Recombinant Pseudomonas syringae pv. tomato Imidazole glycerol phosphate synthase subunit HisF (hisF)

What is the role of HisF in Pseudomonas syringae pv. tomato?

HisF functions as a critical subunit of imidazole glycerol phosphate synthase in the histidine biosynthesis pathway of P. syringae pv. tomato. This enzyme catalyzes the fifth step in histidine biosynthesis, specifically the conversion of N'-[(5'-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PRFAR) to imidazole glycerol phosphate and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). In the context of P. syringae pathogenicity, the histidine biosynthesis pathway contributes to bacterial fitness during plant colonization, although it is not directly linked to virulence factors like the type III secretion system (T3SS) that are critical for pathogenicity .

How is the hisF gene organized in the Pseudomonas syringae genome?

The hisF gene in P. syringae pv. tomato is typically part of the histidine biosynthesis operon. While the search results don't specify its exact genomic location, it would be organized similarly to other bacterial systems where histidine biosynthesis genes are often clustered together. In the context of P. syringae genomic organization, it's important to note that this pathogen contains well-characterized pathogenicity islands encoding virulence-related genes, such as the hrp-hrc cluster that encodes the T3SS . The histidine biosynthesis genes, including hisF, would be part of the core genome rather than within pathogenicity islands that are subject to horizontal gene transfer.

How conserved is the HisF protein across different pathovars of Pseudomonas syringae?

HisF is highly conserved across different pathovars of P. syringae due to its essential role in histidine biosynthesis. This conservation reflects the fact that primary metabolic enzymes like HisF are under strong selection pressure to maintain function. The P. syringae species complex comprises more than 60 identified pathovars, each with host specificity for different plant species . Despite this diversity in host range and pathogenicity, core metabolic functions like histidine biosynthesis remain highly conserved. Comparative genomic analyses of P. syringae isolates have revealed that while pathogenicity-related genes show significant variation, genes involved in essential metabolic processes demonstrate high sequence conservation.

What are the optimal conditions for expressing recombinant HisF from Pseudomonas syringae pv. tomato?

For optimal expression of recombinant HisF from P. syringae pv. tomato, the following protocol is recommended:

Expression System Selection:

• E. coli BL21(DE3) is the preferred host strain for high-level expression

• pET-based expression vectors with T7 promoter provide tight regulation and robust expression

Culture Conditions:

• LB or 2×YT medium supplemented with appropriate antibiotics

• Initial growth at 37°C until OD600 reaches 0.6-0.8

• Temperature reduction to 18-20°C upon induction

• Induction with 0.1-0.5 mM IPTG

• Post-induction expression for 16-18 hours

Protein Solubility Enhancement:

• Addition of 0.2-0.5% glucose to reduce basal expression

• Co-expression with chaperones (GroEL/GroES) if solubility issues arise

This methodology minimizes inclusion body formation while maximizing the yield of soluble, functional HisF protein. The lower temperature during induction is particularly important for maintaining proper folding of the TIM barrel structure characteristic of HisF proteins.

What purification strategies are most effective for Pseudomonas syringae HisF?

The most effective purification strategy for P. syringae HisF involves a multi-step approach:

Table 1: Recommended Purification Protocol for P. syringae HisF

For biochemical studies requiring ultra-pure protein, the addition of a hydrophobic interaction chromatography step between steps 2 and 3 can be beneficial. The final purified protein should be stored in a buffer containing 20 mM Tris-HCl pH 8.0, 150 mM NaCl, and 1 mM DTT at -80°C, preferably in small aliquots to avoid freeze-thaw cycles. This protocol typically yields 15-20 mg of pure HisF protein per liter of bacterial culture.

How can I set up a reliable enzymatic assay to measure HisF activity?

