replicon carries msrA and the surrounding genomic context could provide insights into its evolutionary history and functional integration.

• Correlation with ecological niche: Phylogenetic analysis of msrA genes from various Photobacterium species that inhabit different environments (deep sea, shallow water, associated with host organisms) could reveal adaptation patterns correlated with specific ecological niches .

What functional differences exist between P. profundum MsrA and eukaryotic MsrA enzymes?

Based on studies of MsrA from different organisms, several functional differences likely exist between P. profundum MsrA and eukaryotic counterparts:

• Structural organization: Eukaryotic MsrA enzymes often have different domain organizations compared to bacterial versions. For example, yeast MsrA is a protein of 184 amino acids , while bacterial MsrA proteins may have different sizes and domain structures.

• Subcellular localization: Eukaryotic MsrA proteins may be localized to specific cellular compartments (mitochondria, cytosol, etc.), while bacterial MsrA, including that from P. profundum, would typically be cytosolic or potentially membrane-associated.

• Redox partners: While both bacterial and eukaryotic MsrA typically use thioredoxin systems as electron donors, the specific redox partners and their interactions may differ.

• Pressure adaptation: P. profundum MsrA would have specific adaptations for function at high pressure (28 MPa) that would not be present in eukaryotic MsrA enzymes from non-piezophilic organisms.

• Regulatory mechanisms: Regulation of MsrA expression and activity would differ significantly between P. profundum and eukaryotes. In yeast, for example, msrA mutants show increased sensitivity to oxidative stress , but the regulatory pathways controlling expression would be distinct from those in bacteria.

• Evolutionary history: Eukaryotic MsrA shares a common ancestor with bacterial MsrA but has undergone distinct evolutionary adaptations. Comparative analysis could reveal conserved catalytic mechanisms versus divergent structural features.

What are the key challenges in expressing and studying recombinant P. profundum MsrA in conventional laboratory settings?

Key challenges in expressing and studying recombinant P. profundum MsrA include:

• Pressure-adapted protein expression: P. profundum MsrA is naturally adapted to function at high pressure (28 MPa) . Expressing it at atmospheric pressure in conventional systems may result in improper folding or reduced activity.

• Temperature considerations: P. profundum grows optimally at 15°C , suggesting its proteins may be cold-adapted. Expression in E. coli typically occurs at higher temperatures, potentially affecting folding and solubility.

• Maintaining enzymatic activity: MsrA contains catalytic cysteine residues susceptible to irreversible oxidation during purification. Careful attention to reducing conditions is essential throughout all experimental procedures.

• Measuring activity under pressure: Assessing enzymatic activity under high-pressure conditions requires specialized equipment not commonly available in standard laboratories.

• Replicating physiological conditions: P. profundum's natural environment (deep sea) has distinctive characteristics beyond just high pressure, including low temperature, specific ion concentrations, and oxygen levels that may be difficult to replicate.

• Solubility issues: Recombinant expression may result in inclusion body formation, particularly if the protein's folding is pressure-dependent.

• Post-translational modifications: Any native post-translational modifications in P. profundum may be absent in recombinant systems, potentially affecting function.

Methodological approaches to address these challenges:

• Use cold-adapted expression strains and low-temperature expression protocols

• Employ solubility-enhancing fusion tags

• Include osmolytes that may stabilize pressure-adapted proteins

• Consider cell-free expression systems that can operate under pressure

How can I design experiments to assess the impact of pressure on MsrA activity in vitro?

