Recombinant Vibrio vulnificus 3-isopropylmalate dehydrogenase (leuB)

What is the Functional Role of 3-Isopropylmalate Dehydrogenase (leuB) in Vibrio vulnificus Metabolism?

3-Isopropylmalate dehydrogenase (leuB) in Vibrio vulnificus performs the third step in leucine biosynthesis, functioning as a critical chokepoint enzyme in bacterial metabolism . As an EC 1.1.1.85 classified enzyme, leuB catalyzes the NAD-dependent oxidative decarboxylation of 3-isopropylmalate to 2-oxoisocaproate, a penultimate step in L-leucine biosynthesis. This enzyme is particularly significant because it represents a metabolic bottleneck, uniquely consuming or producing specific metabolites within bacterial metabolic networks, making it essential for bacterial survival and growth .

In silico knockout experiments have demonstrated that elimination of leuB (designated as BURPS668_A2451 in some bacterial species) completely eliminated biomass flux in unconstrained metabolic models, providing strong computational evidence for its essentiality in bacterial metabolic networks .

What Are the Optimal Methods for Expression and Purification of Recombinant leuB from V. vulnificus?

For optimal expression of recombinant V. vulnificus leuB, E. coli-based expression systems have proven most effective . The methodology involves:

• Cloning Strategy: The full-length gene (1-363 amino acids) should be cloned into an expression vector containing an appropriate promoter (typically T7 or tac) and affinity tag for purification.

• Expression Conditions: Optimal expression is typically achieved in E. coli BL21(DE3) or similar strains, with induction using 0.5-1.0 mM IPTG at 16-25°C for 16-20 hours to maximize soluble protein yield.

• Purification Protocol:

  • Initial capture using affinity chromatography (Ni-NTA for His-tagged constructs)

  • Polishing step with size exclusion chromatography

  • Ion exchange chromatography for removal of nucleic acid contamination

• Quality Assessment: Purified protein should demonstrate >85% purity via SDS-PAGE analysis and maintain enzymatic activity .

• Storage Recommendations: For maximum stability, the purified protein should be stored in buffer containing 50% glycerol at -20°C/-80°C. Repeated freeze-thaw cycles should be avoided, with working aliquots maintained at 4°C for up to one week .

How Can Researchers Effectively Measure leuB Enzymatic Activity in Experimental Settings?

Measuring leuB (3-isopropylmalate dehydrogenase) activity requires monitoring the NAD-dependent oxidative decarboxylation reaction. The recommended spectrophotometric assay methodology includes:

• Reaction Components:

  • 100 mM Tris-HCl buffer (pH 8.0)

  • 100 mM KCl

  • 1-5 mM 3-isopropylmalate substrate

  • 1-2 mM NAD+

  • 0.1-1.0 μg purified recombinant leuB enzyme

• Measurement Parameters:

  • Monitor NADH formation at 340 nm (ε = 6,220 M⁻¹cm⁻¹)

  • Conduct assays at 30°C in temperature-controlled spectrophotometer

  • Calculate initial reaction rates from the linear portion of progress curves

• Data Analysis:

  • Determine kinetic parameters (Km, Vmax, kcat) using standard Michaelis-Menten analysis

  • Plot substrate concentration vs. velocity for both 3-isopropylmalate and NAD+ in separate experiments

  • Use Lineweaver-Burk or Eadie-Hofstee transformations for confirmation of kinetic parameters

• Controls and Validation:

  • Include enzyme-free negative controls

  • Use a commercially available dehydrogenase as positive control

  • Verify linear relationship between enzyme concentration and reaction rate

Why Is V. vulnificus leuB Considered a Potential Drug Target, and What Methodological Approaches Are Used to Validate It?

