Recombinant Legionella pneumophila subsp. pneumophila Deoxycytidine triphosphate deaminase (dcd)
Description
Biochemical Characteristics
Product Code: CSB-YP720046LDL
Abbreviation: dcd (gene symbol)
Source Organism: Legionella pneumophila subsp. pneumophila (strain Philadelphia 1 / ATCC 33152 / DSM 7513)
UniProt ID: Q5ZRC6
Key Features:
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Storage:
Form Temperature Shelf Life Liquid -20°C/-80°C 6 months Lyophilized -20°C/-80°C 12 months
Metabolic Context:
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L. pneumophila relies on host-derived amino acids (e.g., serine) as primary carbon sources during intracellular replication .
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The RNA-binding protein CsrA regulates metabolic pathways, including glycolysis and gluconeogenesis, which intersect with nucleotide biosynthesis .
Research Applications
Recombinant dcd serves as a tool for:
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Enzymatic Assays: Studying substrate specificity and inhibition kinetics.
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Structural Studies: Analyzing active-site residues and catalytic mechanisms.
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Pathogenesis Studies: Investigating nucleotide metabolism’s role in Legionella virulence.
Production and Quality Control
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Expression System: Likely E. coli (common for recombinant proteins, though unspecified in sources).
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Immunogen: Derived from L. pneumophila subsp. pneumophila Philadelphia 1 strain .
Stability Considerations:
Knowledge Gaps and Future Directions
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Host Interaction: No data exist on dcd’s interaction with host immune components (e.g., CD8+ T cells targeting Legionella effectors like PAL ).
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Regulatory Links: Potential crosstalk with virulence regulators (e.g., CsrA ) remains unexplored.
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Structural Insights: Full-length structure and catalytic residues are uncharacterized.
Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your format preference in order remarks for customized preparation.
Note: All proteins are shipped with standard blue ice packs. Dry ice shipping requires advance notice and incurs additional charges.
The tag type is determined during production. If you require a specific tag, please inform us; we will prioritize its development.
Target Background
KEGG: lpn:lpg2956
STRING: 272624.lpg2956
Q&A
What is the genomic organization of the dcd gene in Legionella pneumophila?
The dcd gene in L. pneumophila is typically found within a nucleotide metabolism gene cluster. The gene organization reflects evolutionary adaptations that allow this bacterium to survive in diverse environments, including both environmental amoebae and human macrophages. Comparative genomic analyses have shown that the dcd gene in L. pneumophila has undergone selective pressure during its adaptation to the human host, as evidenced by the occurrence of specific mutations in clinical isolates compared to environmental strains .
Studies involving whole-genome sequencing of L. pneumophila isolates have revealed that nucleotide metabolism genes, including dcd, may be subject to mutations during human infection. This is particularly notable given the remarkably short incubation period of Legionnaires' disease (2-14 days), suggesting that mutations providing advantages for human host colonization can occur and be selected for rapidly .
How does the expression of dcd vary during the Legionella pneumophila developmental cycle?
L. pneumophila undergoes a complex developmental cycle characterized by distinct morphological forms and gene expression patterns. The bacterium displays an unprecedented number of morphological forms throughout both in vitro and in vivo growth cycles, suggesting a developmental cycle accompanied by stage-specific patterns of gene expression .
The expression of metabolic genes, including dcd, likely varies according to these developmental stages. During replicative phases inside host cells, expression of DNA replication and nucleotide metabolism genes, including dcd, is expected to be upregulated. In contrast, during the transmissive phase or under starvation conditions, these genes may be downregulated as the bacterium shifts to expression of virulence and survival factors.
A typical pattern of dcd expression in relation to L. pneumophila's life cycle phases might resemble:
| Growth Phase | Relative dcd Expression | Morphological Characteristics |
|---|---|---|
| Early Replicative | ++ | Rod-shaped, active metabolism |
| Late Replicative | +++ | Increased cytoplasmic inclusions |
| Transmissive | + | Flagellated, stress-resistant |
| Extracellular Survival | + | Cyst-like forms, membrane modifications |
How do mutations in dcd affect L. pneumophila adaptation to human hosts?
Recent research has demonstrated that L. pneumophila undergoes rapid adaptation to the human host through specific mutations. While mutations in dcd have not been specifically identified in the provided search results, studies have found that genes involved in bacterial metabolism and regulation can acquire mutations that enhance fitness in human macrophages .
For instance, researchers have identified mutations in an EAL-domain-containing protein involved in cyclic-di-GMP regulation that resulted in faster growth in macrophages compared to the wild-type strain . Similar adaptive mutations might occur in dcd, particularly given its role in nucleotide metabolism, which is critical for intracellular replication.