Designing experiments to assess pressure effects on MsrA activity requires specialized approaches:

Equipment considerations:

• High-pressure reaction vessels capable of maintaining constant pressure during enzymatic reactions

• Pressure-stable cuvettes or reaction containers for spectrophotometric measurements

• Pressure-resistant sampling systems for withdrawing reaction aliquots at different time points

Experimental design:

• Pressure-dependence of kinetic parameters:

  • Measure Km and kcat values at multiple pressure points (e.g., 0.1, 10, 28, 40 MPa)

  • Plot kinetic parameters versus pressure to determine optimal pressure and pressure stability range

• Thermodynamic analysis:

  • Determine activation volume (ΔV‡) by applying transition state theory to pressure-dependent rate data

  • Calculate volume changes associated with substrate binding using pressure-dependent Km values

• Stability measurements:

  • Assess thermal stability (Tm) at different pressures using differential scanning calorimetry

  • Monitor unfolding/aggregation under pressure using light scattering techniques

• Structural changes:

  • Use pressure-resistant FTIR or circular dichroism cells to monitor secondary structure changes with pressure

  • Apply hydrogen-deuterium exchange mass spectrometry at different pressures to identify regions with altered dynamics

Control experiments:

• Include mesophilic MsrA (e.g., from E. coli) as a pressure-sensitive control

• Test activity against both free Met(O) and protein-bound Met(O) substrates

• Verify that the thioredoxin system components (if used) are functional under pressure

• Include pressure-stable internal standards for quantification

Data analysis considerations:

• Use appropriate models that account for pressure effects on equilibria and rate constants

• Consider potential biphasic behavior if the enzyme transitions between conformational states at different pressures

What are effective strategies for studying MsrA-dependent pathways in P. profundum under different pressure conditions?

Effective strategies for studying MsrA-dependent pathways in P. profundum under different pressure conditions include:

Genetic approaches:

• Gene knockout/complementation:

  • Create msrA deletion mutants using marker exchange-eviction mutagenesis with sacB-containing suicide vectors as employed for other P. profundum genes

  • Complement with wild-type or mutant alleles to verify phenotypes

  • Assess growth and survival at different pressures (0.1-90 MPa)

• Reporter gene fusions:

  • Create transcriptional/translational fusions to monitor msrA expression under different pressure conditions

  • Use pressure-resistant fluorescent proteins or luciferase reporters

Physiological approaches:

• Oxidative stress susceptibility:

  • Compare sensitivity of wild-type and msrA mutants to oxidative stress agents (H₂O₂, paraquat) at different pressures

  • Measure intracellular ROS levels using pressure-resistant fluorescent probes

• Metabolomic profiling:

  • Analyze metabolite changes in wild-type versus msrA mutants at different pressures

  • Focus on pathways known to be pressure-regulated (glycolysis, oxidative phosphorylation)

Molecular analyses:

• Protein oxidation state:

  • Measure protein carbonylation levels as markers of oxidative damage

  • Quantify free and protein-bound methionine sulfoxide in wild-type and msrA mutants at different pressures, as done in yeast studies

• Transcriptome/proteome analysis:

  • Perform RNA-seq and quantitative proteomics at different pressures

  • Compare wild-type and msrA mutant profiles to identify affected pathways

  • Account for potential anti-correlation between transcriptome and proteome as observed in previous studies

Technical considerations:

• Culture conditions:

  • Use pressure vessels capable of maintaining constant pressure during growth

  • Employ anaerobic conditions to control for oxygen availability effects

  • Maintain consistent temperature (typically 15-17°C) across experiments

• Data integration:

  • Integrate transcriptomic, proteomic, and phenotypic data to build comprehensive models of MsrA function

  • Compare results with data from other pressure-responsive pathways in P. profundum

What recent advances have been made in understanding the role of MsrA in pressure adaptation of deep-sea bacteria?

Recent advances in understanding MsrA's role in pressure adaptation of deep-sea bacteria include:

• Systems-level understanding: Proteomic studies have revealed comprehensive pressure-dependent protein expression patterns in P. profundum, providing context for how antioxidant systems like MsrA integrate with other cellular processes . These studies show that:

  • Proteins involved in glycolysis/gluconeogenesis are up-regulated at high pressure

  • Proteins involved in oxidative phosphorylation are up-regulated at atmospheric pressure

  • Different pressure conditions represent distinct ecological niches with particular nutrient limitations and abundances

• Pressure sensing and signaling: Research has identified key pressure sensing systems including the ToxR/ToxS transmembrane proteins that regulate pressure responses , potentially including oxidative stress defense systems.