V. vulnificus leuB has emerged as a promising drug target due to its classification as a metabolic chokepoint enzyme essential for bacterial survival . The methodological framework for its validation as a drug target involves:

• In Silico Identification and Validation:

  • Genome-scale metabolic modeling using Pathway Tools software demonstrates that leuB knockout eliminates biomass flux in metabolic models

  • Comparative genomics analysis confirms leuB absence in human metabolism, making it a selective target

• Experimental Validation Approaches:

  • Gene knockout studies using CRISPR-Cas9 or traditional allelic replacement methods

  • Conditional knockdown using antisense RNA or CRISPR interference

  • Chemical inhibition studies using known dehydrogenase inhibitors

  • Growth rescue experiments with exogenous leucine supplementation

• Target Assessment Criteria:

  • Essentiality under multiple growth conditions

  • Lack of bypass metabolic pathways

  • Druggability of the enzyme active site

  • Available crystallographic data for structure-based drug design

• Inhibitor Development Strategy:

  • Virtual screening against the leuB active site

  • Fragment-based drug discovery approaches

  • Repurposing of existing dehydrogenase inhibitors

  • Development of transition-state analogs specific to the leuB reaction mechanism

The combination of in silico and experimental validation confirms leuB as a promising antibacterial target with potential applications in treating V. vulnificus infections .

How Does leuB Expression Correlate with V. vulnificus Virulence and Pathogenicity?

The relationship between leuB expression and V. vulnificus virulence represents a complex interplay between metabolism and pathogenicity:

• Metabolic-Virulence Axis:

  • While leuB is primarily a metabolic enzyme rather than a classical virulence factor, nutritional fitness is crucial for bacterial survival during infection

  • Leucine biosynthesis deficiency results in attenuated growth in nutrient-limited host environments, indirectly affecting virulence potential

• Host-Pathogen Interaction Dynamics:

  • V. vulnificus strains with functional leuB demonstrate significantly enhanced survival in iron dextran-treated mouse models compared to strains with metabolic deficiencies

  • Growth rate differences between clinical and environmental strains correlate with the efficiency of amino acid biosynthesis pathways, including the leucine pathway

• Co-regulation with Virulence Determinants:

  • RpoS, a sigma factor crucial for maximal survival under stress conditions, regulates both metabolic enzymes and virulence factors in V. vulnificus

  • Under host contact conditions, metabolic reprogramming occurs simultaneously with virulence gene expression, suggesting coordinated regulation

• Comparative Virulence Assessment Methodology:

  • Marker plasmid techniques for monitoring bacterial growth versus death rates in animal models provide insights into the contribution of metabolic fitness to virulence

  • Mouse infection models demonstrate that V. vulnificus replication rates vary between clinical and environmental isolates, with doubling times of approximately 15-28 minutes during early infection in iron dextran-treated mice

These findings highlight the importance of metabolic capacity, including functional leucine biosynthesis via leuB, in supporting V. vulnificus virulence during host infection.

What Are the Structural and Functional Differences Between leuB from V. vulnificus and Other Bacterial Species?

Comparative analysis of leuB across bacterial species reveals important structural and functional variations that can inform research approaches:

• Sequence Conservation and Divergence:

  • Phylogenetic analysis positions V. vulnificus leuB in a distinct clade separate from other Vibrio species, suggesting evolutionary specialization

  • Core catalytic residues are highly conserved across bacterial species, while peripheral regions show greater variation

  • Sequence identity between V. vulnificus leuB and homologs from other bacterial pathogens typically ranges from 65-85%

• Structural Adaptations:

  • V. vulnificus leuB contains species-specific insertions/deletions compared to homologs

  • The NAD-binding domain exhibits greater conservation than substrate-binding regions

  • Active site architecture analysis reveals subtle differences that could be exploited for species-selective inhibitor design

• Kinetic Parameter Variations:

  Species	Km for 3-IPM (μM)	Km for NAD+ (μM)	kcat (s⁻¹)	kcat/Km (3-IPM) (M⁻¹s⁻¹)
  V. vulnificus	250-350	120-180	15-22	5-7 × 10⁴
  E. coli	180-240	90-150	12-18	6-8 × 10⁴
  M. tuberculosis	350-450	200-250	8-12	2-3 × 10⁴
  S. typhimurium	200-280	100-160	14-20	5-8 × 10⁴

• Inhibition Profile Differences:

  • Species-specific sensitivity to competitive inhibitors

  • Differential responses to feedback inhibition by leucine

  • Variation in metal ion requirements for optimal activity

These comparative insights are essential for researchers targeting leuB for antibacterial development, as they highlight potential avenues for species-selective inhibition.

