Recombinant Pseudomonas aeruginosa N5-carboxyaminoimidazole ribonucleotide synthase (purK)

What is the biochemical function of PurK in Pseudomonas aeruginosa?

PurK (N5-carboxyaminoimidazole ribonucleotide synthase) is a critical enzyme in the prokaryotic purine biosynthetic pathway. This enzyme specifically catalyzes the conversion of 5-aminoimidazole ribonucleotide (AIR) to N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) in a reaction that requires both ATP and bicarbonate (HCO3-). This conversion represents an essential step in de novo purine biosynthesis unique to prokaryotes, making it absent in eukaryotic organisms. The reaction forms a carbamic acid derivative that undergoes subsequent rearrangement catalyzed by another enzyme, PurE, which converts N5-CAIR to 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). The entire process is crucial for the synthesis of nucleotides required for DNA and RNA production in Pseudomonas aeruginosa and other prokaryotes .

How does the structure of Pseudomonas aeruginosa PurK compare to PurK from other prokaryotes?

While specific structural data for P. aeruginosa PurK is limited in the provided search results, comparative analysis can be drawn from the high-resolution (1.96 Å) structure of Bacillus anthracis PurK (baPurK). The baPurK structure reveals several key features likely conserved across bacterial species, including P. aeruginosa:

• A flexible B-loop (residues 149/150-157) positioned in close proximity to the active site

• An active site capable of binding Mg²⁺ without requiring additional ligands

• Probable ATP-binding pocket consistent with its ATP-dependent enzymatic activity

These structural characteristics are fundamental to understanding PurK function across prokaryotes. Researchers investigating P. aeruginosa PurK should consider these features while recognizing potential species-specific variations in loop regions and substrate-binding domains. Detailed structural comparison requires crystallization of P. aeruginosa PurK and subsequent analysis via X-ray crystallography or cryo-electron microscopy techniques to identify unique structural elements that might influence its catalytic activity or inhibitor binding properties .

What expression systems are most effective for producing recombinant P. aeruginosa PurK?

Based on successful recombinant protein production methodologies for other P. aeruginosa proteins, Escherichia coli represents the most established and efficient heterologous expression system for P. aeruginosa PurK. When designing an expression strategy, researchers should consider:

Expression Vector Selection:

• pET series vectors containing T7 promoters provide high-level expression

• Addition of polyhistidine (His₆) tags facilitates subsequent purification

• Inclusion of solubility-enhancing fusion partners (e.g., MBP, SUMO) may improve folding

Host Strain Optimization:

• BL21(DE3) and derivatives offer reduced protease activity

• Rosetta or CodonPlus strains address potential codon bias issues

• Arctic Express strains with cold-adapted chaperones can assist proper folding

Expression Conditions:

• Induction at lower temperatures (16-22°C) often improves solubility

• Reduced IPTG concentrations (0.1-0.5 mM) may prevent inclusion body formation

• Extended expression periods (overnight) at lower temperatures maximize yield

This approach mirrors successful methodologies employed for other recombinant P. aeruginosa proteins, where E. coli expression systems followed by two-step purification processes have yielded functional proteins suitable for subsequent research applications .

What purification strategy yields the highest purity and activity for recombinant P. aeruginosa PurK?

A multi-stage purification approach is recommended to obtain high-purity, functionally active recombinant P. aeruginosa PurK. Based on established purification protocols for similar recombinant proteins from P. aeruginosa, the following methodology is recommended:

Stage 1: Initial Capture

• For soluble expression: Clarified cell lysate preparation through sonication or high-pressure homogenization in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, and protease inhibitors

• For inclusion bodies: Isolation through sequential washing steps with Triton X-100-containing buffers followed by solubilization in 8M urea or 6M guanidine hydrochloride

Stage 2: Affinity Chromatography

• Ni-Sepharose affinity chromatography utilizing the His-tag

• Stepwise imidazole gradient (50 mM, 100 mM, 250 mM) for washing and elution

• Collection of elution fractions with UV absorbance monitoring

Stage 3: Secondary Purification

• Size exclusion chromatography (Superdex 75/200) to remove aggregates and improve homogeneity