• Genetic approaches to piezophily: Large-scale transposon mutagenesis of P. profundum has identified genes conditionally required for high-pressure growth, with mutations in chromosomal structure, ribosome assembly, and regulatory systems all affecting pressure adaptation .

• Motility-pressure interactions: Studies have demonstrated that P. profundum utilizes separate flagellar systems for swimming and swarming under high-pressure conditions , illustrating how fundamental cellular processes are adapted to pressure.

• Transcriptome-proteome relationships: Comparative analysis of transcriptomic and proteomic data has revealed complex regulation of gene expression at different pressures, including anti-correlation between transcript and protein levels for some genes, suggesting post-transcriptional regulatory mechanisms .

What are the potential applications of recombinant P. profundum MsrA in studying protein oxidation and repair mechanisms?

Recombinant P. profundum MsrA offers several valuable applications for studying protein oxidation and repair mechanisms:

• Pressure-resistant repair system: P. profundum MsrA could provide a pressure-resistant tool for repairing oxidized proteins in high-pressure biochemical applications, potentially with different substrate specificity or stability characteristics compared to mesophilic counterparts.

• Model for structure-function relationships: As a pressure-adapted enzyme, P. profundum MsrA can serve as a model for understanding how protein structure and function adapt to extreme conditions, particularly in relation to catalytic mechanisms involving redox chemistry.

• Biomarker development: Understanding how MsrA activity and expression change under different pressure and oxidative stress conditions could lead to the development of biomarkers for environmental stress in marine ecosystems.

• Comparative biochemistry platform: P. profundum MsrA provides an opportunity to compare mechanistic details of methionine sulfoxide reduction across evolutionary diverse organisms, from piezophiles to mesophiles to psychrophiles.

• Oxidative damage assessment: Recombinant P. profundum MsrA could be used to quantitatively assess methionine oxidation in proteins exposed to different stress conditions, providing insights into oxidation patterns and susceptibility.

• Structure-guided enzyme engineering: Insights from P. profundum MsrA structure and function could guide the engineering of oxidoreductases with enhanced stability under extreme conditions for various biotechnological applications.

What are the most significant unresolved questions regarding P. profundum MsrA that warrant further investigation?

Several significant unresolved questions regarding P. profundum MsrA warrant further investigation:

• Pressure-adapted catalytic mechanism: How does P. profundum MsrA maintain catalytic efficiency at high pressure? Does it employ a different reaction mechanism or transition state compared to mesophilic MsrA enzymes?

• Structural adaptations: What specific structural features allow P. profundum MsrA to function optimally at 28 MPa? High-resolution structures determined under pressure would provide valuable insights.

• Regulatory networks: How is msrA expression regulated in response to pressure changes? Is it directly controlled by pressure-sensing systems like ToxR/ToxS, or through other mechanisms?

• Redox partner interactions: How do the interactions between MsrA and its redox partners (likely thioredoxin systems) adapt to high pressure? Are there pressure-specific redox partners?

• Substrate specificity: Does P. profundum MsrA show different substrate preferences compared to mesophilic counterparts? Are there specific pressure-adaptive proteins that are particularly protected by MsrA?

• Evolutionary history: Did P. profundum msrA evolve from shallow-water ancestors through gradual adaptation, or was it acquired through horizontal gene transfer from other deep-sea organisms?

• Integration with other stress responses: How does the MsrA-mediated oxidative stress response integrate with other pressure-specific adaptations in P. profundum? Does it play roles beyond protein repair, such as in signaling or regulation?

• Multi-stress adaptation: How does MsrA contribute to P. profundum's adaptation to the multiple stresses of the deep sea environment (low temperature, high pressure, potential nutrient limitations)?

• Applied potential: Could P. profundum MsrA be utilized in biotechnological applications requiring pressure-resistant enzymes or in maintaining protein integrity under pressure?

Addressing these questions would significantly advance our understanding of both MsrA biochemistry and deep-sea adaptations.