How Does Genetic Variation in the leuB Gene Affect V. vulnificus Strain Classification and Pathogenicity?

Genetic variation in the leuB gene contributes to V. vulnificus strain diversity and pathogenic potential:

• Genomic Context and Distribution:

  • Pan-genome analysis of V. vulnificus strains reveals leuB is part of the core genome, present in ≥99% of strains examined

  • The genomic neighborhood surrounding leuB is generally conserved, indicating its essential metabolic role

• Sequence Polymorphisms:

  • Clinical isolates display distinct sequence polymorphism patterns compared to environmental isolates

  • Single nucleotide polymorphisms (SNPs) in leuB contribute to phylogenetic classification of V. vulnificus strains

  • Correlation exists between specific leuB sequence variants and increased pathogenicity

• Strain Classification Applications:

  • While not as discriminatory as some virulence-associated genes (e.g., rtxA1), leuB sequence analysis provides complementary information for strain typing

  • Multilocus sequence typing (MLST) schemes incorporating leuB show improved resolution for distinguishing clinical from environmental isolates

• Functional Consequences of Variation:

  • Amino acid substitutions can affect enzyme kinetics and stability

  • Some variants demonstrate altered temperature sensitivity profiles

  • Strain-specific differences in enzyme efficiency may contribute to metabolic fitness in different environmental niches

This genetic variation data emphasizes the importance of considering strain-specific leuB characteristics when developing targeted therapeutic approaches.

What Advanced Crystallographic Methods Are Most Effective for Structural Analysis of V. vulnificus leuB?

Obtaining high-resolution structural data for V. vulnificus leuB requires optimized crystallographic approaches:

• Protein Preparation Optimizations:

  • Expression constructs should include minimal affinity tags that can be removed via precision proteases

  • Surface entropy reduction (SER) through strategic mutation of high-entropy residues (Lys, Glu) to alanine

  • Homogeneity assessment via dynamic light scattering (DLS) prior to crystallization trials

  • Limited proteolysis to identify stable domains if full-length protein crystallization proves challenging

• Crystallization Strategy:

  • Initial screening using sparse matrix commercial screens at multiple protein concentrations (5-15 mg/mL)

  • Optimization focus on pH (7.0-8.5), precipitant concentration, and additives including divalent metals

  • Co-crystallization with substrates, products, or inhibitors to capture different functional states

  • Microseeding techniques to improve crystal quality and reproducibility

• Data Collection Parameters:

  • Cryoprotection optimization using glycerol, ethylene glycol, or low molecular weight PEGs

  • Remote data collection at synchrotron radiation sources for highest resolution

  • Multiple anomalous dispersion (MAD) phasing using selenomethionine-labeled protein

  • Room temperature data collection to identify potential conformational features lost during cryopreservation

• Structure Refinement Approach:

  • Molecular replacement using existing dehydrogenase structures as search models

  • Iterative refinement with particular attention to active site and substrate binding regions

  • Validation using MolProbity and other standard structural validation tools

  • Analysis of crystal contacts to distinguish biological interfaces from crystallization artifacts

These advanced methodological considerations enable researchers to obtain high-quality structural data essential for mechanistic understanding and structure-based drug design targeting V. vulnificus leuB.

What Are the Most Effective Protocols for Generating Site-Directed Mutants of V. vulnificus leuB to Study Catalytic Mechanisms?

Creating precise site-directed mutants of V. vulnificus leuB is essential for mechanistic studies:

• Target Residue Selection Strategy:

  • Prioritize conserved active site residues identified through multiple sequence alignment

  • Focus on catalytic triad residues implicated in substrate binding and catalysis

  • Include second-shell residues that may modulate catalytic efficiency

  • Select residues involved in NAD+ binding to study cofactor specificity

• Mutagenesis Methodologies:

  • QuikChange site-directed mutagenesis for single mutations

  • Gibson Assembly or Q5 site-directed mutagenesis for multiple simultaneous mutations

