Recombinant Raoultella terrigena Malate dehydrogenase (mdh)

What is Raoultella terrigena and why study its malate dehydrogenase?

Raoultella terrigena is a Gram-negative, rod-shaped, facultative anaerobic bacterium belonging to the Enterobacteriaceae family. It is frequently misidentified as Klebsiella species due to similarities in microbiological characteristics . R. terrigena has been isolated from various environments including soil and water, and has been identified as an opportunistic pathogen capable of causing human infections, particularly in immunocompromised individuals .

Studying malate dehydrogenase from R. terrigena is scientifically valuable for several reasons. First, MDH plays a crucial role in central carbon metabolism, particularly in the tricarboxylic acid (TCA) cycle. Second, understanding R. terrigena MDH structure and function provides insights into the organism's metabolic adaptations to diverse environments. Third, from a comparative biochemistry perspective, studying MDH from different organisms helps elucidate evolutionary relationships. Finally, given R. terrigena's emerging clinical significance and reported multidrug resistance patterns, studying its metabolic enzymes might reveal potential targets for novel antimicrobial strategies .

What is the function of malate dehydrogenase in bacterial metabolism?

Malate dehydrogenase catalyzes the NAD+/NADH-dependent reversible reaction between malate and oxaloacetate . In bacterial metabolism, MDH serves several critical functions:

• TCA Cycle: In the oxidative direction, MDH catalyzes the conversion of malate to oxaloacetate while reducing NAD+ to NADH, representing one of the final steps in the TCA cycle.

• Anaplerotic and Cataplerotic Reactions: MDH participates in reactions that replenish or remove TCA cycle intermediates based on cellular needs.

• Redox Balance: Through its involvement in NAD+/NADH interconversion, MDH contributes to maintaining cellular redox homeostasis.

• Gluconeogenesis: In the reverse direction, MDH can contribute to gluconeogenesis by converting oxaloacetate to malate.

• Malate-Aspartate Shuttle: In some bacteria, MDH participates in this shuttle system that transfers reducing equivalents across membranes.

The bidirectional nature of the MDH reaction allows it to function flexibly according to the metabolic demands of the cell, making it a critical enzyme for bacterial adaptation to changing environmental conditions.

How can R. terrigena MDH be identified and characterized molecularly?

Molecular identification and characterization of R. terrigena MDH involves several complementary approaches:

Gene Identification:

• Whole genome sequence analysis using homology-based searches with known MDH sequences from related species

• PCR amplification using primers designed based on conserved regions of MDH genes in Enterobacteriaceae

• Creation of cDNA libraries from R. terrigena RNA, similar to approaches used for other species' MDH genes

Sequence Analysis:

• Multiple sequence alignment with MDH sequences from related organisms to identify conserved catalytic residues and structural motifs

• Phylogenetic analysis to place R. terrigena MDH in evolutionary context with other bacterial MDHs

• In silico prediction of protein properties (molecular weight, isoelectric point, solubility)

Recombinant Expression and Characterization:

• Cloning the MDH gene into an expression vector (such as pET28a+) for expression in E. coli or other suitable hosts

• Purification via affinity chromatography if expressed with a tag (His, GST, etc.)

• Enzymatic assays to determine kinetic parameters and substrate specificity

• Protein modeling software to predict 3D structure based on amino acid sequence

Modern molecular biology approaches such as MALDI-TOF mass spectrometry can also assist in identification of the protein and confirmation of recombinant expression.

What expression systems are suitable for producing recombinant R. terrigena MDH?