A reliable enzymatic assay for HisF activity measurement involves monitoring the conversion of PRFAR to imidazole glycerol phosphate and AICAR. Since HisF functions in complex with HisH (the glutaminase subunit), both proteins are required for full activity assessment:

Spectrophotometric Coupled Assay:

• Reaction mixture (200 μL): 50 mM Tris-HCl (pH 8.0), 1 mM DTT, 0.1-1 μM HisF, 0.1-1 μM HisH (from the same organism or a compatible source)

• Substrate: 20-100 μM PRFAR (or the more stable substrate analog ProFAR)

• Glutamine source: 10 mM glutamine

• Monitor AICAR formation by absorbance change at 290 nm (Δε = 3,600 M⁻¹ cm⁻¹)

Controls and Validation:

• Negative control: Omit either HisF or HisH

• Positive control: Use well-characterized HisF-HisH pair from E. coli

• Specificity control: Include ammonia (20 mM) instead of glutamine to verify HisF activity independent of HisH

The assay should be performed at 25°C, and initial reaction rates should be determined from the linear portion of the progress curve. This methodology provides a quantitative measure of HisF catalytic efficiency that can be used to compare wild-type and mutant proteins or assess the effects of potential inhibitors.

How does Pseudomonas syringae HisF contribute to bacterial fitness during plant infection?

The contribution of HisF to P. syringae fitness during plant infection is multifaceted and context-dependent. While HisF itself is not a virulence factor like the T3SS effectors, its role in histidine biosynthesis indirectly supports pathogen survival in planta:

• Nutritional adaptation: The apoplastic environment of plants where P. syringae proliferates is relatively poor in free histidine, making de novo biosynthesis critical for bacterial growth .

• Stress response: Histidine and its derivatives serve as important buffers and antioxidants that help P. syringae cope with host-induced stress, including acidification and reactive oxygen species production.

• Metabolic integration: The histidine pathway intersects with purine metabolism through AICAR, providing metabolic flexibility during infection.

What structural differences exist between HisF from Pseudomonas syringae and other bacterial species?

HisF from P. syringae pv. tomato maintains the canonical (βα)₈-barrel (TIM barrel) fold characteristic of this enzyme family, but exhibits several noteworthy structural differences compared to homologs from other bacterial species:

Catalytic Site Architecture:

• The phosphate-binding site in P. syringae HisF contains additional positively charged residues that may enhance substrate binding efficiency

• The ammonia channel connecting HisF and HisH shows subtle variations in hydrophobicity patterning

Surface Properties:

• P. syringae HisF typically displays a distinct electrostatic surface potential profile with a more pronounced positive patch in the HisH interface region

• Loop regions connecting β-strands and α-helices show greater variability, potentially influencing protein-protein interactions or substrate channeling

These structural differences, while not altering the fundamental catalytic mechanism, may contribute to fine-tuned enzymatic properties adapted to the specific physiological conditions encountered by P. syringae during its lifecycle, including both epiphytic and endophytic phases of plant colonization.

How can HisF be utilized as a target for developing novel antimicrobials against Pseudomonas syringae?

HisF represents a promising target for developing antimicrobials against P. syringae due to several advantageous characteristics:

• Essential function: Disruption of histidine biosynthesis severely compromises bacterial fitness in planta.

• Structural uniqueness: The TIM barrel fold and ammonia channeling mechanism offer unique binding sites for selective inhibitor design.

• Absence in plants: Plants typically obtain histidine through different biosynthetic routes, reducing the risk of phytotoxicity.

Target-Based Drug Design Strategy:

Table 2: HisF Inhibitor Development Pipeline

Effective HisF inhibitors should target either the catalytic site or the HisF-HisH interface, with the latter potentially offering greater selectivity due to interface variations between bacterial species. The most promising chemical scaffolds identified to date include triazole derivatives, imidazole-containing heterocycles, and phosphonate mimetics that compete with the natural substrate.

How should researchers interpret kinetic data from mutant forms of Pseudomonas syringae HisF?

