Recombinant Ralstonia pickettii Nucleoside diphosphate kinase (ndk)
Description
Overview of NDK Structure and Function
NDK enzymes are oligomeric proteins with a conserved ferredoxin fold. Key structural and catalytic features include (Fig. 1):
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Catalytic His residue: Phosphorylated during the reaction cycle (e.g., His-117 in Mycobacterium tuberculosis NDK) .
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Nucleotide-binding clamp: Formed by the αA-α2 hairpin and Kpn-loop, stabilizing substrates like ATP and GDP .
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Conserved residues: Lys10, Tyr50, Arg86, Thr92, and Arg104 interact with phosphate groups, while Phe58 facilitates aromatic stacking with nucleotide bases .
NDK in Bacterial Pathogenesis
NDK contributes to microbial virulence through:
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ATPase activity: Modulates extracellular ATP (eATP) signaling to evade host immune responses (e.g., blocking P2X7 receptor-mediated ROS production) .
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Biofilm formation: Supports polysaccharide synthesis via NTP generation .
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Stress adaptation: Stabilizes nucleotide pools under nutrient-limiting conditions .
While R. pickettii NDK remains uncharacterized, genomic studies reveal:
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Horizontal gene transfer (HGT): R. pickettii acquires adaptive genes via genomic islands (GIs), potentially including NDK homologs .
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Virulence-associated elements: Secretion systems (T4SS/T6SS) and antimicrobial resistance genes , suggesting NDK could synergize with these pathways.
Recombinant NDK Production and Characterization
Recombinant NDK production typically involves:
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Cloning: NDK genes expressed in E. coli (e.g., pET vectors) .
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Purification: Affinity chromatography (e.g., His-tagged proteins) .
For R. pickettii NDK, analogous protocols could apply, though experimental validation is needed.
Genomic and Evolutionary Context of R. pickettii NDK
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Pan-genome analysis: R. pickettii has a flexible gene repertoire enriched in nucleotide metabolism (COG-F) , aligning with NDK’s role.
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Environmental adaptation: Unique mutations in carbon/energy metabolism genes may influence NDK’s functional regulation.
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Antimicrobial resistance: β-lactamase production (e.g., OXA-22) suggests NDK could augment survival in clinical settings.
Research Gaps and Future Directions
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Structural studies: No crystal structures of R. pickettii NDK exist; homology modeling using M. tuberculosis or P. aerophilum templates is needed .
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Functional assays: Testing recombinant NDK’s ATPase activity and role in biofilm formation.
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Pathogenicity links: Investigating NDK’s interaction with R. pickettii secretion systems .
Product Specs
Target Background
KEGG: rpi:Rpic_1055
STRING: 402626.Rpic_1055
Q&A
What is the primary biochemical role of NDK in Ralstonia pickettii, and how does its activity differ from homologs in other organisms?
Nucleoside diphosphate kinase (NDK) in Ralstonia pickettii catalyzes the reversible transfer of γ-phosphate between nucleoside triphosphate (NTP) and diphosphate (NDP) substrates, maintaining nucleotide homeostasis. Unlike fungal NDKs (e.g., Aspergillus flavus), which regulate spore development and virulence, R. pickettii NDK may play a broader role in stress adaptation due to its genetic plasticity and horizontal gene transfer (HGT) events .
Methodological Consideration:
To compare substrate specificity, use kinetic assays (e.g., pyruvate kinase-lactate dehydrogenase coupled reactions) with diverse NTP/NDP partners. For example, test ATP, GTP, CTP, and UTP as phosphate donors and dTDP, dGDP, dCTP, dUTP as acceptors .
How should recombinant R. pickettii NDK be expressed and purified for structural or functional studies?
Recombinant NDK production typically involves:
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Cloning: Amplify the ndk gene via PCR, ligate into an E. coli expression vector (e.g., pET28a), and verify via Sanger sequencing.
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Expression: Induce with IPTG (0.1–1 mM) at 16–18°C for 16–24 hours to optimize solubility.
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Purification: Use nickel affinity chromatography for His-tagged proteins, followed by gel filtration for oligomeric state analysis (e.g., tetramers or hexamers in A. flavus homologs) .
Challenges:
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Low solubility due to hydrophobic patches.
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Solution: Add 500 mM NaCl or 2 mM β-mercaptoethanol to lysis buffers .
What kinetic parameters define NDK activity, and how are they measured?
Key parameters include:
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K<sub>m</sub> (Michaelis constant): Substrate affinity.
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V<sub>max</sub> (Maximum velocity): Catalytic efficiency.
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K<sub>d</sub> (Binding affinity): Measured via isothermal titration calorimetry (ITC).
Example Protocol:
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Enzyme Assay: Measure ATP-dependent dTDP → dTTP conversion using a coupled pyruvate kinase-lactate dehydrogenase system .
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ITC: Titrate recombinant NDK with ADP/GDP to determine K<sub>d</sub> values (e.g., ~150–160 μM for A. flavus NDK) .
| Parameter | A. flavus NDK (Reference) | R. pickettii NDK (Hypothetical) |
|---|---|---|
| K<sub>m</sub> (ATP) | 50–100 μM | TBD |
| K<sub>d</sub> (ADP) | 153 μM | TBD |
What structural insights can be gained from crystallographic studies of R. pickettii NDK?
