Recombinant Dihydrolipoyl dehydrogenase (lpdA)
Description
Catalytic Mechanism and Mutational Insights
lpdA catalyzes the oxidation of dihydrolipoamide to lipoamide, transferring electrons to NAD⁺/NADP⁺ . Mutations in the NAD⁺-binding site (e.g., H322Y, E354K) reduce NADH inhibition, altering metabolic flux :
Key Mutational Effects
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E354K Mutation: Increases PDH complex activity under anaerobic conditions by reducing NADH sensitivity (Ki for NADH: 10-fold higher than wild-type) .
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H322Y Mutation: Disrupts FAD-NAD⁺ interactions, impairing electron transfer .
| Mutation | Catalytic Impact | Metabolic Outcome |
|---|---|---|
| E354K | Reduced NADH inhibition (Ki↑) | Enhanced ethanol fermentation in E. coli |
| H322Y | Impaired FAD-NAD⁺ coupling | Accumulation of dihydrolipoamide |
Metabolic Rewiring and Adaptive Roles
lpdA mutations enable metabolic bypasses in redox-defective mutants:
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Suppression of trxB gor ahpC Mutants: In E. coli, lpdA mutations accumulate dihydrolipoamide, which replaces glutathione as an electron donor for ribonucleotide reductase (RNR) reactivation . Glutaredoxins mediate this electron transfer, restoring TCA cycle function .
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Anaerobic Ethanol Fermentation: Mutant lpdA variants (E354K) enable anaerobic growth in ldhA pflB double mutants by rerouting pyruvate to ethanol .
Biochemical and Biotechnological Applications
Recombinant lpdA variants are leveraged in:
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Metabolic Engineering: Engineered E. coli strains with lpdA mutations improve ethanol yield under anaerobic conditions .
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Flavin Biosynthesis: Adaptive evolution experiments reveal lpdA mutations enhance fitness in riboflavin auxotrophic strains, suggesting roles in alternative electron transfer pathways .
Comparative Biochemical Properties
lpdA homologs exhibit divergent substrate specificities and redox roles:
| Enzyme | Substrate Preference | Electron Acceptor | Inhibitors |
|---|---|---|---|
| E. coli LpdA | Dihydrolipoamide | NAD⁺ | NADH, FAD analogs |
| M. tuberculosis LpdA | Quinones (e.g., DMQ) | NADP⁺ | NADP⁺, 2'-phospho-ADP-ribose |
| Human DLD | Dihydrolipoamide (mitochondrial) | NAD⁺ | O₂, ubiquinone |
Moonlighting Functions
lpdA homologs exhibit non-canonical roles under stress:
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Proteolytic Activity: Human DLD mutants degrade frataxin under acidic conditions, linking metabolic dysfunction to iron metabolism .
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Diaphorase Activity: Catalyzes NADH oxidation using alternative acceptors (e.g., ubiquinone, nitric oxide), modulating oxidative stress .
Research Challenges and Future Directions
Product Specs
Target Background
KEGG: ecc:c0145
STRING: 199310.c0145
