Recombinant Chlamydophila caviae Uridylate kinase (pyrH)

What is Uridylate kinase (PyrH) and what is its role in Chlamydophila caviae?

Uridylate kinase (PyrH) in Chlamydophila caviae is an essential enzyme that catalyzes the conversion of UMP to UDP, a critical step in the pyrimidine metabolic pathway. This reaction is fundamental for the synthesis of nucleotides required for DNA and RNA production, making it essential for bacterial survival and replication. The enzyme belongs to the UMP kinase family and plays a pivotal role in nucleotide biosynthesis in a variety of bacteria, including those causing respiratory tract infections (RTIs). Due to its essential nature in bacterial metabolism and its absence in mammalian cells, which use a different enzyme for this reaction, PyrH represents a potential target for antimicrobial development . In C. caviae specifically, the pyrH gene is conserved among all completed Chlamydiaceae genomes, underscoring its fundamental importance in chlamydial biology and metabolism .

How does the C. caviae genome compare to other Chlamydiaceae species?

The genome structure comparison between C. caviae and other Chlamydiaceae species is illustrated in the following table:

The largest set of genes shared exclusively with another chlamydial genome were those shared with C. pneumoniae, reflecting their closer phylogenetic relationship . Notably, the replication termination region (RTR) or plasticity zone (PZ) of C. caviae contains unique elements, including a near-full tryptophan operon, which is only partially present in C. trachomatis and C. suis .

Why is C. caviae used as a model organism for chlamydial infections?

Chlamydophila caviae, the agent of guinea pig inclusion conjunctivitis (GPIC), serves as an excellent model for naturally occurring Chlamydia trachomatis infections and diseases in humans despite being phylogenetically distant. The value of C. caviae as a model organism stems from several key similarities to human chlamydial infections:

• Transmission mechanisms: C. caviae shares similar transmission routes with human chlamydial infections, including sexual transmission .

• Disease progression: The chronic immune-mediated disease progression in C. caviae infections, such as pannus formation during ocular infections and tubal salpingitis during female genital infections, closely resembles the progression observed in human chlamydial infections .

• Pathologic endpoints: C. caviae infections result in highly similar pathologic outcomes to human infections, including corneal damage and tubal blockage .

• Genomic conservation: Despite some unique genes, C. caviae shares most functionally assigned genes found in other Chlamydiaceae, making it valuable for studying core chlamydial biology .

These similarities make C. caviae particularly useful for studying immune responses, pathogenesis mechanisms, and potential therapeutic strategies that may be applicable to human chlamydial infections, especially when compared to other animal models that do not as closely resemble the human disease process .

How can recombinant C. caviae PyrH be expressed and purified for functional studies?

Recombinant C. caviae PyrH can be expressed and purified using established molecular biology techniques similar to those used for other chlamydial proteins. Based on methodologies described for related proteins, the following protocol would be effective:

• Gene cloning: Amplify the pyrH gene from C. caviae genomic DNA using PCR with specific primers containing appropriate restriction enzyme sites (e.g., KpnI and SalI) .

• Expression vector construction: Clone the amplified pyrH gene into a protein expression vector such as pQE-80L, which adds a histidine tag to the N-terminus of the recombinant protein to facilitate purification .

• Transformation: Transform the recombinant plasmid into a suitable E. coli expression strain (e.g., E. coli thymidylate synthase-deficient strain for complementation studies if necessary) .

• Protein expression: Induce protein expression with IPTG in LB medium at optimal temperature and time conditions, typically 37°C for 3-4 hours post-induction .

• Cell harvesting and lysis: Harvest bacteria by centrifugation at 7,000 rpm for 10 minutes at 4°C. Resuspend the pellet in binding buffer (e.g., 5 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl pH 7.9) and lyse cells by sonication in the presence of lysozyme (350 μg/mL) .

• Purification: Clarify lysates by ultracentrifugation at 45,000 rpm for 1.5 hours at 4°C. Purify His-tagged recombinant PyrH using nickel-affinity chromatography, washing with binding buffer followed by wash buffer (60 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl pH 7.9), and eluting with elution buffer (200 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl pH 7.9) .

• Storage: Add glycerol to a final concentration of 10% to prevent protein precipitation and dialyze overnight at 4°C against a suitable buffer (e.g., 50 mM Tris-HCl pH 7.9 with 5% glycerol). Store aliquots at -80°C for long-term use .

This methodology ensures the production of pure, active recombinant PyrH protein suitable for enzymatic and structural studies.

What assays can be used to measure the enzymatic activity of recombinant PyrH?

