Recombinant Chlamydophila caviae Deoxyuridine 5'-triphosphate nucleotidohydrolase (dut)

What is the primary function of Deoxyuridine 5'-triphosphate nucleotidohydrolase (dut) in Chlamydophila caviae?

Deoxyuridine 5'-triphosphate nucleotidohydrolase (dut) in Chlamydophila caviae serves two critical functions in nucleotide metabolism. First, it catalyzes the hydrolysis of dUTP to produce dUMP, which is the immediate precursor required for thymidine nucleotide biosynthesis. Second, it plays a crucial role in maintaining DNA integrity by decreasing the intracellular concentration of dUTP, thereby preventing the misincorporation of uracil into DNA during replication and repair processes . This function is particularly important in bacterial systems where maintaining genomic stability is essential for pathogen survival. The enzyme belongs to the broader dUTPase family, which is highly conserved across bacterial species, indicating its evolutionary significance in nucleotide metabolism pathways.

How does Chlamydophila caviae dut compare to orthologous proteins in related species?

Comparative analysis of Chlamydophila caviae dut with orthologous proteins in related Chlamydial species reveals significant insights into evolutionary conservation and adaptation:

The dut gene exists within a core genome that is highly conserved across Chlamydophila species, with most of the variation occurring in membrane proteins and pathogenicity factors rather than metabolic enzymes . While the core enzymatic function remains consistent, subtle variations in sequence and structure may reflect adaptations to different host environments and infection strategies. Significantly, the conservation of dut across these species underscores its essential role in bacterial metabolism and potential value as a therapeutic target.

What expression systems are most effective for producing recombinant Chlamydophila caviae dut?

For optimal expression of recombinant Chlamydophila caviae dut, several expression systems can be considered based on research needs:

• E. coli Expression System: The most common approach uses pET-based vectors with T7 promoter systems in E. coli BL21(DE3) or its derivatives. This system typically yields 10-20 mg of soluble protein per liter of culture under optimized conditions.

• Methodology for Optimized Expression:

  • Culture conditions: Growth at 30°C rather than 37°C after induction often improves solubility

  • Induction: Lower IPTG concentrations (0.1-0.5 mM) and extended expression times (16-18 hours)

  • Buffer composition: Inclusion of 5-10% glycerol and 1-5 mM DTT in lysis buffers enhances stability

• Purification Strategy:

  • His-tagged constructs allow for initial IMAC (immobilized metal affinity chromatography)

  • Size exclusion chromatography as a second step ensures high purity

  • Typical yields of >90% pure protein are achievable following this two-step purification

The recombinant protein can be expressed with various affinity tags (His, GST, MBP) to facilitate purification, with consideration given to the potential impact of these tags on enzymatic activity. For structural studies requiring tag removal, precision protease cleavage sites can be incorporated into the construct design.

What assay methods are used to measure Chlamydophila caviae dut enzymatic activity?

Several complementary methods can be employed to assess the enzymatic activity of Chlamydophila caviae dut:

• Spectrophotometric Assays:

  • Cresol Red Assay: Measures proton release during dUTP hydrolysis by monitoring absorbance changes at 573 nm

  • Coupled Enzyme Assay: Links dUMP production to subsequent enzymatic reactions that generate measurable spectrophotometric signals

• HPLC-Based Methods:

  • Direct quantification of substrate (dUTP) consumption and product (dUMP) formation

  • Provides precise kinetic parameters (Km, kcat, kcat/Km)

  • Typical Km values for dUTPases range from 0.5-5 μM for dUTP

• Isothermal Titration Calorimetry (ITC):

  • Measures heat released during catalysis

  • Provides thermodynamic parameters along with kinetic data

  • Can determine binding affinity for substrates and inhibitors

• Malachite Green Assay:

  • Detects inorganic pyrophosphate (PPi) released during the reaction

  • Simple colorimetric detection at 630 nm

Standard reaction conditions typically include: Tris-HCl buffer (pH 7.5-8.0), 5-10 mM MgCl₂ (cofactor), 1-100 μM dUTP (substrate), and controlled temperature (25-37°C). Proper enzyme dilution is essential to ensure linear reaction rates during initial velocity measurements.

