Recombinant Drosophila simulans Enolase-phosphatase E1 (GD19634)
Description
Functional Analogies
ENOPH1 in humans exhibits:
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Enzymatic activity: Bifunctional phosphatase/enolase activity, requiring Mg²⁺ as a cofactor .
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Pathway involvement: Central to methionine salvage and sulfur amino acid metabolism .
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Disease associations: Implicated in glioma, hepatocellular carcinoma, and blood-brain barrier dysfunction .
Limitations in Available Data
The search results exclusively focus on human ENOPH1, with no references to Drosophila simulans or GD19634. Key gaps include:
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Structural characterization: No crystallographic or biochemical data for GD19634.
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Biological roles: No studies linking GD19634 to specific Drosophila pathways or diseases.
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Therapeutic relevance: No evidence of its use in research or clinical applications.
Recommended Research Directions
To address the knowledge gap, the following steps are suggested:
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Literature mining: Search databases like FlyBase or WormBase for Drosophila ENOPH1 orthologs.
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Protein structure prediction: Use homology modeling to predict GD19634’s structure based on human ENOPH1’s crystal structure .
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Functional assays: Test GD19634’s enzymatic activity in methionine salvage pathways or stress responses.
Product Specs
Target Background
KEGG: dsi:Dsimw501_GD19634
Q&A
What is the molecular function of Enolase-phosphatase E1 (GD19634)?
Enolase-phosphatase E1 (GD19634) is a bifunctional enzyme that catalyzes two sequential reactions in metabolic pathways. First, it performs the enolization of 2,3-diketo-5-methylthiopentyl-1-phosphate (DK-MTP-1-P) to form the intermediate 2-hydroxy-3-keto-5-methylthiopentenyl-1-phosphate (HK-MTPenyl-1-P). Subsequently, it dephosphorylates this intermediate to produce the acireductone 1,2-dihydroxy-3-keto-5-methylthiopentene (DHK-MTPene) . This dual functionality makes it an important enzyme in cellular metabolism within Drosophila simulans.
How should researchers optimize expression systems for GD19634 recombinant protein production?
The choice of expression system significantly impacts the yield and functionality of recombinant GD19634. Based on research findings, four primary expression systems have been utilized:
| Expression System | Typical Yield | Advantages | Limitations |
|---|---|---|---|
| E. coli | Highest | Cost-effective, rapid growth, easy manipulation | Potential improper folding, less post-translational modifications |
| Yeast | Medium-high | Better folding than E. coli, some post-translational modifications | Longer expression time than E. coli |
| Baculovirus (insect cells) | Medium | Close to native folding, good for functional studies | More complex system, higher cost |
| Mammalian cells | Lower | Most authentic folding and modifications | Highest cost, longest production time |
For basic structural studies, E. coli expression may be sufficient, while functional enzymatic studies might benefit from insect or mammalian cell expression systems that better preserve the native conformation and activity .
What purification strategies yield the highest purity and activity of recombinant GD19634?
Effective purification of GD19634 typically involves affinity chromatography utilizing the fusion tags engineered into the recombinant protein. Based on research protocols:
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For His-tagged GD19634: Immobilized metal affinity chromatography (IMAC) using Ni-NTA columns provides >85% purity in a single step.
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Secondary purification steps may include:
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Size exclusion chromatography to remove aggregates and improve homogeneity
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Ion exchange chromatography for removing contaminants with different charge properties
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The purification protocol should be optimized to maintain enzymatic activity, typically including stabilizing buffers containing reducing agents and glycerol. Purity is commonly assessed using SDS-PAGE, with most research protocols aiming for >85% purity .
What are the optimal conditions for measuring GD19634 enzymatic activity?
While specific conditions for GD19634 are not explicitly detailed in the provided research, bifunctional enzymes like enolase-phosphatases typically require carefully controlled assay conditions:
| Parameter | Optimal Range | Notes |
|---|---|---|
| pH | 7.0-7.5 | Activity can decrease significantly outside this range |
| Temperature | 25-30°C | Temperature stability should be assessed separately |
| Buffer | HEPES or Tris-HCl | Phosphate buffers should be avoided for phosphatase activity assays |
| Substrate concentration | 0.1-1.0 mM | Depends on Km of the enzyme |
| Cofactors | Mg²⁺ or Mn²⁺ | Typically at 1-5 mM concentration |
Activity measurements may involve monitoring substrate depletion or product formation using spectrophotometric, fluorometric, or chromatographic methods. For the dual activities, separate assays may be needed to measure each function individually .
How do storage conditions affect GD19634 stability and activity?
