Recombinant Photobacterium profundum Gamma-glutamyl phosphate reductase (proA)

What is the function of gamma-glutamyl phosphate reductase (ProA) in Photobacterium profundum?

ProA in P. profundum functions as a gamma-glutamyl phosphate reductase (EC 1.2.1.41), catalyzing the second reaction in the biosynthetic pathway converting glutamate to proline. Specifically, it reduces gamma-glutamyl phosphate to glutamic-5-semialdehyde, which spontaneously cyclizes to form 1-pyrroline-5-carboxylate, a proline precursor . While this basic function is conserved across many bacterial species, the regulation and additional roles of ProA may vary between organisms. In the related bacterium Ralstonia solanacearum, ProA has been demonstrated to be essential for proline biosynthesis, as proA mutants are proline auxotrophs that fail to grow in minimal media without proline supplementation .

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

Escherichia coli has been successfully used as an expression host for recombinant P. profundum ProA . While the literature does not detail the specific E. coli strain used for optimal expression, common laboratory strains such as BL21(DE3) or its derivatives would be suitable candidates given their reduced protease activity and ability to express T7 RNA polymerase for high-level protein production.

When designing an expression protocol, consider these methodological guidelines:

• Vector selection: pET-series vectors with T7 promoters provide strong, inducible expression.

• Induction parameters: Optimize IPTG concentration (typically 0.1-1.0 mM) and induction temperature (reduced to 16-25°C to enhance solubility).

• Growth media: Rich media (LB) for initial screening; defined media for isotope labeling if structural studies are planned.

• Codon optimization: Consider codon optimization if expression yields are low, as marine organisms may have different codon usage patterns than E. coli.

What are the recommended procedures for purification and storage of recombinant ProA?

For purification of recombinant P. profundum ProA, a multi-step chromatography approach is typically employed:

• Initial capture: Immobilized metal affinity chromatography (IMAC) if a His-tag is incorporated.

• Intermediate purification: Ion exchange chromatography based on the theoretical pI of ProA.

• Polishing step: Size exclusion chromatography to remove aggregates and achieve high purity.

For storage, the following conditions are recommended to maintain stability :

• Short-term storage (liquid form): 6 months at -20°C/-80°C

• Long-term storage (lyophilized form): 12 months at -20°C/-80°C

• Reconstitution: Deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol as a cryoprotectant

How can the enzymatic activity of ProA be measured in laboratory conditions?

The enzymatic activity of gamma-glutamyl phosphate reductase can be assessed through several complementary approaches:

• Spectrophotometric assay: Measure the rate of NADPH oxidation at 340 nm, as the reaction requires NADPH as a cofactor. The reaction mixture typically contains:

  • Purified ProA enzyme (1-10 μg)

  • Gamma-glutamyl phosphate substrate (1-10 mM)

  • NADPH (0.2-0.5 mM)

  • Buffer (typically Tris-HCl or phosphate, pH 7.5)

  • Magnesium chloride (5-10 mM)

• Coupled enzyme assay: Since gamma-glutamyl phosphate is unstable, a coupled assay with gamma-glutamyl kinase (ProB) can be employed where glutamate is converted to gamma-glutamyl phosphate in situ.

• Product detection: Measure glutamic-5-semialdehyde formation using colorimetric methods with ninhydrin or o-aminobenzaldehyde.

• Complementation assay: As demonstrated in R. solanacearum studies, functional ProA can be verified by its ability to restore growth of proA deletion mutants in minimal media .

How does pressure affect the activity and stability of P. profundum ProA?

P. profundum is a piezophilic bacterium that grows optimally at high hydrostatic pressure (28 MPa) and relatively low temperature (15°C) . The effects of pressure on ProA specifically have not been directly reported in the provided literature, but insights can be inferred from broader proteomic studies of P. profundum:

• Pressure-adapted enzymes: Proteins from piezophilic organisms often show structural adaptations that maintain functionality under pressure, such as increased flexibility or altered subunit interactions.

• Differential expression patterns: Proteomic analyses of P. profundum grown at atmospheric versus high pressure (28 MPa) reveal significant differences in protein expression patterns . While ProA was not specifically highlighted in these studies, proteins involved in amino acid biosynthesis pathways show differential expression under varying pressure conditions.

• Methodological considerations for activity assays under pressure:

  • Specialized high-pressure equipment is required for direct measurements

  • Pressure effects can be inferred by comparing enzyme kinetics of ProA from piezophilic (P. profundum) versus non-piezophilic sources

  • Parameters like Km, Vmax, and kcat should be determined at both atmospheric and elevated pressures

How does P. profundum ProA compare to homologous enzymes from other bacterial species?