Several assays can be employed to measure the enzymatic activity of recombinant PyrH, which catalyzes the phosphorylation of UMP to UDP using ATP as a phosphate donor. Based on methodologies used for similar enzymes, the following assays would be appropriate:

• Coupled spectrophotometric assay: This assay links the production of ADP (a product of the PyrH reaction) to the oxidation of NADH through coupling enzymes such as pyruvate kinase and lactate dehydrogenase. The decrease in NADH absorbance at 340 nm can be monitored continuously, providing real-time kinetic data.

• Luminescence-based kinase assay: As mentioned in the search results, a luminescence-based kinase assay can be developed to measure PyrH activity . This typically involves detection of ATP consumption or ADP production using luciferase-based reagents that generate light proportional to the remaining ATP concentration.

• Radiometric assay: Using [γ-32P]ATP or [γ-33P]ATP as a substrate, the transfer of the radioactive phosphate to UMP can be measured. After separating the reaction products by thin-layer chromatography or other methods, the radioactive UDP can be quantified.

• HPLC-based assay: High-performance liquid chromatography can be used to separate and quantify the substrates and products of the PyrH reaction. This method provides direct measurement of UDP formation.

• Malachite green phosphate assay: This colorimetric assay detects inorganic phosphate released in a coupled reaction where the UDP produced by PyrH is further metabolized by a phosphatase.

For optimal results, the reaction conditions should be standardized with respect to pH, temperature, and cofactor concentrations. A typical reaction mixture might contain 50 mM Tris-HCl (pH 7.5-8.0), 5-10 mM MgCl₂, 1-2 mM ATP, 0.5-1 mM UMP, and an appropriate amount of purified recombinant PyrH enzyme. Activity measurements should be performed in the linear range of the enzyme reaction.

How does the structure of C. caviae PyrH compare to PyrH from other bacterial species?

While specific structural information about C. caviae PyrH is not directly provided in the search results, we can infer several features based on the conservation of PyrH across bacterial species and available structural studies of UMP kinases from other organisms.

UMP kinases typically function as hexamers (trimers of dimers) and belong to the amino acid kinase family, structurally distinct from eukaryotic nucleoside monophosphate kinases. Based on sequence alignments and structural studies from other bacterial species, C. caviae PyrH likely contains:

• A core nucleotide-binding domain with characteristic motifs for ATP binding

• UMP-binding site with conserved residues for substrate recognition

• Magnesium-binding sites essential for catalytic activity

• Allosteric regulatory sites that respond to GTP (activator) and UTP (inhibitor)

Comparative sequence analysis could reveal the degree of conservation in catalytic and regulatory sites between C. caviae PyrH and other bacterial homologs. The analysis of ThyX homologs shown in the search results suggests that similar alignments could be performed for PyrH to identify conserved functional motifs.

For a comprehensive structural comparison, X-ray crystallography or cryo-electron microscopy studies of recombinant C. caviae PyrH would be necessary. These studies would provide insights into any unique structural features that might be exploited for species-specific inhibitor design. Molecular modeling approaches, using existing bacterial PyrH structures as templates, could also provide preliminary structural insights prior to experimental structure determination.

What are the implications of targeting PyrH for antimicrobial development against Chlamydophila species?

Targeting PyrH for antimicrobial development against Chlamydophila species presents several promising advantages and considerations:

The compound PYRH-1 mentioned in the search results represents a potential lead structure for developing specific PyrH inhibitors . Future antimicrobial development would benefit from structural studies of C. caviae PyrH to enable structure-based drug design approaches.

What expression systems are most effective for producing functional recombinant C. caviae PyrH?

Several expression systems can be considered for producing functional recombinant C. caviae PyrH, each with distinct advantages and limitations:

• E. coli expression systems:

  • pQE vector series: These vectors, such as pQE-80L mentioned in the search results , provide high-level expression with an N-terminal His-tag for simplified purification. The T5 promoter system allows for tight regulation with IPTG induction.

  • pET vector system: Offers extremely high expression levels under the T7 promoter in E. coli BL21(DE3) or similar strains, with various fusion tag options.

  • pGEX system: Provides GST fusion for enhanced solubility and alternative purification options.

• Optimization strategies:

  • Temperature modulation: Growing cultures at lower temperatures (16-25°C) after induction can enhance proper folding and solubility.

  • Codon optimization: Adapting the C. caviae pyrH gene sequence to E. coli codon preference can significantly improve expression levels.

  • Co-expression with chaperones: Including molecular chaperones like GroEL/GroES can assist in proper protein folding.

  • Solubility tags: Fusion with solubility-enhancing tags such as SUMO, MBP, or TrxA can improve yield of soluble protein.