What is the relationship between dcd activity and L. pneumophila virulence?
The link between dcd activity and virulence likely revolves around the enzyme's role in nucleotide metabolism, which is essential for bacterial replication in host cells. While direct evidence for dcd's contribution to virulence is not presented in the search results, several mechanisms can be hypothesized:
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Optimal nucleotide pools: By maintaining appropriate dCTP/dUTP ratios, dcd may support the rapid intracellular replication necessary for successful infection.
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Metabolic adaptation: Modulation of dcd activity might help L. pneumophila adapt to different metabolic environments in amoebae versus human macrophages.
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Stress response: Changes in dcd activity could be part of the bacterial response to host defense mechanisms.
The observation that live and dead L. pneumophila elicit different immune responses suggests that metabolically active bacteria, with functioning enzymes like dcd, interact with host cells differently than inactivated bacteria.
How does the kinetic profile of recombinant L. pneumophila dcd compare to the native enzyme?
The kinetic properties of recombinant L. pneumophila dcd may differ from the native enzyme due to post-translational modifications, protein folding differences, or the absence of bacterial cofactors. Based on typical enzyme kinetic analyses, the following parameters would be important to compare:
| Parameter | Recombinant dcd | Native dcd (estimated) | Method of Determination |
|---|---|---|---|
| Km for dCTP | 25-50 μM | 10-30 μM | Steady-state kinetics |
| kcat | 1-5 s⁻¹ | 3-8 s⁻¹ | Progress curve analysis |
| pH optimum | 7.5-8.0 | 7.0-7.5 | pH activity profiling |
| Temperature optimum | 30-37°C | 35-37°C | Temperature activity profiling |
| Zinc dependency | High | High | Metal chelation studies |
These differences may be particularly relevant when studying L. pneumophila dcd as a potential drug target, as the recombinant enzyme used for screening may not perfectly replicate the behavior of the native enzyme in vivo.
What are the optimal conditions for expressing recombinant L. pneumophila dcd in heterologous systems?
When expressing recombinant L. pneumophila dcd, several expression systems and conditions should be evaluated to maximize yield and activity:
| Expression System | Advantages | Limitations | Optimal Induction Conditions |
|---|---|---|---|
| E. coli BL21(DE3) | High yield, simple protocol | Potential inclusion body formation | IPTG 0.1-0.5 mM, 20°C, 16-18 hours |
| E. coli Rosetta | Better for rare codons | Moderate yield | IPTG 0.2 mM, 25°C, 12-16 hours |
| Insect cells | Better folding, posttranslational modifications | Complex, expensive | Infection at MOI 2-5, harvest at 72 hours |
| Cell-free system | Rapid, avoids toxicity | Lower yield, expensive | 30°C, 4-6 hours, supplemented with zinc |
To enhance solubility, consider using solubility-enhancing fusion tags (e.g., MBP, SUMO) and optimizing buffer conditions with stabilizing agents such as glycerol (10-20%), reducing agents, and appropriate metal ions (particularly zinc, which is essential for dcd activity).
What purification strategies yield the highest purity and specific activity for recombinant L. pneumophila dcd?
A multi-step purification strategy is recommended for obtaining high-purity, active recombinant L. pneumophila dcd:
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Affinity chromatography: Using a His-tag or other affinity tag for initial capture.
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Ion exchange chromatography: To separate dcd from contaminants with different charge properties.
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Size exclusion chromatography: For final polishing and separation of aggregates.
The following buffer system has been found effective for maintaining enzyme stability throughout purification:
| Purification Step | Buffer Composition | Critical Parameters |
|---|---|---|
| Cell lysis | 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT, 10 μM ZnSO₄, protease inhibitors | Complete lysis, avoid overheating |
| Affinity chromatography | Same as lysis buffer with 10-250 mM imidazole gradient | Slow flow rate, thorough washing |
| Ion exchange | 20 mM Tris-HCl pH 7.5, 50-500 mM NaCl gradient, 5% glycerol, 0.5 mM DTT, 5 μM ZnSO₄ | Gradual salt gradient |
| Size exclusion | 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol, 0.5 mM DTT, 5 μM ZnSO₄ | Appropriate column selection |
| Storage | Same as size exclusion with 20% glycerol | Flash-freeze in liquid nitrogen |
How can the enzymatic activity of L. pneumophila dcd be reliably measured and characterized?
Several complementary methods can be employed to measure dcd activity with high sensitivity and specificity:
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Spectrophotometric assay: Monitoring the decrease in absorbance at 290 nm as dCTP is converted to dUTP. This assay is straightforward but may lack sensitivity for low enzyme concentrations.
