Recombinant Bartonella quintana Carbamoyl-phosphate synthase small chain (carA)

What is the function of Carbamoyl-phosphate synthase in Bartonella quintana?

Carbamoyl-phosphate synthase (CPSase) plays a crucial role in ammonia assimilation in Bartonella quintana, as it does in virtually all organisms. The enzyme catalyzes the synthesis of carbamoyl phosphate (CP), which serves as a common precursor for both pyrimidine nucleotides and arginine biosynthesis. In B. quintana, like other bacteria, CPSase likely consists of a glutaminase (CarA) subunit that hydrolyzes glutamine and transfers ammonia to a synthetase (CarB) subunit. The CarA subunit specifically represents the smaller component of this enzymatic complex, forming a functional heterodimer with CarB to catalyze the complete synthesis of carbamoyl phosphate . This enzymatic activity is essential for nucleotide synthesis and thus for B. quintana replication and survival.

How does Bartonella quintana CarA compare structurally to other bacterial CarA proteins?

While the search results don't provide specific structural information about B. quintana CarA, we can draw inferences based on conserved features of bacterial CPSases. B. quintana CarA likely shares structural similarities with the glutaminase subunits of other bacterial CPSases, particularly those from alphaproteobacteria. Based on sequence analysis of related CPSases, B. quintana CarA would be expected to have a molecular mass of approximately 40 kDa, similar to the glutaminase (CarA, GLN) subunit from E. coli .

The protein likely contains conserved residues involved in glutamine binding and hydrolysis, and would associate with the larger CarB subunit to form a functional enzyme complex. Unlike the smallest known active CPSase from Methanobrevibacter smithii (MS-s) which represents just a portion of the synthetase domain, B. quintana CarA would function as a complete glutaminase domain with the associated catalytic apparatus .

What expression systems are most effective for producing recombinant B. quintana CarA?

For recombinant expression of B. quintana CarA, E. coli-based expression systems have proven effective for similar proteins. Based on methodologies used for other CPSases, the following approach is recommended:

Expression System Selection:

When expressing B. quintana CarA, vector selection should include strong inducible promoters like T7 or tac. Based on successful approaches with other CPSases, expression should be induced with IPTG (0.1-1.0 mM) when cultures reach mid-log phase (OD600 ~0.6-0.8) followed by incubation at reduced temperatures (16-25°C) for 18-24 hours to enhance protein solubility . For purification, incorporating an affinity tag (His6 or GST) at either the N or C-terminus facilitates one-step purification while maintaining enzymatic activity, as demonstrated with other recombinant CPSases .

How can researchers distinguish between the glutaminase and ammonia-dependent activities of recombinant B. quintana CarA?

Distinguishing between glutaminase and ammonia-dependent activities of B. quintana CarA requires specific enzymatic assays targeting different aspects of CPSase function:

Glutaminase Activity Assay:
To measure the glutamine hydrolysis function independent of the synthetase reaction, researchers should monitor glutamate production from glutamine. This can be accomplished using either:

• Spectrophotometric coupling with glutamate dehydrogenase, where glutamate production is linked to NADH oxidation

• HPLC-based quantification of glutamate

• Radiometric assays using [14C]-glutamine

Ammonia-dependent Synthetase Activity:
To assess whether the recombinant CarA can function with free ammonia (bypassing the glutaminase reaction), perform the CPSase reaction using NH4Cl as the nitrogen source instead of glutamine. Based on knowledge of CPSase mechanisms, compare the kinetic parameters (Km, Vmax) between glutamine and ammonia as substrates .

Experimental Approach:
Measure enzyme activity under varying substrate concentrations and compare the apparent Km values for ATP, bicarbonate, and ammonia/glutamine. For B. quintana CarA, researchers would expect to find a high apparent affinity for substrates, similar to what has been observed in the small CPSase from M. smithii which showed high apparent affinity for ATP and ammonia .

What are the critical residues in B. quintana CarA that affect catalytic efficiency, and how can they be identified through site-directed mutagenesis?