Crystallography reveals:
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Active-site residues: Conserved motifs (e.g., P-loop, α4 helix) critical for phosphate transfer.
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Oligomerization state: A. flavus NDK forms hexamers; R. pickettii may adopt similar quaternary structures .
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Inhibitor binding: Docking studies with azidothymidine (AZT) or other nucleoside analogs to block catalytic activity .
Method:
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Crystallization: Use hanging-drop vapor diffusion with polyethylene glycol (PEG) or ammonium sulfate as precipitants.
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Resolution: Aim for ≤2.5 Å to resolve side-chain interactions.
How does R. pickettii NDK contribute to pathogenicity or environmental adaptation?
NDK may:
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Regulate virulence: Modulate macromolecular secretion systems or antimicrobial resistance via nucleotide metabolism .
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Enable stress tolerance: Maintain NTP/NDP balance under nutrient-limiting conditions (e.g., drinking water systems) .
Experimental Design:
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Gene knockout: Compare wild-type and Δndk strains in biofilm formation or stress assays.
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Complementation: Restore NDK activity to validate phenotypic rescue .
What methods are effective for identifying NDK inhibitors in R. pickettii?
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High-throughput screening: Test libraries of nucleoside analogs (e.g., AZT, 3′-phosphorylated derivatives) for IC<sub>50</sub> values.
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Structural-based design: Use crystallographic data to predict binding pockets for small-molecule inhibitors .
Case Study:
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AZT Inhibition: In A. flavus, AZT binds NDK, blocking spore production. Similar approaches could target R. pickettii NDK .
How do horizontal gene transfer (HGT) events influence R. pickettii NDK diversity?
HGT introduces:
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Prophages: May carry ndk homologs with altered substrate specificity.
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Genomic islands: Integrate ndk variants conferring niche-specific adaptations (e.g., water environments) .
Analysis Pipeline:
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Pan-genome analysis: Identify ndk gene clusters in core vs. accessory genomes.
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Phylogenetic trees: Detect recombination breakpoints using tools like Gubbins .
What contradictions exist in the literature regarding NDK’s role in bacterial physiology?
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Virulence vs. Housekeeping: Some studies link NDK to pathogenicity (e.g., A. flavus) , while others emphasize metabolic regulation (e.g., R. pickettii) .
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Oligomerization: Conflict between dimeric and hexameric states in different species.
Resolution:
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Species-specific assays: Compare NDK knockout phenotypes across genera.
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Structural validation: Use cryo-EM for R. pickettii NDK oligomerization states .
How can site-directed mutagenesis inform NDK’s catalytic mechanism in R. pickettii?
Target conserved residues (e.g., Arg-104, His-117, Asp-120 in A. flavus) for functional studies :
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Create mutants: Replace residues with alanine (R104A, H117A, D120A).
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Assess activity: Measure kinetic parameters (V<sub>max</sub>, K<sub>m</sub>) and compare to wild-type.
Hypothetical Data:
| Mutant | Relative Activity (%) | K<sub>m</sub> (ATP) (μM) |
|---|---|---|
| WT | 100 | 50 |
| R104A | 20 | 200 |
| H117A | 5 | 500 |
What computational tools are recommended for analyzing R. pickettii NDK sequences?
| Tool | Application | Source |
|---|---|---|
| BLASTp | Identify virulence/resistance homologs | |
| PHI-base | Map pathogenicity-related genes | |
| Gubbins | Detect recombination in pan-genomes | |
| SWISS-MODEL | Predict NDK structure from homologs |
How does R. pickettii NDK differ from NDKs in non-pathogenic Ralstonia species?
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Genetic context: R. pickettii NDK may be flanked by mobile elements (e.g., IS elements) enabling horizontal transfer .
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Functional specialization: Adaptive mutations in carbon/energy metabolism genes linked to NDK regulation .
Comparative Analysis:
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ANI (Average Nucleotide Identity): Compare ndk sequences between R. pickettii and R. solanacearum .
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COG Enrichment: Identify overrepresented functional categories in R. pickettii NDK-associated genes .
What challenges arise when studying R. pickettii NDK in complex microbial communities?
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Strain diversity: Multiple ndk variants may coexist, complicating functional analysis.
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Environmental interactions: NDK activity may vary under different nutrient or pH conditions.
Mitigation Strategies:
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Metagenomic binning: Isolate R. pickettii genomes from mixed samples.
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In situ assays: Use microfluidic devices to mimic drinking water niches .
How can NDK inhibitors be optimized for R. pickettii-specific targeting?
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Structure-guided design: Use R. pickettii NDK crystal structures to predict inhibitor binding modes.
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Phage display: Screen peptide libraries for high-affinity binders.
Example Workflow:
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Docking: Virtual screen small molecules against R. pickettii NDK active site.
What unresolved questions remain in R. pickettii NDK research?
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Evolutionary origin: Did ndk arise via HGT from other bacteria or eukaryotes?
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Regulatory networks: How does NDK integrate with two-component systems or quorum sensing?
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Therapeutic potential: Can NDK inhibition disrupt R. pickettii biofilms in clinical settings?
How should researchers interpret conflicting data on NDK’s role in Ralstonia species?
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Experimental rigor: Verify findings with complementary methods (e.g., RNA-seq, knockout + complementation).
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Species context: Acknowledge niche-specific adaptations (e.g., water vs. soil habitats) .
