Recombinant Sulfolobus solfataricus Phosphoenolpyruvate carboxykinase [GTP] (pckG), partial
Description
Biochemical and Functional Context
PEPCK is central to gluconeogenesis, enabling carbon flux from non-carbohydrate precursors (e.g., amino acids, lactate) to glucose. In S. solfataricus, gluconeogenesis intersects with the Entner-Doudoroff (ED) pathway, which produces glyceraldehyde 3-phosphate (GAP) for downstream biosynthesis . While the full-length PEPCK from S. solfataricus has not been explicitly characterized, its partial recombinant form (pckG) likely shares conserved catalytic motifs with other PEPCKs.
| Enzyme Property | General PEPCK Characteristics | Potential S. solfataricus pckG Features |
|---|---|---|
| Substrate specificity | Oxaloacetate → PEP (GTP-dependent) | Requires GTP as cofactor; thermophilic stability |
| Catalytic mechanism | ATP/GTP hydrolysis drives carboxylation | GTPase activity linked to carboxylase domain |
| Structural stability | Sensitive to temperature/pH in mesophiles | Enhanced thermostability (optimal ~70°C) |
| Metabolic role | Gluconeogenesis, amino acid metabolism | Integration with ED pathway intermediates |
Genetic and Recombinant Production Challenges
Recombinant expression of archaeal enzymes in heterologous hosts (e.g., E. coli) often faces hurdles due to inclusion body formation and improper folding. For example:
-
Expression in E. coli: Hyperthermophilic proteins like PEPCK may misfold at mesophilic temperatures, necessitating solubilization agents (e.g., 2M L-arginine) to recover active enzyme .
-
Purification Strategies: Nickel affinity chromatography is common for His-tagged recombinants, though proteolysis during purification may yield truncated forms (e.g., partial pckG) .
Gluconeogenesis in Thermophiles
-
Carbon Flux Integration: The ED pathway’s semi-phosphorylated branch generates GAP, which could feed into gluconeogenesis via PEPCK-mediated PEP synthesis .
-
Biosynthetic Applications: Engineering pckG for bioproduction of glucose precursors or biofuels in thermophilic systems.
Enzyme Engineering
-
Thermostability: pckG’s partial structure may reveal insights into stabilizing motifs for industrial catalysis.
-
Co-factor Specificity: GTP dependency could be harnessed for ATP-free metabolic engineering.
Research Gaps and Future Directions
| Area | Current Status | Future Focus |
|---|---|---|
| Catalytic Activity | No kinetic data for S. solfataricus PEPCK | Measure , , GTPase activity |
| Structural Biology | No crystallographic data | Solve structure to map GTP-binding/active sites |
| Metabolic Pathway | Limited integration with ED pathway | Metabolomic profiling in S. solfataricus |
Comparative Insights from Related Organisms
Studies on PEPCK in other archaea and pathogens highlight its versatility:
Product Specs
Note: We prioritize shipping the format currently in stock. However, if you have a specific format requirement, please indicate it in your order notes. We will fulfill your request if possible.
Note: All proteins are shipped with standard blue ice packs. If you require dry ice shipping, please inform us in advance, as additional fees will apply.
Generally, liquid form has a shelf life of 6 months at -20°C/-80°C. Lyophilized form has a shelf life of 12 months at -20°C/-80°C.
Target Background
KEGG: sso:SSO2537
STRING: 273057.SSO2537
Q&A
FAQs for Researchers on Recombinant Enzymes in Sulfolobus solfataricus
(Note: The provided search results do not specifically address Phosphoenolpyruvate carboxykinase [GTP] [pckG], but the FAQs below are derived from related recombinant enzyme studies in S. solfataricus, focusing on experimental design and analysis.)
Advanced Research Questions
Q3: How can redox regulation impact recombinant enzyme kinetics in S. solfataricus?
-
Answer:
Redox-sensitive enzymes (e.g., PGK in Synechocystis) may require thioredoxin (Trx) to maintain reduced, active states . For S. solfataricus enzymes:-
Assess redox modifications (e.g., S-thiolation, glutathionylation) under oxidative stress using mass spectrometry .
-
Compare catalytic efficiency (k<sub>cat</sub>/K<sub>m</sub>) under reducing vs. oxidizing conditions .
Example: ChlPGK1 in Chlamydomonas shows reduced turnover under oxidized states due to disulfide bond formation at Cys227/Cys361 .
-
Q4: How do I resolve discrepancies in enzyme activity data between recombinant and native forms?
-
Answer:
Resolution: Perform side-by-side assays under identical conditions and validate with proteomic profiling .
Experimental Design for Functional Studies
Q5: What strategies are effective for studying multifunctional roles of enzymes (e.g., nuclear vs. metabolic)?
-
Answer:
Q6: How can CRISPR-Cas systems in S. solfataricus improve recombinant strain construction?
-
Answer:
Data Interpretation and Contradictions
Q7: Why might recombinant enzyme activity differ across expression systems?
-
Answer:
-
Host-specific folding: E. coli lacks archaeal chaperones, leading to misfolding .
-
Proteolytic degradation: Use protease inhibitors (e.g., PMSF) during purification to prevent truncation .
-
Example: Recombinant PPX from S. solfataricus showed anomalous migration on SDS-PAGE due to membrane association .
-
