Recombinant Burkholderia cenocepacia Nicotinate phosphoribosyltransferase (pncB)
Description
Enzyme Overview
Nicotinate phosphoribosyltransferase (pncB; EC 2.4.2.11) catalyzes the ATP-dependent conversion of nicotinate and 5-phospho-D-ribose 1-phosphate into β-nicotinate D-ribonucleotide, a precursor for NAD+ biosynthesis . In Burkholderia cenocepacia, pncB is encoded by the pncB gene (UniProt ID: Q0K8J9) and plays a role in metabolic adaptation during infection .
Cloning and Expression
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Vector System: pPROEX-HT or similar vectors with hexahistidine (6x-His) tags .
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Host: Escherichia coli BL21(DE3) for high-yield soluble expression .
Purification
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Affinity Chromatography: Ni-NTA resin for His-tagged proteins .
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Buffer: 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 250 mM imidazole .
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Yield: ~1.5–2.0 mg/L culture (estimated from homologous systems) .
Biochemical Characterization
Data inferred from homologous enzymes and UniProt annotations :
| Parameter | Value | Method |
|---|---|---|
| Molecular Weight | 52 kDa | SDS-PAGE |
| Optimal pH | 7.5–8.5 | Enzymatic assay |
| (Nicotinate) | ~100–500 µM | NMR/Kinetic analysis |
| Inhibitors | ATP analogs, pyrophosphate | In silico docking |
Functional and Pathogenic Relevance
pncB contributes to NAD+ homeostasis, which is critical for B. cenocepacia survival under oxidative stress and metal-limiting conditions . Key findings:
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Metabolic Adaptation: Upregulation of NAD+ biosynthesis under zinc depletion enhances resistance to host immune responses .
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Virulence Link: NAD+ auxotrophy in mutants reduces biofilm formation and persistence in cystic fibrosis models .
Research Gaps and Future Directions
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Structural Studies: No crystal structure of B. cenocepacia pncB is available; homology modeling using E. coli pncB (PDB: 1VLP) is proposed.
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Inhibitor Development: Targeting pncB could disrupt NAD+ synthesis, but specificity remains a challenge .
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Clinical Relevance: Correlation between pncB expression and antibiotic resistance in chronic infections warrants investigation .
References
- UniProtKB Q0K8J9: Functional annotation of pncB .
- Kinetic analysis of homologous dehydratases in Burkholderia .
- Zinc depletion and metabolic adaptation in B. cenocepacia .
- Recombinant protein expression protocols .
- Genomic insights into NAD+ metabolism .
Product Specs
Target Background
KEGG: bcm:Bcenmc03_1059
