Recombinant Photobacterium profundum Uridylate kinase (pyrH)

What is the function of Uridylate kinase (pyrH) in bacterial metabolism?

Uridylate kinase (pyrH) catalyzes the critical phosphorylation of UMP to UDP in the pyrimidine metabolic pathway of bacteria, including Photobacterium profundum. This reaction represents an essential step in nucleotide biosynthesis, providing precursors for DNA and RNA synthesis. The enzyme's activity is particularly vital during bacterial replication, as UDP serves as an important precursor for various cellular processes including capsular polysaccharide synthesis . The reaction can be represented as:

UMP + ATP → UDP + ADP

In pathogenic bacteria like Vibrio vulnificus, pyrH has been demonstrated to be preferentially upregulated during infection, highlighting its importance for successful bacterial replication in host environments .

How conserved is the pyrH gene across bacterial species?

The pyrH gene shows remarkable conservation across bacterial genomes. Sequence analysis reveals that homologs of this gene are present in virtually all bacterial genomes reported to date, with no direct counterparts in eukaryotes . This makes it an attractive target for antimicrobial development.

For example, comparative analysis of pyrH sequences among Vibrio species demonstrates moderate to high sequence conservation:

• Vibrio species showed significant pyrH sequence similarity ranging from 79% to 99.6%

• Photobacterium species exhibited 79% to 99.6% pyrH sequence similarity

This conservation suggests an essential evolutionary role for this enzyme in bacterial metabolism.

What are the key structural features of PyrH protein that contribute to its enzymatic function?

The PyrH protein contains several highly conserved amino acid residues that are critical for substrate binding and catalytic activity. Based on crystallographic studies of E. coli PyrH:

• UMP substrate recognition occurs through simultaneous recognition of its base, sugar, and phosphate moieties

• Key substrate-binding residues include:

  • Arg62: The terminal nitrogen interacts with the terminal oxygen of alpha-phosphate

  • Asp77: The side chain oxygen forms hydrogen bonds with 2'OH of ribose

These amino acid residues are remarkably conserved across multiple bacterial species, including V. vulnificus, E. coli, Salmonella enterica, Bacillus subtilis, and Listeria monocytogenes, with sequence identity ranging from 29% to 85.5% .

What are the optimal expression systems for producing recombinant Photobacterium profundum pyrH?

The choice of expression system for recombinant P. profundum pyrH depends on research objectives and required protein characteristics:

E. coli expression system:

• Advantages: Highest yield, shorter turnaround times, cost-effective

• Methodology: Clone the pyrH gene into a suitable vector (pET systems commonly used) with appropriate tags for purification

• Considerations: May lack post-translational modifications; protein folding might be compromised for this deep-sea bacterial protein

Yeast expression system:

• Advantages: Good yields, some post-translational modifications, suitable for challenging proteins

• Methodology: Clone into vectors like pPICZ with appropriate signal sequences

• Considerations: Longer expression time than E. coli but still relatively efficient

Insect cells with baculovirus:

• Advantages: Better post-translational modifications, improved protein folding

• Methodology: Generate bacmid, transfect insect cells, harvest after 72-96 hours

• Considerations: More complex and time-consuming than prokaryotic systems

Mammalian cells:

• Advantages: Most complete post-translational modifications

• Methodology: Transiently transfect HEK293 or stably express in CHO cells

• Considerations: Lower yields, highest complexity, but potentially necessary if activity depends on specific modifications

For initial characterization studies, E. coli or yeast systems are recommended for their balance of yield and turnaround time.

How can I design effective site-directed mutations to study P. profundum pyrH function?