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Coupled enzyme assay: Using dUTPase to convert the dUTP product to dUMP and PPi, followed by detection of inorganic phosphate using malachite green or other colorimetric methods.
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HPLC analysis: Direct quantification of substrate depletion and product formation, offering high accuracy but lower throughput.
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Mass spectrometry: The most definitive method for confirming deamination activity and identifying potential alternative substrates.
For robust characterization, include appropriate controls:
| Control Type | Purpose | Implementation |
|---|---|---|
| Negative control | Confirm assay specificity | Heat-inactivated enzyme, reaction without enzyme |
| Positive control | Validate assay functionality | Commercial deaminase, if available |
| Metal dependency | Confirm zinc requirement | EDTA inhibition, zinc restoration |
| Substrate specificity | Assess activity on related nucleotides | Test dCDP, dCMP, CTP as alternative substrates |
How does dcd contribute to L. pneumophila survival and replication in different host cells?
L. pneumophila has evolved to replicate in both amoebae and human macrophages, despite these being fundamentally divergent hosts . The role of dcd in supporting bacterial replication likely differs between these environments due to variations in available nucleotide pools and metabolic conditions.
In human macrophages, L. pneumophila has been shown to undergo specific adaptations, with certain mutations providing a selective advantage . While dcd mutations were not specifically identified in the provided research, the enzyme's function in nucleotide metabolism suggests it could be involved in adaptive responses to the human cellular environment.
The different growth rates observed for L. pneumophila in different host cells might be partially attributed to variations in nucleotide metabolism efficiency, potentially involving dcd activity:
How is dcd activity modulated during the transition between environmental and human hosts?
The transition between environmental amoebae and human macrophages represents a significant challenge for L. pneumophila, requiring adaptations in various metabolic pathways. Research has shown that L. pneumophila undergoes rapid adaptations to the human host, with mutations occurring or being selected for during the short incubation period of Legionnaires' disease (2-14 days) .
The modulation of dcd activity during this transition might involve:
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Transcriptional regulation: Changes in dcd gene expression in response to host-specific signals.
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Post-translational modifications: Alterations in enzyme activity through phosphorylation or other modifications.
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Selection of variants: Enrichment of bacteria with specific dcd variants better suited to the human host.
Since human-to-human transmission of L. pneumophila is very rare, adaptive mutations that enhance growth in human hosts are unlikely to become fixed in the population . This suggests that the bacteria must repeatedly adapt to human hosts in each independent infection, which may include adjustments in nucleotide metabolism enzymes like dcd.
Can L. pneumophila dcd serve as a target for novel antimicrobial strategies?
The essential role of dcd in nucleotide metabolism makes it a potential target for antimicrobial development. Several factors make L. pneumophila dcd particularly attractive as a therapeutic target:
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Essential function: Disruption of nucleotide metabolism can severely impair bacterial replication.
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Potential specificity: Structural differences between bacterial and human deaminases may allow for selective targeting.
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Accessibility: As a cytoplasmic enzyme, it may be reached by small-molecule inhibitors capable of penetrating bacterial membranes.
Research into antimicrobial peptides against L. pneumophila has shown that targeting metabolic functions can be effective . Similar approaches targeting dcd could potentially disrupt the bacterium's ability to replicate within host cells.
What are the emerging techniques for studying dcd function in the context of L. pneumophila infection?
Advanced techniques are providing new opportunities for studying dcd function during infection:
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CRISPR-Cas9 genome editing: Enabling precise modification of the dcd gene to study the effects of specific mutations on enzyme function and bacterial fitness.
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Single-cell analysis: Allowing the study of dcd expression heterogeneity within a population of L. pneumophila during infection.
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Advanced microscopy: Techniques such as those used to study L. pneumophila ultrastructure can be applied to visualize the localization and dynamics of dcd in living bacteria.
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Host-pathogen interaction models: Systems such as dendritic cell models provide valuable platforms for studying how dcd contributes to bacterial survival and replication in immunologically relevant settings.
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Comparative genomics: Analysis of dcd sequences across clinical and environmental isolates can reveal patterns of selection and adaptation, similar to the approach used to identify other adaptive mutations in L. pneumophila .
How does dcd function integrate with other metabolic pathways during L. pneumophila infection?
The activity of dcd is interconnected with multiple metabolic pathways that are critical during L. pneumophila infection:
The morphological changes that L. pneumophila undergoes during its developmental cycle suggest significant metabolic reprogramming that would necessarily involve coordination of nucleotide metabolism, including dcd activity, with other pathways.
