Recombinant Chlamydophila caviae Orotate phosphoribosyltransferase (pyrE)

What is Chlamydophila caviae Orotate phosphoribosyltransferase (pyrE)?

Orotate phosphoribosyltransferase (OPRTase, EC 2.2.4.10) is an essential enzyme in the pyrimidine biosynthetic pathway encoded by the pyrE gene in Chlamydophila caviae. This enzyme catalyzes the critical reaction transferring ribose-5'-phosphate from 5'-phosphoribosyl-1'-pyrophosphate (PRPP) to orotate (OA) to form orotidine-5'-monophosphate (OMP), which represents the first pyrimidine nucleotide formed in the pathway. The reaction is a key step in the de novo synthesis of UMP, which is subsequently converted to UTP and CTP—essential pyrimidine nucleotides for RNA synthesis and cellular metabolism . Within C. caviae, this enzyme functions as part of the conserved set of genes that are present across all sequenced Chlamydiaceae genomes, indicating its fundamental importance to chlamydial biology and survival .

How is the pyrE gene conserved across Chlamydiaceae species?

Analysis of the C. caviae genome reveals that pyrE belongs to the core set of genes conserved across all four sequenced Chlamydiaceae genomes (C. caviae, C. muridarum, C. trachomatis, and C. pneumoniae). Of the 1009 annotated genes in C. caviae, approximately 798 (79%) are conserved across all four species, with pyrE being among these highly conserved genes . This high degree of conservation underscores the essential nature of pyrimidine biosynthesis in chlamydial metabolism.

The conservation pattern can be visualized through BLAST Score Ratio (BSR) analysis, which indicates that pyrE in C. caviae shows higher similarity to its ortholog in C. pneumoniae compared to C. muridarum or C. trachomatis. This aligns with the phylogenetic relationships between these species and supports the evolutionary significance of maintaining functional pyrE across the Chlamydiaceae family .

What is known about the genomic context of pyrE in C. caviae?

The pyrE gene in C. caviae exists within the context of the complete 1,173,390 nucleotide genome. Unlike some bacterial species where pyrimidine biosynthesis genes are organized in operons, the genomic organization of pyrE in Chlamydiaceae may differ. In other bacterial species such as Bacillus subtilis and Pseudomonas species, pyrimidine biosynthesis genes are often arranged in specific operon structures with regulatory elements .

While the exact genomic organization around pyrE in C. caviae is not detailed in the provided search results, comparative genomic studies show that C. caviae maintains essential biosynthetic pathways despite its reduced genome size, which is characteristic of obligate intracellular pathogens. The presence of pyrE suggests that C. caviae retains the capability for de novo pyrimidine synthesis rather than relying entirely on host-derived nucleotides .

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

Based on established protocols for working with chlamydial proteins, several expression systems can be considered for recombinant C. caviae pyrE production:

• E. coli Expression Systems: The most commonly used approach involves cloning the pyrE gene into vectors such as pET series (T7 promoter-based) or pBAD (arabinose-inducible) systems. For optimal expression, codon optimization may be necessary given the different codon usage between Chlamydiaceae and E. coli.

• Lambda Phage Expression Systems: The methodology described for constructing genomic DNA expression libraries of C. felis can be adapted for C. caviae pyrE expression. This involves partially digesting genomic DNA with appropriate restriction enzymes (such as EcoRI), ligating into λ-ZAPII phage arms, and packaging into phage particles .

• Cell-Free Expression Systems: For proteins that may be toxic to host cells, cell-free systems based on E. coli extracts supplemented with T7 RNA polymerase can be employed.

The choice of expression system should consider factors such as the need for post-translational modifications, protein solubility, and the desired yield. For functional studies, maintaining proper folding and activity of the recombinant enzyme is critical.

What purification strategies yield highest purity recombinant C. caviae pyrE?

