Recombinant Coxiella burnetii GMP synthase [glutamine-hydrolyzing] (guaA), partial

What is the biological function of GMP synthase in Coxiella burnetii?

GMP synthase (EC 6.3.5.2) in C. burnetii catalyzes the final step in GMP biosynthesis, converting XMP to GMP through the substitution of the C2 oxo-group of the purine base with an amino group. This enzyme plays a critical role in purine metabolism, which is essential for bacterial survival and replication. The reaction involves a two-step process:

• Hydrolysis of glutamine to produce ammonia in the glutamine amidotransferase (GATase) domain

• Formation of adenyl-XMP intermediate in the ATP pyrophosphatase (ATPPase) domain, followed by the reaction of ammonia with this intermediate to generate GMP

The C. burnetii GMP synthase is structurally characterized as having three distinct domains common to other bacterial GMP synthases: the N-terminal ATP-PPase domain, the GATase domain, and a C-terminal dimerization domain .

How does the structure of C. burnetii GMP synthase compare to orthologs from other organisms?

C. burnetii GMP synthase shares structural similarities with other bacterial GMP synthases, but with distinct characteristics. Comparative analysis reveals:

The conserved domain architecture across species highlights the evolutionary importance of this enzyme, while the differences in kinetic parameters suggest potential targets for species-specific inhibitors .

What are the optimal conditions for recombinant expression of C. burnetii GMP synthase?

For successful recombinant expression of C. burnetii GMP synthase, two primary expression systems have been documented:

E. coli Expression System:

• The guaA gene from C. burnetii can be cloned into an expression vector with a histidine tag

• Expression in E. coli yields protein with >85% purity as determined by SDS-PAGE

• The recombinant protein is typically expressed as a partial construct rather than the full-length protein

Yeast Expression System:

• Alternative expression in yeast systems has been reported for applications requiring eukaryotic post-translational modifications

• Similar purity levels (>85%) can be achieved

For optimal activity, expression conditions should be carefully controlled with induction parameters optimized to maximize soluble protein yield while minimizing inclusion body formation.

What purification methods yield the highest purity and activity of recombinant C. burnetii GMP synthase?

Based on established protocols for similar enzymes, the following purification strategy is recommended:

• Initial Capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin to capture the His-tagged protein

• Intermediate Purification: Ion exchange chromatography to separate based on charge differences

• Polishing Step: Size exclusion chromatography to achieve final purity and remove any aggregates

Critical considerations for maintaining enzyme activity during purification:

• Maintain buffer pH between 7.0-8.0

• Include reducing agents (e.g., DTT or β-mercaptoethanol) to prevent oxidation of cysteine residues

• Add glycerol (5-50%) to the final formulation for long-term storage stability

• Store working aliquots at 4°C for up to one week; for extended storage, maintain at -20°C or -80°C

What are the established methods for measuring C. burnetii GMP synthase enzymatic activity?

Several complementary approaches can be used to evaluate the enzymatic activity of recombinant C. burnetii GMP synthase:

Stopped-Flow Spectrophotometry Assay:

• Measures the rate of adenyl-XMP formation using ATP·Mg²⁺ and XMP as substrates

• Allows for real-time monitoring of the reaction kinetics

Glutamate Dehydrogenase Coupling Assay:

• Detects liberated glutamate resulting from the GATase domain activity

• Useful for measuring the allosteric activation of the GATase domain by substrate binding to the ATPPase domain

• Can also be employed to determine binding affinities (Kd values) for ATP·Mg²⁺ and XMP in inactive mutants

Direct Product Analysis:

• GMP formation can be directly quantified using HPLC or coupled enzyme assays

• This approach allows for assessment of complete catalytic activity (both Gln-dependent and NH₄Cl-dependent GMP formation)

When developing these assays for C. burnetii GMP synthase, it's important to consider the enzyme's kinetic parameters and optimize conditions accordingly.

What are the key kinetic parameters of C. burnetii GMP synthase and how do they compare to orthologous enzymes?

While specific kinetic parameters for C. burnetii GMP synthase have not been directly reported in the provided search results, comparison with related bacterial GMP synthases provides a framework for understanding its likely kinetic behavior:

*Values shown are from related bacterial GMP synthases as specific C. burnetii values were not provided in the search results

Understanding these parameters is crucial for designing inhibition studies and characterizing potential antimicrobial compounds targeting C. burnetii GMP synthase. The differences in kinetic parameters between bacterial and human enzymes provide potential avenues for selective inhibition .

