Recombinant Desulfovibrio vulgaris 3-isopropylmalate dehydratase small subunit (leuD)

What expression systems are most effective for producing recombinant Desulfovibrio vulgaris leuD?

E. coli remains the most widely used and effective expression system for producing recombinant D. vulgaris leuD . The methodology typically involves:

• Vector selection: Vectors containing strong promoters (T7, tac) are preferred for high-level expression

• Host strain optimization: BL21(DE3) or Rosetta strains are commonly used, with the latter being advantageous when codon bias is a concern

• Expression conditions:

  • Induction: 0.1-1.0 mM IPTG

  • Temperature: 20-30°C (lower temperatures often improve proper folding)

  • Duration: 4-16 hours post-induction

While E. coli is the standard expression system, researchers should note that D. vulgaris proteins often contain iron-sulfur clusters or other cofactors that may require post-translational modifications. In such cases, anaerobic expression conditions or co-expression with chaperones may be necessary to obtain properly folded, active enzyme .

What are the optimal storage conditions for maintaining stability of recombinant D. vulgaris leuD?

Based on empirical data, the optimal storage conditions for recombinant D. vulgaris leuD are:

For reconstitution, it is recommended to briefly centrifuge the vial prior to opening and reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL with 50% glycerol as a cryoprotectant .

How do mutations in the leuD gene affect D. vulgaris growth under different metabolic conditions?

Recent genome-wide transposon mutagenesis studies of D. vulgaris Hildenborough (DvH) have revealed that leuD is conditionally essential depending on the growth medium and metabolic state . When analyzing over 1,137 non-essential genes with conditional phenotypes, researchers found:

• Leucine availability: In media lacking leucine, leuD mutants show severe growth defects, confirming the enzyme's essential role in leucine biosynthesis

• Metabolic stress conditions: Under sulfate-limiting conditions, leuD mutants exhibit heightened sensitivity, suggesting interconnections between amino acid metabolism and energy conservation pathways in sulfate-reducing bacteria

• Nitrogen source variations: When alternative nitrogen sources are provided, leuD mutation phenotypes vary, indicating potential regulatory connections between leucine biosynthesis and nitrogen assimilation pathways

This conditional essentiality makes leuD an interesting target for studying metabolic network interactions in D. vulgaris and potentially for developing selective inhibitors of sulfate-reducing bacteria .

What techniques are most effective for assessing the enzymatic activity of recombinant D. vulgaris leuD?

The enzymatic activity of recombinant D. vulgaris leuD can be assessed using multiple complementary approaches:

• Spectrophotometric assays: Monitor the conversion of 3-isopropylmalate to 2-isopropylmalate through coupled enzyme systems or direct measurement of substrate depletion at 235-240 nm

• HPLC-based methods:

  • Separate substrate and product using reverse-phase HPLC

  • Typical conditions: C18 column, mobile phase of 0.1% trifluoroacetic acid in water/acetonitrile

  • Detection: UV absorption at 210-220 nm

• Mass spectrometry:

  • LC-MS/MS for direct quantification of substrate and product

  • Isotope-labeled substrates can be used to track reaction progression

• NMR spectroscopy:

  • Real-time monitoring of reaction kinetics

  • Provides structural information about reaction intermediates

When measuring enzyme activity, researchers should ensure that both leuC and leuD subunits are present, as the individual subunits typically show minimal or no activity. The heterodimeric complex may require reconstitution with Fe-S clusters for full activity .

How does the structure of D. vulgaris leuD compare to homologous proteins from other bacteria, and what implications does this have for evolutionary studies?

Comparative structural analysis of D. vulgaris leuD reveals important evolutionary insights:

• Conserved domains: D. vulgaris leuD contains a highly conserved domain structure characteristic of the aconitase superfamily of dehydratases

• Unique adaptations: Several key residues in the active site show adaptations specific to anaerobic bacteria, particularly:

  • Modified metal-binding residues that may reflect the different redox environment

  • Altered substrate-binding pocket residues that may contribute to substrate specificity

• Evolutionary implications:

  • Phylogenetic analysis places D. vulgaris leuD in a distinct clade among sulfate-reducing bacteria

  • The protein shows evidence of horizontal gene transfer events based on codon usage analysis

  • The evolutionary rate of leuD appears slower than many other metabolic genes, suggesting strong selective pressure

This evolutionary conservation, combined with specific adaptations, makes leuD a valuable marker for studying the evolution of metabolic pathways in anaerobic bacteria and particularly in the Desulfovibrio genus .

What role does leuD play in the environmental adaptation and stress responses of D. vulgaris?

Research on D. vulgaris stress responses has revealed that leuD plays unexpected roles beyond leucine biosynthesis:

• Salt stress adaptation: Transcriptomic and metabolomic studies of salt-adapted D. vulgaris strains (ES9-11 and ES10-5) show that mutations affecting branched-chain amino acid metabolism, including the leucine biosynthesis pathway, contribute to enhanced salt tolerance. This suggests leuD may be involved in osmotic stress responses .

• Oxidative stress response: Under oxidative stress conditions, leuD expression is altered, suggesting a connection between branched-chain amino acid metabolism and redox homeostasis.

• pH adaptation: When D. vulgaris experiences acid or alkaline shock, metabolic reconfigurations occur that affect amino acid biosynthesis pathways, including potential regulatory changes in leuD expression .

• Metal resistance: Studies of metal-resistant D. vulgaris strains indicate that alterations in amino acid metabolism pathways, including leucine biosynthesis, may contribute to metal tolerance mechanisms.

These findings suggest that leuD and its metabolic products may serve as metabolic integration points that connect primary metabolism with stress response pathways in D. vulgaris .