• Ion exchange chromatography (if required) to remove remaining contaminants

Stage 4: Final Preparation

• Dialysis against storage buffer (50 mM Tris-HCl pH 9.0, 0.01% Tween 20)

• Filter sterilization through 0.22 μm membrane

• Flash freezing in liquid nitrogen and storage at -80°C in small aliquots

This purification strategy typically yields protein with >95% purity as assessed by SDS-PAGE, with recovered yields of 10-15 mg of purified protein per liter of bacterial culture. Enzyme activity should be confirmed through specific activity assays monitoring ATP hydrolysis and/or N5-CAIR formation .

How can researchers accurately determine the kinetic parameters of recombinant P. aeruginosa PurK?

Accurate determination of P. aeruginosa PurK kinetic parameters requires carefully designed assay systems that monitor either substrate consumption or product formation. A comprehensive kinetic characterization should include:

Primary Assay Methods:

• ATP Consumption Assay

  • Coupling ATP hydrolysis to NADH oxidation via pyruvate kinase and lactate dehydrogenase

  • Monitoring absorbance decrease at 340 nm

  • Reaction conditions: 50 mM HEPES (pH 7.5), 20 mM KCl, 6 mM MgCl₂, 1 mM phosphoenolpyruvate, 0.2 mM NADH, 2 units pyruvate kinase, 2 units lactate dehydrogenase

• Direct N5-CAIR Formation Assay

  • HPLC separation and quantification of reaction products

  • UV detection at 260 nm

  • Comparison against synthesized N5-CAIR standards

Experimental Design for Kinetic Parameters:

Addressing Common Methodological Challenges:

• Maintain anaerobic conditions to prevent spontaneous AIR carboxylation

• Include control reactions without enzyme to account for background rates

• Use freshly prepared substrates to prevent degradation

• Perform reactions at physiologically relevant temperature (37°C)

By systematically varying each substrate concentration while maintaining others at saturating levels, researchers can generate accurate Michaelis-Menten plots for determination of K_m, V_max, and k_cat values, providing crucial insights into the catalytic efficiency of recombinant P. aeruginosa PurK.

What structural features of P. aeruginosa PurK contribute to its catalytic mechanism?

Understanding the catalytic mechanism of P. aeruginosa PurK requires detailed examination of structural elements that facilitate substrate binding and chemical transformation. Based on structural insights from related PurK enzymes like the B. anthracis homolog, several key features likely contribute to catalysis:

Active Site Architecture:
The active site contains essential residues arranged to coordinate Mg²⁺, which plays a critical role in positioning ATP for phosphoryl transfer. The magnesium ion binds without requiring additional ligands, suggesting a unique coordination environment that may influence catalytic efficiency .

Flexible B-loop Region (residues ~149-157):
This mobile loop likely undergoes conformational changes during catalysis, potentially:

• Shielding the active site from solvent during reaction

• Positioning substrate and catalytic residues optimally

• Facilitating product release through dynamic movements

This B-loop flexibility represents a critical structural element for enzyme function, as observed in the B. anthracis PurK structure at 1.96 Å resolution .

Substrate Binding Pockets:
Three distinct binding regions accommodate:

• AIR binding pocket (likely composed of basic and hydrophilic residues)

• ATP binding region (containing conserved P-loop motif)

• Bicarbonate binding site (possibly involving positively charged residues)

Proposed Catalytic Mechanism:

• ATP and Mg²⁺ bind, followed by bicarbonate and AIR

• ATP phosphorylates bicarbonate to form a carboxyphosphate intermediate

• Nucleophilic attack by AIR amine group on the carboxyphosphate

• Formation of carbamic acid derivative (N5-CAIR)

• Release of inorganic phosphate and ADP

Mutagenesis studies targeting conserved residues in these regions would provide experimental validation of their roles in the catalytic mechanism, offering insights into potential inhibition strategies for antimicrobial development.

How can recombinant P. aeruginosa PurK be utilized as a target for antimicrobial development?

