Recombinant Phytophthora parasitica Imidazoleglycerol-phosphate dehydratase (HIS3)
Description
Gene Cloning and Expression in E. coli
The HIS3 gene (encoding IGPD) was isolated from a P. parasitica genomic library and cloned into E. coli strain hisB463, which lacks functional IGPD. Key findings include:
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Complementation: The cloned gene restored histidine prototrophy in E. coli hisB463 mutants, confirming functional expression .
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Promoter Independence: The gene was expressed constitutively in E. coli without reliance on the vector’s promoter, suggesting native regulatory elements in the cloned DNA .
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Plasmid Characteristics: The complementing plasmid (pHS1) contained a 7 kb insert, with transformation efficiency yielding 28 His⁺ colonies from one plasmid pool .
Enzymatic Activity and Inhibition
Recombinant IGPD exhibited identical sensitivity to amitrole as the native enzyme in P. parasitica:
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Amitrole Sensitivity: Growth of E. coli expressing P. parasitica HIS3 was inhibited at 2 mM amitrole, mirroring the pathogen’s sensitivity .
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Histidine Reversal: Inhibition by amitrole was fully reversed by histidine supplementation, confirming IGPD as the primary target .
Table 1: Growth Inhibition by Amitrole
| Amitrole (mM) | E. coli Wild-Type | E. coli + pHS1 | P. parasitica |
|---|---|---|---|
| 0 | +++ | +++ | +++ |
| 2 | +++ | + | + |
| 16 | +++ | - | - |
| Growth: +++ (normal), + (reduced), – (inhibited). Data from Karlovsky (1994) . |
Applications in Inhibitor Screening
The study demonstrated that recombinant IGPD in E. coli can replicate pathogen-specific inhibitor responses, enabling high-throughput screening for targeted herbicides. Advantages include:
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Cost Efficiency: Avoids culturing slow-growing pathogens like P. parasitica .
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Functional Validation: Confirms inhibitor specificity without interference from host metabolic pathways .
Comparative Analysis with Other IGPDs
Research Implications
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Herbicide Development: Amitrole’s non-selectivity and toxicity necessitate IGPD-specific inhibitors, which recombinant P. parasitica enzyme can help identify.
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Evolutionary Insights: The conserved function of IGPD across kingdoms underscores its role in essential metabolism, yet structural divergences explain differential inhibitor potency .
Product Specs
Q&A
Here’s a structured collection of FAQs tailored for academic researchers investigating recombinant Phytophthora parasitica Imidazoleglycerol-phosphate dehydratase (HIS3):
What experimental strategies are optimal for cloning and expressing Phytophthora parasitica HIS3 in heterologous systems?
The HIS3 gene was isolated from a plasmid library by complementing E. coli hisB463 mutants to histidine prototrophy . Key steps include:
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Library construction: Genomic DNA fragments (~7.9 kb inserts) cloned into pUC19 vectors
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Selection: Screening 52,000 colonies via ampicillin resistance and histidine auxotrophy rescue
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Promoter independence: HIS3 expression in E. coli remained constitutive even under lac repressor overexpression (pAC-lacIQ plasmid) .
How does imidazoleglycerol-phosphate dehydratase function in histidine biosynthesis?
The enzyme catalyzes the sixth step in histidine biosynthesis:
This reaction is critical for maintaining histidine pools, as shown by growth inhibition in P. parasitica under amitrole exposure .
What methodologies resolve discrepancies in amitrole sensitivity between native P. parasitica and recombinant HIS3-expressing E. coli?
Despite expressing the same enzyme, P. parasitica exhibits 10-fold greater sensitivity to amitrole than recombinant E. coli . Experimental approaches include:
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Comparative inhibition assays: Dose-response curves for amitrole in minimal media (Table 1) .
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Histidine rescue: Cytotoxicity reversal with exogenous histidine confirms target specificity .
Table 1: Growth inhibition by amitrole
| Organism | Amitrole (mM) | Growth Response | Histidine Rescue |
|---|---|---|---|
| P. parasitica | 2 | ++ | Not applicable |
| E. coli (wild-type) | 16 | +++ | Not required |
| E. coli (pHS1-HIS3) | 4 | + | Yes (0.1 mM) |
How do structural differences in HIS3 orthologs impact inhibitor binding?
Triazole phosphonates inhibit P. parasitica HIS3 with sub-nM affinity (Ki = 8.5–40 nM) , while 3-amino-1,2,4-triazole (amitrole) shows weaker effects . Strategies for analysis:
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Enzyme kinetics: Measure Ki values using purified recombinant HIS3 .
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Site-directed mutagenesis: Compare P. parasitica and Saccharomyces cerevisiae HIS3 isoforms (8 divergent amino acids) .
What controls validate HIS3 activity in complementation assays?
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Negative controls: Use hisB463 E. coli without plasmid or with empty vector .
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Repression controls: Co-transform with pAC-lacIQ to confirm lac promoter independence .
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Substrate depletion: Monitor imidazoleglycerol-phosphate accumulation via HPLC .
How to address incomplete histidine rescue in cytotoxicity assays?
If histidine supplementation only partially restores growth:
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Check for off-target effects via metabolomic profiling.
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Test combinatorial inhibition of HIS3 and other histidine pathway enzymes (e.g., histidinol phosphatase) .
Why might HIS3 expression in E. coli show variable activity?
Potential factors:
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Post-translational modifications: P. parasitica HIS3 may require eukaryotic chaperones absent in E. coli.
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Gene dosage: Multicopy plasmids (e.g., pUC19) may cause toxicity, necessitating titratable promoters .
What metrics prioritize HIS3 inhibitors for herbicide development?
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Selectivity ratio: Compare Ki values for P. parasitica HIS3 vs. human homologs (absent in animals) .
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Cellular permeability: Use radiolabeled inhibitors to quantify uptake in fungal vs. plant cells .
