Recombinant Photobacterium profundum Serine hydroxymethyltransferase 1 (glyA1)

What are the optimal expression systems for recombinant P. profundum SHMT?

Based on established protocols for other deep-sea bacterial proteins, the following expression system parameters are recommended:

Expression System Design:

• Vector choice: pET-based vectors with T7 promoter systems for high-level expression

• Host strain: E. coli BL21(DE3) or Rosetta(DE3) for handling potential rare codons

• Fusion tags: N-terminal 6×His tag to facilitate purification without disrupting the C-terminal domain that is often involved in oligomerization of SHMTs

• Induction conditions: Lower temperatures (15-20°C) to mimic the psychrophilic nature of P. profundum and improve protein folding

Critical Expression Parameters:

This approach aligns with protocols developed for other deep-sea bacterial proteins, including those from P. profundum, which require special considerations due to their adaptation to extreme environmental conditions .

What purification challenges are specific to P. profundum SHMT and how can they be addressed?

Purifying recombinant P. profundum SHMT presents unique challenges due to its adaptation to high pressure and low temperature environments:

Purification Challenges and Solutions:

• Maintaining enzymatic activity:

  • Incorporate pyridoxal-5'-phosphate (PLP, the cofactor for SHMT) in all purification buffers (typically 50-100 μM)

  • Include reducing agents (1-5 mM DTT or β-mercaptoethanol) to protect active site cysteine residues

• Pressure-adapted protein stability:

  • Perform purification steps at lower temperatures (4-10°C)

  • Consider adding osmolytes or pressure-stabilizing compounds (e.g., trimethylamine N-oxide, glycine betaine)

  • Use buffers with higher ionic strength than typically used for mesophilic proteins

• Purification protocol:

  • Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA

  • Intermediate purification: Ion exchange chromatography (typically Q-Sepharose)

  • Polishing step: Size exclusion chromatography to obtain homogeneous oligomeric state

• Storage considerations:

  • Store with PLP cofactor and reducing agents

  • Add glycerol (20-25%) for cryoprotection

  • Flash freeze in liquid nitrogen and store at -80°C in small aliquots

These strategies address the unique properties of piezophilic enzymes while preserving their native structure and function during the purification process.

How can the enzymatic activity of P. profundum SHMT be accurately measured?

Multiple complementary assays can be employed to measure SHMT activity, each with specific advantages:

Spectrophotometric Assays:

• Coupled enzyme assay with NADH oxidation:

  • SHMT reaction coupled to methylenetetrahydrofolate dehydrogenase (MTHFD)

  • Monitors NADH oxidation at 340 nm

  • Reaction mixture contains: Serine, THF, NADH, MTHFD, and purified SHMT

  • Advantage: Continuous real-time monitoring of activity

• Direct measurement of glycine formation:

  • Use 5,10-CH2-THF and glycine as substrates (reverse reaction)

  • Measure serine formation using coupled reactions with D-amino acid oxidase and horseradish peroxidase

  • Advantage: Works well for kinetic parameter determination

• Threonine aldolase activity measurement:

  • Similar to the method described for S. thermophilus SHMT

  • Use L-threonine as substrate and measure acetaldehyde formation

  • Acetaldehyde can be quantified using aldehyde dehydrogenase with NAD+ and spectrophotometric detection at 340 nm

High-Pressure Activity Measurements:

For assessing activity under high hydrostatic pressure, specialized equipment is required:

• High-pressure stopped-flow spectrophotometer

• Pressure-resistant cuvettes or capillaries

• Control experiments at atmospheric pressure (0.1 MPa) compared to elevated pressures (e.g., 28 MPa, the optimal growth pressure for P. profundum SS9)

These methodologies will provide comprehensive characterization of both the SHMT and potential threonine aldolase activities of the enzyme under various experimental conditions.

How does high hydrostatic pressure affect the kinetic parameters of P. profundum SHMT?

P. profundum SHMT, being derived from a piezophilic organism, likely exhibits optimized kinetic parameters under elevated hydrostatic pressure. Based on studies of other enzymes from piezophilic bacteria, including P. profundum, the following patterns might be expected:

Predicted Pressure Effects on Kinetic Parameters:

To accurately determine these parameters, researchers should:

• Measure initial reaction velocities at varying substrate concentrations under different pressure conditions

• Utilize pressure-resistant reaction chambers with real-time monitoring capabilities

• Control for pressure effects on substrate solubility and buffer pH

• Compare results with SHMT from mesophilic bacteria under identical conditions

This approach would reveal how P. profundum SHMT has evolved to maintain optimal activity in its native high-pressure environment .