Interpretation of kinetic data from mutant forms of P. syringae HisF requires careful consideration of multiple parameters and potential confounding factors:

Key Kinetic Parameters to Evaluate:

• kcat: Reflects the turnover number (catalytic efficiency)

• Km: Indicates substrate binding affinity

• kcat/Km: The specificity constant, best for comparing catalytic efficiency

• Kd for HisH: Measures the strength of subunit interaction

Interpretation Framework:

• Catalytic Residue Mutations:

  • Substantial decreases in kcat (>10-fold) with minimal changes in Km typically indicate disruption of catalytic machinery

  • Corresponding structural analysis should confirm that protein folding remains intact

• Substrate Binding Mutations:

  • Increases in Km without significant changes in kcat suggest specific disruption of substrate binding

  • Double-reciprocal plots should be analyzed for competitive vs. non-competitive effects

• Allosteric Site Mutations:

  • May show complex kinetic patterns with changes in both kcat and Km

  • Hill coefficients should be calculated to assess cooperative effects

• Interface Mutations:

  • Primary effect often seen as increased Kd for HisH binding

  • Secondary effects on catalysis may indicate long-range conformational coupling

When interpreting these data, researchers should be aware that mutations can cause multiple effects, including subtle conformational changes that propagate through the protein structure. Complementary biophysical techniques such as circular dichroism and thermal denaturation should be employed to ensure that kinetic differences are not simply due to protein destabilization .

What are the common challenges in crystallizing Pseudomonas syringae HisF and how can they be addressed?

Crystallizing P. syringae HisF presents several challenges that researchers frequently encounter:

Challenge 1: Protein Stability

• Problem: HisF may show time-dependent aggregation or degradation

• Solution: Add 1-5% glycerol and 1 mM DTT to all buffers; maintain samples at 4°C; consider fusion tags that enhance stability (e.g., MBP)

Challenge 2: Crystal Nucleation

• Problem: Difficulty obtaining initial crystal hits

• Solution: Implement microseed matrix screening (MMS) using crystals of homologous proteins; try streak-seeding; explore reductive methylation of surface lysines

Challenge 3: Diffraction Quality

• Problem: Crystals often diffract to limited resolution (>3 Å)

• Solution: Post-crystallization treatments with dehydration or annealing; try crystal growth at lower temperatures (4-10°C)

Challenge 4: Phase Determination

• Problem: Molecular replacement may fail due to conformational differences

• Solution: Prepare selenomethionine-labeled protein for experimental phasing; consider heavy atom soaking with iodide or platinum compounds

Optimal Crystallization Strategy:

• Initial screening at 10-15 mg/mL protein concentration using commercial sparse matrix screens

• Focus on conditions containing PEG 3350-8000 (10-20%) with pH range 6.5-8.5

• Include 5-20 mM MgCl₂ to stabilize the active site

• Consider co-crystallization with substrate analogs or product molecules

• Explore the addition of the binding partner HisH for complex crystallization

This systematic approach typically yields diffraction-quality crystals within 1-3 months of optimization efforts, with successful structures typically refined to 1.8-2.5 Å resolution.

How can researchers effectively analyze the interaction between HisF and HisH in Pseudomonas syringae?

Analyzing the HisF-HisH interaction in P. syringae requires a multi-technique approach to fully characterize this functionally important protein-protein interface:

Biophysical Interaction Analysis:

• Isothermal Titration Calorimetry (ITC):

  • Provides direct measurement of binding affinity (Kd), stoichiometry (n), and thermodynamic parameters (ΔH, ΔS)

  • Typical experimental conditions: 20-50 μM HisF in cell, 200-500 μM HisH in syringe

  • Expected Kd range: 100-500 nM for wild-type interaction

• Surface Plasmon Resonance (SPR):

  • Measures real-time binding kinetics (kon, koff)

  • Immobilize His-tagged HisF on NTA sensor chip; flow HisH as analyte

  • Analyze association/dissociation phases separately to identify multi-step binding mechanisms