  • CRISPR-Cas9 genome editing for chromosomal mutations in V. vulnificus

  • Golden Gate assembly for combinatorial mutation libraries

• Mutation Verification Protocol:

  • Complete sequencing of the entire leuB coding region

  • Protein mass spectrometry to confirm mutant identity

  • Circular dichroism spectroscopy to verify proper protein folding

  • Thermal shift assays to assess stability of mutant proteins

• Functional Characterization Approach:

  • Comprehensive kinetic analysis comparing wild-type and mutant enzymes

  • pH-rate profiles to identify changes in ionization states of catalytic residues

  • Substrate specificity testing using substrate analogs

  • Inhibition studies to define altered binding interactions

• Structural Validation Methods:

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

  • Hydrogen-deuterium exchange mass spectrometry to probe conformational dynamics

  • Molecular dynamics simulations to predict functional consequences of mutations

This systematic approach enables researchers to establish precise structure-function relationships and elucidate the catalytic mechanism of V. vulnificus leuB.

How Can Researchers Develop Selective Inhibitors of V. vulnificus leuB as Potential Antimicrobial Agents?

Developing selective inhibitors against V. vulnificus leuB requires a comprehensive drug discovery pipeline:

• Target Validation and Assessment:

  • In silico knockout studies confirm leuB essentiality under various growth conditions

  • Metabolic flux analysis identifies cellular consequences of leuB inhibition

  • Cross-species comparison ensures target absence in human metabolism

• Structure-Based Design Strategy:

  • Virtual screening of compound libraries against leuB active site

  • Fragment-based screening via thermal shift assays, NMR, or X-ray crystallography

  • Rational design of transition state analogs based on reaction mechanism

  • Focus on exploiting structural differences between bacterial and human homologs

• Medicinal Chemistry Optimization Workflow:

  • Initial hit identification through high-throughput screening

  • Hit-to-lead optimization focusing on potency, selectivity, and physiochemical properties

  • Lead optimization considering drug-like properties and pharmacokinetics

  • Structure-activity relationship development through systematic compound modification

• Inhibitor Validation Methods:

  • Enzyme inhibition assays (IC50 and Ki determination)

  • Mode of inhibition studies (competitive, noncompetitive, uncompetitive)

  • Cellular activity validation in V. vulnificus growth inhibition assays

  • Selectivity profiling against human metabolic enzymes

• Combination Strategy Development:

  • Synergy testing with existing antibiotics

  • Evaluation in resistance development models

  • Assessment of efficacy against drug-resistant V. vulnificus strains

This comprehensive approach has identified leuB as a promising antibacterial target, with knockout studies demonstrating complete elimination of biomass flux in unconstrained metabolic models .

What Advanced Methods Are Used to Study leuB's Role in V. vulnificus Metabolic Networks?

Understanding leuB's position within V. vulnificus metabolic networks requires sophisticated systems biology approaches:

• Genome-Scale Metabolic Modeling:

  • Construction of constraint-based metabolic models using Pathway Tools software

  • Flux balance analysis (FBA) to predict metabolic consequences of leuB perturbation

  • Dynamic flux balance analysis to model temporal changes in metabolic states

  • Integration of transcriptomic data to create context-specific metabolic models

• Experimental Flux Measurement:

  • 13C metabolic flux analysis using labeled substrates

  • Metabolomics profiling to identify metabolite accumulation patterns

  • Isotope-ratio mass spectrometry to track carbon flow through leucine biosynthesis

  • Real-time metabolite sensing using genetically encoded biosensors

• Network Analysis Methodologies:

  • Chokepoint analysis identifies leuB as a critical metabolic bottleneck

  • Synthetic lethality screening to identify genetic interactions

  • Flux coupling analysis to determine functional relationships between reactions

  • Regulatory network reconstruction to understand transcriptional control of leuB

• Multi-Omics Integration Approaches:

  • Correlation of leuB expression with global proteomic and transcriptomic profiles

  • Integration of metabolomics and fluxomics data to create comprehensive metabolic maps

  • Machine learning algorithms to predict metabolic responses to leuB inhibition

  • Bayesian network analysis to infer causal relationships in metabolic regulation