What structural adaptations in P. profundum SHMT likely contribute to its function under high pressure?

P. profundum SHMT, like other proteins from piezophilic organisms, likely contains specific structural adaptations that enable function under high hydrostatic pressure. While the specific crystal structure of P. profundum SHMT has not been reported in the provided search results, comparative analysis with other piezophilic proteins suggests the following probable adaptations:

• Increased flexibility in certain regions:

  • Reduced number of proline residues in loops

  • Potentially fewer salt bridges and hydrogen bonds in specific regions

  • These features would counter the pressure-induced rigidification

• Modified core packing:

  • Potentially fewer large hydrophobic residues in the core

  • Increased presence of small residues that allow for pressure-induced compression

  • Optimized void volumes to accommodate pressure effects

• Active site adaptations:

  • Pressure-resistant binding pocket for PLP cofactor

  • Modified substrate binding regions that maintain optimal geometry under pressure

• Oligomeric interface modifications:

  • If tetrameric (like most SHMTs), specialized interfaces that resist pressure-induced dissociation

  • Potentially increased hydrophilic interactions at subunit interfaces

These structural features would enable P. profundum SHMT to maintain catalytic efficiency under the high-pressure conditions of its native deep-sea environment, similar to adaptations observed in other P. profundum proteins .

How can site-directed mutagenesis be used to investigate pressure adaptation of P. profundum SHMT?

Site-directed mutagenesis offers a powerful approach to investigate the molecular basis of pressure adaptation in P. profundum SHMT. A systematic research strategy would include:

Mutagenesis Strategy for Pressure Adaptation Studies:

• Comparative sequence analysis:

  • Align P. profundum SHMT with homologs from mesophilic bacteria

  • Identify unique residues in P. profundum SHMT, particularly those in:

    • Active site regions

    • Subunit interfaces

    • Surface-exposed loops

    • Core packing regions

• Targeted mutations to investigate:

  • Pressure-sensitive to pressure-resistant mutations:

    • Introduce P. profundum-specific residues into mesophilic SHMT

    • Test if these confer increased pressure resistance

  • Pressure-resistant to pressure-sensitive mutations:

    • Mutate unique P. profundum SHMT residues to corresponding residues in mesophilic homologs

    • Determine if pressure sensitivity increases

• Experimental evaluation of mutants:

  • Measure enzymatic activity at atmospheric and high pressure

  • Determine thermal and pressure stability profiles

  • Assess oligomeric state under varying pressure

  • Perform structural analysis (e.g., circular dichroism) under pressure

• Controls and validation:

  • Create conservative mutations (similar amino acid properties) as controls

  • Verify proper folding of all mutants

  • Test multiple mutations in the same region to confirm effects

This approach has been effectively used to study other pressure-adapted proteins and would provide valuable insights into the molecular mechanisms of SHMT adaptation to high pressure .

How has the glyA gene evolved in Photobacterium species for pressure adaptation?

Evolutionary analysis of the glyA gene in Photobacterium species reveals adaptation patterns specifically related to pressure tolerance. Based on comparative genomic studies of Photobacterium species:

Evolutionary Patterns of glyA in Photobacterium:

• Sequence conservation and divergence:

  • Core catalytic residues show high conservation across all Photobacterium species

  • Greater sequence divergence in regions likely involved in pressure adaptation

  • P. profundum glyA shows specific substitutions not found in shallow-water Photobacterium species

• Selective pressure analysis:

  • Evidence of positive selection in specific regions of piezophilic Photobacterium glyA genes

  • Different patterns between strains adapted to different depth ranges:

    • P. profundum SS9 (optimal at 28 MPa)

    • P. profundum DSJ4 (optimal at 10 MPa)

    • Shallow-water Photobacterium species

• Genomic context analysis:

  • The glyA gene may be located in different genomic contexts in piezophilic vs. non-piezophilic Photobacterium species

  • Potential co-evolution with other genes involved in one-carbon metabolism under pressure

• Horizontal gene transfer considerations:

  • Some Photobacterium genomic islands contain genes acquired through horizontal transfer

  • Analysis should determine whether glyA shows evidence of horizontal acquisition or vertical inheritance

Comparative genomic approaches have revealed that P. profundum contains specific genomic islands and gene clusters associated with pressure adaptation , and the glyA gene may be part of these specialized genetic elements that contribute to the piezophilic lifestyle.

How does the expression of glyA1 in P. profundum change under different pressure conditions?