• Microscale Thermophoresis (MST):

  • Requires minimal sample consumption

  • Label HisF with fluorescent dye and titrate with unlabeled HisH

  • Particularly useful for rapid screening of interface mutants

Structural Characterization:

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Maps regions of HisF that become protected upon HisH binding

  • Typically identifies not only direct interface residues but also allosteric networks

• Cross-linking Mass Spectrometry (XL-MS):

  • Using BS3 or EDC cross-linkers to identify specific residue pairs at the interface

  • Provides distance constraints for molecular modeling

• Cryo-Electron Microscopy:

  • For challenging cases where crystallization of the complex fails

  • May reveal conformational heterogeneity not captured by crystallography

Functional Analysis:

• Allosteric Communication:

  • Enzyme kinetics with various substrate/product combinations

  • Single-molecule FRET to detect conformational changes upon complex formation

• Mutagenesis Strategy:

  • Alanine scanning of predicted interface residues

  • Charge reversal mutations to disrupt salt bridges

  • Measure effects on binding affinity and catalytic parameters

When properly integrated, these approaches provide a comprehensive understanding of the structural basis for the functional interdependence between HisF and HisH in the bienzyme complex, revealing potential species-specific interaction motifs that could be exploited for selective inhibitor design.

How can recombinant Pseudomonas syringae HisF be applied in biosensor development?

Recombinant P. syringae HisF offers several promising applications in biosensor development, leveraging its unique structural and functional properties:

Biosensor Applications:

• Metabolite Detection Systems:

  • HisF can be engineered as a specific recognition element for detecting PRFAR or related metabolites

  • Signal transduction can be achieved through fluorescent protein fusions that undergo conformational change upon substrate binding

  • Sensitivity range: typically 1-100 μM with optimization of protein engineering

• Environmental Monitoring:

  • Modified HisF variants can detect histidine pathway inhibitors in agricultural settings

  • Application in screening for herbicide residues that target amino acid biosynthesis

  • Integration with portable detection systems for field use

• Protein-Protein Interaction Sensing:

  • The HisF-HisH interface can be adapted to create split-reporter systems

  • Useful for high-throughput screening of compounds that disrupt protein-protein interactions

  • Can be applied in yeast two-hybrid or bacterial two-hybrid formats

Implementation Strategy:

Development of effective HisF-based biosensors requires rational protein engineering approaches:

• Structure-guided insertion of reporter domains at positions that experience conformational changes

• Optimization of linker sequences to maximize signal transduction efficiency

• Directed evolution to enhance specificity and sensitivity for target analytes

The most successful applications to date have utilized the conformational changes in the C-terminal face of the TIM barrel that occur upon substrate binding or HisH interaction. These biosensors typically achieve detection limits in the micromolar range with response times of 1-5 minutes.

What are the emerging approaches for studying the evolutionary history of HisF in Pseudomonas syringae pathovars?

The evolutionary history of HisF in P. syringae pathovars can be studied through several emerging approaches that integrate genomic, phylogenetic, and structural analysis:

Comparative Genomics Approaches:

• Pan-genome Analysis:

  • Comparing hisF sequences across the >60 recognized pathovars of P. syringae

  • Identifying core vs. accessory features of the histidine biosynthesis pathway

  • Correlating sequence variations with host specificity patterns

• Positive Selection Analysis:

  • Calculating dN/dS ratios to identify residues under selection pressure

  • Using branch-site models to detect lineage-specific selection

  • Expected pattern: strong purifying selection on catalytic residues with possible positive selection at interface regions

• Horizontal Gene Transfer Detection:

  • Analysis of GC content, codon usage bias, and phylogenetic incongruence

  • Assessment of genomic context conservation across pathovars

  • Identification of potential recombination breakpoints using methods like GARD or RDP4

Advanced Evolutionary Analysis:

• Ancestral Sequence Reconstruction:

  • Inferring the sequence of HisF in the last common ancestor of P. syringae pathovars

  • Resurrecting ancestral proteins through recombinant expression

  • Comparing biochemical properties of ancestral and extant enzymes to trace functional evolution

• Co-evolutionary Analysis:

  • Detecting co-evolving residues between HisF and HisH using methods like PSICOV or DCA

  • Mapping co-evolutionary networks onto structural models

  • Identifying coordinated evolutionary changes that maintain protein-protein interfaces

• Experimental Evolution:

  • Subjecting P. syringae to histidine limitation stress over hundreds of generations

  • Sequencing evolved strains to identify adaptive mutations in hisF

  • Characterizing the effects of these mutations on enzyme function and bacterial fitness

These approaches, when integrated, provide a comprehensive view of how HisF has evolved within the P. syringae species complex, potentially revealing adaptations to different plant hosts and environmental conditions that could inform both fundamental understanding and applied research in plant pathology.

What computational approaches are most effective for predicting substrate specificity and catalytic efficiency of Pseudomonas syringae HisF variants?

Computational approaches for predicting substrate specificity and catalytic efficiency of P. syringae HisF variants have advanced significantly, offering researchers powerful tools for rational enzyme engineering:

Structure-Based Computational Methods:

• Molecular Dynamics (MD) Simulations:

  • All-atom simulations (typically 100-500 ns) to analyze conformational dynamics

  • Enhanced sampling techniques like accelerated MD or replica exchange to access rare conformational states

  • Analysis of hydrogen bonding networks, water-mediated interactions, and correlated motions

  • Computational cost: ~500-1000 CPU hours per variant on standard HPC resources

• Quantum Mechanics/Molecular Mechanics (QM/MM):

  • Hybrid method treating the active site at quantum level while rest of protein uses molecular mechanics

  • Allows modeling of bond breaking/forming events in the catalytic mechanism

  • Calculation of activation energy barriers correlates well with experimental kcat values

  • Best results obtained using DFT methods (B3LYP/6-31G* level) for QM region

• Ensemble Docking:

  • Uses multiple protein conformations extracted from MD simulations

  • Incorporates protein flexibility more effectively than rigid docking

  • Improves prediction of binding modes for substrate and transition state analogs

Machine Learning Approaches:

• Sequence-Based Prediction:

  • Graph neural networks trained on enzyme superfamily sequence-function relationships

  • Feature extraction using position-specific scoring matrices and evolutionary couplings

  • Typical performance: R² = 0.70-0.85 for kcat prediction within enzyme families

• Structure-Based Prediction:

  • 3D convolutional neural networks analyzing active site geometry

  • Integration of electrostatic and hydrophobic interaction maps

  • Feature importance analysis for identifying critical residues

• Transfer Learning Models:

  • Pre-trained on large enzyme datasets and fine-tuned for HisF-specific prediction

  • Incorporation of reaction mechanism knowledge as biased features

  • Cross-validation using experimental mutagenesis data

Integrated Workflow for Optimal Prediction:

The most effective strategy combines multiple computational approaches:

• Initial screening of variants using fast sequence-based ML methods

• Refinement of top candidates using MD simulations

• Detailed energetic analysis of promising mutations using QM/MM

• Experimental validation of top 5-10 predicted variants

This integrated approach typically achieves prediction accuracy of catalytic efficiency (kcat/Km) within 5-10 fold of experimental values for conservative mutations and 10-50 fold for more radical substitutions, providing a valuable tool for rational design of HisF variants with desired properties.

What are the most significant recent advances in understanding Pseudomonas syringae HisF function and applications?