Several expression systems can be considered for recombinant production of R. terrigena MDH:

Optimization Considerations:

• Host strain selection: BL21(DE3) and derivatives are common choices due to reduced protease activity

• Induction conditions: Temperature (typically 15-37°C), inducer concentration, and induction time

• Media composition: Rich media (LB, TB) for high yields or defined media for specific applications

• Codon optimization: May be necessary for efficient expression, as noted in studies with other MDH genes

Alternative Expression Systems:

• Yeast systems (S. cerevisiae, P. pastoris) can be considered if E. coli expression results in insoluble or inactive protein

• Cell-free expression systems allow rapid protein production for screening or producing proteins toxic to host cells

• Insect cell systems may be useful for producing more complex proteins with post-translational modifications

The Gibson Cloning assembly method, as previously used for other MDH genes , is an efficient approach for inserting the R. terrigena MDH gene into appropriate expression vectors.

What are the optimal conditions for expressing recombinant R. terrigena MDH?

Optimizing expression of recombinant R. terrigena MDH requires systematic testing of multiple parameters:

E. coli Strain Selection:

• BL21(DE3): Standard strain for T7 promoter-based expression

• Rosetta or CodonPlus: Provide rare tRNAs if codon bias is an issue

• C41/C43(DE3): Derived from BL21(DE3), better for potentially toxic proteins

• SHuffle: Engineered for improved disulfide bond formation in cytoplasm

Expression Vector Considerations:

• Promoter strength: T7 (strong, inducible) vs. tac (moderate, inducible)

• Fusion tags: His6 (small, minimal impact), MBP (enhances solubility), GST (aids folding)

• Cleavage sites: Include protease recognition sequences (TEV, thrombin) if tag removal is desired

Induction Parameters:

• Temperature: Test range from 15°C (slower expression, better folding) to 37°C (rapid expression)

• IPTG concentration: Typically 0.1-1.0 mM, with lower concentrations sometimes improving solubility

• Induction timing: Mid-log phase (OD600 ~0.6-0.8) is standard

• Duration: 3-4 hours at 37°C or overnight at lower temperatures

Media and Growth Conditions:

• Media types: LB (standard), TB (higher cell density), auto-induction media (no IPTG needed)

• Additives: 0.5-1% glucose to suppress basal expression; glycylglycine to buffer pH

• Aeration: Ensure adequate oxygen supply through proper flask-to-medium ratio and agitation

Codon Optimization:
As noted in research with other MDH genes, codon optimization may be necessary for efficient expression in E. coli . This involves adjusting the coding sequence to match E. coli codon preferences without changing the amino acid sequence.

Experimental Design:
A factorial design testing key parameters (temperature, IPTG concentration, induction time) is recommended, followed by SDS-PAGE and activity assays to determine optimal conditions for soluble, active enzyme production.

How can codon optimization enhance R. terrigena MDH expression in E. coli?

Codon optimization can significantly enhance heterologous protein expression by addressing codon usage differences between the source organism and expression host:

Rationale for Optimization:
Different organisms preferentially use different codons to encode the same amino acid. When expressing R. terrigena genes in E. coli, rare codons in the host can lead to translational pausing, premature termination, or reduced expression levels. Initial experiments with MDH from other organisms have indicated that codon optimization might be required for optimal expression .

Key Optimization Strategies:

• Codon Adaptation Index (CAI) Improvement:

  • Identify rare E. coli codons in the R. terrigena MDH sequence

  • Replace these with synonymous codons more frequently used in highly expressed E. coli genes

  • Target a CAI value of >0.8 for optimal expression efficiency

• GC Content Adjustment:

  • Optimize GC content to 40-60%, similar to highly expressed E. coli genes

  • Avoid extreme GC content that can lead to stable mRNA secondary structures

• mRNA Secondary Structure Optimization:

  • Minimize stable secondary structures, especially near the translation initiation region

  • Eliminate internal Shine-Dalgarno-like sequences that could cause ribosomal pausing

  • Remove potential RNase E cleavage sites to improve mRNA stability

Implementation Approaches:

• Gene synthesis: Design and synthesize a fully optimized gene based on the protein sequence

• Site-directed mutagenesis: Modify specific problematic codons in an existing clone

• Hybrid approach: Optimize only critical regions (N-terminus, rare codon clusters)

Validation Methods:

• Compare expression levels between optimized and native sequences using SDS-PAGE

• Measure soluble vs. insoluble protein fractions to assess proper folding

• Conduct enzyme activity assays to ensure functionality is maintained

Codon optimization tools (OPTIMIZER, JCat, IDT Codon Optimization Tool) can generate optimized sequences based on E. coli codon usage tables. The resulting optimized gene can be synthesized and cloned into the chosen expression vector using methods like Gibson Assembly .

