Recombinant Burkholderia multivorans Phosphoribosyl-AMP cyclohydrolase (hisI)

What is the function of Phosphoribosyl-AMP cyclohydrolase (hisI) in Burkholderia multivorans?

Phosphoribosyl-AMP cyclohydrolase (hisI) is a critical enzyme in the histidine biosynthesis pathway in B. multivorans. The enzyme catalyzes the cyclohydrolysis of N¹-(5-phospho-β-D-ribosyl)-AMP (PRAMP) to N-(5′-phospho-D-ribosylformimino)-5-amino-1-(5′′-phospho-D-ribosyl)-4-imidazolecarboxamide (ProFAR) . As part of a bifunctional enzyme in many bacterial species, hisI functions in the N-terminal domain while the C-terminal domain contains phosphoribosyl-ATP pyrophosphohydrolase (HisE) activity. The complete enzyme (HisIE) catalyzes two sequential steps in histidine biosynthesis, converting phosphoribosyl-ATP (PRATP) to PRAMP, and then PRAMP to ProFAR . This pathway is particularly significant given B. multivorans's emergence as a prominent pathogen in cystic fibrosis patients, where its metabolic capabilities may contribute to virulence and persistence .

What expression systems are most effective for producing recombinant B. multivorans hisI?

E. coli-based expression systems remain the most widely used platform for recombinant B. multivorans hisI production. The enzyme is typically expressed with a polyhistidine tag (usually 6×His) at either the N- or C-terminus to facilitate purification . When designing expression constructs, researchers should consider several factors:

• Codon optimization for E. coli expression, as B. multivorans has a different codon usage pattern

• Inclusion of appropriate promoter systems (T7 or tac promoters are commonly used)

• Induction conditions optimization (IPTG concentration, temperature, and duration)

• Solubility enhancement strategies (fusion partners like MBP or SUMO may improve solubility)

For difficult-to-express variants, alternative expression hosts such as Pseudomonas species may prove advantageous due to their closer phylogenetic relationship to Burkholderia, potentially providing a more suitable cellular environment for proper folding of B. multivorans proteins.

What are the optimal purification strategies for His-tagged B. multivorans hisI?

Purification of His-tagged B. multivorans hisI typically employs immobilized metal affinity chromatography (IMAC) using Ni-NTA resins. Based on the literature, the following methodological approach is recommended:

• Cell lysis in a buffer containing 20-50 mM Tris-HCl (pH 8.0), 300-500 mM NaCl, 10-20 mM imidazole, and protease inhibitors

• Clarification of lysate by centrifugation (15,000-20,000 × g for 30 minutes)

• IMAC purification with step-wise imidazole gradients (20 mM for binding, 50 mM for washing, and 250-300 mM for elution)

• Size-exclusion chromatography as a polishing step

For applications requiring higher purity, additional ion-exchange chromatography may be employed. The purified enzyme should be stored in a buffer containing glycerol (20-25%) and reducing agents like DTT or TCEP to maintain stability . During purification, maintaining the enzyme at 4°C helps preserve activity.

How can I verify the activity of purified recombinant B. multivorans hisI?

Activity verification of recombinant B. multivorans hisI can be accomplished through multiple complementary approaches:

• Spectrophotometric assays: The cyclohydrolase activity can be monitored by following the conversion of PRAMP to ProFAR, which results in a change in absorbance at 290 nm (increase) and 245 nm (decrease) .

• LC-MS analysis: Liquid chromatography-mass spectrometry can be used to detect the conversion of substrate to product, confirming the formation of ProFAR from PRAMP .

• Coupled enzyme assays: The activity can be measured in a coupled reaction with the preceding enzyme (HisE) using PRATP as the starting substrate.

A typical activity assay buffer would contain 50 mM Tris-HCl (pH 7.5-8.0), 100 mM NaCl, and 1-5 mM MgCl₂. The reaction is typically performed at 25-37°C with substrate concentrations in the range of 0.1-1.0 mM.

How does bifunctionality of HisIE affect catalytic efficiency in B. multivorans compared to monofunctional enzymes?

A comparative kinetic analysis between the bifunctional enzyme and separated domains would provide valuable insights into the evolutionary advantages of domain fusion in the histidine biosynthesis pathway.

What approaches can be used to investigate the role of hisI in B. multivorans pathogenicity in cystic fibrosis infections?

B. multivorans has emerged as the most common Burkholderia species isolated from cystic fibrosis patients in several countries, including Canada (44.95% of cases) and the UK . Investigating the role of hisI in pathogenicity requires multifaceted approaches:

• Gene knockout/knockdown studies: CRISPR-Cas9 or homologous recombination-based methods to generate hisI-deficient strains, followed by virulence testing in appropriate models.

• Transcriptomic analysis: RNA-seq comparing gene expression between clinical isolates and environmental strains, focusing on histidine biosynthesis genes including hisI.

• CF infection models: Using cell culture systems (human bronchial epithelial cells) or animal models (mice with CF-like lung conditions) to assess the impact of hisI mutations on bacterial colonization and persistence.