• Alternative expression hosts:

  • Insect cell expression systems: For proteins that require eukaryotic post-translational processing.

  • Cell-free protein synthesis: Allows for rapid production and can accommodate proteins toxic to living cells.

• Induction and growth parameters:

  • IPTG concentration: Typically 0.1-1.0 mM, with lower concentrations often yielding more soluble protein.

  • Media formulation: Rich media like TB (Terrific Broth) often yields higher biomass and protein expression.

  • Induction timing: Inducing at mid-log phase (OD600 ~0.6-0.8) generally provides optimal balance between cell density and expression capacity.

Based on the methodologies described in the search results for similar proteins , an E. coli expression system with a His-tagged construct would likely be most effective for C. caviae PyrH, allowing for straightforward purification via nickel-affinity chromatography while maintaining enzymatic activity.

What purification strategies yield the highest purity and activity of recombinant PyrH?

To achieve the highest purity and activity of recombinant C. caviae PyrH, a multi-step purification strategy can be implemented:

• Initial capture by affinity chromatography:

  • Immobilized metal affinity chromatography (IMAC): For His-tagged PyrH, nickel-charged resin provides excellent initial purification. Based on protocols for similar proteins, a binding buffer containing 5 mM imidazole, 500 mM NaCl, and 20 mM Tris-HCl pH 7.9 can be used, followed by washing with increased imidazole concentration (60 mM) and elution with higher imidazole (200 mM) .

  • Gradient elution: Implementing a linear imidazole gradient (5-250 mM) can help separate PyrH from proteins with different binding affinities.

• Secondary purification:

  • Ion exchange chromatography: Based on the theoretical pI of PyrH, either anion (Q-Sepharose) or cation (SP-Sepharose) exchange chromatography can further remove contaminants.

  • Size exclusion chromatography (SEC): Particularly useful for separating monomeric from aggregated forms and removing remaining impurities while also performing buffer exchange.

• Buffer optimization for stability:

  • Addition of glycerol (5-10%) to prevent precipitation .

  • Inclusion of reducing agents (DTT or β-mercaptoethanol) to maintain reduced state of cysteine residues.

  • Addition of specific cofactors or substrates (Mg²⁺) that might stabilize the enzyme structure.

• Activity preservation strategies:

  • Maintain cold chain (4°C) throughout purification.

  • Include protease inhibitors during initial lysis and early purification steps.

  • Perform overnight dialysis at 4°C to remove imidazole, which can affect enzyme activity .

  • Consider buffer components that mimic physiological conditions (pH 7.5-8.0, 50-100 mM salts).

• Quality control assessments:

  • SDS-PAGE to verify purity (>95% homogeneity).

  • Western blotting to confirm identity.

  • Activity assays to ensure functional integrity throughout purification process.

  • Dynamic light scattering to assess homogeneity and absence of aggregation.

These strategies, based on successful purification of related enzymes, should yield highly pure and active recombinant C. caviae PyrH suitable for enzymatic, structural, and inhibitor studies.

How can site-directed mutagenesis be used to study the catalytic mechanism of C. caviae PyrH?

Site-directed mutagenesis represents a powerful approach to investigate the catalytic mechanism and structure-function relationships of C. caviae PyrH. This methodology can be implemented through the following systematic approach:

This comprehensive mutagenesis approach would provide detailed insights into the catalytic mechanism of C. caviae PyrH, identifying residues essential for substrate binding, catalysis, and regulation, ultimately informing structure-based inhibitor design.

What are the optimal conditions for measuring PyrH activity in vitro?

Establishing optimal conditions for measuring C. caviae PyrH activity in vitro is crucial for accurate kinetic analysis and inhibitor screening. Based on protocols used for similar enzymes, the following parameters should be optimized:

• Buffer composition and pH:

  • Buffer type: Tris-HCl, HEPES, or phosphate buffers in the range of 50-100 mM

  • Optimal pH range: Typically 7.5-8.0 for bacterial kinases, but should be determined experimentally through pH-activity profiling

  • Salt concentration: 50-150 mM NaCl or KCl to maintain ionic strength

• Essential cofactors:

  • Divalent cations: Mg²⁺ (typically 5-10 mM) is essential for nucleotide kinases as it coordinates with phosphate groups of ATP

  • Alternative cations: Mn²⁺ or Ca²⁺ may substitute for Mg²⁺ with altered activity and should be tested

• Substrate concentrations:

  • UMP: Range from 10 μM to 1 mM for kinetic parameter determination

  • ATP: Typically 0.5-2 mM, ensuring it's not limiting during assays

  • For inhibitor studies, substrate concentrations at or below their Km values are recommended