A systematic purification strategy for recombinant C. caviae pyrE typically involves:

• Affinity Chromatography: Expressing pyrE with an affinity tag (His6, GST, or MBP) allows for initial purification using metal affinity (Ni-NTA for His-tagged proteins), glutathione affinity (for GST-fusion proteins), or amylose affinity (for MBP-fusion proteins) chromatography.

• Ion Exchange Chromatography: Based on the theoretical isoelectric point of pyrE, anion or cation exchange chromatography can be employed as a secondary purification step to remove contaminants with different charge properties.

• Size Exclusion Chromatography: As a final polishing step, gel filtration can separate the target protein from aggregates and remaining impurities based on molecular size.

For structural studies requiring extremely high purity, additional techniques such as hydroxyapatite chromatography or hydrophobic interaction chromatography may be necessary. The purification process should be optimized with buffers containing stabilizing agents (such as glycerol or specific metal ions) that maintain enzymatic activity.

How can researchers verify the activity of purified recombinant C. caviae pyrE?

Several complementary approaches can be used to assess the enzymatic activity of recombinant C. caviae pyrE:

• Spectrophotometric Assays: The formation of OMP from orotate and PRPP can be monitored by measuring the change in absorbance at 295-300 nm, which reflects the conversion of orotate to OMP.

• Coupled Enzyme Assays: The pyrE reaction can be coupled with subsequent enzymatic reactions (such as OMP decarboxylase) to produce UMP, which can be monitored through various detection methods.

• Radiometric Assays: Using [14C]-labeled orotate or [32P]-labeled PRPP allows for sensitive detection of product formation through scintillation counting or autoradiography.

• HPLC Analysis: Products and substrates can be separated and quantified using high-performance liquid chromatography, providing detailed kinetic information.

When establishing these assays, it is essential to determine optimal conditions, including pH, temperature, metal ion requirements, and substrate concentrations that may be specific to the C. caviae enzyme.

How does C. caviae pyrE structure compare to orthologs from other bacterial species?

While specific structural information for C. caviae pyrE is not provided in the search results, comparative analysis can be inferred based on conserved features of orotate phosphoribosyltransferases across bacterial species:

Orotate phosphoribosyltransferases typically belong to the Type I PRTase superfamily, characterized by a core α/β structure with a five-stranded parallel β-sheet surrounded by α-helices. Key structural elements include:

• PRPP Binding Site: A conserved region with positively charged residues that interact with the phosphate groups of PRPP.

• Orotate Binding Pocket: Often contains aromatic residues that form stacking interactions with the pyrimidine ring of orotate.

• Flexible Loops: Dynamic regions that undergo conformational changes upon substrate binding, essential for catalysis.

The BSR analysis mentioned in the search results indicates that C. caviae proteins, including pyrE, generally show higher similarity to C. pneumoniae orthologs than to those from C. trachomatis or C. muridarum . This suggests that structural features may be more conserved between C. caviae and C. pneumoniae pyrE enzymes, potentially reflecting their evolutionary relationship and similar functional constraints.

What are the predicted catalytic mechanisms of C. caviae pyrE?

The catalytic mechanism of orotate phosphoribosyltransferase typically involves several steps:

• Ordered Binding: PRPP typically binds first, followed by orotate, inducing conformational changes that bring catalytic residues into optimal positions.

• Nucleophilic Attack: The N1 nitrogen of orotate acts as a nucleophile, attacking the C1' carbon of the ribose in PRPP, displacing the pyrophosphate group.

• Stabilization of Transition State: Conserved residues, often including aspartate or glutamate, coordinate divalent metal ions (typically Mg2+) that stabilize the negatively charged transition state.

• Product Formation and Release: After formation of OMP, pyrophosphate is released first, followed by OMP.

While specific catalytic residues for C. caviae pyrE are not identified in the search results, they are likely conserved with other bacterial orotate phosphoribosyltransferases based on the essential nature of this reaction and the high conservation of pyrE across Chlamydiaceae .

What is the relationship between pyrE function and C. caviae pathogenicity?