• Patient isolate sequence analysis: Comparison of hisI sequences from globally distributed B. multivorans strains to identify potential adaptive mutations. The identification of 64 distinct sequence types of B. multivorans globally, with 12 being associated with human infection, suggests genetic diversity that may affect enzyme function .

• Metabolomic profiling: Analyzing histidine pathway metabolites in clinical vs. environmental isolates to determine if histidine biosynthesis is upregulated during infection.

This research is particularly significant given that certain B. multivorans strain types appear better adapted to human infection, with overlap between strains recovered from environmental sources and clinical samples .

What methodological challenges exist in studying the kinetics of B. multivorans hisI, and how can they be addressed?

Several methodological challenges complicate kinetic studies of B. multivorans hisI:

• Substrate availability: PRAMP is not commercially available and must be enzymatically synthesized using HisE domain activity or chemical methods. Using a truncated version of HisIE containing only the C-terminal (HisE) domain can facilitate PRAMP synthesis .

• Product detection: ProFAR detection requires specialized analytical techniques. UV-VIS spectroscopy (monitoring absorbance changes at multiple wavelengths) and LC-MS are complementary approaches for accurate product quantification .

• Distinguishing domain activities: In the bifunctional enzyme, separating the kinetic contributions of each domain requires careful experimental design, potentially including domain truncation studies and intermediate isolation.

• Proper controls: Researchers should include controls for non-enzymatic hydrolysis of substrates and account for potential contaminating enzymatic activities from the expression host.

A recommended approach is to employ multiple detection methods in parallel and utilize computational modeling to interpret complex kinetic data, especially when investigating reaction mechanisms and inhibitor interactions.

What are the most effective methods for detecting His-tagged B. multivorans hisI in Western blots?

The detection of His-tagged B. multivorans hisI in Western blots can be accomplished through several methods, each with distinct advantages:

• Nickel-NTA conjugate detection: Direct detection using HisDetector™ Nickel-HRP or Nickel-AP conjugates offers a one-step approach with high specificity. These conjugates can detect His-tagged proteins at nanogram levels with minimal background and shorter processing times (approximately 1-2 hours) compared to antibody-based methods .

• Anti-His antibody detection: Two-step detection using unconjugated anti-His antibodies followed by secondary antibody-enzyme conjugates. While effective, this approach requires 4-5 hours to complete with multiple washing steps and may produce higher background .

• Direct labeled anti-His antibody: One-step detection using HRP-labeled anti-His antibodies (like Penta-His™ HRP) offers intermediate time efficiency but may still have specificity limitations depending on the structural context of the His-tag .

The table below compares these methods based on experimental data:

For most research applications, Nickel-NTA conjugates provide the best balance of sensitivity, specificity, and workflow efficiency for detecting His-tagged B. multivorans hisI .

What assays can be used to measure the cyclohydrolase activity of B. multivorans hisI?

Several complementary assays can be employed to measure the cyclohydrolase activity of B. multivorans hisI:

When designing these assays, researchers should control reaction temperature (typically 25-37°C), pH (optimal range 7.5-8.0), and buffer composition (50 mM Tris-HCl, 100 mM NaCl, 1-5 mM MgCl₂). Additionally, the inclusion of appropriate blanks and controls is essential to account for non-enzymatic substrate degradation and background signal.

How can I optimize expression conditions for soluble recombinant B. multivorans hisI in E. coli?

Optimizing expression conditions for soluble B. multivorans hisI requires systematic evaluation of multiple parameters:

• Expression strain selection: BL21(DE3), Rosetta, or SHuffle strains are recommended depending on the codon usage and disulfide bond formation requirements.

• Temperature optimization: Lower induction temperatures (16-25°C) often increase the proportion of soluble protein compared to standard 37°C induction.

• Induction parameters: Testing various IPTG concentrations (0.1-1.0 mM) and induction durations (4-16 hours) to find optimal conditions.

• Media formulation: Enriched media (TB or 2YT) typically yield higher biomass and protein levels than standard LB medium.

• Co-expression with chaperones: GroEL/GroES, DnaK/DnaJ/GrpE, or trigger factor co-expression can enhance proper folding.

A systematic optimization approach would include:

Additionally, fusion tags such as MBP, SUMO, or Trx may significantly enhance solubility. A His-SUMO-hisI construct, for example, would allow for both enhanced solubility and tag removal using SUMO protease after purification.

How can I determine if mutations in the B. multivorans hisI gene affect enzyme function?

Assessing the functional impact of mutations in the B. multivorans hisI gene requires a comprehensive approach combining computational, biochemical, and structural analyses:

• Sequence analysis: Aligning the mutant sequence with wild-type and orthologous hisI sequences from other organisms to determine conservation of the mutated residue. Highly conserved residues are more likely to be functionally important.

• Structural prediction: Using homology modeling based on available HisI crystal structures to predict how mutations might affect active site geometry, substrate binding, or protein stability.