PurK represents a promising antimicrobial target due to several advantageous characteristics that make it suitable for drug development efforts:

Target Validation Rationale:

• PurK is essential for de novo purine biosynthesis in prokaryotes

• The enzyme is absent in humans and other eukaryotes, offering selectivity

• Inhibition would disrupt nucleotide synthesis, affecting bacterial growth and virulence

Methodological Approaches for Inhibitor Discovery:

Validation and Optimization Workflow:

• Primary screening against recombinant P. aeruginosa PurK using ATP consumption assays

• Counter-screening against human enzymes to confirm selectivity

• Measurement of binding affinity via isothermal titration calorimetry or surface plasmon resonance

• Co-crystallization or soaking experiments to determine binding mode

• Structure-activity relationship studies for potency optimization

• Assessment of inhibitors in bacterial growth assays and animal infection models

Successful inhibitor development would potentially yield new antimicrobial agents against P. aeruginosa, which is particularly valuable given this pathogen's increasing resistance to conventional antibiotics and its classification as a critical priority pathogen by the World Health Organization .

What role can recombinant P. aeruginosa proteins play in vaccine development?

Recombinant P. aeruginosa proteins, including potentially PurK, can serve as critical components in next-generation vaccine development strategies. These approaches build upon demonstrated successes with other recombinant P. aeruginosa antigens:

Immunization Strategies Using Recombinant Proteins:

Recombinant P. aeruginosa proteins can be utilized in multiple vaccine formats:

• Subunit Vaccines: Purified recombinant proteins formulated with adjuvants have shown promising results in experimental models. For example, a recombinant vaccine using two immunogenic P. aeruginosa proteins (OprF and detoxified exotoxin A) demonstrated significant immunogenicity when absorbed with aluminum hydroxide .

• Outer Membrane Vesicle (OMV) Vaccines: Engineered P. aeruginosa strains can produce recombinant OMVs containing targeted antigens. An OMV-based approach using recombinant P. aeruginosa delivering a bivalent antigen (PcrV-HitA fusion) demonstrated 70% protection against intranasal challenge in an animal model .

Optimization Parameters for Recombinant Vaccine Production:

Preclinical Evaluation Requirements:

• Assessment of specific antibody production (IgG, IgA) via ELISA

• Functional antibody testing through opsonophagocytic or neutralization assays

• Challenge studies in appropriate animal models

• Safety evaluation including local and systemic reactogenicity

These approaches reflect successful methodologies with recombinant P. aeruginosa antigens, where targeted immunization strategies have demonstrated significant protection in experimental models. While PurK specifically has not been reported as a vaccine antigen in the provided search results, the methodologies established with other recombinant P. aeruginosa proteins provide a framework for evaluating its potential in this capacity .

What crystallization methods are most effective for structural determination of P. aeruginosa PurK?

Successful crystallization of recombinant P. aeruginosa PurK requires systematic screening and optimization of conditions. Based on the crystallization approaches used for related PurK enzymes, the following methodology is recommended:

Initial Crystallization Screening:

• Sample Preparation

  • Highly purified protein (>98% purity by SDS-PAGE)

  • Concentration range: 8-15 mg/ml in crystallization buffer

  • Buffer composition: 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM DTT

  • Optional addition of 2-5 mM MgCl₂ to stabilize structure

• Primary Screening

  • Commercial sparse matrix screens (Hampton Research, Molecular Dimensions)

  • Sitting drop vapor diffusion method (typical drop: 1 μl protein + 1 μl reservoir)

  • Incubation at constant temperature (18-20°C)

  • Monitoring crystal formation for 2-4 weeks

Optimization Strategies for Diffraction-Quality Crystals:

Co-crystallization with Ligands:
For mechanistic studies, co-crystallization with substrates, products, or substrate analogs is recommended:

• ATP/ADP at 2-5 mM concentration with equimolar MgCl₂

• AIR analogs or stable N5-CAIR mimics

• Potential inhibitors for structure-based drug design

Data Collection and Structure Determination:

• Crystals should be cryoprotected (typically 20-25% glycerol or ethylene glycol)

• X-ray diffraction data collection at synchrotron radiation facilities

• Molecular replacement using B. anthracis PurK structure (PDB: 3Q2O) as search model

• Refinement and validation using standard crystallographic software

This systematic approach, which mirrors the methodology used to determine the B. anthracis PurK structure at 1.96 Å resolution, provides the best chance of obtaining high-quality structural data for P. aeruginosa PurK .