When designing site-directed mutations for functional studies of P. profundum pyrH, follow this methodological approach:

• Identify conserved residues by performing multiple sequence alignment with homologous proteins from related species

• Target substrate-binding regions based on existing crystal structures of UMP kinases (e.g., E. coli PyrH)

• Mutation strategy:

  • For substrate binding studies: Change Arg62 to His (R62H) and/or Asp77 to Asn (D77N) to disrupt UMP binding

  • For catalytic activity studies: Target residues in the active site

  • For allosteric regulation: Focus on amino acids that interact with potential regulatory molecules

Laboratory protocol:

• Design primers for site-directed mutagenesis with appropriate nucleotide substitutions

• Perform PCR-based mutagenesis (e.g., QuikChange protocol)

• Verify mutations by restriction enzyme digestion and DNA sequencing

• Express both wild-type and mutant proteins

• Purify using affinity chromatography (e.g., intein-fusion protein expression system)

• Confirm mutation effects through enzymatic activity assays comparing wild-type vs. mutant

Based on studies with V. vulnificus pyrH, mutations at R62H, D77N, and the double mutant R62H/D77N resulted in dramatic decreases in UMP kinase activity (>97% activity loss), confirming these residues are critical for enzymatic function .

What enzymatic assay methods can be used to measure PyrH activity?

Several complementary methods can be employed to measure PyrH enzymatic activity:

1. Luminescence-based kinase assay:

• Principle: Detection of ATP consumption during the phosphorylation reaction

• Methodology:

  • Mix purified PyrH with UMP substrate and ATP

  • Add luminescence reagent that produces light proportional to remaining ATP

  • Measure signal using a luminometer

• Advantages: High sensitivity, suitable for high-throughput screening of inhibitors

2. Coupled spectrophotometric assay:

• Principle: Coupling UDP production to NADH oxidation via pyruvate kinase and lactate dehydrogenase

• Methodology:

  • Reaction mixture: PyrH + UMP + ATP + phosphoenolpyruvate + NADH + pyruvate kinase + lactate dehydrogenase

  • Monitor decrease in absorbance at 340nm as NADH is consumed

• Advantages: Continuous real-time monitoring of enzyme activity

3. Radiometric assay:

• Principle: Direct measurement of 32P incorporation from [γ-32P]ATP to UMP

• Methodology:

  • Incubate PyrH with [γ-32P]ATP and UMP

  • Separate reaction products by thin-layer chromatography

  • Quantify radioactive UDP by phosphorimager or scintillation counting

• Advantages: Direct measurement, high sensitivity

The specific activity can be calculated in units per microgram of protein (U/μg), with one unit defined as the amount of enzyme that catalyzes the formation of 1 μmol of UDP per minute under defined conditions .

How can pyrH be used for molecular identification and phylogenetic analysis of Vibrio and Photobacterium species?

The pyrH gene serves as an excellent phylogenetic marker for Vibrio and Photobacterium species due to its high conservation and appropriate level of sequence variation. The methodological approach for using pyrH in phylogenetic studies includes:

1. PCR amplification of pyrH:

• Use degenerate primers targeting conserved regions:

  • Forward primer: pyrH-04-F (5'-ATGASNACBAAYCCWAAACC-3')

  • Reverse primer: pyrH-02-R (5'-GTRAABGCNGMYARRTCCA-3')

• These primers bind to positions 1 and 599 of the pyrH gene, respectively

2. Sequence the PCR product using standard Sanger sequencing methods

3. Sequence analysis:

• Perform multiple sequence alignment using MUSCLE or ClustalW

• Calculate sequence similarity percentages between species

• Construct phylogenetic trees using maximum likelihood or Bayesian methods

4. Interpretation of results:

• Photobacterium species typically show 79-99.6% pyrH sequence similarity

• Different strains within the same species show much lower variation (often <2%)

• Example: Photobacterium damselae strains show only 1.0% pyrH sequence variation

A significant advantage of pyrH over 16S rRNA for discriminating closely related species is its higher resolution while maintaining sufficient conservation for reliable amplification across diverse species.

How does high hydrostatic pressure affect the structure and function of P. profundum pyrH?