• Enzyme kinetics comparison: Determining and comparing kinetic parameters (KM, kcat, kcat/KM) of wild-type and mutant enzymes under standardized conditions to quantify functional changes:

  • Decreased kcat suggests impaired catalytic mechanism

  • Increased KM indicates reduced substrate binding affinity

  • Altered pH profile may reflect changes in catalytic residues

• Stability assessment: Comparing thermal stability (Tm) through differential scanning fluorimetry or circular dichroism to determine if mutations affect protein folding or stability.

• Complementation studies: Testing whether the mutant gene can rescue a hisI-deficient bacterial strain's ability to grow in histidine-free medium.

Comparing the pH dependence and solvent isotope effects between wild-type and mutant enzymes can provide particular insight into changes affecting the proton transfer steps known to be important in the reaction mechanism .

What is known about the epidemic potential of B. multivorans strains and how might this relate to metabolic enzymes like hisI?

The epidemiology of B. multivorans infections, particularly in cystic fibrosis patients, presents interesting questions about the relationship between metabolic capabilities and virulence:

B. multivorans has emerged as the most prevalent Burkholderia species in CF infections in several countries, representing 49% of all Burkholderia isolates and 44.95% of individual patient infections in a Canadian study . Multilocus sequence typing (MLST) has identified 64 distinct B. multivorans sequence types globally, with 12 sequence types being globally distributed and associated with human infection .

Several epidemiological patterns suggest potential links to metabolic adaptation:

• Environmental reservoir: The overlap between strains recovered from environmental sources (water, industrial products) and human infections suggests environmental adaptability may contribute to virulence . Metabolic versatility, potentially including robust histidine biosynthesis pathways, may be important for survival in diverse niches.

• Global distribution: Certain sequence types (e.g., ST-16, the French epidemic clone) have demonstrated epidemic potential , suggesting they may possess metabolic or virulence advantages.

• Species shift: The transition from B. cenocepacia to B. multivorans as the dominant Burkholderia species in CF patients in many countries coincides with improved infection control measures, suggesting B. multivorans may have distinct transmission or persistence mechanisms.

Research investigating the relationship between metabolic enzymes like hisI and virulence could focus on comparing enzyme variants across clinical and environmental isolates, particularly examining whether globally distributed strains share common metabolic adaptations that might explain their success in human infection.

What computational approaches can help predict structure-function relationships in B. multivorans hisI?

Computational approaches offer valuable insights into structure-function relationships in B. multivorans hisI without requiring extensive laboratory resources:

• Homology modeling: Using structures of homologous HisI enzymes as templates (particularly from other Gram-negative bacteria) to predict the 3D structure of B. multivorans hisI. The resulting models can identify putative active site residues and substrate binding pockets.

• Molecular dynamics simulations: Simulating the dynamic behavior of the enzyme under various conditions (with/without substrate, different pH environments, presence of potential inhibitors) to understand flexibility, substrate approach pathways, and conformational changes during catalysis.

• Quantum mechanics/molecular mechanics (QM/MM): For detailed reaction mechanism studies, particularly focusing on the proton transfer steps known to be rate-limiting in the cyclohydrolase reaction .

• Virtual screening: Docking potential inhibitors to identify compounds that might selectively target B. multivorans hisI for antibiotic development.

• Sequence-based analysis: Using tools like ConSurf to identify evolutionarily conserved residues likely to be functionally important, or PROVEAN to predict the functional impact of amino acid substitutions.

When applying these approaches, researchers should account for the bifunctional nature of HisIE in B. multivorans, potentially modeling both the individual HisI domain and its context within the full bifunctional enzyme to understand domain interactions and substrate channeling effects.

What are the future research directions for B. multivorans hisI?

The study of B. multivorans hisI presents several promising research avenues:

• Inhibitor development: Given B. multivorans's clinical significance in cystic fibrosis patients , the essential nature of histidine biosynthesis, and the absence of the pathway in humans, hisI represents a potential antibiotic target. Structure-based drug design approaches could yield selective inhibitors.

• Comparative genomics: Analyzing hisI sequence variations across the 64 identified B. multivorans sequence types , particularly focusing on the 12 globally distributed lineages associated with human infection, may reveal selection pressures and adaptations relevant to pathogenicity.

• Metabolic network integration: Understanding how histidine biosynthesis interacts with other metabolic pathways during infection, potentially through systems biology approaches combining transcriptomics, proteomics, and metabolomics.

• Domain communication: Further investigation of the bifunctional HisIE enzyme to understand domain interactions, substrate channeling, and the evolutionary advantages of domain fusion compared to separate enzymes .

• Environmental adaptation: Exploring how histidine biosynthesis capabilities may contribute to B. multivorans's survival in diverse environments, from natural water sources to industrial products to the CF lung .

These research directions not only advance our understanding of a fundamental metabolic pathway but may also contribute to developing new therapeutic strategies against an increasingly prevalent pathogen in vulnerable populations.