What purification strategies yield the highest purity and activity for R. terrigena MDH?

Purification of recombinant R. terrigena MDH requires a strategic approach to achieve high purity while maintaining enzymatic activity:

Initial Considerations:

• All purification steps should be performed at 4°C to minimize protein denaturation

• Buffer composition should include stabilizing agents (glycerol, reducing agents)

• Activity assays should be performed at each step to monitor purification efficiency

Recommended Purification Workflow:

• Cell Lysis:

  • Mechanical methods: Sonication (pulsed to prevent overheating) or French press

  • Chemical methods: Lysozyme treatment (0.2-1 mg/mL) followed by detergent

  • Addition of protease inhibitors (PMSF, EDTA, or commercial cocktails)

• Initial Clarification:

  • Centrifugation (20,000-30,000 × g for 30-60 minutes)

  • Filtration through 0.45 μm membrane filters

• Affinity Chromatography (for tagged proteins):

  • Ni-NTA or TALON resin for His-tagged proteins

  • Typical binding buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10-20 mM imidazole

  • Wash with increasing imidazole (20-50 mM) to remove non-specific binding

  • Elution with high imidazole (250-500 mM)

• Ion Exchange Chromatography:

  • Based on the predicted pI of R. terrigena MDH

  • Anion exchange (Q Sepharose) if pI < 7.0

  • Cation exchange (SP Sepharose) if pI > 7.0

  • Gradient elution with increasing salt concentration

• Size Exclusion Chromatography:

  • Final polishing step to achieve high purity

  • Separation based on molecular size

  • Typical buffer: 50 mM phosphate or Tris pH 7.5, 150 mM NaCl

• Tag Removal (if necessary):

  • Treatment with appropriate protease (TEV, thrombin)

  • Second affinity step to remove cleaved tag and protease

Quality Assessment:

• SDS-PAGE to evaluate purity (target >95%)

• Western blot to confirm identity

• Activity assays to verify functional enzyme

• Mass spectrometry to confirm protein identity and integrity

• Dynamic light scattering to assess homogeneity

Specific Considerations for MDH:

• Include cofactor (NAD+) in buffers to stabilize the enzyme

• Consider adding substrate analogs to stabilize the enzyme conformation

• Test different buffer systems (HEPES, phosphate, Tris) for optimal activity retention

The final purification protocol should be optimized empirically for R. terrigena MDH to maximize both yield and specific activity.

What methods are recommended for determining the kinetic parameters of R. terrigena MDH?

Accurate determination of kinetic parameters (Kcat, Vmax, Km) for R. terrigena MDH requires carefully designed assays and analytical approaches:

Spectrophotometric Assays:
The standard method for MDH activity measurement is a continuous spectrophotometric assay monitoring NADH absorbance at 340 nm (ε = 6,220 M⁻¹·cm⁻¹):

• Forward Reaction (Malate → Oxaloacetate):

  • Monitor increase in absorbance as NAD⁺ is reduced to NADH

  • Typical assay components: 50-100 mM buffer (pH 7.5-8.5), 0.1-0.5 mM NAD⁺, variable malate concentrations (0.05-10 mM), purified enzyme

• Reverse Reaction (Oxaloacetate → Malate):

  • Monitor decrease in absorbance as NADH is oxidized to NAD⁺

  • Typical components: 50-100 mM buffer (pH 7.0-7.5), 0.1-0.2 mM NADH, variable oxaloacetate concentrations (0.01-2 mM), purified enzyme

