Recombinant Staphylococcus aureus Enolase (eno)
Description
Definition and Biological Significance
Recombinant Staphylococcus aureus Enolase (eno) is a genetically engineered form of the glycolytic enzyme enolase produced in heterologous expression systems such as Escherichia coli. Enolase catalyzes the dehydration of 2-phosphoglycerate (2-PGA) to phosphoenolpyruvate (PEP) in glycolysis but also exhibits "moonlighting" functions, including binding host extracellular matrix (ECM) proteins like plasminogen (Plg), laminin, and fibronectin, thereby enhancing bacterial virulence .
Protein Sequence and Domains
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Sequence: 434 amino acids with conserved catalytic motifs (e.g., residues E168, E211, K345) .
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Oligomeric State: Exists as catalytically active dimers and fragile octamers .
Table 1: Key Biochemical Properties
| Property | Value/Description | Source |
|---|---|---|
| Optimal pH | 7.5 | |
| Kinetic Parameters | , | |
| Metal Ion Effects | Mg²⁺ stimulates activity; Hg²⁺, Cr²⁺ inhibit completely | |
| Plasminogen Binding |
Host-Pathogen Interactions
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Plasminogen Activation: Binds Plg via lysine residues, facilitating its conversion to plasmin by host tissue plasminogen activator (tPA). This enhances bacterial invasion through fibrinolytic activity .
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ECM Adhesion: Mediates attachment to laminin () and fibronectin, promoting colonization .
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Immune Evasion: Surface-localized enolase binds complement inhibitors (e.g., C4b-binding protein), reducing opsonization .
Metabolic and Virulence Regulation
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Biofilm Formation: Associated with 66.6% of clinical S. aureus isolates, though no direct correlation with eno gene presence was observed .
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Stress Adaptation: Upregulated during nutrient deprivation (e.g., iron limitation) to sustain glycolysis .
Therapeutic Development
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Vaccine Targets: Elicits IgG responses in S. aureus-infected patients .
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Inhibitor Screening: Fluorides and neurotoxic compounds inhibit enzymatic activity ( for NaF) .
Table 2: Research Applications
Expression Systems
Table 3: Production Workflow
| Step | Conditions | Outcome |
|---|---|---|
| Induction | 0.4 mM IPTG, 4 hours at 37°C | Soluble expression |
| Purification | Ni-Sepharose affinity chromatography | >90% purity (SDS-PAGE) |
| Storage | Tris buffer, 50% glycerol, -80°C | Stable for 12 months |
Challenges and Future Directions
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Phenotypic Heterogeneity: Environmental factors (pH, temperature) and genetic variability limit consistent in vitro biofilm models .
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Therapeutic Barriers: Cross-reactivity with human enolase (46% sequence homology) risks autoimmune responses .
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Advanced Models: 3D organoid systems are needed to better mimic in vivo host-pathogen dynamics .
Product Specs
Target Background
KEGG: sau:SA0731
Q&A
What is Staphylococcus aureus Enolase (eno) and what is its primary function?
Staphylococcus aureus Enolase (eno) is an evolutionarily conserved enzyme that catalyzes the reversible conversion of 2-phosphoglycerate into phosphoenolpyruvate, a critical step in glycolysis. This enzyme is essential for the degradation of carbohydrates via glycolysis for energy production . Beyond its metabolic function, S. aureus enolase also serves as a surface-localized protein involved in pathogenesis and host-pathogen interactions .
What structural forms does S. aureus enolase adopt?
S. aureus enolase exists in two distinct structural conformations: a catalytically active octamer and a robust dimer. Crystal structures have been determined at high resolutions (1.6 Å with bound phosphoenolpyruvate and 2.45 Å without) . Importantly, biochemical and structural studies have demonstrated that only the octameric variant is enzymatically active, while the dimeric form lacks catalytic activity but may be involved in other biological processes .
What are the specifications of recombinant S. aureus enolase?
Recombinant S. aureus enolase is typically produced with the following characteristics:
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Molecular weight: 63.1 kDa (with N-terminal 6xHis-SUMO tag)
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Full protein length: 434 amino acids
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Purity: Greater than 90% as determined by SDS-PAGE
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Expression system: Commonly E. coli
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Common strain source: Staphylococcus aureus strain Mu50 / ATCC 700699
How stable is recombinant S. aureus enolase under laboratory conditions?
The shelf life of recombinant S. aureus enolase depends on storage conditions and formulation. In liquid form, stability is typically around 6 months at -20°C/-80°C, while the lyophilized form maintains stability for approximately 12 months at the same temperatures. Repeated freeze-thaw cycles should be avoided. Working aliquots can be stored at 4°C for up to one week .
