Recombinant Isocitrate dehydrogenase [NADP], mitochondrial (icdA), partial

Introduction to Recombinant Isocitrate Dehydrogenase [NADP], Mitochondrial (icdA), Partial

Recombinant Isocitrate Dehydrogenase [NADP], mitochondrial (icdA), partial refers to a genetically engineered form of the mitochondrial NADP$^+$-dependent isocitrate dehydrogenase (IDH2 in mammals), which catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (αKG) while reducing NADP$^+$ to NADPH. This enzyme is critical for mitochondrial redox balance, antioxidant defense, and cellular metabolism . The "partial" designation indicates that the recombinant protein may lack specific domains or residues compared to the full-length enzyme, often used for structural or functional studies .

Catalytic Mechanism

IDH2 operates via a two-step reaction:

1. Dehydrogenation: Isocitrate → Oxalosuccinate

2. Decarboxylation: Oxalosuccinate → αKG + CO$_2$ .

The reaction generates NADPH, essential for glutathione reductase and thioredoxin reductase to maintain mitochondrial glutathione (GSH) levels and combat oxidative stress .

Expression Systems

• Escherichia coli: Widely used for recombinant IDH2 production. For example, porcine IDH2 was expressed as a maltose-binding protein (MBP) fusion and purified via thrombin cleavage .

• Yeast Systems: Saccharomyces cerevisiae models (e.g., IDP1 R148H) mimic human IDH2 mutations to study 2HG-associated pathologies .

Functional Assays

• Activity Measurement: NADPH production monitored at 340 nm .

• Redox Balance Analysis: Mitochondrial NADPH/NADP$^+$ ratios assessed via HPLC or enzymatic cycling .

Role in Antioxidant Defense

• Calorie Restriction (CR): Sirt3 deacetylates IDH2, enhancing NADPH production and glutathione recycling, thereby reducing ROS and delaying age-related hearing loss .

• Ischemia-Reperfusion Injury: Idh2$^{-/-}$ mice exhibit exacerbated kidney damage due to NADPH depletion and impaired GPx activity .

Cancer and Metabolic Disorders

• 2HG Accumulation: Mutant IDH2 (R140/R172) produces 2HG, linked to gliomas and leukemias .

• Metabolic Reprogramming: IDH2 knockdown in Mortierella alpina alters lipid biosynthesis by disrupting NADPH supply .

Table 2: Mitochondrial vs. Cytosolic NADP+^++-IDHs

Challenges and Future Directions

• Structural Optimization: Improving thermostability and catalytic efficiency of partial recombinant IDH2 for industrial applications (e.g., biofuel production) .

• Therapeutic Targeting: Developing IDH2 inhibitors (e.g., AG-221) to block 2HG production in cancers .