DsbE E.Coli
Description
Structure and Biochemical Properties
DsbE is a monomeric protein with a molecular mass of 18.1 kDa, containing 161 amino acids . It belongs to the thioredoxin superfamily and features a conserved WCPTC active-site motif critical for its reductive activity . Key structural and biochemical properties include:
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Source: Expressed in E. coli and purified via chromatography .
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Formulation: Typically stored in 20 mM Tris-HCl (pH 7.5), 2 mM EDTA, and 10% glycerol .
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Active-Site Cysteines: Cys80 and Cys83 in the WCPTC motif are essential for reducing disulfide bonds .
Functional Role in E. coli
DsbE operates in the reductive pathway of the E. coli disulfide bond (DSB) system, working downstream of DsbD to maintain a reduced environment in the periplasm . Its primary functions include:
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Cytochrome c Maturation: Reduces disulfide bonds in apocytochrome c to allow heme attachment by CcmF and CcmH .
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Electron Transfer: Maintained in a reduced state by DsbD, a transmembrane protein that transfers electrons from the cytoplasm .
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Substrate Specificity: Acts on cysteine residues in apocytochrome c, ensuring proper thioether bond formation for heme ligation .
Comparative Analysis of DsbE with Other Dsb Proteins
DsbE differs mechanistically from other E. coli Dsb proteins involved in disulfide bond management:
Active-Site Mutagenesis
Mutagenesis studies highlight the necessity of Cys80 and Cys83 for DsbE’s function:
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C80S or C83S: Partially restored cytochrome c maturation, suggesting residual thiol-reducing activity .
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C80S/C83S: Minimal cytochrome c production, indicating a critical reliance on both cysteines .
| Mutation | Cytochrome c Yield | Complementation by Thiols |
|---|---|---|
| Wild-type | Normal | Not required |
| C80S | Low | Partial recovery with cysteine |
| C83S | Low | Partial recovery with cysteine |
| C80S/C83S | Minimal | No recovery |
Dependence on DsbD
DsbE requires DsbD to maintain its reduced state. In dsbD-deficient mutants, DsbE becomes oxidized, leading to:
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Loss of Cytochrome c: Complete absence of c-type cytochromes in ccmG deletion mutants .
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Thiol Supplementation: Rescue of cytochrome c maturation in active-site mutants via exogenous thiols (e.g., cysteine) .
Evolutionary and Pathogenic Relevance
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Gram-Positive Homologs: DsbE-like proteins in Mycobacterium tuberculosis (DsbE and DsbF) exhibit oxidase activity, contrasting with E. coli DsbE’s reductive role .
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Virulence: While DsbA is linked to bacterial virulence, DsbE’s role in pathogenesis remains underexplored .
Applications and Research Challenges
Product Specs
Product Science Overview
Thiol Disulfide Interchange Protein DsbE, also known as CcmG, is a crucial protein in the periplasm of Escherichia coli (E. coli). It plays a significant role in the formation and maintenance of disulfide bonds in proteins, which are essential for their proper folding and stability. The recombinant form of DsbE is produced using genetic engineering techniques to express the protein in E. coli.
DsbE is a periplasmic thioredoxin-like protein that is involved in the maturation of cytochrome c, a component of the electron transport chain. The protein has a thioredoxin fold and contains a CXXC motif, which is critical for its redox activity. The primary function of DsbE is to reduce disulfide bonds in apocytochrome c, facilitating the correct formation of disulfide bonds before the attachment of heme groups by other proteins such as CcmF and CcmH .
Cytochrome c maturation in E. coli involves a complex pathway requiring multiple proteins, including DsbE. DsbE is one of the 12 proteins necessary for the assembly of cytochrome c in the periplasm. It acts as a reducing agent, ensuring that the cysteine residues in apocytochrome c are in the correct redox state for heme attachment. This process is vital for the proper functioning of the electron transport chain and cellular respiration .
The recombinant form of DsbE is produced by cloning the gene encoding DsbE into an expression vector, which is then introduced into E. coli cells. The cells are cultured, and the protein is expressed and purified using conventional chromatography techniques. The recombinant DsbE protein is typically purified to a high degree of purity (>95%) and validated using SDS-PAGE .
Recombinant DsbE is used in various research applications, including studies on protein folding, redox biology, and cytochrome c maturation. It serves as a model protein for understanding the mechanisms of disulfide bond formation and is also used in the development of biotechnological applications that require the correct folding of disulfide-bonded proteins .