Identifying critical residues in B. quintana CarA requires a strategic approach combining computational analysis with experimental validation:

Step 1: Computational Analysis
First, perform sequence alignment of B. quintana CarA with well-characterized CPSase small subunits, focusing on highly conserved residues. Similar to how active site residues were identified in MS-s CPSase, look for conservation patterns across species . Use homology modeling based on crystallized CPSase structures to predict the three-dimensional structure and identify potential catalytic and substrate-binding residues.

Step 2: Site-Directed Mutagenesis Strategy
Based on computational predictions, design a systematic mutagenesis approach targeting:

• Predicted catalytic residues (typically Asp, Glu, His, Lys, Arg)

• Substrate binding pocket residues

• Residues implicated in subunit interaction with CarB

Step 3: Functional Analysis
For each mutant, assess:

• Enzyme kinetics (Km, kcat, kcat/Km) for all substrates

• Thermal stability using differential scanning fluorimetry

• Protein-protein interactions with CarB using size exclusion chromatography and chemical cross-linking

Example Mutation Strategy Table:

When analyzing results, researchers should compare the effects of mutations to naturally occurring CPSase variants across species to gain insight into evolutionary conservation of function .

How does the B. quintana CarA-CarB complex assembly differ from other bacterial CPSase complexes?

The assembly of the B. quintana CarA-CarB complex likely follows patterns seen in other bacterial CPSases but may have unique features related to its pathogenic lifestyle:

Complex Assembly Analysis Methodology:

• Size determination: Use analytical ultracentrifugation, native PAGE, and size exclusion chromatography to determine whether B. quintana CPSase forms heterodimers (CarA-CarB) or higher-order structures like heterotetramers, similar to the E. coli CPSase which can form both configurations .

• Interface mapping: Employ chemical cross-linking followed by mass spectrometry to identify contact regions between CarA and CarB subunits. Cross-linkers of various spacer arm lengths can help determine spatial relationships.

• Electron microscopy: Negative-stain and cryo-EM techniques can visualize the complex architecture and confirm oligomeric state.

Expected Structural Features:
Based on known CPSase structures, researchers would expect to find:

• A glutaminase domain (CarA) interacting with the synthetase domain (CarB)

• A tunnel connecting the active sites of CarA and CarB for ammonia channeling

• Potential regulatory sites at the interface

Evolutionary Considerations:
Unlike metabolically diverse free-living bacteria, B. quintana has a restricted host range and reduced genome. This specialization may have led to specific adaptations in its CPSase complex assembly that optimize function in its unique ecological niche within human hosts and louse vectors . Comparative analysis with CPSases from other α-proteobacteria could reveal lineage-specific features of the B. quintana enzyme complex.

What are the optimal conditions for measuring the enzymatic activity of recombinant B. quintana CarA?

Establishing optimal conditions for B. quintana CarA activity requires systematic evaluation of multiple parameters:

Buffer Optimization Table:

Reaction Monitoring:
The enzymatic activity should be monitored using one of these approaches:

• Colorimetric detection of inorganic phosphate released during ATP hydrolysis

• Coupled enzyme assays linking ADP production to NADH oxidation

• Radiometric assays measuring incorporation of 14C-bicarbonate into carbamoyl phosphate

Stability Assessment:
To ensure reliable measurements, determine protein stability under assay conditions by:

• Testing activity retention after various pre-incubation times

• Evaluating the effects of stabilizing agents (glycerol, BSA, reducing agents)

• Monitoring time-dependent activity to ensure linearity

Based on studies of related CPSases, the recombinant B. quintana CarA would likely show optimal activity at physiological temperatures (30-37°C) and slightly alkaline pH (7.5-8.0), reflecting its adaptation to the human host environment .

How can researchers efficiently co-express and co-purify the B. quintana CarA-CarB complex for structural studies?

For structural studies of the complete B. quintana CPSase complex, co-expression and co-purification of both subunits is recommended:

Co-expression Strategy:

• Dual-plasmid system: Clone CarA and CarB into compatible vectors with different antibiotic resistance markers

• Polycistronic expression: Clone both genes in a single vector with a dicistronic arrangement

Expression Construct Design:

Purification Protocol:

• Affinity chromatography (IMAC or Strep-Tactin)

• Ion exchange chromatography to separate fully assembled complex

• Size exclusion chromatography for final polishing and buffer exchange

Complex Stability Assessment:
Monitor complex integrity using:

• Analytical size exclusion chromatography

• Native gel electrophoresis

• Dynamic light scattering

• Thermal shift assays

For structural studies, it's crucial to verify that the purified complex is catalytically active and properly assembled. Based on chemical cross-linking studies of other CPSases, researchers should expect the B. quintana CPSase to form a stable complex with a defined stoichiometry, likely similar to the heterodimeric/heterotetrameric arrangements observed in E. coli CPSase .

What approaches can be used to identify potential inhibitors specific to B. quintana CarA that might serve as antimicrobial leads?

Developing inhibitors specific to B. quintana CarA requires a structured drug discovery approach focusing on selective targeting:

Target Validation Strategy:

• Confirm essentiality of CarA in B. quintana through genetic approaches

• Identify structural or sequence differences between human and B. quintana CPSases

• Focus on unique binding pockets or regulatory mechanisms

Screening Methodologies:

Selectivity Assessment:
To ensure specificity for B. quintana CarA over human CPSase:

• Counter-screen against human CPSase

• Evaluate cytotoxicity in human cell lines

• Test activity against other bacterial species

Structure-Activity Relationship Development:
For promising hits, develop SAR by:

• Synthesizing analogs with systematic modifications

• Co-crystallizing enzyme-inhibitor complexes when possible

• Using molecular dynamics simulations to optimize binding interactions

This approach leverages the unique features of B. quintana, which has evolved as a specialized human pathogen with restricted host range , potentially resulting in targetable differences in its essential metabolic enzymes compared to human counterparts.

How should researchers interpret kinetic data for B. quintana CarA when the results differ from expected values based on other bacterial CPSases?

When B. quintana CarA exhibits unexpected kinetic parameters compared to other bacterial CPSases, researchers should consider several factors:

Systematic Analysis Approach:

• Confirm protein quality: Verify protein purity, proper folding, and absence of degradation by SDS-PAGE, circular dichroism, and activity retention over time.

• Validate assay conditions: Test whether the unusual kinetics are due to suboptimal assay conditions by systematically varying buffers, pH, temperature, and ionic strength.

• Consider biological context: B. quintana has evolved as a specialized human pathogen with restricted niche , which may have selected for kinetic parameters optimized for its unique environment compared to free-living bacteria.

Data Interpretation Table:

Evolutionary Interpretation:
Analyze the sequence and predicted structure of B. quintana CarA in comparison with other bacterial CPSases to identify unique features that might explain kinetic differences. Consider that smaller enzymes like the 41 kDa CPSase from M. smithii have been shown to be catalytically active with unique kinetic properties optimized for their specific biological contexts .

What are common issues in the expression and purification of recombinant B. quintana CarA, and how can they be addressed?

Researchers working with recombinant B. quintana CarA may encounter several challenges:

Expression Issues and Solutions:

Purification Troubleshooting:

Activity Reconstitution:
If purified B. quintana CarA shows limited activity, consider:

• Testing whether activity depends on association with CarB

• Adding potential cofactors or metal ions

• Evaluating protein refolding protocols if recovered from inclusion bodies

Based on experiences with other CPSases, researchers should be particularly attentive to maintaining the native quaternary structure, as CPSases typically function as multimeric complexes with specific inter-subunit interactions that are essential for catalytic activity .

How can researchers resolve contradictions in structural data when comparing B. quintana CarA with homologous proteins from other species?

