Recombinant Burkholderia multivorans Anthranilate phosphoribosyltransferase (trpD)

What is Anthranilate phosphoribosyltransferase (TrpD) and why is it significant in B. multivorans research?

Anthranilate phosphoribosyltransferase (TrpD, EC2.4.2.18) is an enzyme that catalyzes the second step in tryptophan biosynthesis. It transfers a phosphoribosyl group to anthranilate, generating phosphoribosyl anthranilate (PRA), which forms the basic skeleton of tryptophan . TrpD belongs to the phosphoribosyltransferase (PRT) superfamily and is the only member of the structural class IV PRT .

In B. multivorans, TrpD is particularly significant because this organism is a member of the Burkholderia cepacia complex (BCC), which is notorious for its pathogenicity in persons with cystic fibrosis . Understanding the structural and functional aspects of key metabolic enzymes like TrpD could potentially provide insights into bacterial survival mechanisms and identify novel therapeutic targets.

How does B. multivorans TrpD fit within the genomic context of clinical isolates?

B. multivorans isolates typically have three large replicons (chromosomes) that are highly conserved in their structure . The genomic sequences across these chromosomes in clinical isolates share >99.8% nucleotide sequence identity . Within this genomic context, genes involved in tryptophan biosynthesis, including trpD, may be subject to adaptive evolution in the lungs of cystic fibrosis patients.

Genomic analysis of B. multivorans isolates from cystic fibrosis patients has revealed that different patients infected with the same strain (e.g., ST-742) show peculiar patterns of genomic diversity, including small nucleotide polymorphisms indicative of low rates of adaptive evolution within patients, and well-defined segments of high mutation enrichment between patients . This genomic variability may extend to genes like trpD, potentially affecting protein structure and function.

What expression systems are typically used for recombinant B. multivorans TrpD?

While the search results don't specifically detail expression systems for B. multivorans TrpD, research on related TrpD enzymes provides methodological guidance. For instance, recombinant TrpD from Thermococcus kodakarensis (TkTrpD) has been successfully expressed and characterized .

Typical methodology involves:

• Gene cloning into suitable expression vectors

• Transformation into E. coli expression hosts

• Culture in media such as Luria-Bertani (LB)

• Induction of protein expression

• Cell harvesting and lysis

• Protein purification using chromatographic techniques

When working with B. multivorans TrpD, researchers should consider the organism's genomic characteristics, including the high GC content typical of Burkholderia species, which may require codon optimization for efficient expression in heterologous hosts.

How do divalent cations influence the activity of TrpD, and what methodological approaches should researchers use to investigate this in B. multivorans TrpD?

Research on TrpD from T. kodakarensis (TkTrpD) has revealed unique divalent cation dependencies that may serve as a model for investigating B. multivorans TrpD. While most characterized TrpD enzymes are Mg²⁺-dependent, TkTrpD showed maximum activity in the presence of Zn²⁺ (1580 μmol·min⁻¹·mg⁻¹), followed by Ca²⁺ (948 μmol·min⁻¹·mg⁻¹) and Mg²⁺ (711 μmol·min⁻¹·mg⁻¹) .

Methodological approach for investigating cation influence:

• Perform enzyme activity assays with different divalent cations (Mg²⁺, Ca²⁺, Zn²⁺, Mn²⁺) at various concentrations

• Measure reaction rates using spectrophotometric methods to track substrate consumption or product formation

• Determine kinetic parameters (Kₘ, Vₘₐₓ, kcat) under different ionic conditions

• Complement activity studies with structural analysis (X-ray crystallography or cryo-EM) in the presence of different metal ions

• Conduct site-directed mutagenesis of potential metal-binding residues to confirm their role

The crystallographic analysis of TkTrpD-Zn²⁺ identified multiple zinc binding sites, including at the expected zinc-binding motif DE(217-218), a new dimer interface, binding to Glu118 at crystal lattice contacts, and ligated with Glu235 . Researchers working with B. multivorans TrpD should investigate similar potential binding sites.

What kinetic parameters should be investigated for B. multivorans TrpD, and what substrate inhibition phenomena might researchers encounter?

Key kinetic parameters for investigation include:

• Kₘ and Vₘₐₓ for both substrates (anthranilate and PRPP)

• Optimal temperature and pH

• Effect of divalent cations on activity

• Substrate inhibition characteristics

• Thermal stability profiles

Researchers should be aware of potential substrate inhibition phenomena, as observed with TkTrpD where anthranilate concentrations above 4 μM resulted in reduced enzymatic activity . This phenomenon has also been observed in M. tuberculosis TrpD .

