Recombinant Bacillus pseudofirmus ATP synthase subunit c (atpE)
Description
Introduction
Recombinant Bacillus pseudofirmus ATP synthase subunit c (atpE) is a bioengineered protein derived from the alkaliphilic bacterium Bacillus pseudofirmus OF4. This subunit is a critical component of the ATP synthase’s c-ring, which drives proton translocation across membranes to synthesize ATP. The recombinant form is produced via heterologous expression systems, often in E. coli, enabling structural and functional studies of this extremophile enzyme’s adaptations to high pH environments .
Functional Role in ATP Synthase
The c-ring facilitates proton (or sodium ion) translocation, directly coupling ion movement to ATP synthesis. In B. pseudofirmus OF4, the c₁₃ ring optimizes the ion-to-ATP ratio for growth in pH >10 environments. Mutations altering the c-ring stoichiometry (e.g., c₁₂) reduce ATP synthesis efficiency and growth yields under alkaline conditions .
Functional Adaptations
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Ion-to-ATP Ratio: c₁₃ ensures efficient ATP synthesis at high pH by maintaining a 13:1 H⁺/ATP ratio .
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Stability: AxAxAxA motif prevents helix unfolding under alkaline stress, critical for extreme environments .
Impact of C-ring Mutations
Mutants with alanine-to-glycine substitutions (e.g., A→G) form c₁₂ rings, leading to:
| Parameter | Wild Type (c₁₃) | Mutant (c₁₂) |
|---|---|---|
| Molar Growth Yield | Optimal at high pH | Reduced by 30–50% at pH >10 |
| ATP Synthesis Rate | High efficiency | Lower efficiency |
| C-ring Stability | Stable at pH 11 | Reduced stability |
Role of Assembly Factors
The atpI gene (encoding a chaperone) and yidC-like proteins (YqjG, SpoIIIJ) are critical for c-ring assembly:
| Factor | Role in Assembly | Dependency |
|---|---|---|
| AtpI | Stabilizes c-ring assembly; deletion reduces ATP synthase yield and stability | Partial |
| YqjG/SpoIIIJ | Complement bacterial YidC in E. coli, enabling membrane protein maturation | Overlapping |
Bioenergetic Insights
The c₁₃ ring’s adaptation highlights evolutionary strategies for extremophiles to optimize bioenergetics. Studies on recombinant atpE provide a model for understanding:
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Alkaline Stress Resistance: Mechanisms for maintaining membrane integrity and proton gradients under high pH .
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ATP Synthase Engineering: Designing synthetic c-rings with tailored stoichiometry for biofuel production or biomedical applications .
Experimental Tools
Recombinant atpE is used in:
Product Specs
Note: We will prioritize shipping the format currently in stock. However, if you have a specific format requirement, please indicate it in your order notes. We will accommodate your request if possible.
Note: Our proteins are typically shipped with standard blue ice packs. If you require dry ice shipping, please inform us in advance as additional fees will apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
Target Background
KEGG: bpf:BpOF4_06875
STRING: 398511.BpOF4_06875
Q&A
What is the unique structural feature of ATP synthase subunit c in Bacillus pseudofirmus OF4?
The ATP synthase of Bacillus pseudofirmus OF4 possesses a tridecameric (13-subunit) c-subunit rotor ring, which is a key adaptation for functioning in alkaline environments . The most distinctive feature is the presence of an AxAxAxA motif near the center of the inner helix of each c-subunit, specifically the A16xAxAxA22 sequence . This contrasts with neutralophilic bacteria, which typically have a GxGxGxG motif in the same position . This alanine motif is critical for maintaining the c13 ring stoichiometry, which enables optimal ATP synthesis at extremely high pH levels (>10) .
How does the structure of the c-ring affect the bioenergetics of B. pseudofirmus OF4?
The c-ring stoichiometry directly determines the number of ions transferred during enzyme operation, which has profound effects on the ion-to-ATP ratio - a cornerstone parameter of cell bioenergetics . Research shows that the c13 ring configuration in B. pseudofirmus OF4 is specifically adapted for optimal growth in highly alkaline conditions. When alanine-to-glycine mutations cause the formation of smaller c12 rings, there is a measurable reduction in the organism's capacity to grow on limiting malate at high pH . This provides evidence of a direct connection between the precisely adapted ATP synthase c-ring stoichiometry and cell physiology in extreme environments .
What methods can be used to analyze c-ring stoichiometry and structure?
Researchers employ multiple complementary techniques to analyze c-ring structure:
| Technique | Application | Key Information Obtained |
|---|---|---|
| Atomic Force Microscopy | Topological analysis | Ring diameter and subunit arrangement |
| X-ray Crystallography | High-resolution structure | Atomic-level details of protein conformation |
| SDS-PAGE | Stability and mobility analysis | c-ring integrity and c-monomer mobility |
| Trichloroacetic Acid Treatment | Subunit isolation | Separation of monomeric c-subunits |
These techniques were successfully employed to determine that alanine-to-glycine mutations in the c-subunit of B. pseudofirmus OF4 result in smaller c12 rings compared to the wild-type c13 rings . The structural data from these studies was deposited in the Protein Data Bank (PDB ID code 3zo6) .