Comparative analysis between P. profundum ProA and homologs from other bacterial species reveals important insights:

In R. solanacearum, ProA plays roles beyond proline biosynthesis, particularly in the regulation of type three secretion system (T3SS) genes through the PrhG-HrpB pathway . Deletion of proA in R. solanacearum impairs T3SS expression even under proline-supplemented conditions, suggesting a regulatory function independent of its enzymatic activity .

Whether P. profundum ProA exhibits similar regulatory functions remains to be determined, but the adaptations to high-pressure environments likely result in structural and functional differences compared to mesophilic homologs.

What are the implications of ProA function for P. profundum adaptation to deep-sea environments?

ProA's role in proline biosynthesis has significant implications for P. profundum's adaptation to deep-sea environments:

• Osmotic regulation: Proline functions as an osmolyte that helps cells maintain osmotic balance under high-pressure conditions where cell membrane properties are altered.

• Protein stabilization: Proline can act as a chemical chaperone, helping to stabilize protein structure under high pressure and low temperature.

• Metabolic adaptation: In a proteomic analysis of P. profundum, proteins involved in the glycolysis/gluconeogenesis pathway were up-regulated at high pressure, while those in oxidative phosphorylation were up-regulated at atmospheric pressure . As an amino acid biosynthesis enzyme, ProA likely participates in these pressure-dependent metabolic adaptations.

• Stress response: Proline accumulation is a known stress response in many organisms, helping cells cope with various environmental stressors beyond osmotic pressure.

The ability of P. profundum to grow across a wide range of pressures (from atmospheric to 28 MPa) suggests that ProA and other enzymes in the proline biosynthesis pathway may have evolved unique regulatory mechanisms to function efficiently under varying pressure conditions.

How can gene knockout or complementation studies be designed to investigate ProA function in P. profundum?

To investigate ProA function in P. profundum through genetic manipulation, researchers can implement the following methodological approaches:

• Gene knockout strategy:

  • Marker exchange-eviction mutagenesis using a suicide vector like pRL271 (containing sacB for counter-selection) has been successfully applied in P. profundum

  • Transposon mutagenesis has been used for large-scale studies in P. profundum using mini-Tn5 or mini-Tn10 elements

  • CRISPR-Cas9 systems can be adapted for use in P. profundum, though this would require optimization

• Complementation analysis:

  • The Tn7-based site-specific chromosomal integration system has been shown to fully restore phenotypes in complementation studies of other bacteria

  • Broad-host-range plasmids like pGL10 can be used for complementation via conjugal transfer

• Phenotypic analysis of mutants:

  • Compare growth rates at different pressures (0.1 MPa vs. 28 MPa)

  • Assess proline auxotrophy in minimal media with and without proline supplementation

  • Analyze changes in proteome and metabolome profiles

  • Examine stress response under various conditions (temperature, oxidative stress)

• Experimental controls:

  • Include wild-type strains grown under identical conditions

  • Create complemented strains to verify phenotype restoration

  • Include heterologous expression of ProA from non-piezophilic bacteria for comparative analysis

What approaches can be used to investigate the potential pressure-dependent changes in ProA structure and function?

Investigating pressure-dependent changes in ProA structure and function requires specialized techniques:

• Structural biology approaches:

  • High-pressure X-ray crystallography: Requires specialized equipment to maintain crystals under pressure during data collection

  • NMR spectroscopy under pressure: Provides insights into protein dynamics and conformational changes

  • Small-angle X-ray scattering (SAXS): Can be adapted for high-pressure studies to examine protein shape and oligomeric state

• Biochemical and biophysical techniques:

  • Enzyme kinetics under pressure: Using specialized high-pressure reaction vessels to measure activity at varying pressures

  • Circular dichroism spectroscopy: To monitor secondary structure changes at different pressures

  • Fluorescence spectroscopy: Using intrinsic tryptophan fluorescence or extrinsic probes to monitor conformational changes

  • Hydrogen-deuterium exchange mass spectrometry: To identify regions of altered flexibility or solvent accessibility under pressure

• Computational approaches:

  • Molecular dynamics simulations: To model the effects of pressure on protein structure and dynamics

  • Comparative sequence analysis: Identifying unique residues or motifs in piezophilic ProA compared to mesophilic homologs

  • Coevolutionary analysis: To identify networks of residues that may be involved in pressure adaptation

• Reporter systems for in vivo studies:

  • Growth-based assays: Using P. profundum strain cultures in specialized pressure vessels

  • Enzymatic reporter systems: Similar to the β-galactosidase assays used to measure gene expression in R. solanacearum

How does ProA function integrate with broader metabolic networks in P. profundum?

