Recombinant Shewanella sediminis Malate dehydrogenase (mdh)
Description
Introduction to Shewanella sediminis and its Malate Dehydrogenase
Shewanella sediminis is a psychrophilic (cold-loving) rod-shaped marine bacterium originally isolated from Halifax Harbour sediment. It has garnered scientific interest for its remarkable ability to degrade hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) . The bacterium belongs to the Na+-requiring group of Shewanella species and possesses distinctive metabolic capabilities that set it apart from other members of the genus .
Malate dehydrogenase (EC 1.1.1.37) is a critical enzyme in central metabolism that catalyzes the reversible conversion of malate to oxaloacetate using NAD+ as a cofactor . In the Shewanella genus, this enzyme plays vital roles beyond the citric acid cycle, including aspartate biosynthesis, the malate-aspartate shuttle, gluconeogenesis, and lipogenesis . The recombinant form of S. sediminis MDH has been produced to study its unique properties and potential applications.
2.1. Protein Structure
While the exact crystal structure of S. sediminis MDH has not yet been definitively determined, bacterial MDHs typically form oligomeric structures. Based on comparative analysis of other bacterial MDHs, S. sediminis MDH likely adopts a tetrameric quaternary structure composed of identical subunits, each with a molecular weight of approximately 30-35 kDa . The enzyme's structure includes regions involved in substrate binding, cofactor binding, and dimer-dimer interface formation.
2.2. Gene Organization
The mdh gene in S. sediminis encodes the malate dehydrogenase enzyme. Like other members of the Shewanella genus, the gene organization and transcriptional regulation of mdh in S. sediminis is likely integrated into metabolic networks controlling central metabolism . Comparative genomic studies of the Shewanella genus have revealed conservation of key metabolic pathways, with species-specific adaptations reflecting their diverse ecological niches .
3.1. Catalytic Reaction
Recombinant S. sediminis MDH catalyzes the following reversible reaction:
L-Malate + NAD+ ⇌ Oxaloacetate + NADH + H+
While the thermodynamic equilibrium favors malate formation, in vivo conditions and metabolic demands can drive the reaction in either direction .
3.2. Kinetic Properties
Based on comparative data from bacterial MDHs, the kinetic parameters for S. sediminis MDH likely fall within the following ranges, though specific values for this species would require direct experimental determination:
| Parameter | Probable Range | Notes |
|---|---|---|
| Km for Oxaloacetate | 20-189 μM | Expected to be optimized for lower temperatures |
| Km for NADH | 14-90 μM | Cofactor binding likely adapted to cold environments |
| Km for Malate | 0.12-9 mM | Higher than for oxaloacetate due to reaction equilibrium |
| Km for NAD+ | 0.1-2.7 mM | Less affinity compared to reduced form |
| pH Optimum (Reduction) | 8.0-8.75 | For oxaloacetate reduction |
| pH Optimum (Oxidation) | 9.0-10.0 | For malate oxidation |
As a psychrophilic organism, S. sediminis MDH is expected to demonstrate higher catalytic efficiency at lower temperatures compared to mesophilic counterparts, with possible structural modifications that enhance flexibility at low temperatures .
3.3. Temperature and pH Dependence
Being derived from a psychrophilic organism, recombinant S. sediminis MDH likely exhibits maximum activity at temperatures lower than those of mesophilic bacteria. By comparison with other cold-adapted enzymes, its temperature optimum might be in the range of 15-25°C, with significant activity retained even at temperatures close to 0°C . This cold adaptation would be a valuable property for biotechnological applications requiring low-temperature reactions.
The pH profile of bacterial MDHs typically shows optimal activity for oxaloacetate reduction at slightly alkaline pH (8.0-8.75) and for malate oxidation at more alkaline pH (9.0-10.0) . S. sediminis MDH likely follows this pattern, though specific adaptations to marine sediment environments may exist.