What role does Chlamydophila caviae dut play in pathogen survival and replication?

Deoxyuridine 5'-triphosphate nucleotidohydrolase plays a multifaceted role in Chlamydophila caviae survival and pathogenicity:

• Genomic Integrity Maintenance: By preventing uracil incorporation into DNA, dut helps maintain the integrity of the chlamydial genome during the rapid replication that occurs in reticulate bodies (RBs). This is particularly critical in Chlamydophila species, which undergo unique biphasic developmental cycles with intensive DNA replication phases .

• Nucleotide Pool Regulation: The enzyme regulates the balance between dUTP and dTTP pools, which is essential for proper DNA replication and repair. This regulation is especially important in intracellular pathogens that rely on host nucleotide precursors.

• Host-Pathogen Interface: Research suggests that dut activity may influence host inflammatory responses by affecting nucleotide-sensing pathways. The enzyme's presence in the context of Chlamydophila caviae infection may modulate host cell responses through altered nucleotide signaling.

• Developmental Cycle Progression: Proteomic studies of related Chlamydia species indicate that dUTPase activity varies between the infectious elementary body (EB) and the replicative reticulate body (RB) forms, suggesting stage-specific roles in the chlamydial developmental cycle .

The importance of dut is underscored by its conservation across Chlamydophila species despite the considerable genome reduction these obligate intracellular pathogens have undergone through evolution. This conservation suggests strong selective pressure to maintain dut function, making it a potential target for antibacterial strategies.

How do mutations in key residues affect Chlamydophila caviae dut activity and structure?

Site-directed mutagenesis studies provide critical insights into structure-function relationships of Chlamydophila caviae dut:

Mutations in the predicted catalytic residues typically result in proteins that retain structural integrity but show dramatically reduced catalytic efficiency. Circular dichroism spectroscopy can confirm that these mutations do not significantly alter secondary structure content, while thermal denaturation studies often reveal subtle changes in protein stability.

The most informative mutations target: (1) the predicted catalytic aspartate that coordinates magnesium, (2) the conserved arginine involved in triphosphate binding, and (3) residues forming the uracil-binding pocket that confer nucleotide specificity. These structure-function studies not only elucidate the catalytic mechanism but also provide valuable information for the rational design of specific inhibitors.

What are the challenges in crystallizing Chlamydophila caviae dut and determining its structure?

Researchers face several specific challenges when attempting to crystallize Chlamydophila caviae dut:

• Protein Stability Issues:

  • The enzyme may exhibit conformational heterogeneity, particularly in the C-terminal region

  • Buffer optimization critical: screening with various pH ranges (6.5-8.5) and salt concentrations (50-300 mM NaCl)

  • Addition of divalent cations (5-10 mM MgCl₂) and nucleotide analogs often enhances stability

• Crystallization Bottlenecks:

  • Initial crystallization hits frequently yield microcrystals requiring extensive optimization

  • Successful approaches include:

    • Sitting-drop vapor diffusion with PEG-based precipitants (PEG 3350, 20-25%)

    • Inclusion of nucleotide analogs (dUMP, dUDP, or non-hydrolyzable dUTP analogs)

    • Seeding techniques to improve crystal quality

• Structural Analysis Complexities:

  • Potential conformational changes between apo, substrate-bound, and product-bound states

  • Order-disorder transitions in functional loop regions

  • Oligomerization state may differ between solution and crystal

Successful structural determination typically requires multiple approaches, including co-crystallization with substrate analogs, product molecules, or inhibitors to stabilize specific conformational states. Molecular replacement using related dUTPase structures as search models is generally successful for phase determination, as the core fold is well conserved across the dUTPase family.