• Enzyme concentration and reaction time:

  • Enzyme should be diluted to ensure linear reaction rates over the measurement period

  • Reaction times should be established to stay within the linear range of product formation (typically 5-20 minutes)

• Temperature considerations:

  • Standard temperature of 37°C mimics physiological conditions

  • Temperature stability studies (25-45°C) can provide insights into thermal stability

  • For thermolabile compounds, lower temperatures may be necessary

• Reducing conditions:

  • Addition of reducing agents like DTT or β-mercaptoethanol (0.5-1 mM) may enhance stability

  • Determine if the enzyme contains essential cysteine residues that need to be maintained in reduced state

• Assay validation parameters:

  • Signal-to-background ratio: Aim for >10:1 for robust assay performance

  • Z'-factor: Calculate to ensure assay quality (Z' > 0.5 indicates excellent assay)

  • Positive and negative controls should be included in each assay plate

• Specialized considerations:

  • Protection from light if using photosensitive reagents

  • Oxygen-free environment for oxygen-sensitive components

  • Use of carrier proteins (BSA, 0.1%) to prevent enzyme adsorption to surfaces

These optimized conditions would provide a robust and reproducible assay for measuring C. caviae PyrH activity, suitable for both basic enzymatic characterization and high-throughput inhibitor screening.

How can kinetic parameters of recombinant C. caviae PyrH be accurately determined?

Accurate determination of kinetic parameters for recombinant C. caviae PyrH requires rigorous experimental design and data analysis approaches:

• Experimental design for kinetic studies:

  • Initial velocity measurements: Ensure reactions are measured in the linear range where less than 10% of substrate is consumed

  • Substrate variation: For each substrate (UMP and ATP), vary concentration across a wide range (0.1-10 × Km) while keeping the other substrate at saturating levels

  • Replicate measurements: Perform at least triplicate determinations for statistical validity

  • Controls: Include enzyme-free and substrate-free controls to account for background reactions

• Michaelis-Menten kinetics determination:

  • Plot initial velocity (v) versus substrate concentration [S]

  • Fit data to the Michaelis-Menten equation: v = Vmax × [S] / (Km + [S])

  • Determine Km (substrate concentration at half-maximal velocity) and Vmax (maximal velocity)

  • Calculate kcat = Vmax/[E]t where [E]t is the total enzyme concentration

  • Determine catalytic efficiency using the kcat/Km ratio

• Alternative plot methods for improved accuracy:

  • Lineweaver-Burk plot (1/v vs. 1/[S]): Useful for identifying inhibition patterns

  • Eadie-Hofstee plot (v vs. v/[S]): Less sensitive to errors at high substrate concentrations

  • Hanes-Woolf plot ([S]/v vs. [S]): Often provides more reliable parameter estimates

• Multi-substrate kinetic analysis:

  • Perform product inhibition studies to distinguish between sequential or ping-pong mechanisms

  • For bi-substrate reactions like PyrH catalysis, use initial velocity patterns to determine reaction order

  • Consider global fitting approaches for simultaneous analysis of multiple datasets

• Software tools for kinetic analysis:

  • GraphPad Prism, SigmaPlot, or DynaFit for curve fitting and parameter estimation

  • R or Python with specialized biochemical kinetics packages for custom analysis

  • Monte Carlo simulations to estimate confidence intervals

• Advanced kinetic considerations:

  • Test for substrate inhibition at high concentrations

  • Evaluate product inhibition patterns

  • Assess allosteric regulation by nucleotides like GTP or UTP

  • Determine temperature and pH dependence of kinetic parameters

• Reporting standards:

  • Include standard errors or confidence intervals for all parameters

  • Report conditions precisely (temperature, pH, buffer composition)

  • Provide raw data plots alongside fitted curves

  • Compare parameters with those of PyrH from related organisms

These approaches would provide comprehensive and reliable kinetic characterization of recombinant C. caviae PyrH, essential for understanding its catalytic mechanism and for rational inhibitor design.

What computational approaches can be used to predict inhibitors of C. caviae PyrH?