4.1. Expression Systems
Recombinant expression of S. sediminis MDH has been achieved using heterologous expression systems. The most common approach involves cloning the mdh gene into expression vectors suitable for Escherichia coli hosts . Based on techniques used for other Shewanella proteins, the expression typically employs vectors with strong promoters (such as T7) and may include affinity tags (His-tag, intein-based systems) to facilitate purification .
4.2. Purification Protocol
A typical purification protocol for recombinant S. sediminis MDH may include:
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Cell lysis by sonication or mechanical disruption
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Clarification of lysate by centrifugation
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Affinity chromatography (e.g., Ni-NTA for His-tagged proteins)
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Ion exchange chromatography (DEAE or similar)
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Gel filtration chromatography
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Assessment of purity by SDS-PAGE (>85% purity desired)
For specific applications, additional purification steps may be necessary to achieve higher purity .
5.1. Biocatalysis at Low Temperatures
The psychrophilic nature of S. sediminis MDH makes it particularly valuable for biocatalytic processes that require low-temperature reactions. These applications benefit from reduced energy requirements and the ability to convert thermolabile compounds without degradation .
5.2. Bioremediative Applications
As S. sediminis is known for its ability to degrade environmental contaminants like RDX, its enzymes, including MDH, may play roles in metabolic pathways relevant to bioremediation . While MDH itself may not directly catalyze degradation of pollutants, it supports the metabolic network necessary for these processes.
5.3. Diagnostic Applications
Recombinant bacterial MDHs have been used in diagnostic applications. For example, MDH has been employed in indirect enzyme-linked immunoassays for differentiating between vaccination-induced and infection-induced antibodies in bovine brucellosis . The stable nature of recombinant MDH makes it suitable for similar diagnostic applications.
5.4. Drug Discovery Platform
MDH enzymes are emerging as potential targets for drug discovery, particularly for treatments targeting metabolic and neurological disorders, cancer, and infectious diseases . Recombinant S. sediminis MDH could serve as a model for studying inhibitors that may have therapeutic applications, particularly against psychrophilic pathogens.
6.1. Taxonomic Relationships
S. sediminis MDH shares evolutionary relationships with other bacterial MDHs but has distinct features reflecting its adaptation to marine sediment environments. Phylogenetic analysis based on 16S rRNA gene sequencing places S. sediminis in proximity to bioluminescent species like Shewanella hanedai and Shewanella woodyi, though its gyrB sequence shows less than 85% similarity to other Shewanella species .
7.2. Protein Engineering
Using site-directed mutagenesis and directed evolution approaches, recombinant S. sediminis MDH could be engineered for enhanced stability, altered substrate specificity, or improved catalytic efficiency at various temperatures .
7.3. Metabolic Engineering Applications
As a key enzyme in central metabolism, MDH could be integrated into metabolically engineered organisms for various biotechnological applications, including biofuel production and biosynthesis of high-value compounds .
References
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Recombinant Shewanella baltica Malate dehydrogenase (mdh) Product Information
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Shewanella sediminis sp. nov., a novel Na+-requiring psychrophilic rod-shaped marine bacterium
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Identification of a reductive tetrachloroethene dehalogenase in Shewanella sediminis
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The acid-tolerant L-arabinose isomerase from the mesophilic Shewanella sp. ANA-3
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Malate dehydrogenase as a multi-purpose target for drug discovery
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Isolation of Shewanella sp. from Algeria and characterization of chromate resistance and reduction mechanisms
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Function, kinetic properties, crystallization, and regulation of microbial malate dehydrogenases
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Comparative genomic reconstruction of transcriptional networks controlling central metabolism in the Shewanella genus
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Kinetic characterization and thermostability of C. elegans malate dehydrogenase
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Use of recombinant malate dehydrogenase (MDH) in diagnostic applications
Product Specs
Target Background
KEGG: sse:Ssed_0952
STRING: 425104.Ssed_0952
Q&A
What is Malate dehydrogenase (MDH) from Shewanella sediminis?