How does GMP synthase contribute to C. burnetii virulence and pathogenesis?

While the direct contribution of GMP synthase to C. burnetii virulence hasn't been explicitly detailed in the provided search results, several lines of evidence suggest its importance in pathogenesis:

• Essential Metabolic Function: GMP synthase catalyzes the final step in GMP biosynthesis, producing a nucleotide essential for DNA/RNA synthesis and various signaling pathways. This metabolic function is critical for bacterial replication within host cells .

• Carbon Metabolism Integration: C. burnetii has been shown to utilize both glucose and glutamate during infection. As a glutamine-hydrolyzing enzyme, GMP synthase is integrated with the pathogen's broader nitrogen and carbon metabolism, which adapts to the intracellular environment .

• Survival in Lysosomal Environment: C. burnetii uniquely thrives in a lysosome-like intracellular niche. The pathogen's metabolic adaptations, including nucleotide biosynthesis pathways, contribute to its ability to survive in this degradative environment .

• Precedent in Other Pathogens: GMP synthase has been identified as essential for virulence in other intracellular pathogens. For example, GMP synthase mutants of Candida albicans and Aspergillus fumigatus were avirulent in murine infection models , suggesting a similar role might exist in C. burnetii.

Understanding GMP synthase's role in C. burnetii pathogenesis may provide insights into developing targeted therapeutic approaches for Q fever treatment.

How does C. burnetii adapt its metabolic pathways, including nucleotide synthesis, during intracellular infection?

C. burnetii exhibits remarkable metabolic flexibility during intracellular infection, adapting its metabolic pathways to the unique environment of the Coxiella-containing vacuole (CCV):

• Nutrient Acquisition: C. burnetii can assimilate both [¹³C]glucose and [¹³C]glutamate during infection, with concomitant labeling of intermediates in glycolysis, gluconeogenesis, and the TCA cycle . This demonstrates the pathogen's ability to utilize multiple carbon sources simultaneously.

• Metabolic Pathway Adaptation: Intracellular and axenically cultured bacteria show distinct metabolic profiles reflecting the different nutrient availabilities in their respective environments . This suggests that purine biosynthesis pathways, including GMP synthesis, may be differentially regulated during infection.

• Lysosomal Degradation Products: C. burnetii can utilize lysosomal degradation products as nutrients. The bacterium detects specific amino acids present in the lysosome using a two-component system that upregulates expression of genes required for its Type IV secretion system .

• Glucose Transport Redundancy: The disruption of individual glucose transporters (CBU0265 or CBU0347) leads to decreased [¹³C]glucose utilization but does not abolish glucose usage or impact intracellular replication, suggesting metabolic redundancy and flexibility .

This metabolic adaptability likely extends to nucleotide biosynthesis pathways, including those involving GMP synthase, allowing C. burnetii to maintain essential functions even under varying nutrient conditions within the host cell.

How can recombinant C. burnetii proteins, including GMP synthase, be utilized for developing improved diagnostic tools?

Several recombinant C. burnetii proteins have shown promise as diagnostic antigens, providing a framework for how GMP synthase might be similarly employed:

• Recombinant ELISA Development: Recombinant C. burnetii proteins can be used to develop ELISAs with improved sensitivity and specificity. For example, the recombinant Ybgf protein demonstrated 81.8% sensitivity and 90.1% specificity in detecting C. burnetii antibodies in ruminants .

• Differentiation of Infection vs. Vaccination: Certain recombinant antigens, such as Ybgf, show minimal reactivity with antibodies induced by vaccination, potentially allowing for the differentiation of infected from vaccinated animals (DIVA) .

• Phase-Specific Detection: Recombinant proteins can be developed to detect antibodies against specific phases of C. burnetii (Phase I vs. Phase II), providing information about acute versus chronic infection status .

• Multiplexed Diagnostic Approaches: Combining multiple recombinant antigens, potentially including GMP synthase, could enhance diagnostic accuracy. For example, the Com1 outer membrane protein, when used in an ELISA format, showed varying sensitivities and specificities across different host species: sheep (85% sensitivity, 68% specificity), goats (94% sensitivity, 77% specificity), and cattle (71% sensitivity, 70% specificity) .