How can researchers effectively compare purine biosynthesis pathways between P. aeruginosa and other pathogens?

Comprehensive comparison of purine biosynthesis pathways across pathogenic species requires integrative approaches combining genomic, biochemical, and structural analyses. This multifaceted methodology enables identification of conserved features and species-specific adaptations:

Genomic and Bioinformatic Approach:

• Comparative Genomic Analysis

  • Identification of all purine biosynthesis genes through BLAST searches against reference genomes

  • Synteny analysis to determine gene cluster organization

  • Phylogenetic analysis of PurK sequences from diverse pathogens to establish evolutionary relationships

• Structural Bioinformatics

  • Homology modeling of P. aeruginosa PurK based on available crystal structures (like B. anthracis PurK)

  • Identification of conserved catalytic residues and variable regions

  • Analysis of sequence conservation in substrate binding regions

Biochemical Characterization:

Metabolic Flux Analysis:

• Isotope labeling studies using ¹³C-labeled precursors

• Quantification of metabolic intermediates by LC-MS/MS

• Pathway modeling to predict effects of enzyme inhibition

Comparative Pathway Analysis Dashboard:

For P. aeruginosa, special attention should be given to the AIR to CAIR conversion, which involves the sequential action of PurK and PurE. This two-step process converts AIR to N5-CAIR (catalyzed by PurK) and then N5-CAIR to CAIR (catalyzed by PurE). The pathway represents a unique prokaryotic feature, as eukaryotes perform this conversion in a single step catalyzed by a different enzyme .

Understanding these pathway differences is crucial for:

• Identifying selective targets for antimicrobial development

• Predicting potential metabolic adaptations under treatment pressure

• Designing multi-target inhibition strategies for enhanced efficacy

By systematically comparing these pathways across pathogens, researchers can identify the most promising conserved targets with minimal potential for resistance development through alternative pathway utilization .

How can structural data from P. aeruginosa PurK inform rational drug design approaches?

Structural information about P. aeruginosa PurK provides crucial insights that can significantly accelerate rational drug design efforts. A comprehensive structure-based approach should include:

Analysis of Key Binding Sites:

• ATP Binding Pocket

  • Typically contains conserved P-loop motif

  • Offers opportunities for competitive inhibitors

  • Analysis of residue conservation compared to human ATP-utilizing enzymes to ensure selectivity

• Substrate (AIR) Binding Site

  • Species-specific variations may exist in pocket architecture

  • Target for substrate-competitive inhibitors

  • Potential for allosteric modulation

• Bicarbonate Binding Region

  • Often less conserved than ATP site

  • Potential for selective inhibition

• Flexible B-loop Region (residues ~149-157)

  • Dynamic element critical for catalysis

  • Target for inhibitors that disrupt conformational changes

  • Analysis of B. anthracis PurK B-loop provides valuable comparative data

Computer-Aided Drug Design Workflow:

Integration with Experimental Validation:

• Surface plasmon resonance or isothermal titration calorimetry to confirm binding

• X-ray crystallography of enzyme-inhibitor complexes

• Enzymatic inhibition assays to determine mechanism and potency

• Antimicrobial susceptibility testing against P. aeruginosa

The unique structural features observed in PurK, such as the magnesium binding without additional ligands and the flexible B-loop region identified in the B. anthracis enzyme, provide specific targets for inhibitor design. By exploiting these structural elements, researchers can develop compounds that selectively inhibit bacterial purine biosynthesis while avoiding interaction with human enzymes .

What novel experimental approaches are emerging for studying recombinant P. aeruginosa proteins?

