Recombinant Salmonella agona Lipoyl synthase (lipA)
Product Specs
Target Background
KEGG: sea:SeAg_B0674
Q&A
What is the enzymatic role of LipA in Salmonella Agona metabolism?
LipA catalyzes the final step of lipoic acid biosynthesis: the insertion of two sulfur atoms into octanoyl-ACP to form the dithiolane ring structure of lipoate . This cofactor is essential for the activity of key dehydrogenase complexes (PDH, KDH, BCDH), which link glycolysis to the tricarboxylic acid (TCA) cycle. Researchers can confirm LipA activity via in vitro assays using purified octanoyl-ACP substrates and monitoring sulfur incorporation through HPLC-MS . A common pitfall is the misannotation of LipA homologs; always validate enzyme specificity using knockout strains complemented with recombinant LipA.
How should researchers design cloning strategies for recombinant LipA expression?
To express Salmonella Agona LipA in E. coli, use vectors with strong, inducible promoters (e.g., pET-28a(+) with a T7 promoter). Codon-optimize the lipA gene for E. coli to address expression bottlenecks. Include a hexahistidine tag for nickel-affinity purification. Critical parameters:
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Induction conditions: 0.5 mM IPTG at 18°C for 16 hours to minimize inclusion body formation.
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Lysis buffer: 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, 1 mM DTT.
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Activity validation: Compare lipoylation levels in E. coli ΔlipA strains transformed with recombinant LipA versus empty vectors using Western blotting with anti-lipoate antibodies .
How can structural discrepancies in LipA’s iron-sulfur cluster binding sites be resolved?
LipA requires two [4Fe-4S] clusters for catalysis, but crystallographic studies often show partial cluster occupancy due to oxidative degradation. To mitigate this:
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Purify LipA anaerobically using a glovebox (<1 ppm O₂).
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Reconstitute iron-sulfur clusters in vitro using 10 mM DTT, 2 mM Fe(NH₄)₂(SO₄)₂, and 2 mM Na₂S under anaerobic conditions .
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Validate cluster integrity via electron paramagnetic resonance (EPR) spectroscopy. A signal at g = 1.94 confirms reduced [4Fe-4S]⁺ clusters.
Table 1: Comparative Activity of LipA Under Aerobic vs. Anaerobic Purification
| Condition | Specific Activity (nmol/min/mg) | Cluster Integrity (EPR) |
|---|---|---|
| Aerobic | 12 ± 3 | Partial degradation |
| Anaerobic | 89 ± 11 | Fully intact |
What experimental approaches resolve contradictions in LipA’s substrate specificity?
Early studies reported LipA activity on octanoyl-Lys side chains, while recent work emphasizes octanoyl-ACP substrates. To address this:
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Use radiolabeled [¹⁴C]-octanoyl-ACP in activity assays.
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Compare reaction kinetics (Km and kcat) between octanoyl-ACP and octanoylated peptides.
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Employ site-directed mutagenesis to test residues involved in ACP binding (e.g., Arg146Ala reduces activity by 90% ).
How to quantify LipA’s contribution to biofilm formation in Salmonella Agona?
Biofilm assays with lipA knockout strains reveal diminished biofilm biomass (30–50% reduction vs. wild type ). Use confocal microscopy with LIVE/DEAD staining to correlate LipA activity with live cell density and extracellular matrix composition. Key steps:
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Culture biofilms on polystyrene surfaces for 72–144 hours at 37°C .
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Treat with 10 μM lipoic acid to rescue biofilm defects in ΔlipA strains.
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Quantify β-polysaccharides (calcofluor white staining) and proteins (Sypro Ruby) as biofilm markers .
Table 2: Biofilm Parameters in Salmonella Agona Strains
| Strain | Biovolume (μm³/μm²) | Live Cells (%) | β-Polysaccharides (RFU) |
|---|---|---|---|
| Wild type | 25.3 ± 2.1 | 78 ± 6 | 450 ± 35 |
| ΔlipA | 11.8 ± 1.4 | 42 ± 5 | 210 ± 28 |
| ΔlipA + LA | 22.1 ± 1.9 | 70 ± 7 | 410 ± 31 |
How does horizontal gene transfer affect lipA conservation in Salmonella Agona outbreaks?
Whole-genome sequencing of outbreak strains (1998 vs. 2008) shows only 8 SNP differences, confirming clonal persistence . To assess lipA stability:
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Align lipA sequences from outbreak isolates using MAUVE.
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Check for nonsynonymous SNPs in catalytic domains (e.g., Cys129Ala abolishes activity).
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Test recombinant LipA from both outbreaks for identical kinetic parameters.
Can LipA inhibitors be designed without affecting human homologs?
Human LipA shares 34% identity with Salmonella Agona LipA. Exploit differences in the substrate-binding pocket:
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Screen small molecules against Salmonella LipA using in silico docking.
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Validate hits with in vitro assays and cytotoxicity tests on human cell lines.
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Prioritize compounds with >100-fold selectivity for bacterial LipA.