Recent advances in understanding P. syringae HisF function and applications have expanded our knowledge in several key areas:

• Structural Biology Breakthroughs:

  • High-resolution structures of the complete HisF-HisH bienzyme complex have revealed dynamic conformational changes during the catalytic cycle

  • Neutron diffraction studies have mapped proton transfer networks essential for ammonia channeling

  • Cryo-EM analysis has captured intermediates previously inaccessible to crystallography

• Systems Biology Integration:

  • Metabolic flux analysis has positioned HisF at a critical junction connecting amino acid and nucleotide metabolism in P. syringae

  • Transcriptomic studies have revealed differential expression of hisF during specific phases of plant infection

  • Proteome-wide interaction mapping has identified unexpected protein partners beyond HisH

• Technological Applications:

  • Development of HisF-based biosensors for environmental monitoring

  • Engineering of HisF variants with enhanced thermostability for biocatalytic applications

  • Design of selective inhibitors targeting the HisF-HisH interface as potential antimicrobials

These advances have been facilitated by technological improvements in structural biology, computational modeling, and high-throughput experimental methods. The integration of these diverse approaches has provided a more comprehensive understanding of HisF's role in both bacterial metabolism and pathogenesis, opening new avenues for both fundamental research and applied biotechnology.

What key questions remain unanswered in the field of Pseudomonas syringae HisF research?

Despite significant progress, several key questions remain unanswered in P. syringae HisF research:

• Mechanistic Questions:

  • How do conformational dynamics coordinate catalysis between the HisF and HisH active sites?

  • What is the precise path of ammonia channeling through the protein complex?

  • How do allosteric signals propagate between distant sites in the enzyme?

• Physiological Questions:

  • How does histidine biosynthesis integrate with virulence-associated metabolic networks?

  • Does HisF activity respond to plant-derived signals during infection?

  • What is the relationship between histidine availability and expression of pathogenicity factors?

• Evolutionary Questions:

  • How has the HisF-HisH interface co-evolved across different P. syringae pathovars?

  • Are there host-specific adaptations in HisF function among strains with different plant preferences?

  • What selective pressures drive HisF sequence conservation despite diverse ecological niches?

• Applied Research Gaps:

  • Can HisF inhibitors be developed with sufficient specificity to target P. syringae without affecting beneficial microbes?

  • What structural features could be exploited to create pathovar-specific inhibitors?

  • How can HisF engineering be leveraged for developing environmentally friendly crop protection strategies?

Addressing these questions will require interdisciplinary approaches combining structural biology, biochemistry, molecular genetics, plant pathology, and computational biology. The answers will not only enhance our fundamental understanding of enzyme function and evolution but may also lead to novel strategies for managing plant diseases caused by P. syringae.

How might research on Pseudomonas syringae HisF contribute to broader understanding of bacterial pathogenesis and metabolic adaptation?

Research on P. syringae HisF has broader implications that extend beyond this specific enzyme system:

• Models for Metabolic Adaptation:

  • HisF provides a well-defined system for studying how core metabolic enzymes adapt to the nutritional landscape of different plant hosts

  • Comparative studies across pathovars with different host ranges can reveal metabolic signatures of host adaptation

  • Understanding metabolic requirements during infection can identify new vulnerability points in bacterial pathogens

• Enzyme-Engineering Principles:

  • The TIM barrel fold of HisF represents one of nature's most versatile enzyme scaffolds

  • Structure-function studies reveal fundamental principles of enzyme evolution and engineering

  • Insights gained can be applied to designing novel biocatalysts for biotechnological applications

• Protein-Protein Interaction Networks:

  • The HisF-HisH interface exemplifies how protein-protein interactions can create emergent catalytic properties

  • Studies of this interface contribute to understanding molecular recognition principles

  • Methodologies developed can be applied to other protein complexes in various bacterial systems

• Antimicrobial Development Strategy:

  • HisF research provides a template for targeting metabolic bottlenecks in bacterial pathogens

  • The approach of inhibiting protein-protein interfaces rather than active sites offers new possibilities for antimicrobial specificity

  • Lessons learned can inform discovery efforts for other plant, animal, and human pathogens