The relationship between pyrE function and C. caviae pathogenicity can be understood in several contexts:

• Essential Metabolism: As a key enzyme in pyrimidine biosynthesis, pyrE is essential for nucleotide production, DNA replication, and RNA synthesis, all critical for chlamydial growth and virulence.

• Model Organism Significance: C. caviae (GPIC) serves as an excellent model for naturally occurring C. trachomatis infections in humans, with similarities in transmission mechanisms and disease progression . The conservation of pyrE across these species suggests its fundamental role in the chlamydial life cycle.

• Potential Therapeutic Target: Given its essential nature and differences from host enzymes, pyrE represents a potential target for developing anti-chlamydial therapeutics that could disrupt pyrimidine metabolism.

The search results indicate that C. caviae provides a valuable model for studying chlamydial infections, particularly for ocular and genital infections that show similar pathologic endpoints to human C. trachomatis infections, including corneal damage and tubal blockage . The role of pyrE in supporting bacterial growth and replication would be integral to these pathogenic processes.

How do nucleotide metabolism enzymes like pyrE integrate with other metabolic pathways in C. caviae?

Orotate phosphoribosyltransferase functions at a critical intersection of metabolic pathways in C. caviae:

• Connection to Central Carbon Metabolism: The substrate PRPP links pyrE activity to the pentose phosphate pathway, which generates ribose-5-phosphate from glucose metabolism.

• Integration with Amino Acid Metabolism: The pyrimidine biosynthetic pathway begins with carbamoyl phosphate synthesis from glutamine, connecting pyrimidine synthesis to amino acid metabolism.

• Relationship to Energy Metabolism: ATP is required for various steps in the pyrimidine biosynthetic pathway, linking nucleotide synthesis to energy generation in the cell.

C. caviae, like other Chlamydiaceae, has a reduced genome with streamlined metabolic capabilities due to its obligate intracellular lifestyle. The genome sequence reveals that C. caviae retains 19 genes involved in amino acid biosynthesis, compared to 15 in C. muridarum and 14 in C. pneumoniae . This suggests that C. caviae may have more robust biosynthetic capabilities, potentially including a more self-sufficient pyrimidine biosynthetic pathway where pyrE plays a key role.

What challenges exist in designing selective inhibitors of C. caviae pyrE?

Designing selective inhibitors for C. caviae pyrE presents several research challenges:

• Structural Similarity to Host Enzymes: Human cells also possess orotate phosphoribosyltransferase activity as part of the UMP synthase bifunctional enzyme. Selective targeting requires identifying structural or mechanistic differences between bacterial and host enzymes.

• Conservation Across Bacterial Species: The high conservation of pyrE across bacterial species means that inhibitors might have broad-spectrum activity, potentially affecting the host microbiome.

• Access to Intracellular Targets: As C. caviae is an obligate intracellular pathogen, inhibitors must penetrate host cell membranes and the bacterial cell envelope to reach the target.

• Resistance Development: Mutations in pyrE could potentially confer resistance to inhibitors, necessitating strategies to minimize resistance development.

Rational design approaches would benefit from detailed structural information about C. caviae pyrE, particularly in complex with substrates or product analogs, to identify unique binding sites or conformational states that could be exploited for selective inhibition.

How might gene expression data inform understanding of pyrE regulation in C. caviae?

Gene expression analysis of pyrE in C. caviae could provide valuable insights into regulatory mechanisms:

• Developmental Cycle Regulation: Chlamydiae undergo a biphasic developmental cycle, transitioning between elementary bodies (EBs) and reticulate bodies (RBs). Temporal expression patterns of pyrE during this cycle could indicate when pyrimidine biosynthesis is most active.

• Response to Nutrient Availability: Expression changes in response to varying nucleotide or precursor availability could reveal regulatory mechanisms that coordinate pyrimidine synthesis with cellular needs.

• Stress Responses: Changes in pyrE expression under stress conditions (antibiotics, immune factors, nutrient limitation) might indicate roles in adaptation or persistence.