Experimental Design for Kinetic Analysis:

• Initial Rate Determination:

  • Use sufficiently low enzyme concentration to ensure linear progress curves

  • Record initial velocities (first 10% of reaction)

  • Include a range of substrate concentrations spanning 0.2-5× expected Km

  • Maintain constant temperature (typically 25°C or 37°C)

• Data Analysis Approaches:

  • Direct fitting to Michaelis-Menten equation: v = Vmax[S]/(Km + [S])

  • Linear transformations (Lineweaver-Burk, Eadie-Hofstee, Hanes-Woolf) for visualization

  • Non-linear regression using enzyme kinetics software (GraphPad Prism, DynaFit)

• Parameter Calculation:

  • Km: Substrate concentration at half-maximal velocity

  • Vmax: Maximum velocity at saturating substrate

  • kcat: Turnover number (Vmax/[E]total)

  • kcat/Km: Catalytic efficiency

Additional Kinetic Characterization:

• pH Dependence:

  • Determine activity across pH range (6.0-9.0)

  • Plot V/Vmax vs. pH to identify optimal pH and pKa values

• Temperature Effects:

  • Determine activity across temperature range (15-50°C)

  • Arrhenius plot (ln k vs. 1/T) to determine activation energy

  • Thermal stability assessment using activity retention after pre-incubation

• Cofactor Specificity:

  • Test alternative cofactors (NADP⁺/NADPH vs. NAD⁺/NADH)

  • Determine Km values for cofactors

• Inhibition Studies:

  • Product inhibition (oxaloacetate, NADH)

  • Substrate inhibition at high concentrations

  • Effect of divalent cations (Mg²⁺, Mn²⁺, Ca²⁺)

These methods would provide a comprehensive kinetic characterization of recombinant R. terrigena MDH, enabling comparison with MDH from other organisms and providing insights into its metabolic role in R. terrigena.

How does the structure of R. terrigena MDH compare to MDH from other species?

Structural comparison of R. terrigena MDH with homologs from other species provides valuable insights into evolutionary relationships and functional adaptations:

Expected Structural Features:
Based on other bacterial MDHs, R. terrigena MDH likely features:

• Homodimeric or homotetrameric quaternary structure

• Subunit molecular weight of approximately 30-35 kDa

• Rossmann fold for nucleotide binding

• Substrate-binding domain containing conserved catalytic residues

Comparative Analysis Approaches:

• Sequence-Based Structural Predictions:

  • Multiple sequence alignment with characterized MDHs

  • Identification of conserved catalytic residues and structural motifs

  • Protein modeling software can predict 3D structure based on homology to solved structures

  • Analysis of conservation patterns to identify functionally important regions

• Homology Modeling:

  • Use of experimentally determined MDH structures as templates

  • Quality assessment using metrics such as QMEAN, DOPE score

  • Refinement through energy minimization

  • Validation through Ramachandran plot analysis and PROCHECK

• Structural Analysis Focus Areas:

  • Active site architecture and substrate binding residues

  • Cofactor binding pocket and specificity determinants

  • Subunit interfaces and oligomerization

  • Surface properties (electrostatics, hydrophobicity)

Expected Comparative Insights:

R. terrigena MDH likely shares highest structural similarity with MDH from closely related Enterobacteriaceae. It may possess unique features related to its environmental adaptations or substrate preferences. Given the frequent misidentification of R. terrigena as Klebsiella species , comparative structural analysis could potentially identify distinctive features that differentiate MDHs from these closely related bacteria.

The multidrug resistance observed in clinical R. terrigena isolates raises interesting questions about potential structural adaptations that might indirectly affect drug efflux systems or cell wall permeability, which could be investigated through comparative structural genomics.