Cutting-edge experimental approaches are transforming research on recombinant P. aeruginosa proteins, including PurK. These innovative methodologies enhance our understanding of enzyme function and accelerate therapeutic applications:

Advanced Structural Biology Techniques:

• Cryo-Electron Microscopy (Cryo-EM)

  • Enables visualization of proteins without crystallization

  • Particularly valuable for conformational heterogeneity analysis

  • Can capture different catalytic states of PurK

• Time-Resolved Crystallography

  • Captures structural changes during catalysis

  • Reveals transient intermediate states

  • Provides dynamic view of enzyme mechanism

• Neutron Diffraction

  • Locates hydrogen atoms in active site

  • Distinguishes protonation states of catalytic residues

  • Complements X-ray crystallography data

Protein Engineering and Functional Analysis:

Systems Biology Approaches:

• CRISPR interference for conditional knockdown in P. aeruginosa

• Metabolomics analysis of purine pathway perturbations

• Proteomics to identify interaction partners

• Transcriptomics to determine regulatory networks

Vaccine and Therapeutic Development:
Novel approaches for utilizing recombinant P. aeruginosa proteins in vaccinology include:

• mRNA vaccine technology encoding antigenic proteins

• Self-assembling nanoparticles displaying multiple epitopes

• Outer membrane vesicle (OMV) engineering for antigen delivery

The OMV approach has shown particular promise, with recombinant P. aeruginosa OMVs delivering bivalent antigens demonstrating 70% protection in challenge models. This represents a significant advancement over traditional subunit vaccines and illustrates the potential of engineered delivery systems for recombinant P. aeruginosa proteins .

These emerging techniques collectively provide researchers with unprecedented capabilities to understand the structural, functional, and immunological properties of recombinant P. aeruginosa proteins, accelerating both fundamental research and therapeutic applications.

What are the critical knowledge gaps in our understanding of P. aeruginosa PurK?

Despite significant advances in understanding purine biosynthesis in prokaryotes, several critical knowledge gaps remain regarding P. aeruginosa PurK that warrant focused research attention:

Structural Determinants of Function:

• High-resolution structure of P. aeruginosa PurK remains unavailable

• Molecular basis for substrate specificity poorly understood

• Conformational dynamics during catalysis largely unexplored

• Species-specific structural features that may influence inhibitor binding undefined

Regulatory Mechanisms:

• Transcriptional and post-translational regulation of PurK expression under different growth conditions

• Impact of environmental factors (pH, oxygen tension, nutrient availability) on enzyme activity

• Potential moonlighting functions beyond purine biosynthesis

• Integration with broader metabolic networks in P. aeruginosa

Therapeutic Development Challenges:

• Identification of selective inhibitors that distinguish bacterial from human enzymes

• Understanding of potential resistance mechanisms

• Pharmacokinetic properties required for effective targeting in infection models

• Synergistic potential with existing antibiotic classes

Methodological Limitations:

• Difficulties in obtaining stable, active enzyme preparations

• Challenges in developing high-throughput screening assays

• Limited in vivo validation models for target engagement

Addressing these knowledge gaps will require interdisciplinary approaches combining structural biology, enzymology, genetics, and medicinal chemistry to fully elucidate the role and therapeutic potential of P. aeruginosa PurK.

What are the recommended research priorities for advancing P. aeruginosa PurK as an antimicrobial target?

Based on current knowledge and critical gaps, the following research priorities are recommended to advance P. aeruginosa PurK as an antimicrobial target:

Short-term Research Priorities (1-2 years):

• Structural Characterization

  • Determination of high-resolution crystal structure of P. aeruginosa PurK

  • Comparative analysis with PurK structures from other pathogens

  • Identification of unique structural features for selective targeting

• Assay Development and Validation

  • Establishment of robust, high-throughput screening assays

  • Development of cellular assays to confirm target engagement

  • Creation of reporter strains for pathway inhibition monitoring

Medium-term Research Priorities (2-5 years):

Long-term Research Priorities (5+ years):

• Clinical candidate selection and development

• Investigation of broader applications against other pathogens

• Development of multi-target inhibition strategies

• Exploration of alternative delivery mechanisms

Interdisciplinary Integration:
Successful advancement requires integration of multiple research disciplines:

• Structural biology for target characterization

• Medicinal chemistry for inhibitor design

• Microbiology for resistance assessment

• Pharmacology for drug delivery strategies

• Immunology for host response studies