How can functional ATP synthesis be measured in alkaliphilic bacteria?
ATP synthesis in B. pseudofirmus OF4 can be measured using ADP and Pi-loaded membrane vesicles. In published studies, researchers prepared right-side-out (RSO) membrane vesicles from wild-type and mutant strains, loaded them with ADP and inorganic phosphate (Pi), and then energized them with ascorbate-phenazine methosulfate (PMS) . The reaction mixtures were continuously aerated by vortex mixing during incubation with the electron donor . This approach allows for measurement of ATP synthesis at both neutral (pH 7.5) and alkaline (pH 10.5) conditions, enabling direct comparison of enzymatic efficiency across pH ranges and between wild-type and mutant variants .
How do specific Ala-to-Gly mutations affect ATP synthase function?
Studies have examined multiple single and combined Ala-to-Gly mutations in the A16xAxAxA22 motif of the c-subunit, revealing distinct patterns of functional impact:
| Mutation Type | Impact on Function | Effect on c-ring Structure |
|---|---|---|
| A16G (single) | Severe functional defects | Increased mobility on SDS-PAGE |
| A16/20G (double) | Greater functional deficit than A16G alone | Altered c-ring structure |
| A16/18G (double) | Less severe than A16/20G | Modified c-ring properties |
| Multiple mutations | Variable effects depending on positions | Often forms c12 rings instead of c13 |
The most severe functional deficits occurred in mutants containing an Ala16-to-Gly mutation . Interestingly, the functional impact depended not only on the presence of this mutation but also on additional Ala-to-Gly changes and their specific positions. For example, the A16/20G double mutant exhibited a larger functional deficit than both the A16G single mutant and the A16/18G double mutant .
How does the c-ring stoichiometry influence the ion-to-ATP ratio and adaptive advantage?
The c-ring stoichiometry is a critical determinant of the ion-to-ATP ratio in ATP synthase. For each complete rotation of the c-ring, three ATP molecules are synthesized by the F1 sector . Therefore, a c13 ring configuration requires the translocation of 13 ions (protons or sodium ions) to synthesize 3 ATP molecules, resulting in an ion-to-ATP ratio of approximately 4.3:1 .
When mutations cause the formation of c12 rings, the ion-to-ATP ratio changes to 4:1. This seemingly small change has significant physiological consequences in alkaline environments where proton availability is limited. The higher ion-to-ATP ratio of the c13 ring appears to be better adapted for growth at pH >10, where the organism must maximize energy yield from each captured proton . This example demonstrates the fine-tuning of bioenergetic parameters through evolutionary adaptation to extreme environments.
What molecular interactions stabilize the c-ring structure in alkaliphiles?
The AxAxAxA motif in B. pseudofirmus OF4 plays a crucial role in stabilizing the c-ring structure through specific helix-helix packing interactions. The methyl groups of alanine residues are believed to provide optimal spacing and hydrophobic interactions between adjacent helices, maintaining the larger c13 ring configuration . This differs from the glycine-based packing (GxGxGxG) found in neutralophiles, which typically results in tighter helix packing and smaller c-rings (c8-11) .
X-ray crystallography and modeling studies suggest that these structural differences affect not only ring size but also influence proton binding and translocation properties that are essential for ATP synthesis under alkaline conditions .
How does the ATP synthase adaptation relate to the ecological niche of B. pseudofirmus OF4?
B. pseudofirmus OF4 thrives in highly alkaline environments (pH >10) where most organisms cannot survive. The adaptation of its ATP synthase, particularly the c-ring structure, represents a crucial evolutionary innovation that enables energy production under extreme conditions . Growth experiments with malate as a limiting carbon source demonstrated that wild-type cells with c13 rings grow significantly better at high pH than mutants with c12 rings .
This physiological advantage explains why the AxAxAxA motif has been selected and maintained in alkaliphiles despite the fact that c12 rings can support ATP synthesis. The precise tuning of c-ring stoichiometry to environmental conditions highlights the sophisticated bioenergetic adaptations that enable life in extreme environments and provides insights into the evolutionary pressures shaping cellular energetics .
How might knowledge of B. pseudofirmus ATP synthase inform research on other extremophiles?
The insights gained from studying B. pseudofirmus OF4 ATP synthase provide a valuable framework for understanding adaptations in other extremophiles. The direct connection between protein structure (c-ring stoichiometry) and bioenergetic parameters (ion-to-ATP ratio) demonstrates how molecular-level adaptations translate to ecological fitness .
Similar structural adaptations might be expected in organisms facing other types of bioenergetic challenges, such as thermophiles, acidophiles, or halophiles. For instance, the identification of specific binding sites in the c-ring that are involved in inhibitor binding (as seen in mycobacterial ATP synthase) suggests that structural variations in this region might contribute to diverse functional adaptations across species .