The expression of glyA1 in P. profundum likely exhibits pressure-dependent regulation as part of the organism's adaptation to varying hydrostatic pressures. Based on transcriptomic and proteomic studies of P. profundum under different pressure conditions:

Pressure-Dependent Expression Patterns:

• Transcriptomic evidence:

  • Comprehensive transcriptome analysis of P. profundum has revealed pressure-responsive gene expression

  • While glyA-specific expression data isn't explicitly mentioned in the search results, genes involved in amino acid metabolism and one-carbon transfer pathways show differential expression under varying pressure

• Regulatory mechanisms:

  • The ToxR transcriptional regulator influences pressure-responsive gene expression in P. profundum

  • Expression of glyA1 may be directly or indirectly regulated by ToxR or other pressure-sensing regulatory systems

• Proteomic evidence:

  • Differential proteomic analysis between atmospheric (0.1 MPa) and high pressure (10-30 MPa) conditions shows changes in multiple metabolic enzymes

  • Proteins involved in amino acid metabolism and transport are particularly affected by pressure changes

• Metabolic context:

  • High pressure affects the glycolysis/gluconeogenesis and oxidative phosphorylation pathways in P. profundum

  • SHMT's role in one-carbon metabolism likely positions it as an important enzyme whose expression needs to be modulated based on pressure conditions

A complete analysis of glyA1 expression would require:

• qRT-PCR validation at different pressures

• Western blot quantification of protein levels

• Reporter gene assays to identify pressure-responsive promoter elements

• Determination of the role of ToxR and other regulators in controlling glyA1 expression

This approach would provide insights into how P. profundum adjusts its one-carbon metabolism in response to changing hydrostatic pressure .

What specialized equipment and methods are required to study P. profundum SHMT function under high pressure?

Investigating P. profundum SHMT under high hydrostatic pressure requires specialized equipment and methodological considerations:

Essential Equipment for High-Pressure Enzyme Studies:

• High-pressure vessels and systems:

  • Pressure-resistant reaction chambers (typically stainless steel)

  • Hydraulic pressure generation systems with precise pressure control

  • Pressure range capability of 0.1-100 MPa to cover relevant biological pressures

• Spectroscopic equipment for high-pressure measurements:

  • High-pressure spectrophotometric cells with sapphire windows

  • Fiber optic-coupled spectrophotometers for remote monitoring

  • High-pressure stopped-flow systems for kinetic measurements

• Structural analysis under pressure:

  • High-pressure NMR tubes and probes

  • High-pressure circular dichroism cells

  • Diamond anvil cells for extreme pressure studies

Methodological Considerations:

Controls and Standards:

• Establish baseline measurements at atmospheric pressure

• Include pressure-sensitive enzymes as positive controls

• Test mesophilic SHMT homologs as comparative standards

• Monitor buffer pH changes under pressure using pressure-insensitive indicators

These methodologies have been employed in studies of other P. profundum proteins and are essential for understanding how SHMT functions in its native high-pressure environment .

How can SHMT from P. profundum be utilized in biotechnological applications requiring pressure tolerance?

P. profundum SHMT offers unique potential for biotechnological applications that require pressure tolerance, taking advantage of its natural adaptation to deep-sea environments:

Potential Biotechnological Applications:

• Biocatalysis under high-pressure conditions:

  • Pressure enhances certain chemical reactions by influencing reaction volumes and equilibria

  • P. profundum SHMT could catalyze stereospecific reactions under pressure

  • Applications in fine chemical and pharmaceutical synthesis requiring high selectivity

• Development of pressure-resistant enzyme systems:

  • Engineering SHMT fusion proteins with other enzymes for multistep catalysis

  • Creating immobilized enzyme systems for high-pressure continuous reactors

  • Potential applications in deep-sea bioremediation technologies

• Structure-guided enzyme engineering:

  • Use P. profundum SHMT as a scaffold for engineering other enzymes for pressure resistance

  • Identify and transfer pressure-adaptive motifs to mesophilic enzymes

  • Combine pressure and temperature adaptations for multi-extreme conditions

Methodological Approach for Application Development:

• Characterization phase:

  • Establish activity and stability profiles across pressure ranges (0.1-100 MPa)

  • Determine substrate scope under pressure conditions

  • Identify optimal reaction conditions (pH, ionic strength, temperature) at elevated pressures

• Engineering phase:

  • Create chimeric enzymes combining domains from P. profundum SHMT with other enzymes

  • Perform directed evolution under pressure conditions

  • Develop immobilization strategies preserving pressure resistance

• Application testing:

  • Pilot-scale reactions under industrially relevant conditions

  • Comparative analysis with conventional catalysts

  • Lifecycle and reusability assessment under repeated pressure cycles

This approach leverages the natural pressure adaptations of P. profundum SHMT for applications that could benefit from enzymatic catalysis under high-pressure conditions, particularly in industrial processes where pressure is used to enhance reaction rates or selectivity.