What are the challenges and solutions for expressing active recombinant D. vulgaris leuD in heterologous systems?

Expressing active D. vulgaris leuD presents several challenges that require specific methodological solutions:

A particularly effective approach involves expressing leuD and leuC simultaneously using a bicistronic construct, which enables the formation of the active heterodimeric complex in vivo. When purifying the protein, a step-wise refolding protocol with controlled introduction of iron under anaerobic conditions has been shown to improve recovery of active enzyme .

How can recombinant D. vulgaris leuD be used as a model system for studying protein-protein interactions in metabolic complexes?

Recombinant D. vulgaris leuD provides an excellent model system for studying protein-protein interactions in metabolic complexes for several reasons:

• Heterodimeric nature: The requirement for leuC and leuD to form a functional complex makes this system ideal for studying subunit interactions and assembly

• Methodological approaches:

  • Bimolecular Fluorescence Complementation (BiFC): Tagging leuC and leuD with complementary fragments of fluorescent proteins to visualize interactions

  • Surface Plasmon Resonance (SPR): Quantifying binding kinetics between leuD and leuC

  • Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): Mapping interaction interfaces

  • Crosslinking studies: Identifying spatial relationships between subunits

• Integration with larger complexes: Recent studies suggest that leuD may participate in metabolite channeling as part of a larger supramolecular structure

For example, research on lactate utilization in D. vulgaris has revealed the existence of multi-enzyme complexes that optimize metabolic efficiency . Similar principles may apply to leucine biosynthesis enzymes, where leuD could be part of a metabolic assembly that includes other enzymes in the pathway, facilitating substrate channeling and enhancing metabolic efficiency .

What recent technological advances have improved our understanding of leuD function in D. vulgaris?

Several cutting-edge technologies have recently advanced our understanding of leuD function:

• Randomly Barcoded Transposon Mutant Library (RB-TnSeq): This technique has allowed genome-wide fitness profiling of D. vulgaris, revealing conditional essentiality of leuD under various growth conditions .

• Cryo-Electron Microscopy: High-resolution structural studies have begun to elucidate the molecular details of leuD-leuC interactions, providing insights into the catalytic mechanism.

• Metabolic Flux Analysis: By combining stable isotope labeling with mass spectrometry, researchers have mapped the metabolic fluxes through the leucine biosynthesis pathway, revealing regulatory points and connections to central metabolism.

• CRISPR-Cas9 Genome Editing: Precise genome modification techniques have enabled the creation of specific leuD variants in D. vulgaris, allowing in vivo functional studies.

• Single-Cell Techniques: New methods to study heterogeneity in bacterial populations have revealed that leuD expression can vary significantly between individual cells, suggesting complex regulatory mechanisms.

These technologies have collectively transformed our understanding of leuD from a simple biosynthetic enzyme to a multifunctional protein integrated within complex metabolic and regulatory networks in D. vulgaris .

How does leuD function intersect with the unique energy metabolism of sulfate-reducing bacteria like D. vulgaris?

The function of leuD intersects with sulfate-reducing bacteria's unique energy metabolism in several significant ways:

• Lactate metabolism connection: Studies of the lactate utilization operon (luo) in D. vulgaris have revealed that branched-chain amino acid metabolism, including leucine biosynthesis, is coordinated with central energy metabolism. The operon structure and transcriptional regulation suggest that amino acid biosynthesis is tightly linked to the organism's primary energy-generating pathways .

• Redox balance: The isomerization reaction catalyzed by leuD requires proper redox balance. In D. vulgaris, this balance is heavily influenced by the unique electron transport chains and energy conservation mechanisms of sulfate-reducing bacteria.

• Metabolic integration: Metabolomic data from experiments with D. vulgaris demonstrate that leucine and other branched-chain amino acids serve as key metabolic nodes that connect:

  • Carbon flow from lactate oxidation

  • Nitrogen assimilation

  • Sulfate reduction pathways

  • Membrane phospholipid fatty acid composition

• Adaptive significance: Changes in leuD activity may contribute to the metabolic flexibility observed in D. vulgaris during adaptation to different growth conditions. For example, salt-adapted strains (ES10-5) show alterations in branched-chain fatty acid content of membrane phospholipids, which may be linked to changes in leucine metabolism .

This integration highlights the importance of studying leuD not in isolation, but as part of the broader metabolic network that defines the unique physiology of D. vulgaris .

What are the implications of D. vulgaris leuD research for understanding pathogenic Desulfovibrio species?

Research on D. vulgaris leuD has important implications for understanding pathogenic Desulfovibrio species:

• Comparative genomics: Analysis of leuD sequences across Desulfovibrio species reveals conservation patterns that may correlate with pathogenicity potential. For example, Desulfovibrio species associated with Parkinson's disease may show distinctive variations in metabolic enzyme sequences .

• Metabolic adaptation in host environments: Understanding how leuD functions in D. vulgaris provides insights into how pathogenic Desulfovibrio species might adapt their metabolism within host environments, particularly in:

  • Nutrient-limited gut microenvironments

  • Environments with varying pH and redox conditions

  • Competitive microbial communities

• Biomarker potential: The discovery that all Parkinson's disease patients harbor Desulfovibrio bacteria in their gut microbiota, with concentrations correlating with disease severity, suggests that enzymes like leuD might serve as biomarkers or therapeutic targets .

• Antibiotic resistance connection: Studies have shown that some Desulfovibrio strains exhibit antibiotic resistance, and metabolism-related genes like leuD may be involved in adaptation mechanisms that contribute to persistence during antibiotic treatment .

• Potential for targeted interventions: Understanding the structural and functional details of leuD could enable the development of specific inhibitors that target pathogenic Desulfovibrio species while minimizing impacts on beneficial gut microbiota .