Methodological approach for kinetic studies:

• Keep one substrate (e.g., PRPP) at a saturating concentration (e.g., 1 mM) while varying the concentration of the second substrate (anthranilate)

• Similarly, keep anthranilate constant at an optimal concentration (below inhibitory levels) when measuring kinetic parameters for PRPP

• Use spectrophotometric assays to measure reaction rates

• Fit data to appropriate kinetic models, accounting for substrate inhibition where observed

What structural features of B. multivorans TrpD might influence its function in the context of cystic fibrosis infections?

While specific structural data for B. multivorans TrpD is not provided in the search results, insights can be drawn from structural studies of TrpD from other organisms. TrpD displays the typical PRT fold with a small N-terminal α-helical domain and a larger C-terminal α/β domain .

In the context of cystic fibrosis infections, several aspects warrant investigation:

• Structural adaptations to the lung environment

  • Changes in pH optima

  • Altered metal ion preferences

  • Modified substrate binding affinities

• Potential structural variations resulting from genomic adaptations observed in clinical isolates

  • Parallel adaptations identified in B. multivorans across multiple patients

  • Effects of mobile genetic elements and insertion sequences on gene expression

• Thermal stability considerations

  • TkTrpD displayed unusually low thermostability compared to other proteins from the same organism

  • B. multivorans TrpD stability might similarly affect persistence in infection

How can experimental and quasi-experimental designs be applied to study the role of TrpD in B. multivorans virulence and adaptation?

Implementation science approaches can be valuable for studying TrpD's role in B. multivorans infection and adaptation. Both experimental and quasi-experimental designs offer distinct advantages :

Experimental Designs:

• Randomized controlled trials (RCTs) may be used to test:

  • Effects of TrpD inhibitors on bacterial growth

  • Impact of trpD gene knockout on virulence

  • Comparative efficacy of different antimicrobial approaches targeting the tryptophan pathway

• Optimization trials for:

  • Fine-tuning expression conditions for recombinant protein

  • Determining optimal parameters for enzyme activity assays

Quasi-Experimental Designs:

• Interrupted time series (ITS) to:

  • Track adaptive changes in TrpD expression or structure during long-term infections

  • Monitor emergence of resistance to TrpD-targeting compounds

• Stepped wedge designs for:

  • Progressive introduction of genetic modifications to study TrpD domains

  • Sequential testing of different metal cofactors or substrate concentrations

• Pre-post designs with non-equivalent control groups to:

  • Compare TrpD characteristics between clinical isolates from different patient cohorts

  • Evaluate treatment outcomes for infections with different TrpD variants

What techniques should be employed for crystallographic analysis of B. multivorans TrpD, and what structural insights might be anticipated?

Based on successful crystallographic studies of TkTrpD , researchers should consider:

Methodology for Crystallization and Structure Determination:

• Protein purification to >95% homogeneity using chromatographic techniques

• Crystallization screening with and without substrates/metal ions

• Data collection using synchrotron radiation

• Structure determination via molecular replacement using other TrpD structures as templates

• Refinement and validation of the structural model

Anticipated Structural Insights:

• Identification of key catalytic residues

• Characterization of metal binding sites

• Substrate binding pocket architecture

• Conformational changes upon substrate/metal binding

• Potential dimerization interfaces influenced by metal ions

The TkTrpD-Zn²⁺ structure revealed multiple zinc binding sites, including at the DE(217-218) motif in each subunit of the dimer, two additional Zn²⁺ at a new dimer interface, a fifth Zn²⁺ bound to Glu118, and a sixth ligated with Glu235 . Similar metal-binding sites might be present in B. multivorans TrpD, with potential implications for function and stability.

What genomic analysis approaches are recommended for studying trpD gene variations in clinical B. multivorans isolates?

For comprehensive genomic analysis of trpD variations in clinical isolates, researchers should employ:

• Sequencing Strategy:

  • Combine short-read (e.g., Illumina) and long-read (e.g., PacBio, Oxford Nanopore) sequencing technologies

  • This combination allows detection of both small nucleotide polymorphisms and larger structural variations

• Comparative Genomic Analysis:

  • Examine conservation of trpD across isolates from different patients

  • Identify mutation hotspots within the gene

  • Look for evidence of selective pressure (dN/dS ratios)

• Core-genome Phylogenomic Analysis:

  • Place the study isolates in the context of the broader B. multivorans population structure

  • Build a core-genome phylogeny using conserved genes across isolates

The genomic analysis of B. multivorans ST-742 isolates from four cystic fibrosis patients demonstrated peculiar patterns of genomic diversity, including low mutation rates within patients but well-defined segments of high mutation enrichment between patients . Similar patterns might affect the trpD gene.