Computational approaches offer powerful tools for predicting potential inhibitors of C. caviae PyrH, accelerating the drug discovery process. The following integrated computational strategies can be employed:

• Structure-based virtual screening:

  • Homology modeling: Generate a 3D model of C. caviae PyrH based on crystal structures of homologous bacterial UMP kinases if experimental structures are unavailable

  • Molecular docking: Screen virtual compound libraries against the ATP-binding site, UMP-binding site, or allosteric regulatory sites

  • Ensemble docking: Use multiple protein conformations to account for receptor flexibility

  • Binding free energy calculations: Employ MM-GBSA or MM-PBSA methods to refine docking predictions

• Ligand-based approaches:

  • Pharmacophore modeling: Develop pharmacophore hypotheses based on known UMP kinase inhibitors or natural substrates

  • Quantitative structure-activity relationship (QSAR): Build predictive models using activity data from related kinase inhibitors

  • Similarity searching: Identify compounds structurally similar to known UMP kinase inhibitors

  • Fragment-based design: Identify molecular fragments that bind to different sites and link them to create novel inhibitors

• Machine learning methods:

  • Deep learning models: Train neural networks on kinase inhibitor datasets to predict activity against PyrH

  • Support vector machines: Classify compounds based on predicted activity

  • Random forest models: Identify important molecular features for PyrH inhibition

  • Transfer learning: Leverage knowledge from other bacterial kinase inhibitor data

• Molecular dynamics simulations:

  • Binding mode analysis: Simulate protein-ligand complexes to assess stability of binding modes

  • Identification of cryptic binding sites: Reveal transient pockets not evident in static structures

  • Water displacement analysis: Identify energetically favorable displacement of water molecules

  • Allostery investigation: Study how binding at one site affects dynamics at distant sites

• Integration with experimental data:

  • Structure-activity relationship (SAR) analysis: Iteratively refine computational models based on experimental testing of predictions

  • Target-specific scoring functions: Calibrate scoring functions using known PyrH inhibitors

  • Active learning: Prioritize compounds for experimental testing based on model uncertainty

• Specific considerations for PyrH:

  • Selectivity modeling: Compare binding site characteristics between bacterial PyrH and human nucleoside diphosphate kinases to design selective inhibitors

  • Lead compound PYRH-1: Use the structure mentioned in search result as a starting point for analog design

  • Scaffold hopping: Design novel chemical scaffolds with similar pharmacophoric properties to known inhibitors

These computational approaches, used in an integrated fashion, would significantly enhance the efficiency of identifying promising inhibitors of C. caviae PyrH for experimental validation.

How can structural data be integrated with functional assays to understand PyrH mechanism?

Integrating structural data with functional assays provides a comprehensive approach to understanding the catalytic mechanism of C. caviae PyrH. This multidisciplinary strategy combines atomic-level insights with functional characterization:

• Structure-guided mutagenesis:

  • Identify residues at the active site or substrate-binding regions based on structural data

  • Design mutations to test specific hypotheses about their roles in catalysis

  • Correlate structural positions with kinetic effects of mutations

  • Use alanine scanning followed by more specific mutations to probe side chain contributions

• Substrate analog studies:

  • Design substrate analogs based on structural binding modes

  • Test modified substrates with specific changes to probe recognition determinants

  • Correlate structural binding predictions with experimental kinetic parameters

  • Use substrate analogs as mechanistic probes or inhibitors

• Transition state analysis:

  • Use structural data to model potential transition states during phosphoryl transfer

  • Design transition state analogs as potential inhibitors

  • Measure binding affinity of transition state analogs and correlate with structural models

  • Calculate activation energies based on temperature-dependent kinetic studies

• Conformational dynamics investigation:

  • Identify potential conformational changes during catalysis from structures in different states

  • Use site-directed spin labeling and EPR or FRET to measure conformational changes

  • Correlate observed conformational changes with steps in the catalytic cycle

  • Apply molecular dynamics simulations to model conformational transitions

• Ligand binding studies:

  • Use isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR) to measure binding parameters

  • Correlate thermodynamic binding parameters with structural interactions

  • Perform competition assays to map binding sites of substrates and inhibitors

  • Use fluorescent probes to monitor binding events in real-time

• pH and ionic strength dependencies:

  • Determine pH-rate profiles to identify catalytic ionizable groups

  • Compare with structural data to identify candidate residues

  • Test the role of identified residues through site-directed mutagenesis

  • Measure effects of ionic strength on catalysis and correlate with electrostatic maps from structures

• Crystallographic and spectroscopic studies of enzyme-substrate complexes:

  • Obtain crystal structures with bound substrates, products, or substrate analogs

  • Use time-resolved methods to capture catalytic intermediates

  • Apply NMR spectroscopy to map chemical shift perturbations upon ligand binding

  • Correlate spectroscopic observations with structural models

This integrated approach would provide a detailed mechanistic understanding of C. caviae PyrH, revealing the structural basis for substrate specificity, catalysis, and regulation, which could be exploited for the rational design of specific inhibitors.