For optimal diagnostic performance, recombinant proteins should be carefully characterized for antigenicity, cross-reactivity, and stability. The recombinant protein-based assays should be validated against established diagnostic methods and across diverse sample sets.

What challenges exist in developing effective vaccines against C. burnetii, and could recombinant proteins like GMP synthase be potential vaccine candidates?

The development of effective vaccines against C. burnetii faces several significant challenges:

• Historical Limitations: Previous attempts to develop Q fever vaccines have resulted in either unacceptable side effects or insufficient protection . Currently, Q-Vax is a licensed vaccine that has shown effectiveness, but improved options are needed.

• Mixed Results with Recombinant Proteins: An immunization experiment using a mixture of eight recombinant C. burnetii proteins (Omp, Pmm, HspB, Fbp, Orf410, Crc, CbMip, and MucZ) failed to induce a protective immune response in mice against challenge infection with C. burnetii Nine Mile RSA493 . This suggests that not all recombinant proteins are equally effective as vaccine antigens.

• Antigenicity without Protection: The recombinant proteins tested were largely antigenic in BALB/c mice when administered as protein mixtures (with the exception of rPmm), but antigenicity did not translate to protection . This highlights the complexity of developing an effective immune response against this intracellular pathogen.

• Need for Appropriate Animal Models: While mouse models are commonly used, they may not fully recapitulate human disease. The variable response across different host species (as seen with diagnostic antigens) suggests that vaccine efficacy might also vary across species .

Regarding GMP synthase as a potential vaccine candidate:

Future vaccine development strategies might benefit from combining multiple antigens, including both structural proteins and metabolic enzymes, with appropriate adjuvants to enhance immunogenicity.

What is known about inhibitors of bacterial GMP synthases and their potential application against C. burnetii?

While specific inhibitors of C. burnetii GMP synthase are not extensively described in the provided search results, research on GMP synthases from other pathogens provides valuable insights:

• Established Target Status: GMP synthase has been validated as a drug target in multiple infectious pathogens. The enzyme is "indispensable for the growth and survival of many organisms including infectious pathogens where the enzyme is pursued as drug targets" .

• Structural Basis for Selective Inhibition: Structural and kinetic differences between bacterial and human GMP synthases provide a foundation for developing selective inhibitors. For example:

  • The human GMP synthase has a significantly higher (~12×) turnover number than fungal GMP synthase

  • The concentration of glutamine required to reach saturation is almost three times higher for fungal GMP synthase (1130 ± 162 μM) compared to human GMP synthase (406 ± 49 μM)

  • These kinetic differences suggest opportunities for selective targeting

• Key Structural Features for Inhibitor Design:

  • The ATP-PPase domain contains conserved helices (α11 and α12) at the interdomain interface that are critical for catalysis

  • The C-terminal loop with its conserved signature motif KPPXTXE(F/W)X is involved in substrate binding

  • The interdomain interface mediates allosteric activation and is essential for enzyme function

• Potential Inhibition Strategies:

  • Targeting the adenyl-XMP formation in the ATP-PPase domain

  • Disrupting domain crosstalk between GATase and ATP-PPase domains

  • Interfering with ammonia tunneling between domains

  • Developing competitive inhibitors of substrate binding sites

Given C. burnetii's intracellular lifestyle, effective inhibitors would need to penetrate both host cell and bacterial membranes to reach their target, adding an additional challenge for drug development.

How might structural differences between C. burnetii and human GMP synthase be exploited for selective drug design?

Selective inhibition of C. burnetii GMP synthase requires exploiting structural and functional differences between the bacterial and human enzymes:

• Unique Regulatory Mechanisms: Bacterial GMP synthases may be subject to different regulatory mechanisms compared to human enzymes. Understanding these differences could reveal additional targets for selective inhibition.

• Structure-Based Drug Design Approach:

  • Determine the crystal structure of C. burnetii GMP synthase

  • Perform comparative analysis with human GMP synthase structure

  • Identify unique pockets or conformational states in the bacterial enzyme

  • Use computational methods to screen for compounds that selectively bind to these unique features

  • Validate hits with enzymatic assays and optimize for cellular activity and selectivity

The development of selective inhibitors would need to account for C. burnetii's unique intracellular environment and ensure sufficient penetration into the Coxiella-containing vacuole.