Photobacterium profundum is a piezophilic (pressure-loving) deep-sea bacterium , and the adaptations of its pyrH enzyme to high-pressure environments represent an important research area. When investigating pressure effects on P. profundum pyrH:

Methodological approaches:

• Comparative structural analysis:

  • Express recombinant pyrH from P. profundum and mesophilic counterparts

  • Perform circular dichroism spectroscopy at atmospheric and elevated pressures

  • Compare thermostability and pressure stability profiles

• High-pressure enzyme kinetics:

  • Utilize specialized high-pressure vessels equipped with spectroscopic windows

  • Determine kinetic parameters (Km, Vmax) at increasing pressure levels (0.1-100 MPa)

  • Analyze the pressure dependence of catalytic efficiency (kcat/Km)

• Molecular dynamics simulations:

  • Create models of P. profundum pyrH based on homologous crystal structures

  • Simulate protein behavior under various pressure conditions

  • Identify structural elements potentially responsible for pressure adaptation

Research hypothesis: P. profundum pyrH likely contains adaptations that maintain flexibility and catalytic function under high pressure, possibly including increased hydrophobic interactions, reduced void volumes, or altered surface charge distributions compared to mesophilic homologs.

What is the role of pyrH in bacterial virulence and how can this be experimentally assessed?

Studies with V. vulnificus have demonstrated that pyrH plays an essential role in bacterial virulence . While P. profundum is not a primary pathogen, understanding the contribution of pyrH to bacterial fitness provides important insights for antimicrobial development.

Methodological approaches to assess pyrH's role in virulence:

• Construction of pyrH mutant strains:

  • Site-directed mutagenesis targeting conserved residues (R62H/D77N approach)

  • Clean deletion mutants are challenging due to gene essentiality

  • Use conditional expression systems where pyrH expression can be controlled

• In vitro virulence/fitness assays:

  • Growth curve analysis in standard media and under stress conditions

  • Survival in serum or other host-mimicking environments

  • Biofilm formation capacity

  • Resistance to environmental stresses

• Cellular infection models:

  • Assess bacterial replication in cell culture (e.g., using HeLa cell lysate models)

  • Measure cytotoxicity using LDH release assays

  • Test survival in the presence of immune cells

• In vivo infection models:

  • Mouse models with altered pyrH compared to wild-type

  • Determine 50% lethal dose (LD50) for each strain

  • Track bacterial load in tissues over time

In V. vulnificus studies, the R62H/D77N pyrH mutant showed dramatically reduced virulence, with the intraperitoneal LD50 increasing by 26-fold in normal mice and 238,000-fold in iron-overloaded mice . The mutant also demonstrated significantly reduced growth in 50% HeLa cell lysate, 100% human ascitic fluid, and 50% human serum compared to wild-type .

What makes pyrH an attractive target for antimicrobial drug development?

PyrH represents a compelling antimicrobial target for several key reasons:

• Essentiality: pyrH is essential for bacterial survival and replication, making it difficult for bacteria to develop resistance through simple target modification

• Conservation: The gene is highly conserved across bacterial species, suggesting potential for broad-spectrum activity

• Absence in humans: Bacterial PyrH has no direct counterpart in eukaryotes, allowing for selective toxicity

• Structural information: Crystal structures of PyrH from multiple bacteria provide templates for structure-based drug design

• In vivo validation: Studies with V. vulnificus confirm that pyrH is essential for in vivo survival and growth of bacteria during infection

These characteristics align with ideal criteria for antimicrobial targets, as compounds inhibiting PyrH could potentially be both effective and selective.

How can high-throughput screening approaches be optimized for discovering pyrH inhibitors?

A methodological framework for high-throughput screening of pyrH inhibitors includes:

1. Assay development and optimization:

• Implement a luminescence-based kinase assay as described for PYRH-1 evaluation

• Optimize reaction conditions (buffer composition, pH, temperature, enzyme concentration)

• Validate with known inhibitors such as UTP (a natural allosteric inhibitor)

• Calculate Z' factor to ensure assay robustness

2. Compound library selection:

• Diverse commercial libraries (10,000-100,000 compounds)

• Focused libraries based on existing kinase inhibitors

• Virtual screening results based on pyrH structure

• Natural product extracts

3. Primary screening protocol:

• Screen at single concentration (10-20 μM)

• Include positive controls (PYRH-1, UTP) and negative controls (DMSO)

