Recombinant Escherichia coli Malate dehydrogenase (mdh)
Description
Definition and Basic Properties
Malate dehydrogenase (MDH; EC 1.1.1.37) catalyzes the reversible oxidation of malate to oxaloacetate using NAD/NADH as a cofactor. The recombinant form is produced by cloning and expressing the mdh gene (GeneID: 57728493) in E. coli strains, typically with purification tags such as N-terminal 6xHis for affinity chromatography .
Key Biochemical Properties
| Property | Value | Source |
|---|---|---|
| Molecular Weight | 36.3 kDa (theoretical) | |
| Subunit Structure | Dimeric | |
| Optimal pH | 8.0 (Tris/PBS-based buffer) | |
| Catalytic Activity | 130.5 µmol NADH/min/mg | |
| Storage Stability | 12 months at -80°C |
Gene Expression and Regulation
The mdh gene in E. coli is located at 70.6 min on the chromosome and is regulated by:
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ArcA-ArcB system: Represses transcription under anaerobic conditions .
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Carbon sources: Expression varies by ~11-fold depending on substrate availability .
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Heme availability: Heme limitation increases expression fivefold .
Recombinant production typically uses plasmids (e.g., pLC32-38) under strong promoters to bypass native regulatory mechanisms .
Functional Roles in Metabolism
In E. coli, MDH operates in:
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TCA cycle: Converts malate to oxaloacetate under aerobic conditions .
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Gluconeogenesis: Supports oxaloacetate synthesis for glucose production .
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Redox balance: Regulates NAD/NADH ratios, crucial during oxidative stress .
Deletion Mutant Phenotypes
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Δmdh strains exhibit severe growth defects on acetate or C₄ substrates .
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Δmqo (malate:quinone oxidoreductase) mutants show no growth impairment, indicating MDH’s dominance in malate oxidation .
Industrial and Research Applications
Recombinant MDH is employed in:
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Enzyme assays: Quantifying metabolites in metabolic pathways .
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Structural studies: Crystallography and molecular dynamics simulations .
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Biocatalysis: NADH regeneration systems for synthetic biology .
Production Workflow
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Cloning: mdh gene insertion into expression vectors (e.g., pKO3) .
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Expression: Induced in E. coli (e.g., strain K12) under optimized conditions .
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Purification: Affinity chromatography (6xHis tag), yielding >90% purity .
Challenges and Innovations
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Stability: Recombinant MDH requires glycerol (5–50%) for long-term storage .
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Activity optimization: Directed evolution improves thermostability and catalytic efficiency .
Future Directions
Research priorities include engineering MDH for:
Product Specs
Target Background
KEGG: ecj:JW3205
STRING: 316385.ECDH10B_3413
