Recombinant Geobacter uraniireducens Glucose-6-phosphate isomerase (pgi), partial
Description
Enzyme Characteristics and Function
Catalytic Role:
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PGI is essential for central carbon metabolism, enabling interconversion of G6P and F6P. This reaction is pivotal in glycolysis, gluconeogenesis, and the pentose phosphate pathway .
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In G. uraniireducens, PGI likely supports metabolic flexibility, allowing the organism to thrive in subsurface environments with fluctuating nutrient availability .
Cloning and Expression:
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The pgi gene (GenBank accession not specified) is cloned into a plasmid (e.g., pBAD) and expressed in Escherichia coli . This method mirrors protocols used for P. furiosus PGI, which achieved ~95% purity post-affinity chromatography .
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G. uraniireducens genomic DNA serves as the template for PCR amplification, followed by ligation into an expression vector .
Key Steps:
Biochemical and Kinetic Properties
Activity Metrics:
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Homologous PGIs exhibit substrate affinity (Km) in the micromolar range. For example:
Thermostability:
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Archaeal PGIs are thermostable (optimal activity at >80°C) , but G. uraniireducens PGI likely operates at mesophilic temperatures (~32°C) .
Applications and Research Implications
Bioremediation Potential:
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G. uraniireducens is renowned for uranium (U(VI)) and arsenic reduction . PGI-mediated glycolysis supplies ATP for extracellular electron transfer (EET) pathways, which drive metal reduction .
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Recombinant PGI could enhance metabolic modeling to optimize bioremediation strategies .
Metabolic Engineering:
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Partial PGIs may serve as tools to dissect glycolytic flux control. For instance, PGI inhibition in cardiomyocytes elevates G6P levels, activating mTOR signaling and protein synthesis .
Research Gaps and Future Directions
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Structural Analysis: Cryo-EM or X-ray crystallography is needed to resolve the enzyme’s 3D architecture.
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Functional Studies: Assays comparing recombinant vs. native PGI kinetics and thermostability are lacking.
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Biotechnological Optimization: Codon usage adjustments or fusion tags may improve soluble yield in E. coli .
Product Specs
Target Background
KEGG: gur:Gura_3752
STRING: 351605.Gura_3752
