Recombinant Photobacterium profundum Probable allantoicase (alc)

What is the genomic context of the allantoicase gene in Photobacterium profundum?

The allantoicase gene in P. profundum is part of the organism's metabolic gene repertoire. While the exact genomic context isn't specified in the available search results, P. profundum SS9 has a genome consisting of two chromosomes and an 80 kb plasmid . When working with any P. profundum gene, it's important to note its position relative to other functional gene clusters. For instance, like the Orf6 thioesterase gene that is located directly upstream of the pfaA gene , the genomic neighborhood of the allantoicase gene could provide insights into its functional relationships within metabolic networks. Researchers should employ comparative genomic approaches to analyze synteny with related species like Moritella marina, Vibrio splendidus, and Colwellia psychrerythraea, which share high ortholog similarity in other functional genes .

How does high pressure influence the expression of metabolic enzymes like allantoicase in P. profundum?

High hydrostatic pressure significantly alters protein expression patterns in P. profundum. Proteomic analyses have demonstrated that pressure influences the expression of various metabolic pathways; for example, proteins involved in glycolysis/gluconeogenesis are up-regulated at high pressure, while oxidative phosphorylation proteins are up-regulated at atmospheric pressure . Similarly, pressure likely influences the expression of nitrogen metabolism enzymes including allantoicase. To investigate this specifically, researchers should conduct comparative proteomics using label-free quantitation and mass spectrometry analysis of P. profundum grown at atmospheric versus high pressure (28 MPa) conditions . Research designs should account for how different hydrostatic pressures represent distinct ecological niches with specific nutrient limitations and abundances that might influence nitrogen metabolism enzyme expression.

What are the expected basic properties of P. profundum allantoicase compared to mesophilic bacterial allantoicases?

P. profundum allantoicase likely exhibits adaptations to function optimally under high-pressure, low-temperature conditions. Based on the pressure adaptations observed in other P. profundum enzymes, the allantoicase might demonstrate:

These properties should be experimentally determined using purified recombinant enzyme with allantoate as substrate under varying pressure and temperature conditions, similar to methodologies used for characterizing other P. profundum enzymes .

What expression system is optimal for producing recombinant P. profundum allantoicase?

For efficient expression of recombinant P. profundum allantoicase, an E. coli-based expression system with specific adaptations for cold-adapted proteins is recommended. Based on successful approaches with other P. profundum proteins:

• Use BL21-CodonPlus (DE3)-RIL E. coli strain to address potential codon bias issues, as was successful for Orf6 thioesterase from P. profundum .

• Employ a pGEX or similar vector system for expression as a GST-fusion protein to enhance solubility .

• Culture conditions should include:

  • Growth at 37°C until OD600 reaches 0.4

  • Temperature reduction to 15-22°C before induction

  • Induction with low IPTG concentrations (0.1-0.5 mM)

  • Extended expression period (18-24 hours) at reduced temperature

This approach minimizes inclusion body formation common with cold-adapted enzymes expressed at higher temperatures. For challenging cases, consider specialized vectors like pFL190 (arabinose-inducible) that have been successful with other P. profundum proteins .

What purification strategy yields the highest activity for recombinant P. profundum allantoicase?

A multi-step purification approach is essential for obtaining highly active recombinant P. profundum allantoicase:

• Initial capture: If expressed as a GST-fusion protein, use glutathione-Sepharose affinity chromatography with gentle elution using 10-20 mM reduced glutathione in HEPES buffer (pH 7.5) containing at least 150 mM NaCl to maintain stability .

• Tag removal: Cleave the fusion tag using PreScission protease (for GST-tag) and re-pass through the affinity column.

• Polishing steps:

  • Size exclusion chromatography using Superdex 200 in HEPES buffer (pH 7.5) containing 150 mM NaCl and potentially 5% glycerol as a stabilizer

  • Optional ion exchange chromatography if additional purity is required

• Critical considerations:

  • Maintain temperature at 4-10°C throughout purification

  • Include protease inhibitors in initial lysis buffers

  • Consider adding reducing agents (1-5 mM DTT or β-mercaptoethanol) to prevent oxidation of cysteine residues

  • For long-term storage, flash-freeze purified enzyme in small aliquots and store at -80°C

Enzyme activity should be monitored at each purification step using a spectrophotometric assay measuring allantoate degradation or product formation.