• Set threshold (>50% inhibition) for hit selection

4. Secondary validation:

• Dose-response curves to determine IC50 values

• Counter-screening against mammalian kinases to assess selectivity

• Surface plasmon resonance to confirm direct binding

5. Hit characterization:

• Mechanism of inhibition studies (competitive vs. allosteric)

• Antimicrobial activity testing against bacterial panel

• Cytotoxicity assessment against mammalian cell lines

For reference, PYRH-1 (sodium {3-[4-tert-butyl-3-(9H-xanthen-9-ylacetylamino)phenyl]-1-cyclohexylmethylpropoxycarbonyloxy}acetate) has been identified as a PyrH inhibitor with IC50 values of 48 μM against S. pneumoniae PyrH and 75 μM against H. influenzae PyrH .

What structure-activity relationship studies are needed to optimize pyrH inhibitors?

To develop improved pyrH inhibitors based on initial hits like PYRH-1, a systematic structure-activity relationship approach should be implemented:

1. Pharmacophore identification:

• Map key interaction points between lead compounds and pyrH

• Identify essential structural features for activity

• Determine which portions of the molecule can be modified while maintaining activity

2. Systematic structural modifications:

• Vary substitution patterns on the aromatic rings

• Modify the linker length and composition

• Explore bioisosteric replacements for key functional groups

• Adjust stereochemistry at chiral centers

3. Structure-guided design:

• Use available crystal structures of bacterial PyrH proteins

• Perform molecular docking to predict binding modes

• Design compounds to exploit specific binding pocket features

• Target both active site and allosteric sites

4. Testing cascade:

• Primary biochemical assay: IC50 determination against multiple bacterial PyrH proteins

• Antimicrobial activity: MIC determination against panel of bacterial pathogens

• Selectivity: Counter-screening against human kinases

• Physicochemical properties: Solubility, stability, permeability

• ADME/Tox profiling: Metabolic stability, protein binding, cytotoxicity

5. Optimization cycles:

• Iterative synthesis and testing

• Refinement based on multiparameter optimization

• Progression of compounds with balanced profiles

For P. profundum pyrH specifically, consider adaptations required for activity under high-pressure conditions, as this might provide insights into designing inhibitors effective against deep-sea bacteria or structurally similar enzymes in pathogenic species.

How can researchers overcome the challenges of studying an essential gene like pyrH?

Studying essential genes like pyrH presents unique experimental challenges. Here are methodological approaches to address these difficulties:

1. Conditional expression systems:

• Implement inducible promoters (e.g., arabinose or tetracycline-inducible)

• Create temperature-sensitive mutants that function normally at permissive temperatures

• Use degron-based systems for controlled protein degradation

• Methodology: Clone pyrH under control of an inducible promoter while deleting the native copy

2. Site-directed mutagenesis approach:

• Generate partial loss-of-function mutations rather than complete knockouts

• Target substrate-binding residues (R62H/D77N) to maintain minimal activity

• Example from V. vulnificus: The R62H/D77N double mutant retained approximately 1.13% of wild-type activity, allowing cellular survival while significantly impacting function

3. Antisense RNA and CRISPR interference:

• Use inducible antisense RNA to partially repress pyrH expression

• Implement CRISPRi with catalytically inactive Cas9 (dCas9) to reduce transcription

• Advantages: Tunable repression levels, allows titration of gene expression

4. Chemical biology approaches:

• Utilize specific inhibitors as chemical probes

• Apply synthetic lethal screening to identify compensatory pathways

• Couple with transcriptomic or proteomic analysis to understand cellular responses

5. Heterologous complementation:

• Express pyrH from other species to assess functional conservation

• Create chimeric proteins to identify functional domains

• Example: Express mesophilic pyrH in P. profundum to identify pressure-adaptation regions

These approaches can be combined to develop a comprehensive understanding of pyrH function while overcoming the intrinsic challenges of studying essential genes.

What controls and validation experiments are essential when studying recombinant P. profundum pyrH?