How does P. profundum SHMT compare functionally to SHMTs from other piezophilic and mesophilic bacteria?

A comprehensive comparison between P. profundum SHMT and homologs from other bacteria reveals important adaptations and functional differences:

Comparative Analysis of Bacterial SHMTs:

Unique Features of P. profundum SHMT:

• Pressure adaptation strategies:

  • Modified active site geometry that maintains optimal catalysis under pressure

  • Potentially unique surface charge distribution compared to mesophilic homologs

  • Specialized regions that accommodate pressure-induced conformational changes

• Evolutionary considerations:

  • May share features with other deep-sea bacterial enzymes through convergent evolution

  • Could contain unique adaptations specific to the Photobacterium genus

  • Likely preserves ancestral catalytic mechanism while adapting to pressure

This comparative analysis highlights how P. profundum SHMT has likely evolved specific adaptations to function optimally in the high-pressure, low-temperature environment of the deep sea, while maintaining the core catalytic mechanism shared among all SHMTs .

What can we learn about enzyme evolution by studying P. profundum SHMT?

P. profundum SHMT provides a valuable model for understanding fundamental principles of enzyme evolution under extreme environmental conditions, offering insights into both general evolutionary mechanisms and specific adaptations to high pressure:

Evolutionary Insights from P. profundum SHMT:

• Mechanisms of pressure adaptation:

  • Reveals how proteins maintain function under high hydrostatic pressure

  • Demonstrates the balance between structural stability and catalytic flexibility

  • Illustrates convergent vs. divergent evolution in piezophilic organisms

• Evolutionary constraints in essential enzymes:

  • SHMT is a highly conserved enzyme essential for one-carbon metabolism

  • Studying P. profundum SHMT reveals how essential functions are preserved while adapting to extreme conditions

  • Highlights which protein regions are flexible for adaptation vs. which must be conserved

• Multi-stress adaptation principles:

  • P. profundum is adapted to both low temperature and high pressure

  • Its SHMT provides a model for understanding how enzymes adapt to multiple simultaneous stressors

  • Reveals potential synergistic or antagonistic adaptations to different environmental extremes

• Methodological approach for studying enzyme evolution:

  • Phylogenetic analysis:

    • Compare SHMT sequences across bacteria from different depth habitats

    • Identify lineage-specific adaptations vs. convergent evolution

    • Reconstruct ancestral sequences to trace evolutionary trajectories

  • Structural analysis:

    • Map sequence differences to structural elements

    • Identify hotspots of evolutionary change associated with pressure adaptation

    • Compare with other pressure-adapted enzymes to identify common principles

  • Functional validation:

    • Express ancestral or chimeric SHMT variants

    • Test activity under various pressure conditions

    • Use directed evolution under pressure to identify critical adaptive mutations

These approaches would provide valuable insights into not only the specific evolution of P. profundum SHMT but also broader principles of protein adaptation to extreme environments, contributing to our understanding of life's capacity to colonize the full range of habitats on Earth .

How can researchers optimize the design of experiments to study P. profundum SHMT's adaptation to high pressure?

Designing robust experiments to study the pressure adaptation of P. profundum SHMT requires careful consideration of multiple factors:

Experimental Design Optimization:

• Pressure range selection:

  • Include multiple pressure points (0.1, 10, 28, 40, 60 MPa)

  • 28 MPa represents the optimal growth pressure for P. profundum SS9

  • Include pressures beyond physiological range to determine limits of adaptation

• Control selection:

  • Positive controls: Known pressure-resistant proteins

  • Negative controls: Homologous SHMT from mesophilic bacteria

  • Internal controls: Pressure-insensitive reaction components

• Parameter interdependence:

  • Design factorial experiments to test pressure × temperature interactions

  • Account for pressure effects on pH (typically 0.14-0.18 pH units/100 MPa)

  • Consider pressure effects on substrate and cofactor binding

• Statistical power and replication:

  • Minimum of 3-5 biological replicates

  • Multiple technical replicates at each pressure point

  • Power analysis to determine required sample sizes for detecting pressure effects

Methodological Framework:

This experimental framework incorporates the specific challenges of high-pressure biochemistry while maintaining statistical rigor and appropriate controls, ensuring reliable and reproducible results when studying P. profundum SHMT adaptation .