What considerations should guide the design of site-directed mutagenesis experiments for B. multivorans TrpD functional studies?

When designing site-directed mutagenesis experiments for B. multivorans TrpD:

• Target Selection Based on Structural Information:

  • Focus on predicted catalytic residues

  • Investigate potential metal-binding sites (e.g., motifs similar to the DE(217-218) in TkTrpD)

  • Examine substrate binding pocket residues

  • Consider residues at potential dimerization interfaces

• Mutation Design Strategy:

  • Conservative mutations to probe functional importance (e.g., Asp→Glu)

  • Non-conservative mutations to abolish function (e.g., Asp→Ala)

  • Introduction of residues found in TrpD from other organisms to test functional theories

• Functional Characterization of Mutants:

  • Compare kinetic parameters (Kₘ, Vₘₐₓ, kcat) with wild-type enzyme

  • Assess metal ion preferences and binding affinities

  • Evaluate thermal stability and pH optima

  • Examine oligomerization state and potential changes in quaternary structure

How can researchers effectively study substrate inhibition mechanisms in B. multivorans TrpD?

To investigate substrate inhibition mechanisms observed in TrpD enzymes:

• Kinetic Analysis Approach:

  • Conduct detailed enzyme assays across a wide range of substrate concentrations

  • Fit data to appropriate inhibition models (competitive, uncompetitive, non-competitive, mixed)

  • Determine inhibition constants (Ki)

• Structural Investigation:

  • Attempt co-crystallization with inhibitory concentrations of substrate

  • Identify potential secondary binding sites that could mediate inhibition

  • Use molecular dynamics simulations to model substrate binding events

• Site-directed Mutagenesis Strategy:

  • Target residues in potential secondary binding sites

  • Modify residues at the entrance to the active site that might influence substrate access

  • Create variants with altered substrate inhibition profiles

The observation that anthranilate concentrations above 4 μM resulted in reduced TkTrpD activity provides a starting point for similar investigations in B. multivorans TrpD.

What statistical approaches are most appropriate for analyzing kinetic data from B. multivorans TrpD experiments?

For robust statistical analysis of kinetic data:

• Model Selection:

  • Standard Michaelis-Menten equation for simple kinetics

  • Substrate inhibition models when appropriate

  • Allosteric models if cooperative behavior is observed

• Parameter Estimation:

  • Non-linear regression for direct parameter determination

  • Linearization methods (Lineweaver-Burk, Eadie-Hofstee) for visual inspection but not for primary parameter determination

• Statistical Validation:

  • Calculate confidence intervals for all parameters

  • Perform residual analysis to assess model fit

  • Use Akaike Information Criterion (AIC) or similar metrics for model comparison

• Reproducibility Considerations:

  • Conduct experiments in triplicate at minimum

  • Report both technical and biological replicates

  • Use appropriate controls for enzyme activity and stability

How should researchers interpret thermal stability data for B. multivorans TrpD in the context of infection environments?

When analyzing thermal stability data:

• Methodological Considerations:

  • Employ multiple techniques (differential scanning fluorimetry, circular dichroism, differential scanning calorimetry)

  • Assess stability under various pH conditions relevant to infection sites

  • Evaluate effects of potential stabilizing agents (ligands, metals, osmolytes)

• Contextual Interpretation:

  • Compare stability profiles with growth temperature ranges of B. multivorans

  • Consider temperature fluctuations in cystic fibrosis lung environments

  • Assess implications for enzyme function during infection

• Comparative Analysis:

  • Contrast with stability data from other B. multivorans enzymes

  • Compare with TrpD from other Burkholderia species

  • Consider evolutionary implications of stability profiles

The unusually low thermostability observed in TkTrpD compared to other proteins from T. kodakarensis suggests that thermal stability variations might be functionally significant and should be carefully interpreted in B. multivorans TrpD studies.

What comparative analysis approaches should be used when studying B. multivorans TrpD in relation to TrpD from other bacterial species?