Recent investigations of novel Raoultella species provide an opportunity to compare MDH structures within the genus to identify genus-specific features versus species-specific adaptations.

What role might R. terrigena MDH play in the organism's environmental adaptation?

Malate dehydrogenase likely contributes significantly to R. terrigena's ability to adapt to diverse environmental conditions:

Metabolic Flexibility and Energy Production:

• As a key TCA cycle enzyme, MDH enables efficient energy production under aerobic conditions

• The reversible nature of the MDH reaction allows metabolic flexibility in fluctuating environments

• In soil and aquatic habitats where R. terrigena naturally occurs, this metabolic flexibility may provide competitive advantages

Response to Environmental Stressors:

• MDH activity directly influences NAD⁺/NADH ratios, affecting cellular redox state

• Under oxidative stress conditions, modulation of MDH activity could help maintain redox homeostasis

• The TCA cycle intermediates generated through MDH activity may serve as precursors for stress-protective compounds

Host Colonization Capabilities:

• R. terrigena has been identified as an opportunistic pathogen capable of causing septicemia and urinary tract infections

• MDH activity may support growth in nutrient-limited host environments

• Central metabolism often connects to virulence factor expression in opportunistic pathogens

Ecological Interactions:

• Recent research has identified ecological roles for R. terrigena in plant-insect interactions, including degradation of plant defense compounds

• MDH might indirectly support these interactions by providing metabolic intermediates or energy for specialized metabolic pathways

• Comparative analysis with newly described Raoultella species associated with plant bleeding cankers could reveal niche-specific adaptations

Research Approaches to Investigate MDH's Role:

• Transcriptomics to examine MDH expression under diverse environmental conditions

• Metabolomics to trace carbon flux through MDH under different growth conditions

• Gene knockout or knockdown studies to assess phenotypic effects

• Comparative analysis of MDH properties from R. terrigena strains isolated from different niches

Understanding MDH's contribution to environmental adaptation could provide insights into R. terrigena's ecological success and emergence as an opportunistic pathogen with concerning multidrug resistance profiles .

How can site-directed mutagenesis be used to study R. terrigena MDH catalytic mechanism?

Site-directed mutagenesis represents a powerful approach for investigating the catalytic mechanism of R. terrigena MDH by systematically altering specific amino acid residues:

Strategic Target Selection for Mutagenesis:

• Catalytic Residues:

  • Based on sequence alignment with well-characterized MDHs, identify putative catalytic residues

  • Typical catalytic residues in MDHs include conserved Arg, His, and Asp residues

  • Create both conservative mutations (maintain charge/polarity) and non-conservative mutations

• Substrate-Binding Pocket:

  • Identify residues likely involved in malate/oxaloacetate binding

  • Target hydrogen-bonding residues that coordinate substrate hydroxyl and carboxyl groups

  • Modify residues that contribute to substrate specificity

• Cofactor-Binding Site:

  • Target residues in the Rossmann fold that interact with NAD⁺/NADH

  • Investigate determinants of cofactor specificity (NAD⁺ vs. NADP⁺)

  • Examine the role of residues involved in transition state stabilization

Mutagenesis Methodology:

• Primer Design Considerations:

  • Design primers containing the desired mutation with 15-20 matching nucleotides flanking each side

  • Optimize codon usage for the expression host

  • Include silent mutations to create diagnostic restriction sites when possible

• Mutagenesis Techniques:

  • QuikChange or Q5 site-directed mutagenesis for single mutations

  • Gibson Assembly or overlap extension PCR for multiple or complex mutations

  • CRISPR-Cas9 methods for genomic modification in vivo

• Mutant Verification:

  • DNA sequencing to confirm the intended mutation and absence of unwanted changes

  • Expression testing to verify protein production

  • Circular dichroism to ensure proper folding

Functional Analysis Approaches:

• Enzyme Kinetics Assessment:

  • Determine kinetic parameters (Km, kcat, kcat/Km) for each mutant

  • Compare substrate and cofactor binding affinities with wild-type

  • Analyze pH-dependence profiles to identify shifts in optimal pH

• Stability Analysis:

  • Thermal denaturation assays to assess structural stability

  • Chemical denaturation with urea or guanidinium hydrochloride

  • Limited proteolysis to examine conformational changes

• Structural Investigation:

  • X-ray crystallography of key mutants to directly observe structural changes

  • Molecular dynamics simulations to predict effects of mutations on protein dynamics

  • NMR studies for solution-phase structural analysis

Expected Insights:
This systematic approach would reveal critical residues for catalysis, substrate binding, and structural integrity, elucidating the precise catalytic mechanism of R. terrigena MDH. Understanding these molecular details could potentially inform the design of specific inhibitors, which might be particularly relevant given the multidrug resistance observed in clinical R. terrigena isolates .

What challenges exist in crystallizing recombinant R. terrigena MDH for structural studies?

Crystallizing recombinant R. terrigena MDH for structural studies presents several technical challenges that researchers must systematically address:

Sample Preparation Challenges:

• Protein Purity Requirements:

  • Ultra-high purity (>95%) is essential for successful crystallization

  • Contaminants can inhibit crystal formation or lead to non-specific crystal packing

  • Final purification typically requires polishing steps like size-exclusion chromatography

• Protein Stability Considerations:

  • MDH may exhibit limited stability under crystallization conditions

  • Thermal shift assays (Thermofluor) can identify stabilizing buffer compositions

  • Addition of substrates, cofactors, or substrate analogs often enhances protein stability

• Homogeneity Assessment:

  • Verify monodispersity through dynamic light scattering (DLS)

  • Assess oligomeric state through analytical ultracentrifugation or native PAGE

  • Confirm protein integrity through mass spectrometry

Crystallization Process Challenges:

• Initial Screening Approaches:

  • Commercial sparse matrix screens provide starting conditions

  • Systematically test variables: pH (5.0-9.0), precipitants (PEG, salts), additives

  • Screen multiple crystallization methods: vapor diffusion, batch, microdialysis

• Optimization Strategies:

  • Fine grid screening around promising conditions

  • Streak seeding or microseeding from initial crystals

  • Additive screening to improve crystal quality

  • Control of nucleation through temperature fluctuation or oil barriers

• Crystal Quality Issues:

  • Twinning or disorder may require extensive optimization

  • Small crystal size may necessitate synchrotron radiation

  • Crystal fragility may require careful harvesting and cryoprotection

MDH-Specific Considerations:

• Conformational Flexibility:

  • MDH undergoes domain movement during catalysis

  • Co-crystallization with substrates/inhibitors can stabilize specific conformations

  • Surface entropy reduction (replacing surface lysines/glutamates with alanines) may promote crystal contacts

• Ligand Complexes:

  • Substrate binding may be necessary for stable crystal formation

  • NAD⁺/NADH binding can induce conformational changes affecting crystallization

  • Substrate analogs or transition state mimics can trap catalytically relevant conformations

Alternative Approaches:

• Construct Engineering:

  • N- or C-terminal truncations to remove flexible regions

  • Fusion proteins (T4 lysozyme, BRIL) to provide crystal contacts

  • Surface entropy reduction mutations to promote crystallization

• Alternative Structural Methods:

  • Cryo-electron microscopy if crystallization proves challenging

  • Small-angle X-ray scattering (SAXS) for low-resolution envelope

  • NMR for dynamics studies of specific domains

• Computational Approaches:

  • Homology modeling based on related MDH structures

  • Molecular dynamics simulations to explore conformational space

  • Integration of biochemical data with computational models

By systematically addressing these challenges, researchers can increase the likelihood of obtaining diffraction-quality crystals of R. terrigena MDH, potentially revealing unique structural features related to this organism's metabolic adaptations and environmental niche.