How can the catalytic activity of P. profundum allantoicase be accurately measured under high-pressure conditions?

Measuring enzymatic activity under high pressure requires specialized equipment and methodologies:

• High-pressure reaction chambers: Use stainless-steel pressure vessels similar to those employed for studying flagellar motility in P. profundum , but adapted for biochemical assays. These vessels should allow for rapid sample collection or real-time measurement.

• Coupled enzyme assays: For real-time measurements, develop a coupled assay system where allantoicase activity produces a measurable signal (e.g., fluorescence or absorbance change) that can be detected through pressure-resistant windows.

• Quenched assays: Alternatively, perform reactions under pressure for defined periods, rapidly decompress, and immediately quench with a stop solution before analysis by HPLC or LC-MS to quantify substrate depletion or product formation.

• Controls: Include pressure-stable internal standards to normalize for any pressure effects on measurement parameters. Additionally, use pressure-insensitive enzymes as controls to differentiate between pressure effects on the reaction versus effects on measurement.

• Data analysis: Apply appropriate kinetic models that account for pressure effects on reaction volumes and transition states. The apparent Km and kcat values should be determined across a pressure range of 0.1-50 MPa to establish pressure-activity profiles.

This approach allows for determining whether the enzyme exhibits pressure adaptation, with optimal activity at pressures corresponding to the native habitat of P. profundum (approximately 28 MPa) .

What are the substrate specificity profiles of P. profundum allantoicase compared to other characterized allantoicases?

The substrate specificity of P. profundum allantoicase should be comprehensively characterized using:

• Primary substrate kinetics: Determine Km, kcat, and catalytic efficiency (kcat/Km) for allantoate using standardized assay conditions (buffer, pH, temperature, and pressure).

• Substrate panel testing: Evaluate activity against a panel of structurally related compounds including:

  • Allantoate (natural substrate)

  • Ureidoglycolate (potential product/substrate)

  • Structural analogs with modified functional groups

• Comparative analysis: Test substrate specificity under various conditions:

• Inhibition studies: Test product inhibition and identify potential competitive inhibitors.

The substrate profile should be compared with allantoicases from mesophilic organisms to identify adaptations specific to deep-sea environments. Based on observations from other P. profundum enzymes, expect potential broader substrate tolerance as a adaptation to the deep-sea environment, similar to how the Orf6 thioesterase showed flexibility in accepting various fatty acid substrates of different chain lengths .

What structural features of P. profundum allantoicase contribute to its pressure adaptation?

To elucidate the pressure-adaptive structural features of P. profundum allantoicase:

• High-resolution crystal structure determination:

  • Purify the recombinant enzyme to >95% homogeneity

  • Screen crystallization conditions at low temperatures (4-10°C)

  • Solve structure using molecular replacement or experimental phasing

  • Compare with mesophilic allantoicase structures

• Key structural elements to analyze:

  • Active site architecture and substrate binding pocket

  • Oligomerization interfaces (if multimeric)

  • Surface charge distribution and hydrophobic core packing

  • Presence of increased flexibility elements

• Molecular dynamics simulations under varying pressure:

  • Perform simulations at 0.1 MPa, 28 MPa, and 50 MPa

  • Analyze conformational changes, water penetration, and cavity volumes

  • Identify pressure-sensitive regions that may contribute to adaptation

Based on studies of other pressure-adapted proteins, expect to observe increased internal cavities, reduced hydrophobic packing, and increased flexibility in loop regions compared to mesophilic counterparts. These features potentially allow the enzyme to maintain sufficient flexibility for catalysis under high-pressure conditions that would normally rigidify protein structures.

How does temperature interact with pressure to affect the structural stability of P. profundum allantoicase?

