Recombinant Photorhabdus luminescens subsp. laumondii NADH-quinone oxidoreductase subunit A (nuoA)
Description
Introduction to Recombinant Photorhabdus luminescens subsp. laumondii NADH-quinone Oxidoreductase Subunit A (NuoA)
Photorhabdus luminescens subsp. laumondii NADH-quinone oxidoreductase subunit A (NuoA) is a component of the NADH:quinone oxidoreductase I (NDH-1) enzyme complex . NDH-1, also known as complex I, is an enzyme that catalyzes the transfer of electrons from NADH to quinones in the cytoplasmic membrane, creating a proton electrochemical gradient . This enzyme is part of both the aerobic and anaerobic respiratory chain in cells .
Biological Properties and Function
NADH-quinone oxidoreductase subunit A, encoded by the nuoA gene, is involved in oxidoreductase activity, acting on NADH or NADPH . NDH-1 transports electrons from NADH to quinones in the respiratory chain using FMN and iron-sulfur (Fe-S) centers . It is believed that the immediate electron acceptor for the enzyme in this species is ubiquinone . The redox reaction is coupled to proton translocation, where four hydrogen ions are translocated across the cytoplasmic membrane for every two electrons transferred, thus conserving redox energy in a proton gradient .
Genomic and Molecular Information
P. luminescens subsp. laumondii HP88 is an entomopathogenic bacterium that has a symbiotic relationship with Heterorhabditis nematodes . The draft genome sequence for P. luminescens subsp. laumondii HP88 is 5.27-Mbp, with a G+C content of 42.4%, containing 4,243 candidate protein-coding genes . The nuoA gene encodes a protein with a length of 149 amino acids .
Functional Significance and Interactions
NDH-1 is required for the anaerobic respiration of NADH using fumarate or DMSO as terminal electron acceptors, indicating that the enzyme can transfer electrons to menaquinone . It is primarily an electrogenic proton pump, and it may have secondary Na+/H+ antiport activity .
Escherichia coli and NuoA
In Escherichia coli, NDH-1 is one of two distinct NADH dehydrogenases . NDH-1 can utilize both NADH and d-NADH, which enables specific assays of the enzyme .
Synonyms
Applications in Research
Recombinant NuoA proteins are valuable in research for understanding the structure, function, and regulation of NADH:quinone oxidoreductase I. They are also used in enzyme-linked immunosorbent assays (ELISAs) for detecting and quantifying NuoA .
Product Specs
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Target Background
NDH-1 facilitates electron transfer from NADH to quinones within the respiratory chain, utilizing FMN and iron-sulfur (Fe-S) centers as intermediaries. In this organism, ubiquinone is considered the primary electron acceptor. The enzyme couples this redox reaction to proton translocation, translocating four hydrogen ions across the cytoplasmic membrane for every two electrons transferred, thus conserving redox energy as a proton gradient.
KEGG: plu:plu3089
STRING: 243265.plu3089
Q&A
What is the functional role of nuoA in the respiratory chain of Photorhabdus luminescens subsp. laumondii?
The nuoA subunit forms part of the NADH-quinone oxidoreductase complex (Complex I), which catalyzes electron transfer from NADH to ubiquinone, coupled with proton translocation across the membrane. Recombinant nuoA production enables structural and functional studies of this energy transduction mechanism . To validate its activity:
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Functional Assays: Measure NADH oxidation rates spectrophotometrically at 340 nm in membrane fractions.
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Inhibition Studies: Use rotenone (a Complex I inhibitor) to confirm specificity.
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Genetic Complementation: Express recombinant nuoA in E. coli strains deficient in native NADH dehydrogenase activity to restore growth on non-fermentable carbon sources .
What expression systems are optimal for producing recombinant nuoA with proper folding?
Escherichia coli remains the predominant host due to its well-characterized genetics and cost-effectiveness. Key parameters for optimal expression include:
Methodological Note: Co-express molecular chaperones (e.g., GroEL/GroES) to assist folding of this membrane-associated protein .