Malate dehydrogenase from Shewanella sediminis (strain HAW-EB3) is an enzyme that catalyzes the reversible oxidation of malate to oxaloacetate . It belongs to the LDH/MDH superfamily, specifically the MDH type 1 family . The protein consists of 311 amino acids with a molecular mass of approximately 32 kDa . This enzyme plays a crucial role in the tricarboxylic acid (TCA) cycle, facilitating energy metabolism in S. sediminis under various environmental conditions.
How should I design experiments to express recombinant S. sediminis MDH?
When designing experiments for recombinant expression of S. sediminis MDH, consider the following methodological approach:
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Expression system selection: While E. coli BL21 is commonly used for recombinant protein expression, Shewanella proteins may require optimization of codon usage and growth conditions .
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Vector design: Include an affinity tag (such as His-tag) to facilitate purification. The pET-28a vector has been successfully used for expression of recombinant MDH from other bacterial species .
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Growth conditions: Optimize temperature (typically 16-30°C), induction timing, and inducer concentration to maximize soluble protein yield.
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Verification: Confirm expression using SDS-PAGE analysis with Coomassie blue staining .
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Activity preservation: Include appropriate cofactors and stabilizing agents in purification buffers to maintain enzymatic activity.
Recent advances in transformation methods for Shewanella species include a high-efficiency electroporation protocol (~4.0 × 10^6 transformants/μg DNA) that may be adapted for S. sediminis .
What assay systems are recommended for measuring S. sediminis MDH activity?
To effectively measure MDH activity from S. sediminis:
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Spectrophotometric assay: Monitor the conversion of oxaloacetate (OAA) to L-malate by following the oxidation of NADH at 340 nm . This approach allows for continuous measurement of enzyme kinetics.
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Experimental conditions:
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Buffer: Typically 50-100 mM phosphate or Tris-HCl (pH 7.0-7.5)
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Substrate: 0.1-1 mM oxaloacetate
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Cofactor: 0.1-0.2 mM NADH
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Temperature: 25-37°C depending on experimental goals
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Controls: Include enzyme-free and substrate-free controls to account for background oxidation of NADH.
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Data analysis: Calculate initial rates from the linear portion of progress curves and fit to appropriate kinetic models (Michaelis-Menten, etc.).
How can I use site-directed mutagenesis to investigate S. sediminis MDH function?
A systematic approach to site-directed mutagenesis of S. sediminis MDH includes:
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Target selection: Identify conserved residues through sequence alignment with well-characterized MDH enzymes. Focus on residues in the active site, substrate binding pocket, and cofactor binding regions.
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Primer design: Create primers containing the desired mutations following these guidelines:
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Mutations should be centered within the primer sequence
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Primers should have ~10-15 nucleotides of correct sequence on either side of the mutation
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GC content of 40-60% is optimal
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Terminal G or C bases help anchoring
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Expression and purification: Express wild-type and mutant proteins under identical conditions to enable direct comparison .
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Activity analysis: Determine enzyme kinetic parameters (kcat, Km) for each mutant compared to wild-type enzyme .
How can recombineering techniques be applied to study MDH in Shewanella species?
Recent advances in genetic manipulation of Shewanella species offer powerful approaches for MDH research:
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Prophage-mediated genome engineering: A recently developed recombineering system using a λ Red Beta homolog from Shewanella sp. W3-18-1 allows precise genome editing with single-stranded DNA oligonucleotides . This system achieves approximately 5% recombinants among total cells, enabling markerless mutations in Shewanella genomes .
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Implementation methodology:
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Express the λ Red Beta homolog from Shewanella sp. W3-18-1
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Design single-stranded DNA oligonucleotides (50-70 nt) with the desired mutation centered
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Transform cells using the optimized electroporation protocol
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Screen for successful recombinants
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Applications for MDH research:
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Introduce point mutations in chromosomal mdh gene
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Create tagged versions of MDH for in vivo localization studies
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Generate conditional expression systems
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This recombineering technology represents a significant advance for high-throughput genome modification in Shewanella species, including S. sediminis .