How does Chlamydophila caviae dut compare functionally with human dUTPase?

Comparative analysis between Chlamydophila caviae dut and human dUTPase reveals important differences that can be exploited for therapeutic targeting:

Despite sharing the core dUTPase fold and catalytic mechanism, the bacterial and human enzymes exhibit subtle differences in active site architecture that can be exploited for selective inhibitor design. The bacterial enzyme typically shows distinct patterns of sensitivity to nucleotide analogs and small molecule inhibitors compared to its human counterpart.

These differences provide the structural basis for developing pathogen-specific inhibitors that target Chlamydophila caviae dut without affecting human dUTPase activity, potentially minimizing side effects in therapeutic applications.

What is the significance of Chlamydophila caviae dut in bacterial evolution and genomic adaptation?

Evolutionary analysis of Chlamydophila caviae dut provides important insights into bacterial adaptation and genome evolution:

• Conservation Patterns:

  • The dut gene is part of the 842 conserved coding sequences shared between Chlamydophila caviae and Chlamydophila abortus, indicating its essential role in chlamydial biology

  • The high conservation of dut contrasts with significant variation in other gene families like membrane proteins and secretion systems

• Selective Pressure Analysis:

  • dut shows evidence of purifying selection (dN/dS ratios typically <1), emphasizing its essential metabolic function

  • This conservation occurs despite considerable genome reduction in Chlamydophila species, highlighting its indispensable role

• Genomic Context:

  • Unlike some metabolic genes that are lost in host-adapted bacteria, dut is retained across Chlamydophila species

  • This retention contrasts with the loss of other nucleotide metabolism genes in some Chlamydophila species, such as guaB which is a pseudogene in Chlamydophila abortus

• Functional Adaptation:

  • The maintained functionality of dut across diverse Chlamydophila species with different host tropisms suggests it represents a core metabolic function essential for intracellular survival

  • This contrasts with variability in other gene families such as those encoding polymorphic membrane proteins (Pmps) and transmembrane head (TMH) proteins that show greater diversification

The retention of functional dut in Chlamydophila caviae and related species that have undergone significant genome reduction highlights the essential nature of uracil exclusion from DNA even in highly host-adapted bacterial pathogens. This evolutionary conservation makes dut an attractive target for broad-spectrum therapeutic development against chlamydial infections.

What biomedical applications could target Chlamydophila caviae dut?

Targeting Chlamydophila caviae dut presents several promising biomedical applications:

• Antimicrobial Development:

  • Nucleotide analogs that competitively inhibit dut could disrupt bacterial DNA replication

  • Structure-based drug design strategies focusing on active site differences between bacterial and human enzymes

  • Potential inhibitor classes:

    • Modified uracil derivatives with altered sugar or phosphate moieties

    • Transition state analogs mimicking the pentacoordinate phosphorus intermediate

    • Allosteric inhibitors targeting trimeric interface regions

• Diagnostic Applications:

  • Development of nucleic acid amplification tests targeting the dut gene for detecting Chlamydophila caviae

  • Particularly valuable for differentiating C. caviae-like strains from C. trachomatis in clinical specimens

  • Potential application in diagnosing C. caviae-like infections in humans, which were found in 8.9% of specimens from patients with cervicitis or urethritis

• Vaccine Development:

  • Recombinant dut as a potential component in subunit vaccines against chlamydial infections

  • Chimeric constructs linking dut to immunogenic epitopes from surface antigens to enhance immune recognition

• Research Tools:

  • Recombinant dut as a tool for studying nucleotide metabolism in bacterial systems

  • Reporter constructs utilizing dut activity for high-throughput screening of inhibitors

The biological significance of dut, combined with its structural differences from the human ortholog, positions it as a promising target for next-generation antibiotics against Chlamydophila infections, which could address the growing concern of antimicrobial resistance in these pathogens.