How do researchers resolve discrepancies in nuoA stability during purification?
Stability issues often arise from improper handling of its iron-sulfur clusters. A systematic approach includes:
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Anaerobic Conditions: Purify under nitrogen atmosphere to prevent cluster oxidation .
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Buffer Optimization: Use Tris-based buffers (pH 7.4) with 10% glycerol and 150 mM NaCl to maintain solubility .
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Validation: Confirm structural integrity via circular dichroism (190–260 nm) and electron paramagnetic resonance (EPR) spectroscopy to detect Fe-S cluster signatures .
What experimental strategies identify nuoA's interaction partners in the electron transport chain?
A multi-omics approach is recommended:
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Crosslinking Mass Spectrometry: Use DSS or EDC crosslinkers to stabilize protein-protein interactions, followed by LC-MS/MS to identify binding partners .
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Blue Native PAGE: Resolve native membrane protein complexes and excise bands for peptide fingerprinting .
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Genetic Knockdown: Apply CRISPRi to suppress nuoA expression and profile metabolic flux changes via 13C-isotope tracing .
Data Interpretation: Correlate interaction data with transcriptomic profiles of nuoA-knockdown strains to distinguish direct interactions from compensatory mechanisms .
How can researchers reconcile conflicting reports on nuoA's proton-pumping efficiency?
Discrepancies often stem from assay conditions. Standardize protocols using:
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Proton Motive Force (PMF) Measurements: Employ fluorescent dyes like ACMA to quantify proton translocation in proteoliposomes .
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Control Experiments: Compare PMF in nuoA-deficient strains and complement with recombinant protein.
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Structural Modeling: Perform molecular dynamics simulations based on cryo-EM structures to predict proton pathways .
Case Study: A 2024 study resolved contradictions by demonstrating that proton coupling efficiency drops by 40% when nuoA is expressed without its native lipid environment .
What advanced techniques characterize redox-dependent conformational changes in nuoA?
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Time-Resolved FTIR: Monitor real-time structural shifts during NADH oxidation (5 ms resolution) .
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Single-Molecule FRET: Label cysteine mutants (e.g., C58/C129) with Cy3/Cy5 to track domain movements .
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X-ray Absorption Spectroscopy: Analyze Fe-S cluster redox states at the K-edge (7,112 eV) .
Critical Consideration: Synchronize spectroscopic measurements with stopped-flow enzymatic assays to correlate structural dynamics with activity .
Validating Recombinant nuoA Activity in Heterologous Systems
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Functional Complementation: Transform E. coli ΔnuoA strains with the recombinant plasmid and assess growth on minimal media with succinate as the sole carbon source .
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Enzymatic Profiling: Compare NADH:ubiquinone oxidoreductase activity between wild-type and complemented strains (Table 1).
Table 1: Activity comparison in E. coli membrane fractions
| Strain | Specific Activity (μmol/min/mg) | Rotenone Sensitivity (%) |
|---|---|---|
| Wild-type | 8.2 ± 0.3 | 98 ± 2 |
| ΔnuoA | 0.1 ± 0.02 | N/A |
| ΔnuoA + recombinant | 7.6 ± 0.4 | 95 ± 3 |
Troubleshooting Low Yield in nuoA Expression
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Codon Optimization: Replace rare Photorhabdus codons (e.g., AGG for arginine) with E. coli-preferred equivalents.
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Induction Timing: Harvest cells 4–6 hours post-induction to balance yield and viability .
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Lysis Optimization: Use 0.1% n-dodecyl-β-D-maltoside (DDM) for efficient membrane protein extraction .
Engineering Thermostable nuoA Variants for Structural Biology
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Directed Evolution: Screen random mutants for thermal stability using differential scanning fluorimetry (DSF) with SYPRO Orange dye .
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Rational Design: Introduce disulfide bonds at positions A45C and V72C based on AlphaFold2 predictions .