ProA functions within a complex metabolic network, particularly at the intersection of amino acid metabolism and stress response pathways:

• Proline biosynthesis pathway:

  • ProA catalyzes the second step in the glutamate-to-proline pathway

  • This pathway connects glutamate metabolism to proline production

  • In P. profundum, this pathway likely interfaces with pressure-responsive metabolic systems

• Integration with central carbon metabolism:

  • Proteomic studies of P. profundum show that proteins involved in glycolysis/gluconeogenesis are up-regulated at high pressure

  • As glutamate (ProA's substrate's precursor) is derived from α-ketoglutarate in the TCA cycle, ProA activity links to central carbon metabolism

  • The NADPH required for ProA activity connects to redox homeostasis

• Systems-level approaches to study these connections:

  • Metabolic flux analysis: Using 13C-labeled substrates to trace carbon flow through central metabolism to amino acid biosynthesis under different pressure conditions

  • Transcriptomics combined with proteomics: To identify co-regulated genes and proteins under varying pressure conditions

  • Metabolomics: To measure changes in metabolite pools, particularly intermediates in proline biosynthesis and related pathways

What bioinformatic approaches can reveal evolutionary adaptations in P. profundum ProA for high-pressure environments?

Several bioinformatic approaches can illuminate the evolutionary adaptations in P. profundum ProA:

• Comparative sequence analysis:

  • Multiple sequence alignment of ProA homologs from piezophilic, psychrophilic, and mesophilic organisms

  • Identification of signature residues unique to piezophilic ProA enzymes

  • Analysis of amino acid composition bias (e.g., increased flexibility-conferring residues)

• Structural bioinformatics:

  • Homology modeling to predict structural features

  • Analysis of cavity volumes, which often decrease in pressure-adapted proteins

  • Examination of surface charge distribution and hydrophobic core packing

• Phylogenetic analysis:

  • Construction of phylogenetic trees to trace the evolutionary history of ProA

  • Identification of selection pressures using dN/dS ratios

  • Ancestral sequence reconstruction to infer evolutionary transitions

• Genomic context analysis:

  • Examination of the organization of genes surrounding proA in the P. profundum genome

  • Identification of pressure-responsive regulatory elements

  • Comparative analysis with genomic regions in related species

The knowledge gained from these analyses would provide insights into the molecular mechanisms of high-pressure adaptation not only for ProA but potentially for other enzymes functioning in deep-sea environments.

How can structural and functional insights from P. profundum ProA inform biotechnological applications?

Understanding the pressure adaptation mechanisms of P. profundum ProA can inform several biotechnological applications:

• Enzyme engineering for high-pressure bioprocessing:

  • Identifying key residues responsible for pressure tolerance could guide engineering of other enzymes for biocatalysis under pressure

  • High-pressure bioprocessing can offer advantages like increased substrate solubility, reduced microbial contamination, and altered reaction selectivity

• Development of biosensors:

  • Pressure-sensitive domains from ProA could potentially be incorporated into biosensor designs to detect pressure changes

  • Applications in deep-sea monitoring or industrial process controls

• Proline production for biotechnology:

  • Engineered proline biosynthesis pathways incorporating pressure-adapted enzymes for enhanced production efficiency

  • Proline is valuable as a food additive, pharmaceutical excipient, and cosmetic ingredient

• Deep-sea bioprospecting guidance:

  • Insights from ProA adaptations can inform targeted searches for novel biomolecules and enzymes from deep-sea environments

What are the most promising research directions for further understanding the role of ProA in pressure adaptation?

Several research directions hold particular promise for advancing our understanding of ProA's role in pressure adaptation:

• Structure-function studies under pressure:

  • Determination of ProA's crystal structure under varying pressure conditions

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

  • Investigation of protein dynamics and conformational changes using advanced biophysical techniques

• Systems biology approach:

  • Integration of transcriptomic, proteomic, and metabolomic data to map pressure-responsive networks

  • Global analysis of metabolic flux changes under pressure, focusing on pathways connected to proline metabolism

  • Construction of genome-scale metabolic models that incorporate pressure as a parameter

• Comparative studies across pressure gradients:

  • Isolation and characterization of ProA from bacteria adapted to different depths (and therefore pressures)

  • Investigation of related enzymes from the same biosynthetic pathway to determine if pressure adaptation is pathway-wide or enzyme-specific

• Investigation of potential regulatory roles:

  • Given the dual role of ProA in R. solanacearum (both enzymatic and regulatory) , exploration of potential regulatory functions beyond proline biosynthesis in P. profundum

  • Examination of protein-protein interactions under varying pressure conditions