Recombinant Photobacterium profundum Octanoyltransferase (lipB)

What is Photobacterium profundum Octanoyltransferase (lipB) and what is its biological function?

Photobacterium profundum Octanoyltransferase (lipB) is an enzyme involved in lipoic acid biosynthesis in the deep-sea piezophilic bacterium P. profundum. Similar to homologous proteins in other bacteria, it likely catalyzes the transfer of octanoyl groups from octanoyl-acyl carrier protein (ACP) to target proteins during lipoic acid synthesis . Lipoic acid is an essential cofactor for several key metabolic enzyme complexes, including pyruvate dehydrogenase (PDH) and oxoglutarate dehydrogenase (OGDH), which are critical for respiratory growth . In P. profundum, which grows optimally at high hydrostatic pressure (28 MPa) and low temperature (15°C), this enzyme may have unique adaptations that allow it to function efficiently under these extreme conditions .

What growth and culturing conditions are recommended for P. profundum before isolating lipB?

P. profundum SS9 requires specific growth conditions that reflect its deep-sea origin:

• Temperature: Optimal growth at 15-17°C

• Pressure: For true physiological conditions, grow at 28 MPa (optimal pressure), though it can grow at atmospheric pressure (0.1 MPa) for easier handling

• Medium: Marine broth (28 g/liter 2216 medium) supplemented with:

  • 20 mM glucose

  • 100 mM HEPES buffer (pH 7.5)

• Growth mode: Anaerobic conditions are recommended for optimal growth

For high-pressure cultivation, the following approach is effective:

• Inoculate cultures into sealed containers (e.g., Pasteur pipettes, heat-sealed bulbs) excluding air

• Incubate in pressure vessels at 28 MPa and 15-17°C for 5 days or until desired growth phase

• For atmospheric pressure controls, incubate identically sealed containers at 0.1 MPa

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

Based on successful heterologous expression of other P. profundum proteins:

• E. coli expression system: Though not specifically documented for lipB, other P. profundum proteins have been successfully expressed in E. coli. For example, P. profundum SiaQM was successfully expressed in E. coli TOP10 cells using a pBAD-HisA vector with a hexahistidine tag .

• Expression conditions:

  • Growth at 37°C to log phase (OD600 of 1.7-1.9) in Terrific Broth

  • Induction with 0.2% arabinose

  • Expression for 3-4 hours

  • Cell lysis via ultrasonication in phosphate-buffered saline with protease inhibitors

• Recommended vector features:

  • Affinity tag (hexahistidine) for purification

  • Temperature-inducible or chemical-inducible promoter for controlled expression

  • Appropriate antibiotic resistance markers

How can I verify the enzymatic activity of recombinant P. profundum lipB?

To verify octanoyltransferase activity of recombinant P. profundum lipB, consider the following assay approach:

• Substrate preparation:

  • Use octanoyl-ACP or octanoyl-CoA as the octanoyl donor

  • Use appropriate acceptor protein (typically a lipoyl domain from a lipoate-dependent enzyme)

• Activity assay:

  • Monitor the transfer of the octanoyl group from donor to acceptor

  • This can be detected by:

    • Mass spectrometry to identify the modified acceptor protein

    • Gel-shift assays showing mobility changes after modification

    • Radioactive labeling using [³H] or [¹⁴C]-labeled octanoyl substrates

• Pressure effects assessment:

  • Compare enzyme activity at atmospheric pressure (0.1 MPa) and high pressure (28 MPa)

  • Use high-pressure chambers adapted for enzymatic assays

• Controls:

  • Negative control: heat-inactivated enzyme

  • Positive control: well-characterized octanoyltransferase from another organism (if available)

How might the pressure adaptation mechanisms of P. profundum affect lipB function compared to mesophilic homologs?