What are the optimal methods for measuring kinetic parameters of Chlamydophila caviae dut?

Rigorous determination of Chlamydophila caviae dut kinetic parameters requires careful experimental design:

• Steady-State Kinetics Protocol:

  • Sample Preparation: Recombinant enzyme at 10-50 nM concentration, depending on specific activity

  • Reaction Conditions: 50 mM Tris-HCl (pH 7.8), 5 mM MgCl₂, 1 mM DTT, 25°C

  • Substrate Range: dUTP concentrations from 0.1-100 μM (spanning 0.1× to 10× expected Km)

  • Data Collection: Initial rates measured at ≤10% substrate conversion to ensure steady-state conditions

• Analytical Methods Comparison:

• Data Analysis Framework:

  • Michaelis-Menten equation: v = Vmax[S]/(Km+[S])

  • Lineweaver-Burk, Eadie-Hofstee, and Hanes-Woolf plots for graphical validation

  • Non-linear regression using enzyme kinetics software (GraphPad Prism, DynaFit)

  • Statistical validation through residual analysis and confidence interval determination

• Parameter Reporting Standards:

  • kcat (s⁻¹): Turnover number calculated from Vmax/[E]t

  • Km (μM): Substrate concentration at half-maximal velocity

  • Catalytic efficiency: kcat/Km (M⁻¹s⁻¹)

  • Experimental conditions clearly specified (temperature, pH, buffer composition)

For inhibition studies, similar approaches apply with the addition of varying inhibitor concentrations and determination of inhibition constants (Ki) and mechanisms (competitive, non-competitive, uncompetitive, or mixed).

How can proteomic approaches be applied to study dut expression in Chlamydophila caviae?

Advanced proteomic techniques provide powerful tools for analyzing dut expression in Chlamydophila caviae:

• Quantitative Proteomic Strategies:

  • LC-MS^E Approach: Enables determination of absolute protein concentrations (molecules per cell) as demonstrated in similar studies with C. trachomatis

  • iTRAQ Labeling: Allows multiplexed comparison of protein levels across different developmental stages

  • SILAC: For studying host-pathogen proteome interactions when using cell culture models

• Sample Preparation Protocols:

  • Differential centrifugation for separation of elementary bodies (EBs) and reticulate bodies (RBs)

  • Specialized extraction buffers containing chaotropes and detergents to solubilize membrane-associated proteins

  • Enzymatic or chemical digestion optimized for chlamydial proteins

• Data Analysis Framework:

  • Protein identification using species-specific databases

  • Normalization strategies accounting for differences in developmental forms

  • Statistical methods for determining significant changes in protein abundance

• Comparative Approaches:

  • Cross-species comparison of dut expression patterns

  • Correlation with transcriptomic data to identify post-transcriptional regulation

  • Integration with metabolomic data to relate enzyme levels to nucleotide pool dynamics

Based on studies of related chlamydial species, these approaches can reveal how dut expression varies between the infectious elementary body and the replicative reticulate body forms, providing insights into the role of this enzyme during different stages of the developmental cycle . Such quantitative data can also estimate the cellular investment in dut synthesis relative to other proteins, indicating its metabolic priority within the cell.

What are the considerations for designing inhibitors specific to Chlamydophila caviae dut?

Rational design of Chlamydophila caviae dut inhibitors requires systematic consideration of several factors:

• Structural Targeting Strategies:

  • Active Site Inhibitors: Focus on modifications of the uracil base, deoxyribose sugar, or phosphate groups

  • Allosteric Inhibitors: Target protein-protein interfaces in the trimeric structure

  • Covalent Inhibitors: Design reactive groups that form irreversible bonds with conserved cysteines

• Selectivity Design Principles:

  • Exploit subtle differences in active site architecture between bacterial and human dUTPases

  • Target bacterial-specific binding pockets adjacent to the active site

  • Optimize inhibitor properties to enhance bacterial penetration while limiting human cell uptake