A rigorous experimental design for studying recombinant P. profundum pyrH should include these essential controls and validation steps:

1. Expression and purification controls:

• Include a well-characterized protein (e.g., GFP) to validate expression system

• Prepare enzyme-dead mutant as negative control

• Use a homologous pyrH from a related species (e.g., E. coli) as comparison

• Multiple purification methods to ensure native conformation preservation

2. Enzymatic activity validation:

• Measure activity using multiple independent assays (luminescence, spectrophotometric, radiometric)

• Include substrate specificity controls (test related nucleotides)

• Establish Michaelis-Menten kinetics for wild-type enzyme

• Compare with published values for related enzymes

3. Structural validation:

• Circular dichroism spectroscopy to confirm secondary structure

• Size exclusion chromatography to verify oligomeric state

• Thermal shift assays to measure protein stability

• Limited proteolysis to assess proper folding

4. Environmental condition controls:

• Assess activity across relevant pressure ranges (0.1-60 MPa)

• Test temperature optima and stability

• Evaluate salt concentration effects

• Measure pH dependency of activity

5. Site-directed mutagenesis validation:

6. Biological relevance experiments:

• Complementation studies in pyrH-deficient strains

• Growth rate measurements under relevant conditions

• Stress response analysis

• Comparison with native enzyme where possible

How can researchers integrate pyrH sequence data with phenotypic information for comprehensive bacterial classification?

Modern bacterial taxonomy benefits from integrating multiple data types. For pyrH-based classification:

Methodological framework:

• Multi-locus sequence typing (MLST) integration:

  • Include pyrH alongside other housekeeping genes (recA, rpoA, gyrB)

  • Calculate concatenated phylogenies for increased resolution

  • Compare individual gene trees to identify horizontal gene transfer events

  • Example data format:

  Species	pyrH similarity (%)	recA similarity (%)	rpoA similarity (%)	Combined similarity (%)
  P. damselae strains	1.0	0.1	0.1	0.4
  P. rosenbergii strains	3.0	5.5	0.4	3.0
  V. parahaemolyticus strains	6.0	6.0	0.3	4.1

• Correlation of molecular and phenotypic data:

  • Collect standardized phenotypic information (biochemical tests, growth conditions)

  • Perform statistical analyses (principal component analysis, hierarchical clustering)

  • Identify phenotype-genotype associations

  • Create integrated taxonomic frameworks

• Whole genome correlation:

  • Compare pyrH-based phylogenies with whole-genome approaches

  • Calculate average nucleotide identity (ANI) between genomes

  • Determine whether pyrH phylogeny predicts genome-wide relationships

  • Use as a rapid screening tool before whole-genome sequencing

• Bioinformatic tools and databases:

  • Develop specialized databases for pyrH sequences with associated metadata

  • Create automated pipelines for sequence analysis and species identification

  • Implement machine learning approaches for classification

  • Use All by All tables approach for comprehensive association studies

This integrated approach produces more robust bacterial classification systems and provides insights into evolutionary relationships that single-gene approaches might miss.

What statistical approaches are most appropriate for analyzing pyrH inhibitor screening data?

When analyzing high-throughput screening data for pyrH inhibitors, appropriate statistical methods are crucial:

1. Primary screening data analysis:

• Calculate Z' factor to assess assay quality: Z' = 1 - [(3σp + 3σn)/|μp - μn|]
  where σp and σn are standard deviations, μp and μn are means of positive and negative controls

• Apply robust Z-score normalization: Z = (xi - median)/MAD
  where MAD is median absolute deviation

• Set hit threshold: typically >3 standard deviations from mean or >50% inhibition

• Correct for systematic errors (edge effects, plate position bias) using B-score normalization

2. Dose-response analysis:

• Fit data to four-parameter logistic model: y = Bottom + (Top-Bottom)/(1+(x/IC50)^Hill)

• Calculate IC50 with 95% confidence intervals

• Determine Hill slope for binding cooperativity

• Evaluate quality of curve fit using R² and residual analysis

3. Structure-activity relationship analysis:

• Implement hierarchical clustering of compounds by structural features

• Apply principal component analysis to identify key activity-driving chemical properties