P. profundum proteins show several adaptations to high pressure that might also apply to lipB:

• Structural adaptations: Research on other P. profundum proteins suggests that pressure adaptation involves modifications to protein flexibility and compressibility. The lipB enzyme likely incorporates:

  • Altered amino acid composition favoring pressure resistance

  • Modified secondary structure elements that maintain stability under compression

  • Adaptations in the active site that preserve catalytic function under pressure

• Pressure effects on catalysis: Unlike mesophilic enzymes that typically show decreased activity under pressure, P. profundum lipB may exhibit:

  • Optimal catalytic efficiency at elevated pressures (around 28 MPa)

  • Pressure-dependent conformational changes that enhance substrate binding or product release

  • Altered reaction kinetics that favor the reaction under high pressure conditions

• Comparative enzyme kinetics data: Studies with other P. profundum enzymes show:

  • Increased or maintained activity at high pressure

  • Different pressure optima for different enzymes, with some showing maximum activity at 30 MPa

For example, pressure effects on ATP synthesis in P. profundum reveal that the ATPase systems respond differently to pressure, with ATPase-II being more abundant at elevated pressure compared to ATPase-I, suggesting specialized roles under different pressure conditions .

What methodologies are most suitable for studying pressure effects on recombinant P. profundum lipB?

To effectively study pressure effects on P. profundum lipB:

• High-pressure enzymatic assays:

  • Use specialized high-pressure optical cells or chambers that allow spectrophotometric measurements

  • Employ time-resolved assays to measure reaction kinetics under pressure

  • Consider using pressure-cycling approaches to assess reversibility of pressure effects

• Structural analysis under pressure:

  • High-pressure NMR spectroscopy to monitor structural changes

  • High-pressure X-ray crystallography (where facilities exist)

  • Molecular dynamics simulations incorporating pressure parameters

• Comparative analysis across pressure conditions:

  • Generate activity profiles across a pressure range (0.1 MPa to 150 MPa)

  • Determine pressure optima and compare with known growth optimum (28 MPa)

  • Calculate thermodynamic parameters (ΔV‡) for the reaction under different pressures

• Specifically designed equipment:

  • Pressure-retaining samplers for maintaining proteins at in situ pressure

  • High-pressure bioreactors for protein expression under native conditions

  • Custom pressure vessels adapted for biological samples

These approaches have been successfully used with other P. profundum proteins, such as in motility studies where direct swimming velocity measurements were obtained using high-pressure microscopic chambers .

How does the fatty acid metabolism pathway in P. profundum relate to lipB function at different pressures?

The relationship between lipB and fatty acid metabolism in P. profundum at different pressures reflects important adaptations:

• Pressure-dependent fatty acid composition:

  • P. profundum alters its membrane fatty acid composition in response to pressure

  • At high pressure (30 MPa), P. profundum increases production of polyunsaturated fatty acids (PUFAs) like eicosapentaenoic acid (EPA, 20:5n-3)

  • This adaptation maintains membrane fluidity under high pressure

• Role of lipB in pressure adaptation:

  • As an octanoyltransferase, lipB likely plays a role in lipoic acid synthesis

  • Lipoic acid is essential for key metabolic enzymes including those involved in fatty acid metabolism

  • The activity of these enzymes is pressure-dependent, as shown in proteomic studies

• Metabolic pathway shifts:

  • Proteomic analysis reveals that proteins involved in glycolysis/gluconeogenesis are up-regulated at high pressure

  • Conversely, several proteins involved in oxidative phosphorylation are up-regulated at atmospheric pressure

  • These shifts may influence the demand for lipoic acid and thus lipB activity

• Experimental evidence from related pathways:

  • Fatty acid biosynthesis mutants in P. profundum show pressure-dependent growth defects

  • For example, ΔfabB mutants cannot grow at high pressure (30 MPa) unless they acquire suppressor mutations

  • The fatty acid composition table below shows how suppressor mutations restore growth at high pressure:

Table from MAP1002 suppressor strain derived from ΔfabB mutant

This interdependence between fatty acid metabolism and pressure adaptation suggests that lipB function may be critically regulated in response to pressure changes.

What approaches can be used to engineer P. profundum lipB for enhanced stability or function in heterologous systems?