While the search results do not provide specific information about pyrE regulation in C. caviae, the techniques described for working with chlamydial species, including purification of elementary bodies and genomic DNA extraction methods , provide a foundation for gene expression studies that could elucidate these regulatory aspects.

How does the genomic context of pyrE differ among Chlamydiaceae species?

The genomic context of pyrE across Chlamydiaceae species can provide insights into evolutionary adaptations and regulatory mechanisms:

While specific details about the genomic neighborhood of pyrE in each species are not provided in the search results, the comparative genomic analysis indicates that about three-quarters of C. caviae genes, including pyrE, encode functions conserved across all four species . The remaining genes appear to encode "niche-specific" functions necessary for survival and virulence in specific sites or hosts.

What functional differences in pyrE might contribute to host specificity among Chlamydiaceae?

While the core catalytic function of pyrE is conserved across Chlamydiaceae, subtle variations in enzyme properties might contribute to host adaptation:

• Substrate Affinity: Differences in kinetic parameters (Km, kcat) for orotate or PRPP could reflect adaptation to varying nucleotide precursor availability in different host cells or tissues.

• Regulatory Mechanisms: Species-specific regulation of pyrE expression or activity might facilitate adaptation to different host environments.

• Protein-Protein Interactions: Variations in surface residues might enable different interactions with other metabolic enzymes or regulatory proteins.

The BSR analysis mentioned in the search results indicates that C. caviae proteins show a bias toward greater similarity to C. pneumoniae versus C. trachomatis and C. muridarum . This pattern, while not specific to pyrE in the results, suggests evolutionary divergence that might reflect adaptation to different hosts (guinea pigs for C. caviae, humans for C. pneumoniae and C. trachomatis, and mice for C. muridarum).

How can recombinant C. caviae pyrE be used to understand evolutionary relationships in Chlamydiaceae?

Recombinant C. caviae pyrE can serve as a valuable tool for evolutionary studies in several ways:

• Biochemical Characterization: Comparative analysis of kinetic parameters, substrate specificity, and inhibitor sensitivity across recombinant pyrE enzymes from different Chlamydiaceae species can provide insights into functional evolution.

• Structural Comparisons: Solving structures of pyrE from multiple Chlamydiaceae species would allow identification of conserved catalytic cores versus variable regions that might reflect species-specific adaptations.

• Complementation Studies: Cross-species complementation experiments using recombinant pyrE could test functional conservation and reveal species-specific requirements.

The search results indicate that C. caviae provides a good model for the Chlamydiaceae family and a point of comparison against human-associated species like C. pneumoniae . Studies with recombinant pyrE could extend this comparative approach to the molecular level, potentially revealing evolutionary signatures that correlate with host range or tissue tropism.

What quality control measures ensure reliable research with recombinant C. caviae pyrE?

To ensure reproducible and reliable research with recombinant C. caviae pyrE, several quality control measures should be implemented:

• Sequence Verification: Confirm the cloned pyrE sequence matches the reference genome sequence of C. caviae strain (e.g., Fe/C-56 as mentioned in search result ), accounting for any introduced tags or mutations.

• Purity Assessment: Analyze purified protein by SDS-PAGE, mass spectrometry, and size exclusion chromatography to ensure homogeneity and correct molecular weight.

• Activity Benchmarking: Establish standard assay conditions and determine baseline kinetic parameters (Km, Vmax, kcat) for wild-type enzyme to serve as reference for subsequent studies.

• Stability Testing: Assess enzyme stability under various storage conditions (temperature, buffer composition, presence of stabilizing agents) to ensure consistent activity across experiments.

• Batch-to-Batch Consistency: Implement quality metrics to compare protein batches, including specific activity measurements and thermal stability profiles.

These measures are particularly important when working with enzymes from fastidious organisms like C. caviae, where the native protein would be difficult to isolate in sufficient quantities for direct study.

What bioinformatic approaches can predict substrate specificity in C. caviae pyrE?

Several bioinformatic approaches can provide insights into the substrate specificity of C. caviae pyrE:

• Multiple Sequence Alignment: Aligning pyrE sequences from diverse species can identify conserved residues in substrate binding regions and species-specific variations that might affect specificity.