• Develop quantitative structure-activity relationship (QSAR) models

• Use machine learning algorithms (random forest, support vector machines) for activity prediction

4. Selectivity and specificity analysis:

• Calculate selectivity index: SI = IC50(off-target)/IC50(pyrH)

• Perform correlation analysis between different bacterial pyrH orthologs

• Use heat maps to visualize cross-reactivity patterns

5. Advanced techniques for in-depth analysis:

• Apply Bayesian statistics for hit probability assessment

• Implement Monte Carlo simulations for error propagation

• Use bootstrapping for robust confidence interval estimation

• Develop decision trees for compound progression

These statistical approaches ensure rigorous evaluation of screening data and guide rational optimization of pyrH inhibitors.

What are the emerging applications of pyrH beyond antimicrobial development?

While antimicrobial development remains a primary focus, pyrH research has several emerging applications:

1. Synthetic biology and metabolic engineering:

• Manipulating pyrH expression to control nucleotide pools and modulate growth rates

• Engineering pyrH variants with altered regulatory properties for biotechnology applications

• Using pyrH as a biosensor component for detecting nucleotide imbalances

• Methodology: Create synthetic pyrH variants with modified allosteric regulation sites

2. Environmental monitoring and microbial ecology:

• Developing pyrH-based molecular markers for tracking specific bacterial populations

• Assessing bacterial community responses to environmental stressors through pyrH expression

• Creating pyrH-targeted environmental DNA (eDNA) assays for species identification

• Application: Monitor bacterial adaptations in response to climate change in marine environments

3. Vaccine development:

• Using attenuated pyrH mutants as live vaccine candidates

• Example from V. vulnificus research: The R62H/D77N mutant showed potential as a replication-controllable live attenuated vaccine due to its significant attenuation while maintaining immunogenicity

• Methodology: Introduce specific mutations to create temperature-sensitive pyrH variants

4. Extremophile adaptation studies:

• Investigating how pyrH from extremophiles like P. profundum adapts to environmental pressures

• Comparing structural modifications across bacteria from diverse habitats

• Application: Engineering proteins with enhanced stability under extreme conditions

5. Fundamental understanding of bacterial physiology:

• Using pyrH as a model to study essential gene networks and bacterial fitness landscapes

• Investigating coordination between nucleotide metabolism and other cellular processes

• Methodology: Apply systems biology approaches combining transcriptomics, proteomics, and metabolomics

These diverse applications demonstrate how fundamental research on pyrH contributes to broader scientific advances beyond antimicrobial development.

How might machine learning and computational approaches advance pyrH research?

Integrating machine learning and computational methods into pyrH research offers several promising avenues:

1. Inhibitor discovery and optimization:

• Implement deep learning models to predict pyrH-binding compounds

• Train neural networks on existing inhibitor data (e.g., PYRH-1)

• Use reinforcement learning for de novo molecule generation

• Apply transfer learning from related kinase inhibitor datasets

• Methodology: Utilize tabular foundation models like TabPFN for small dataset prediction tasks

2. Structural biology applications:

• Employ AlphaFold or RoseTTAFold to predict structures of pyrH orthologs lacking crystal structures

• Simulate protein dynamics under various conditions (pressure, temperature)

• Model protein-ligand interactions through molecular dynamics

• Predict effects of mutations on protein stability and function

• Output: Detailed maps of conformational changes during catalytic cycles

3. Systems biology integration:

• Model metabolic networks with pyrH as a key node

• Predict system-wide effects of pyrH inhibition using flux balance analysis

• Identify synthetic lethal interactions through in silico genome-scale modeling

• Methodology: Integrate pyrH-centered networks with genome-scale metabolic models

4. Evolutionary analysis:

• Detect signatures of selection on pyrH across bacterial lineages

• Predict functionally important residues through co-evolution analysis

• Reconstruct ancestral pyrH sequences to study evolutionary trajectories

• Output: Identification of unique adaptations in specialized bacteria like P. profundum

5. Clinical and environmental applications:

• Develop rapid identification systems based on pyrH sequences

• Create prediction models for antimicrobial resistance emergence

• Design optimal combination therapies targeting multiple essential pathways

• Methodology: Apply ensemble machine learning methods to integrate diverse data types

These computational approaches can accelerate research progress while providing novel insights that might be difficult to obtain through traditional experimental methods alone.