The interplay between temperature and pressure on P. profundum allantoicase stability should be investigated through:

• Differential scanning calorimetry (DSC) at various pressures:

  • Measure thermal denaturation profiles at pressures ranging from 0.1 to 50 MPa

  • Determine the melting temperature (Tm) as a function of pressure

  • Calculate thermodynamic parameters (ΔH, ΔS, ΔV)

• Pressure perturbation calorimetry:

  • Measure volume changes associated with thermal unfolding

  • Determine the pressure-temperature phase diagram for enzyme stability

• Spectroscopic techniques under pressure:

  • Circular dichroism to monitor secondary structure

  • Fluorescence spectroscopy to track tertiary structure changes

  • FTIR to detect subtle conformational shifts

• Expected results based on other P. profundum proteins:

These analyses would provide insights into the enzyme's adaptation to the cold, high-pressure deep-sea environment, particularly how it maintains catalytic flexibility under conditions that typically rigidify protein structures .

How does P. profundum allantoicase expression change in response to varying pressure and temperature conditions?

To investigate the expression regulation of P. profundum allantoicase:

• Quantitative transcriptomics:

  • Culture P. profundum at combinations of different pressures (0.1, 15, 28, and 45 MPa) and temperatures (4, 15, and 25°C)

  • Perform RNA-seq to quantify allantoicase gene expression

  • Identify co-regulated genes under pressure/temperature combinations

• Promoter analysis:

  • Clone the promoter region of allantoicase into reporter constructs similar to methods used for phr gene studies

  • Test activity under different pressure-temperature regimes

  • Identify transcription factors involved using techniques similar to those used for identifying the role of RNA polymerase sigma factor (rpoX) in other P. profundum genes

• Protein-level analysis:

  • Use targeted proteomics with mass spectrometry to quantify allantoicase protein levels under various conditions

  • Perform pulse-chase experiments to determine protein turnover rates

This approach would reveal whether allantoicase is regulated as part of a pressure-responsive regulon and whether its expression follows patterns similar to other pressure-regulated genes in P. profundum, such as the up-regulation of glycolysis/gluconeogenesis pathway proteins at high pressure .

What genetic engineering approaches can optimize recombinant P. profundum allantoicase for biotechnological applications?

Strategic genetic engineering can enhance recombinant P. profundum allantoicase for specific applications:

• Structure-guided mutagenesis:

  • Target residues involved in substrate binding to alter specificity

  • Modify surface charges to enhance stability in different buffer systems

  • Introduce disulfide bridges to stabilize flexible regions while maintaining activity

• Directed evolution approaches:

  • Develop high-throughput screening assay for allantoicase activity

  • Create random mutagenesis libraries using error-prone PCR

  • Perform selective screening under desired conditions (temperature, pH, solvent presence)

• Domain swapping:

  • Identify domains from homologous enzymes with desirable properties

  • Create chimeric enzymes combining optimal domains

  • Test using expression systems similar to those for P. profundum Orf6

• Immobilization optimization:

  • Design fusion constructs with affinity tags positioned to not interfere with catalytic activity

  • Test various immobilization matrices for optimal enzyme performance

  • Evaluate stability and reusability under application-specific conditions

Genetic engineering should aim to maintain the advantageous pressure-adaptive features of the enzyme while optimizing other parameters like thermostability or pH tolerance. Use marker exchange-eviction mutagenesis techniques with suicide vectors like pRL271, similar to methods used for other P. profundum proteins , to validate mutations in the native organism when necessary.

How does P. profundum allantoicase differ from homologous enzymes in related marine bacteria from various depths?

A systematic comparative analysis should be conducted between P. profundum allantoicase and homologs from bacteria inhabiting different ocean depths:

• Homolog identification and phylogenetic analysis:

  • Identify allantoicase homologs in related genera (Vibrio, Moritella, Colwellia, Shewanella)

  • Perform phylogenetic analysis to establish evolutionary relationships

  • Correlate sequence differences with habitat depth

• Comparative biochemistry:

  • Express and purify homologous enzymes from:

    • Shallow-water relatives (0-10 m depth)

    • Mid-depth relatives (100-1000 m)

    • Deep-sea relatives (>1000 m)

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

  • Analyze temperature optima and stability profiles

• Expected comparative properties:

This comparative approach would reveal the molecular signatures of depth adaptation across the allantoicase enzyme family and identify key amino acid substitutions responsible for pressure adaptation, similar to the evolutionary patterns observed in other P. profundum proteins that have homologs across depth-differentiated bacterial species .