When structural studies of B. quintana CarA yield results that contradict known structures of homologous proteins, a systematic approach to resolution is needed:

Contradiction Analysis Framework:

• Data Quality Assessment:

  • Evaluate resolution limits of structural techniques used

  • Assess completeness of data and potential experimental artifacts

  • Validate refinement statistics for computational models

• Comparative Analysis Methodology:

  • Perform structural alignments with homologous proteins using multiple algorithms

  • Identify regions of greatest divergence and evaluate their functional significance

  • Use molecular dynamics simulations to assess whether observed differences represent stable conformations

• Functional Correlation:

  • Test whether structural differences correlate with functional differences using mutagenesis

  • Compare catalytic parameters of regions showing structural divergence

  • Assess whether differences might reflect species-specific adaptations

Resolution Strategies Table:

When analyzing such contradictions, researchers should consider that B. quintana has evolved as a specialized pathogen with a restricted host range , potentially leading to unique structural adaptations in its enzymes compared to homologs from free-living bacteria. Additionally, the functional constraints on CPSases may allow for structural variations while preserving core catalytic function, as evidenced by the diversity of CPSase structures across species while maintaining the same series of reactions .

How can isothermal titration calorimetry be optimized for studying substrate binding to B. quintana CarA?

Isothermal titration calorimetry (ITC) provides valuable thermodynamic data on substrate binding, but requires careful optimization for B. quintana CarA studies:

Experimental Design Considerations:

Sample Preparation Protocol:

• Purify B. quintana CarA to >95% homogeneity

• Dialyze protein and all substrates/ligands against identical buffer to minimize heat of dilution

• Degas all solutions to prevent bubble formation during experiments

Optimization Parameters Table:

Data Analysis Strategy:

• Subtract reference injections (ligand into buffer)

• Fit to appropriate binding models (one-site, sequential, or cooperative)

• Extract thermodynamic parameters (ΔH, ΔS, ΔG, Kd)

• Perform measurements at different temperatures to determine ΔCp

For B. quintana CarA, researchers should design experiments to examine binding of ATP, bicarbonate, and glutamine individually and sequentially to understand the order of substrate binding and potential cooperativity, similar to studies performed with other multifunctional enzymes .

What strategies can researchers employ to study the in vivo function of B. quintana CarA in the context of host infection?

Studying B. quintana CarA function during host infection requires specialized approaches due to the pathogen's restricted host range and fastidious nature :

Genetic Manipulation Strategies:

• Conditional knockdown systems: Develop inducible antisense RNA or CRISPR interference systems to modulate CarA expression

• Complementation studies: Create point mutations in conserved residues and evaluate their effects on virulence

• Reporter fusions: Generate CarA-reporter fusions to monitor expression during different infection phases

Infection Model Systems:

Functional Assessment Approaches:

• Metabolomic analysis: Compare arginine and pyrimidine levels in wild-type vs. CarA-attenuated strains

• Transcriptomic profiling: Identify genes co-regulated with CarA during infection

• In vivo protein crosslinking: Capture CarA interaction partners during infection

Therapeutic Implications:
Since B. quintana is responsible for various clinical conditions including trench fever, endocarditis, and bacillary angiomatosis , understanding CarA function during infection could reveal new therapeutic targets. Researchers should correlate CarA activity with bacterial persistence and host response, particularly in immunocompromised patients where B. quintana infections are most problematic.

How can cryo-electron microscopy be applied to resolve the structure of the B. quintana CarA-CarB complex in different functional states?

Cryo-electron microscopy (cryo-EM) offers powerful capabilities for visualizing the B. quintana CPSase complex in various conformational states:

Sample Preparation Protocol:

• Complex purification: Co-purify CarA-CarB to homogeneity (>95%) using affinity chromatography followed by size exclusion

• Grid preparation: Apply 2-4 μL of sample (0.5-5 mg/mL) to glow-discharged grids

• Vitrification: Blot for 2-6 seconds and plunge-freeze in liquid ethane

• Screening: Evaluate grid quality using low-dose imaging

Functional State Trapping Strategies:

Data Collection and Processing Workflow:

• Collect movies using direct electron detector with motion correction

• Perform CTF estimation and correction

• Select particles automatically with manual inspection

• Conduct 2D classification to eliminate poor particles

• Generate ab initio model followed by 3D classification

• Perform 3D refinement with focused refinement on flexible regions

• Validate structure using FSC curves, local resolution estimation, and model-to-map correlation

Comparative Structural Analysis:
Compare the B. quintana CPSase structures in different states to identify:

• Conformational changes associated with substrate binding

• Channel formation for ammonia transfer between subunits

• Allosteric regulation mechanisms

The structural insights from cryo-EM would complement biochemical studies and potentially reveal unique features of the B. quintana enzyme related to its function in this specialized human pathogen .

What are emerging approaches for studying the evolutionary relationship between B. quintana CarA and homologous proteins in other pathogens?

The evolutionary relationship between B. quintana CarA and homologous proteins in other pathogens can be explored using cutting-edge approaches:

Phylogenomic Analysis Strategy:

• Perform comprehensive sequence retrieval from diverse bacterial lineages

• Construct phylogenetic trees using maximum likelihood and Bayesian methods

• Test alternative evolutionary hypotheses using statistical approaches

• Correlate CarA evolutionary patterns with pathogen lifestyle and host range

Molecular Evolution Analyses:

• Calculate site-specific evolutionary rates to identify conserved vs. variable regions

• Test for signatures of positive selection at specific sites

• Reconstruct ancestral sequences to trace functional evolution

• Correlate evolutionary changes with structural and functional domains

Similar to how CPSase phylogenetic relationships were analyzed across diverse species , researchers can place B. quintana CarA in its proper evolutionary context, considering its specialized niche as a human pathogen transmitted by lice .

Experimental Validation Approaches:

By combining computational and experimental approaches, researchers can gain insights into how B. quintana CarA has evolved in the context of the organism's restricted host range and specialized lifestyle as a human pathogen .

How might structural knowledge of B. quintana CarA inform novel antimicrobial development strategies?

Structural insights into B. quintana CarA can drive antimicrobial development through several strategic approaches:

Structure-Guided Drug Design Workflow:

• Identify unique structural features in B. quintana CarA not present in human CPSases

• Define druggable pockets using computational solvent mapping

• Employ virtual screening against these targets

• Validate hits with biochemical and biophysical assays

• Optimize lead compounds through medicinal chemistry

Targeting Approaches Table:

Translational Considerations:
Given that B. quintana causes trench fever, endocarditis, and bacillary angiomatosis, particularly in immunocompromised patients , developing targeted antimicrobials could address an important medical need. CPSase inhibitors could be particularly valuable for treating persistent B. quintana infections that have proven difficult to eradicate with current antibiotics.

Combination Therapy Approaches:
By understanding the structural basis of CPSase function in B. quintana, researchers could design inhibitors that synergize with existing antibiotics, potentially through:

• Blocking metabolic bypass pathways

• Preventing adaptation to nutrient-limited environments

• Disrupting bacterial persistence mechanisms

What are the implications of understanding B. quintana CarA for developing diagnostic tools for Bartonella infections?

Understanding B. quintana CarA at the molecular level could enable novel diagnostic approaches for Bartonella infections:

Diagnostic Development Strategy:

• Identify unique epitopes in B. quintana CarA not present in other bacteria

• Develop monoclonal antibodies against these specific regions

• Create recombinant antigens for serological testing

• Design species-specific molecular detection methods

Diagnostic Application Table:

Clinical Significance:
Current diagnosis of B. quintana infections relies on serologic analysis, culture, and molecular biology techniques . Improved diagnostics based on CarA could enable:

• Earlier detection of infections

• Differentiation between active and past infections

• Monitoring treatment response

• Epidemiological surveillance, particularly in homeless populations where B. quintana has reemerged

Implementation Considerations:
For diagnostic translation, researchers should consider:

• Necessary sensitivity and specificity for clinical utility

• Sample types (blood, tissue) and processing requirements

• Integration with existing diagnostic workflows

• Cost and technical requirements for implementation in various settings