• Homology Modeling: Using known crystal structures of orotate phosphoribosyltransferases (e.g., from E. coli or Salmonella) as templates to model C. caviae pyrE structure, with particular focus on substrate binding pocket geometry.

• Molecular Docking: In silico docking of orotate, PRPP, and potential substrate analogs to predicted structures can estimate binding affinities and identify key interaction residues.

• Molecular Dynamics Simulations: Simulating enzyme-substrate complexes can reveal dynamic aspects of binding and conformational changes that may influence specificity.

• Evolutionary Trace Analysis: Mapping sequence conservation patterns onto predicted structures can identify functionally important regions that may determine specificity.

These computational approaches can generate testable hypotheses about residues that determine substrate specificity, guiding experimental design for site-directed mutagenesis studies.

How might CRISPR-based tools advance functional studies of pyrE in C. caviae?

While genetic manipulation of Chlamydiaceae has been challenging due to their obligate intracellular lifestyle, CRISPR-based approaches offer promising avenues for pyrE functional studies:

• Conditional Knockdown Systems: CRISPR interference (CRISPRi) could be adapted to create conditional pyrE knockdown strains, allowing assessment of growth phenotypes when pyrimidine synthesis is compromised.

• Base Editing: CRISPR-based base editors could introduce specific point mutations in the native pyrE gene to study structure-function relationships without complete gene disruption.

• Gene Replacement: CRISPR-Cas9 could facilitate homology-directed repair to replace wild-type pyrE with variant alleles or tagged versions for localization studies.

• Transcriptional Reporters: CRISPR systems could be used to introduce reporter constructs linked to the pyrE promoter to monitor expression dynamics during the developmental cycle.

Implementation of these approaches would require optimization of transformation methods for C. caviae, potentially adapting techniques from the growing toolkit for genetic manipulation of other Chlamydia species.

What potential exists for repurposing existing nucleotide metabolism inhibitors against C. caviae pyrE?

The investigation of existing nucleotide metabolism inhibitors for activity against C. caviae pyrE could accelerate therapeutic development:

• Anticancer Compounds: Several orotate phosphoribosyltransferase inhibitors developed as anticancer agents target human UMPS and could be assessed for activity against C. caviae pyrE.

• Antimalarial Agents: Compounds targeting plasmodial pyrimidine metabolism enzymes might show cross-reactivity with chlamydial enzymes due to conserved catalytic mechanisms.

• Nucleoside/Nucleotide Analogs: FDA-approved nucleoside analogs could be assessed as potential substrates or inhibitors of C. caviae pyrE.

• Fragment-Based Screening: Libraries of small molecular fragments could be screened against recombinant C. caviae pyrE to identify novel chemical scaffolds with inhibitory activity.

Structure-based methods would be valuable for predicting potential interactions and guiding medicinal chemistry efforts to optimize selectivity for the bacterial enzyme over human counterparts.

How might advanced imaging techniques enhance understanding of pyrE localization and dynamics in C. caviae?

Advanced imaging approaches could provide unprecedented insights into pyrE function in the context of the intracellular chlamydial development cycle:

• Fluorescent Protein Fusions: Creating functional fluorescent protein fusions with pyrE could allow real-time tracking of enzyme localization during different developmental stages.

• Super-Resolution Microscopy: Techniques such as STORM or PALM could resolve the subcellular distribution of pyrE within the chlamydial inclusion at nanometer resolution.

• FRET-Based Biosensors: Developing biosensors for pyrimidine metabolites could allow visualization of metabolic dynamics in relation to pyrE activity and localization.

• Correlative Light and Electron Microscopy: Combining fluorescence imaging of tagged pyrE with ultrastructural analysis could relate enzyme localization to specific chlamydial structures.

These approaches would require adaptation of the methods described in the search results for culturing chlamydial species in cell lines and purifying elementary bodies , along with development of genetic tools for expressing tagged proteins in C. caviae.