Engineering P. profundum lipB for enhanced stability or function in heterologous systems could employ several strategies:

• Structure-guided mutagenesis:

  • Identify and modify key residues responsible for pressure adaptation

  • Target flexible regions that might destabilize the protein at atmospheric pressure

  • Introduce stabilizing interactions (salt bridges, disulfide bonds) to maintain active conformation

  • This approach would require structural information, potentially from homology modeling based on related octanoyltransferases

• Directed evolution strategies:

  • Random mutagenesis combined with selection for improved activity in the desired expression system

  • Screen libraries under varying pressure conditions to identify variants with broader pressure tolerance

  • Use DNA shuffling between P. profundum lipB and mesophilic homologs to create chimeric enzymes

• Expression optimization:

  • Codon optimization for the host organism

  • Co-expression with chaperones to aid proper folding

  • Use of solubility-enhancing fusion partners (e.g., SUMO, MBP)

  • Expression at reduced temperatures to mimic natural conditions (15-17°C)

• Experimental design for optimization:

  • Establish baseline kinetic parameters at various pressures (0.1-50 MPa)

  • Determine thermal and pressure stability profiles of wild-type enzyme

  • Compare engineered variants using standardized assays that include:

    • Activity measurements at varying pressures

    • Stability assessments under storage conditions

    • Performance in the intended application

• Potential applications of engineered variants:

  • Enzymes with broader pressure-temperature activity profiles

  • Catalysts for high-pressure industrial bioprocesses

  • Model systems for studying pressure effects on protein function

What is known about the genomic context of lipB in P. profundum and how does it compare to other bacteria?

The genomic context of genes can provide valuable insights into their regulation and functional relationships:

• Genomic organization in P. profundum:

  • P. profundum SS9 has a complex genome consisting of two chromosomes and an 80 kb plasmid

  • While the specific location of lipB isn't directly mentioned in the search results, genomic analysis of P. profundum reveals that many metabolic genes are organized differently compared to related bacteria

• Comparative genomics with other Photobacterium species:

  • The Photobacterium genus shows high genomic diversity

  • Genomic islands and horizontal gene transfer have played significant roles in the evolution of this genus

  • This suggests that the genetic context of lipB might vary across Photobacterium species

• Regulation under pressure:

  • P. profundum adapts to pressure through differential gene expression

  • Proteomic analysis has identified numerous proteins that are differentially expressed at high versus atmospheric pressure

  • Given its likely role in metabolism, lipB expression might be similarly regulated by pressure

• Predicted operonic structure:

  • In other bacteria, genes involved in lipoic acid metabolism are often arranged in operons

  • For example, in some bacteria lipB is co-transcribed with genes involved in related metabolic pathways

  • Identifying genes co-regulated with lipB could provide insights into its function and regulation in P. profundum

How can differential pressure experiments be designed to investigate lipB function in vivo in P. profundum?

Designing differential pressure experiments to investigate lipB function in vivo requires specialized approaches:

• Gene knockout or knockdown studies:

  • Create lipB deletion mutants in P. profundum using established genetic tools

  • Design complementation vectors expressing wild-type or mutated lipB

  • Compare growth of wild-type and mutant strains at different pressures (0.1 MPa vs. 28 MPa)

• Pressure-controlled cultivation system:

  • Use high-pressure cultivation systems as described in the literature:

    • Heat-sealed bulbs or Pasteur pipettes excluding air

    • Water-cooled pressure vessels maintaining desired pressure (0.1-40 MPa)

    • Temperature control at 15-17°C

  • Maintain parallel cultures at different pressures for comparative analysis

• Growth measurements under pressure:

  • Monitor growth using specialized equipment adapted for high-pressure conditions

  • High-throughput monitoring can be achieved using adapted microplate readers

  • Calculate pressure-specific growth parameters (growth rate, lag phase, yield)

• Metabolic analysis:

  • Measure lipoic acid content in wild-type and lipB mutant cells grown at different pressures

  • Analyze activity of lipoic acid-dependent enzymes (PDH, OGDH) under different pressure conditions

  • Perform metabolomic analysis to identify metabolic bottlenecks in lipB mutants

• Proteomic approach:

  • Use label-free quantitative proteomics to compare protein expression in wild-type vs. lipB mutants

  • Identify compensatory changes in protein expression that occur in response to lipB mutation

  • This approach has been successfully used to study pressure adaptation in P. profundum

• Experimental design considerations:

  • Include appropriate controls (positive and negative)

  • Ensure biological replicates (typically triplicates)

  • Maintain consistent conditions other than pressure

  • Control for growth phase effects by harvesting at equivalent physiological states

The expertise gained from studying other P. profundum proteins under pressure, such as flagellar proteins and ATPases , provides a valuable foundation for designing these experiments.