Proper storage is crucial for maintaining GD19634 activity. The research suggests the following guidelines:
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Long-term storage: Store at -20°C or preferably -80°C.
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Working aliquots: Store at 4°C for up to one week.
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Avoid repeated freeze-thaw cycles as they can significantly reduce enzyme activity.
For lyophilized protein preparations, reconstitution should be performed in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Adding glycerol to a final concentration of 20-50% helps maintain stability during freezing .
How does GD19634 compare to homologous enzymes in other Drosophila species?
Comparative analysis of enolase-phosphatase E1 across Drosophila species can provide evolutionary insights. While the search results don't provide explicit comparative data, researchers investigating evolutionary conservation might consider:
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Sequence alignment analysis to identify conserved catalytic domains
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Phylogenetic analysis to determine evolutionary relationships
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Functional comparison of kinetic parameters (Km, Vmax, kcat) between homologs
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Structural modeling to identify species-specific variations in active sites
Such comparative studies could reveal how selective pressures have shaped the enzyme's function across closely related species and potentially identify regions critical for catalytic activity.
What are the potential applications of GD19634 in metabolic pathway research?
As a bifunctional enzyme in metabolic pathways, GD19634 presents several research opportunities:
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Metabolic flux analysis: Using recombinant GD19634 to study rate-limiting steps in metabolic pathways
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Inhibitor development: Designing and testing specific inhibitors to understand metabolic regulation
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Structural biology: Investigating the structural basis for dual catalytic activities
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Enzyme engineering: Modifying GD19634 for enhanced stability or altered substrate specificity
Researchers may use GD19634 as a model enzyme to study the evolution and advantages of bifunctional enzymes in metabolic efficiency .
How can researchers effectively design mutation studies to understand GD19634 structure-function relationships?
Structure-function studies of GD19634 require strategic approaches to mutation design:
This systematic approach would help elucidate the molecular basis of the dual catalytic activities and potentially identify residues that could be targeted for inhibitor design .
What considerations are important when selecting expression tags for GD19634 purification?
The choice of expression tags can significantly impact GD19634 purification and functionality:
| Tag Type | Advantages | Potential Issues | Recommended Applications |
|---|---|---|---|
| His-tag | Small size, minimal interference, effective purification | May affect metal-dependent enzyme activity | General purification, structural studies |
| GST-tag | Enhanced solubility, simple detection | Large size may affect function, additional cleavage step | Improving solubility of difficult-to-express constructs |
| MBP-tag | Significant solubility enhancement | Large size, potential interference with activity | Expression of insoluble constructs |
| FLAG-tag | High specificity, good for detection | More expensive resins for purification | Co-immunoprecipitation studies |
Researchers should consider:
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Tag position (N- or C-terminal) based on predicted structure
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Inclusion of protease cleavage sites if tag-free protein is needed
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Validation of enzymatic activity with and without tags
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Testing multiple tag options for optimal expression and purification
How can researchers address common issues in GD19634 recombinant expression?
When expressing recombinant GD19634, researchers might encounter several challenges:
| Issue | Potential Causes | Troubleshooting Strategies |
|---|---|---|
| Low expression yield | Codon bias, toxicity to host, protein instability | Optimize codon usage, use slower induction, lower expression temperature (16-20°C), add stabilizing components |
| Inclusion body formation | Rapid expression, improper folding | Reduce induction temperature, co-express with chaperones, use solubility-enhancing tags (MBP, SUMO) |
| Loss of enzymatic activity | Improper folding, missing cofactors, oxidation | Include reducing agents, optimize buffer composition, ensure proper metal ion availability |
| Protein aggregation during purification | Hydrophobic interactions, improper buffer conditions | Add mild detergents, increase salt concentration, include glycerol, optimize pH |
A systematic approach to optimization is recommended, changing one parameter at a time and assessing its impact on yield and activity .
What analytical methods are most effective for characterizing GD19634 structure and function?
Comprehensive characterization of GD19634 requires multiple analytical approaches:
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Structural characterization:
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Circular dichroism (CD) spectroscopy for secondary structure content
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Differential scanning fluorimetry for thermal stability
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Limited proteolysis to identify flexible regions
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X-ray crystallography or cryo-EM for high-resolution structure (if feasible)
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Functional characterization:
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Enzyme kinetics (Km, Vmax, kcat) for both catalytic activities
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Substrate specificity profiling
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pH and temperature activity profiles
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Inhibition studies
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Interaction studies:
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Size exclusion chromatography to determine oligomeric state
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Surface plasmon resonance for binding studies
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Isothermal titration calorimetry for thermodynamic parameters of substrate binding
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These methods provide complementary information that can be integrated to develop a comprehensive understanding of GD19634 structure-function relationships .