Can functional substitution experiments between allantoicases from different pressure environments reveal critical residues for pressure adaptation?

Functional substitution experiments can identify key residues for pressure adaptation:

• Site-directed mutagenesis strategy:

  • Identify non-conserved residues between deep-sea and shallow-water allantoicases

  • Create single and multiple point mutations in both enzymes

  • Focus on:

    • Surface-exposed charged residues

    • Hydrophobic core packing residues

    • Flexible loop regions

    • Active site coordination residues

• Pressure-activity profiling:

  • Test each mutant's activity across a pressure range (0.1-50 MPa)

  • Determine if mutations shift pressure optima toward the donor organism's habitat

  • Measure kinetic parameters to distinguish effects on binding versus catalysis

• Structural validation:

  • Obtain crystal structures of key mutants

  • Perform molecular dynamics simulations under pressure

  • Correlate structural changes with functional effects

• Complementation studies:

  • Create gene deletion mutants using techniques similar to those employed for flagellin gene deletions in P. profundum

  • Complement with wild-type and mutant alleles

  • Test function under various pressure conditions

This approach can identify individual residues or combinations that serve as "pressure switches," similar to how specific protein domains have been identified as critical for pressure adaptation in other P. profundum proteins through comparative analyses with their mesophilic counterparts .

How does allantoicase function integrate into the broader nitrogen metabolism network of P. profundum under pressure?

To understand the metabolic context of allantoicase in P. profundum:

• Metabolic flux analysis:

  • Culture P. profundum with isotope-labeled nitrogen sources

  • Track metabolite flux through the allantoicase pathway under different pressures

  • Identify pressure-dependent shifts in nitrogen utilization

• Comparative transcriptomics:

  • Analyze co-expression patterns of allantoicase with other nitrogen metabolism genes

  • Identify pressure-responsive regulatory networks

  • Compare with expression patterns of proteins involved in other metabolic pathways that show pressure-dependent regulation, such as glycolysis/gluconeogenesis

• Gene deletion studies:

  • Create allantoicase knockout strains using marker exchange-eviction mutagenesis

  • Characterize growth phenotypes under various nitrogen sources and pressures

  • Identify compensatory metabolic pathways under different conditions

• Metabolomic profiling:

  • Compare metabolite profiles between wild-type and allantoicase mutants

  • Identify accumulated precursors and depleted products

  • Measure changes in related metabolic pathways

This integrated approach would reveal whether allantoicase function is essential under specific pressure conditions and how its activity coordinates with other nitrogen metabolism enzymes to adapt to the deep-sea environment, potentially showing similar pressure-responsive patterns to those observed in other metabolic pathways in P. profundum .

What role might the allantoicase pathway play in P. profundum adaptation to fluctuating nutrient conditions in the deep sea?

To investigate the ecological significance of allantoicase in P. profundum's adaptation to deep-sea nutrient fluctuations:

• Simulated environmental stress responses:

  • Culture P. profundum under combinations of pressure and nutrient limitation

  • Monitor allantoicase expression and activity during nutrient shifts

  • Compare responses between wild-type and allantoicase mutants

• Comparative analysis of nutrient utilization:

  • Test growth on different nitrogen sources under varying pressures

  • Determine if allantoicase provides a competitive advantage under specific conditions

  • Measure growth rates and yields in defined minimal media

• Mixed culture competition experiments:

  • Create fluorescently labeled wild-type and allantoicase mutant strains

  • Co-culture under fluctuating nutrient conditions

  • Monitor population dynamics using flow cytometry

• Ecological modeling:

  • Integrate experimental data into models of deep-sea nitrogen cycling

  • Predict the contribution of allantoicase-mediated metabolism to microbial community function

  • Estimate ecological significance under various oceanographic scenarios

This ecological perspective would determine whether allantoicase represents a specific adaptation to the deep-sea environment where different hydrostatic pressures represent distinct ecosystems with their own particular nutrient limitations and abundances, as noted in previous studies of P. profundum .