How does S. aureus enolase contribute to bacterial pathogenesis?
S. aureus enolase plays a dual role in pathogenesis:
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As a glycolytic enzyme in its octameric form, it supports bacterial metabolism and energy production.
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As a moonlighting protein in its dimeric form, it contributes to virulence through interaction with host plasminogen on the bacterial surface .
This interaction with host plasminogen has several pathophysiological implications:
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Mediates bacterial adherence to host tissues, facilitating colonization
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Contributes to the activation of plasminogen with the help of plasminogen activators
These mechanisms collectively enhance S. aureus tissue penetration and dissemination during infection.
What methodological approaches are used to study S. aureus enolase structure-function relationships?
Researchers employ several complementary approaches to investigate S. aureus enolase:
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Structural analysis: X-ray crystallography at high resolutions (1.6 Å and 2.45 Å) with and without bound phosphoenolpyruvate
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Oligomeric state characterization:
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Host interaction studies:
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Functional analysis:
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Biochemical assays to measure catalytic activity
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Surface localization studies
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Pathogenesis studies in infection models
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How can researchers target the interaction between S. aureus enolase and host plasminogen?
Inhibiting the S. aureus enolase-plasminogen interaction represents a promising therapeutic approach. Researchers have demonstrated two effective strategies:
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Mutagenesis approach: Creating mutant variants of enolase that disrupt plasminogen binding but maintain enzymatic function.
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Synthetic peptide inhibitors: Developing small peptides that compete for the plasminogen binding site on enolase.
Both approaches have successfully inhibited the interactions and their associated pathophysiological consequences in experimental settings . This provides potential avenues for developing novel anti-virulence therapies that don't rely on conventional antibiotics.
What is the role of S. aureus enolase in immune responses?
Recombinant S. aureus enolase has demonstrated immunogenic properties that could be leveraged for vaccine development. When used in combination with other staphylococcal proteins such as phosphoglycerate kinase (PGK) and elongation factor-G (EF-G), immunization elicited a type 3 immune response characterized by:
This type 3 cell immunity environment is considered crucial for protection against S. aureus infections, as it promotes neutrophil recruitment and activation - a primary defense mechanism against staphylococcal pathogens .
What challenges exist in utilizing S. aureus enolase as a vaccine candidate?
Despite promising immunological properties, several challenges must be addressed:
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Cross-reactivity concerns: The high conservation of enolase across species raises concerns about potential autoimmune reactions.
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Redundant virulence factors: S. aureus possesses numerous virulence factors, meaning targeting enolase alone may provide insufficient protection.
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Complex immune requirements: Optimal protection against S. aureus likely requires balanced humoral and cellular immune responses.
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Strain variation: Different S. aureus strains may exhibit variations in enolase expression or structure.
Current research suggests that combination approaches, such as chimeric vaccines incorporating multiple S. aureus antigens with selective immune-stimulatory properties, may offer the most promising path forward .
How should researchers optimize expression and purification of recombinant S. aureus enolase?
For optimal expression and purification:
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Tagging strategy: N-terminal 6xHis-SUMO tag facilitates purification and may enhance solubility
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Purification approach:
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Storage considerations:
What experimental approaches can differentiate between the metabolic and moonlighting functions of S. aureus enolase?
To distinguish between the dual functions of S. aureus enolase, researchers can employ:
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Oligomeric state separation:
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Site-directed mutagenesis:
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Target catalytic site residues to disrupt enzymatic function
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Modify surface-exposed residues involved in plasminogen binding
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Create mutations at oligomerization interfaces to stabilize specific forms
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Functional assays:
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Enzymatic activity measurements (phosphoenolpyruvate formation)
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Plasminogen binding assays
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Bacterial adherence to host cell models
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How can researchers develop inhibitors targeting S. aureus enolase?
A systematic approach to inhibitor development includes:
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Binding site characterization:
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Crystallographic analysis of enolase-inhibitor complexes
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Computational docking studies to identify potential binding pockets
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Rational design strategies:
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Peptide mimetics based on plasminogen binding regions
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Small molecule screening targeting either catalytic activity or protein-protein interactions
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Validation approaches:
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In vitro binding and inhibition assays
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Cell-based infection models to assess functional inhibition
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Animal models to evaluate in vivo efficacy and safety
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Current research has demonstrated that both mutant variants of enolase and synthetic peptide inhibitors can effectively block the enolase-plasminogen interaction and reduce associated virulence mechanisms .