For comprehensive comparative analysis:

• Sequence-Based Comparisons:

  • Multiple sequence alignment of TrpD from diverse species

  • Phylogenetic analysis to establish evolutionary relationships

  • Identification of conserved motifs and variable regions

• Structural Comparisons:

  • Superposition of available crystal structures

  • Root mean square deviation (RMSD) calculation for structural alignments

  • Analysis of active site conservation and variation

• Functional Comparisons:

  • Side-by-side kinetic parameter analysis

  • Metal ion preference comparison

  • Substrate specificity assessment

  • Inhibition profile characterization

• Context-Specific Interpretation:

  • Relate observed differences to ecological niches

  • Consider pathogenicity and virulence implications

  • Assess potential as drug targets based on unique features

This comparative approach can reveal unique features of B. multivorans TrpD that might be exploited for therapeutic intervention, particularly in the context of cystic fibrosis infections.

What evidence supports TrpD as a potential therapeutic target in B. multivorans infections, particularly in cystic fibrosis patients?

Several lines of evidence support TrpD as a potential therapeutic target:

• Essential Metabolic Role:

  • TrpD catalyzes a critical step in tryptophan biosynthesis

  • Disruption of tryptophan biosynthesis could inhibit bacterial growth in tryptophan-limited environments

• Pathogen-Specific Considerations:

  • B. multivorans is a member of the Burkholderia cepacia complex (BCC), notorious for its pathogenicity in persons with cystic fibrosis

  • B. multivorans infections are difficult to treat due to multiple mechanisms of bacterial resistance

• Therapeutic Validation:

  • B. multivorans meningitis has been successfully treated with trimethoprim/sulfamethoxazole

  • Some B. multivorans isolates from cystic fibrosis patients develop resistance to multiple antibiotics, with only trimethoprim/sulfamethoxazole remaining effective

• Structural Targetability:

  • Unique features of TrpD, such as metal-binding sites and substrate binding pockets, could be exploited for selective inhibition

  • The TrpD enzyme structure has been solved for multiple species, providing templates for structure-based drug design

What challenges might researchers face when studying recombinant B. multivorans TrpD, and how can these be addressed methodologically?

Potential challenges and methodological solutions include:

• Protein Expression and Solubility:

  • Challenge: Heterologous expression may result in inclusion bodies

  • Solution: Optimize expression conditions (temperature, induction, host strains), use solubility tags, or explore refolding protocols

• Enzyme Stability:

  • Challenge: TrpD may exhibit low stability, as observed with TkTrpD

  • Solution: Include stabilizing agents in buffers, optimize storage conditions, and consider protein engineering approaches to enhance stability

• Substrate Inhibition:

  • Challenge: Anthranilate concentrations above 4 μM resulted in reduced TkTrpD activity

  • Solution: Carefully design kinetic assays with appropriate substrate concentrations, consider alternative assay methods that can function at lower substrate concentrations

• Metal Ion Requirements:

  • Challenge: Determining optimal metal cofactors for activity

  • Solution: Systematically test various divalent cations (Mg²⁺, Ca²⁺, Zn²⁺, Mn²⁺) at different concentrations and under various pH conditions

• Crystallization Difficulties:

  • Challenge: Obtaining diffraction-quality crystals

  • Solution: Employ high-throughput crystallization screening, explore co-crystallization with substrates or inhibitors, consider surface entropy reduction mutations

How might genomic variation in the trpD gene observed in clinical B. multivorans isolates impact enzyme function and therapeutic targeting?

Genomic analysis of B. multivorans isolates from cystic fibrosis patients has revealed several patterns that may impact TrpD function and therapeutic targeting:

• Mutation Patterns:

  • Clinical isolates exhibit low rates of adaptive evolution within patients but high diversity between patients

  • A set of 30 parallel adaptations was observed across multiple patients, suggesting that specific genomic backgrounds may dictate adaptation routes

  • Similar adaptive patterns might affect the trpD gene, potentially leading to functional variations

• Structural Genomic Variations:

  • Clinical isolates displayed large structural genomic variations, including different plasmid contents and active roles for transposases in gene deactivation

  • Such variations could potentially affect trpD expression or regulation

• Environmental Adaptation:

  • Limited within-patient B. multivorans evolution contrasted with high between-patient strain diversity suggests an environmental microdiverse reservoir for endemic strains

  • This environmental reservoir might harbor TrpD variants with pre-existing differences in function or inhibitor susceptibility

• Therapeutic Implications:

  • Genomic diversity between patients suggests that TrpD inhibitors might need to account for strain-specific variations

  • The identification of parallel adaptations across patients indicates potential hotspots for mutation that might affect drug binding