What are the most critical considerations when designing a research project focused on P. profundum pyrH?

When planning a comprehensive research project on P. profundum pyrH, researchers should consider these critical factors:

1. Experimental design fundamentals:

• Implement randomized controlled experiments with appropriate replication

• Include positive and negative controls for all assays

• Design experiments with sufficient statistical power

• Consider biological relevance of experimental conditions (pressure, temperature, salinity)

2. Technical challenges specific to P. profundum:

• Growth requirements: P. profundum is a piezophilic deep-sea bacterium requiring specialized equipment

• Expression systems: Choose systems capable of producing functional deep-sea bacterial proteins

• Activity assays: Develop methods that can function under high-pressure conditions

• Stability concerns: Account for potential instability of the recombinant protein

3. Comparative framework:

• Include pyrH from related species as references (other Photobacterium species, Vibrio species)

• Consider both closely related shallow-water and deep-sea species

• Analyze both pathogenic and non-pathogenic strains to understand virulence contributions

4. Interdisciplinary approach:

• Combine structural biology, biochemistry, microbiology, and computational methods

• Engage collaborators with specialized equipment (high-pressure cultivation systems)

• Consider ecological and evolutionary contexts of P. profundum adaptations

• Integrate findings with broader understanding of deep-sea microbial physiology

5. Translation potential:

• Identify aspects with applications in biotechnology or medicine

• Consider how pressure adaptations might inform protein engineering

• Evaluate potential as antimicrobial target despite non-pathogenic nature of P. profundum

By addressing these considerations from the outset, researchers can design more robust and impactful studies on P. profundum pyrH while avoiding common pitfalls in experimental design and interpretation.

How can researchers effectively troubleshoot common problems in pyrH expression and activity assays?

When working with recombinant pyrH, researchers frequently encounter several challenges. Here are methodological approaches to troubleshoot common problems:

1. Low expression yield:

• Problem: Poor expression of soluble P. profundum pyrH

• Troubleshooting steps:

  • Optimize codon usage for expression host

  • Lower induction temperature (16-20°C)

  • Use solubility-enhancing fusion tags (MBP, SUMO, TrxA)

  • Screen multiple expression vectors with different promoter strengths

  • Try autoinduction media instead of IPTG induction

  • Consider cell-free expression systems

2. Protein instability:

• Problem: Purified protein shows rapid activity loss

• Troubleshooting steps:

  • Add stabilizing agents (glycerol 10-20%, reducing agents like DTT or β-ME)

  • Optimize buffer conditions (pH, salt concentration)

  • Include protease inhibitors throughout purification

  • Determine thermal stability profile to identify optimal storage temperature

  • Test addition of substrate or product analogs as stabilizers

3. Inconsistent activity assays:

• Problem: High variability in enzymatic assay results

• Troubleshooting steps:

  • Control reaction temperature precisely

  • Ensure consistent enzyme/substrate concentrations

  • Verify ATP quality (ATP hydrolyzes during storage)

  • Pre-incubate components to reach temperature equilibrium

  • Determine linear range of the assay

  • Use internal standards for normalization

4. Pressure-related challenges:

• Problem: Difficulty measuring activity under high pressure

• Troubleshooting steps:

  • Use pressure-resistant fluorescent probes

  • Develop stopped-flow methods compatible with pressure chambers

  • Implement specialized high-pressure equipment with optical windows

  • Consider indirect assays where activity is measured after pressure treatment

5. Inactive recombinant protein:

• Problem: Protein expresses but shows no detectable activity

• Troubleshooting steps:

  • Verify correct protein folding via circular dichroism

  • Test multiple purification strategies to find gentle conditions

  • Include cofactors that might be required (metal ions)

  • Refolding from inclusion bodies if necessary

  • Co-express with molecular chaperones

  • Verify expression construct sequence is correct