What mutagenesis approaches can be used to study the structure-function relationship of C. burnetii GMP synthase?

Several mutagenesis strategies can be employed to investigate the structure-function relationship of C. burnetii GMP synthase:

• Site-Directed Mutagenesis of Catalytic Residues:

  • Target conserved residues in the ATP-PPase domain, such as those on helices α11 and α12, which are critical for catalysis

  • Mutate residues in the C-terminal loop containing the conserved signature motif KPPXTXE(F/W)X

  • Substitute key residues involved in substrate binding, such as homologs of Lys547, Glu553, and Arg539

• Domain Interface Mutations:

  • Create mutations at the interface between GATase and ATP-PPase domains to study domain crosstalk

  • Investigate how perturbing interdomain interactions affects allosteric activation and ammonia tunneling

• Complementation Studies:

  • Express C. burnetii GMP synthase in E. coli or other bacterial mutants lacking functional GMP synthase (similar to studies of other C. burnetii enzymes)

  • Assess the ability of wild-type and mutant forms to restore growth or function

• Chimeric Protein Construction:

  • Create chimeric proteins by swapping domains between C. burnetii GMP synthase and orthologs from other species

  • This approach can help identify species-specific functional elements

For effective mutagenesis studies, it's crucial to:

• Verify that mutations don't disrupt protein folding using circular dichroism and size-exclusion chromatography

• Assess multiple aspects of enzyme function (adenyl-XMP formation, GATase activation, GMP synthesis)

• Compare kinetic parameters of mutants with wild-type enzyme

What advanced analytical techniques are most suitable for characterizing the enzymatic mechanism of C. burnetii GMP synthase?

To fully elucidate the enzymatic mechanism of C. burnetii GMP synthase, a combination of advanced analytical techniques is recommended:

• Transient Kinetic Analysis:

  • Stopped-flow spectrophotometry to measure rapid reaction kinetics

  • Pre-steady-state kinetics to determine individual rate constants for each step of the reaction

  • Quench-flow techniques coupled with HPLC to identify reaction intermediates

• Spectroscopic Methods:

  • Nuclear Magnetic Resonance (NMR) spectroscopy to monitor structural changes during catalysis

  • Fluorescence spectroscopy with strategically placed fluorophores to track domain movements

  • Circular dichroism to assess conformational changes upon substrate binding

• Structural Biology Approaches:

  • X-ray crystallography of the enzyme in different catalytic states

  • Cryo-electron microscopy to visualize conformational dynamics

  • Hydrogen-deuterium exchange mass spectrometry to identify regions with altered solvent accessibility during catalysis

• Computational Methods:

  • Molecular dynamics simulations to model conformational changes and substrate binding

  • Quantum mechanics/molecular mechanics (QM/MM) calculations to model the chemical reaction mechanism

  • Bioinformatic analysis comparing C. burnetii GMP synthase with orthologs from diverse species

• Isotope Labeling Studies:

  • Use of 13C, 15N, or 18O labeled substrates to track atom transfer during catalysis

  • Kinetic isotope effect measurements to identify rate-limiting steps

• Advanced Binding Studies:

  • Isothermal titration calorimetry (ITC) to determine thermodynamic parameters of substrate binding

  • Surface plasmon resonance (SPR) to measure association and dissociation kinetics

  • Microscale thermophoresis to assess binding under near-native conditions

These techniques should be applied in a complementary manner to build a comprehensive understanding of the C. burnetii GMP synthase mechanism, from substrate binding through product release, with particular attention to the allosteric communication between domains that is characteristic of this enzyme family.

How has the guaA gene evolved in C. burnetii compared to other bacterial species?

The evolution of the guaA gene in C. burnetii can be examined through several complementary perspectives:

Understanding the evolutionary trajectory of the guaA gene in C. burnetii provides valuable context for interpreting its functional properties and potential as a drug target or diagnostic marker.

What does comparative genomic analysis reveal about GMP synthase conservation across C. burnetii strains and its implications for diagnostic or therapeutic applications?

Comparative genomic analysis of GMP synthase across C. burnetii strains offers important insights with significant implications: