Recombinant Rhizobium leguminosarum bv. viciae Malate dehydrogenase (mdh)
Description
Enzymatic Function and Metabolic Role
Malate dehydrogenase (EC 1.1.1.37) is a NAD⁺-dependent oxidoreductase central to:
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TCA cycle: Converts malate to oxaloacetate, generating NADH for ATP synthesis .
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Symbiotic nitrogen fixation: Facilitates carbon exchange between rhizobia and legume hosts (e.g., Pisum sativum) by metabolizing malate, a primary carbon source for bacteroids .
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Redox balance: Maintains cytoplasmic NAD⁺/NADH equilibrium, critical for stress resistance and energy homeostasis .
In Rhizobium leguminosarum, MDH operates in tandem with other TCA enzymes (e.g., succinyl-CoA synthetase, 2-oxoglutarate dehydrogenase) to optimize metabolic flux under symbiotic conditions .
Recombinant Expression and Activity
Recombinant MDH is typically produced via plasmid-based expression in E. coli, yielding His-tagged proteins for purification . Key findings from functional studies include:
Table 1: Kinetic Properties of Recombinant MDH
| Parameter | Value (Wild-Type) | Value (Recombinant) | Conditions | Source |
|---|---|---|---|---|
| Malate transport | 100% | 21–31%* | pH 7.0, 30°C | |
| Km (oxaloacetate) | 52 μM | 54 μM | pH 8.5, 37°C | |
| Optimal pH | 8.5–9.0 | 8.5–9.0 | Forward/reverse reactions |
*When expressed with maeP (malate-specific transporter) in dct mutants .
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Substrate specificity: Recombinant MDH from R. leguminosarum exclusively recognizes malate, distinguishing it from fumarate/succinate transporters .
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Regulation: Expression is modulated by carbon sources (e.g., succinate, arabinose) and the ArcA-ArcB redox-sensing system .
Symbiotic Relevance
Recombinant MDH mutants reveal critical insights into SNF efficiency:
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R. leguminosarum dct mutants expressing maeP (malate transporter) achieve ~50% wild-type nitrogenase activity with Pisum sativum .
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Transcriptomic data suggest nodules prioritize malate over succinate/fumarate during symbiosis .
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Disruption of mdh reduces TCA cycle intermediates, impairing bacteroid development and plant nitrogen assimilation .
Applications and Future Directions
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Agricultural biotechnology: Engineering MDH variants with enhanced malate affinity could improve nitrogen fixation in legume crops .
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Industrial enzymology: Thermostable MDH variants are candidates for biocatalysis in alkaline environments .
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Metabolic modeling: Integrated RNA-seq and enzyme kinetics data enable predictive models of carbon flux in symbiosis .
Product Specs
Target Background
KEGG: rle:RL4439
STRING: 216596.RL4439