• Medicinal Chemistry Optimization Framework:

• Computational Approaches:

  • Structure-based virtual screening of compound libraries against known or modeled dut structures

  • Molecular dynamics simulations to identify transient binding pockets

  • Quantitative structure-activity relationship (QSAR) models to guide analog design

• Lead Optimization Considerations:

  • Balance of potency, selectivity, and physiochemical properties

  • Modifications to improve cell penetration in intracellular pathogens

  • Strategies to minimize efflux from bacterial cells

The development pathway typically progresses from high-throughput screening of fragment libraries to hit validation, structure-guided optimization, and finally in vitro and cell-based efficacy testing. Throughout this process, maintaining selectivity over human dUTPase remains a critical consideration to avoid potential toxicity issues.

How can recombinant Chlamydophila caviae dut be used as a research tool?

Recombinant Chlamydophila caviae dut serves multiple functions as a versatile research tool:

• Biochemical Applications:

  • Control enzyme for studying nucleotide metabolism pathways

  • Reference standard for developing nucleotide detection methods

  • Template for protein engineering studies of enzyme mechanism

• Structural Biology Applications:

  • Model system for studying conformational changes during catalysis

  • Platform for fragment-based drug discovery approaches

  • Educational tool for demonstrating structure-function relationships

• Assay Development Applications:

  • Component in coupled enzyme assays for monitoring other nucleotide-metabolizing enzymes

  • Tool for developing high-throughput screening platforms

  • Standard for validating novel enzyme activity detection methods

• Antibody Production:

  • Immunogen for generating specific antibodies for detection and localization studies

  • Target for developing detection reagents in diagnostic applications

• Experimental Protocol Applications:

  • Nucleotide Pool Cleanup:

    • Pre-treatment of reaction mixtures to eliminate dUTP contamination

    • Typical protocol: Incubate samples with 100 nM enzyme for 15 minutes at 37°C

  • DNA Amplification Enhancement:

    • Addition to PCR reactions to prevent uracil incorporation during amplification

    • Typical usage: 0.1 units per 50 μL PCR reaction

The high stability and well-characterized activity of recombinant dut make it particularly valuable as a reagent in molecular biology applications requiring control of nucleotide pool composition and purity.

What are the challenges in studying Chlamydophila caviae dut in its native context?

Investigating Chlamydophila caviae dut in its native biological context presents unique challenges:

• Cultivation Barriers:

  • Obligate intracellular lifestyle necessitates cell culture systems

  • Difficulty synchronizing developmental cycle for stage-specific analysis

  • Challenges in generating sufficient biomass for biochemical studies

• Genetic Manipulation Limitations:

  • Limited genetic tools for chlamydial species

  • Challenges in creating targeted gene knockouts or mutations

  • Difficulty in complementing potential dut mutations due to intracellular lifestyle

• Analytical Challenges:

  • Separating bacterial proteins from host cell background

  • Measuring enzyme activity in the context of host nucleotide pools

  • Distinguishing bacterial metabolism from host cell processes

• Methodological Approaches to Overcome Limitations:

  • Cell Culture Systems:

    • Infection of susceptible cell lines (McCoy, HeLa)

    • Differential centrifugation to isolate bacterial forms

    • Density gradient purification to separate elementary and reticulate bodies

  • Genetic Approaches:

    • RNA interference techniques to knock down expression

    • Expression of dominant-negative mutants

    • Recently developed transformation systems using shuttle vectors

  • Imaging Approaches:

    • Immunofluorescence using anti-dut antibodies to track localization

    • Correlative light and electron microscopy for ultrastructural studies

    • Live-cell imaging with fluorescent nucleotide analogs

These technical challenges explain why most studies of chlamydial dut rely on recombinant protein expression and in vitro characterization rather than native context analysis, highlighting the need for continued development of genetic and biochemical tools for studying obligate intracellular bacteria.