What is the role of MDH in Shewanella's metabolic adaptation to different environments?
MDH plays a pivotal role in Shewanella's remarkable metabolic versatility:
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Respiratory flexibility: Shewanella species are known for their diverse respiratory capabilities, ranging from using metals such as Cr(VI) to electrodes to solvents like dimethyl sulfoxide (DMSO) as electron acceptors . MDH contributes to this flexibility by:
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Maintaining TCA cycle function under varied growth conditions
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Contributing to redox balance during anaerobic respiration
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Supporting carbon flow between different metabolic pathways
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Metal reduction connections: Shewanella's ability to reduce toxic metals like chromium involves multiple enzymatic systems . While the direct role of MDH in metal reduction hasn't been fully characterized, it likely supports the energetic requirements of these processes.
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Biofilm formation: Some Shewanella strains form efficient air-liquid interface biofilms (pellicles) that demonstrate enhanced metal reduction capabilities . The metabolic activity of MDH may contribute to the energy requirements of biofilm formation and maintenance.
How should I design experiments to investigate MDH function in different Shewanella strains?
When investigating MDH function across Shewanella strains, consider this experimental framework:
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Strain selection: Include diverse Shewanella species with varying metabolic capabilities (e.g., S. sediminis, S. oneidensis MR-1, S. fidelis H76, S. algidipiscicola H111) .
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Growth conditions:
Condition Variables to Control Measurements Aerobic Temperature, media composition, growth phase MDH activity, growth rate Microaerobic O₂ concentration, redox potential MDH expression levels, metabolite profiles Anaerobic Electron acceptor type, redox potential MDH activity, respiratory rates -
Independent variables: Electron acceptor type, carbon source, temperature, salinity .
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Dependent variables: MDH activity, mdh gene expression levels, growth rates .
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Controls: Include wild-type strains and defined mutants (e.g., mdh deletion strains if available) .
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Data collection: Record enzymatic activity, protein expression levels, growth parameters, and metabolite profiles at defined intervals .
This comprehensive approach will provide insights into how MDH function varies across Shewanella strains and environmental conditions.
How can I resolve issues with low activity of recombinant S. sediminis MDH?
Researchers often encounter challenges with enzymatic activity of recombinant MDH. Consider these methodological solutions:
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Expression optimization:
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Lower induction temperature (16-20°C)
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Reduce inducer concentration
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Extend expression time (overnight)
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Test different E. coli strains (BL21, Rosetta, Arctic Express)
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Protein solubility issues:
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Add solubility-enhancing tags (SUMO, MBP, GST)
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Include folding chaperones during expression
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Optimize lysis conditions (sonication parameters, buffer composition)
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Activity preservation during purification:
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Include stabilizing agents (glycerol 10-20%, reducing agents)
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Add cofactors (NAD⁺/NADH)
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Maintain cold temperature throughout purification
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Consider rapid purification methods to minimize time
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Assay troubleshooting:
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Verify substrate quality (prepare fresh oxaloacetate)
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Check cofactor integrity (NADH oxidizes over time)
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Optimize buffer conditions (pH, ionic strength)
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Test for inhibitors in the protein preparation
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How can contradictory results in MDH activity assays be reconciled?
When facing inconsistent or contradictory results in MDH activity measurements:
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Methodological variations:
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Standardize assay conditions across experiments
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Document all buffer components, including minor additives
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Control temperature precisely during measurements
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Ensure consistent protein handling procedures
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Data analysis approaches:
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Result interpretation framework:
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Compare relative activities rather than absolute values
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Consider enzyme stability over time in different conditions
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Evaluate potential inhibitory compounds in reaction mixtures
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Assess protein quality via multiple methods (activity, circular dichroism, thermal shift)
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