Recombinant Vibrio vulnificus 1-deoxy-D-xylulose 5-phosphate reductoisomerase (dxr)

What is the role of Dxr in Vibrio vulnificus metabolism?

Dxr (1-deoxy-D-xylulose 5-phosphate reductoisomerase) is a critical enzyme in the viability of V. vulnificus, catalyzing the second step of the MEP (2-C-methylerythritol 4-phosphate) pathway. It performs a crucial rearrangement and reduction, converting 1-deoxy-D-xylulose 5-phosphate (Dxp) to 2-C-methylerythritol 4-phosphate (MEP) . This pathway is essential for the synthesis of isoprenoids, specifically isopentenyl diphosphate and dimethylallyl pyrophosphate, which serve as fundamental building blocks for various cellular components including cell membranes and certain metabolic cofactors . The MEP pathway's absence in humans but presence in many pathogenic bacteria makes Dxr an attractive target for antimicrobial development .

What are the optimal conditions for recombinant expression of V. vulnificus Dxr?

For optimal recombinant expression of V. vulnificus Dxr, the gene should be cloned into an expression vector with an appropriate promoter (typically T7) and affinity tag (His-tag is commonly used). Expression in E. coli BL21(DE3) or similar strains at reduced temperatures (16-25°C) after IPTG induction (0.1-0.5 mM) has shown good results . The expression conditions should be optimized considering:

• Media composition: Rich media (LB) for initial screening, while defined media may be required for isotopic labeling

• Induction timing: Mid-log phase (OD₆₀₀ of 0.6-0.8) typically yields best results

• Post-induction incubation time: 16-20 hours at lower temperatures improves protein folding

• Codon optimization: May be necessary due to codon usage differences between V. vulnificus and E. coli

What purification strategy yields the highest activity for recombinant V. vulnificus Dxr?

A multi-step purification approach yields the highest activity for recombinant V. vulnificus Dxr:

• Initial capture using immobilized metal affinity chromatography (IMAC) with Ni²⁺ or Co²⁺ resins for His-tagged protein

• Optional tag cleavage using specific proteases if required for structural or functional studies

• Ion exchange chromatography (typically Q-Sepharose) for removing nucleic acid contaminants

• Size-exclusion chromatography to isolate properly folded dimeric enzyme and remove aggregates

Throughout purification, buffer conditions should maintain:

• pH 7.5-8.0 (typically HEPES or Tris buffer)

• 100-200 mM NaCl to maintain solubility

• 1-5 mM MgCl₂ or MnCl₂ to ensure metal cofactor availability

• 1-5 mM DTT or 0.5-1 mM TCEP to maintain reduced cysteine residues

• 10% glycerol to improve stability during storage

How does metal ion coordination affect the catalytic activity of V. vulnificus Dxr?

Metal ion coordination is essential for V. vulnificus Dxr catalytic activity, with significant implications for both mechanism and inhibitor design. The enzyme naturally co-purifies with Mg²⁺ ions when expressed in E. coli, but can also utilize Mn²⁺ . These divalent metal cations serve multiple crucial functions:

• Coordination of substrate positioning in the active site

• Stabilization of reaction intermediates during the complex rearrangement-reduction reaction

• Facilitation of hydride transfer from NADPH to the substrate

Experimental studies have shown that metal substitution affects both the catalytic efficiency (kcat/Km) and inhibitor binding. Specifically, Mn²⁺ can substitute for Mg²⁺ with retention of activity, though kinetic parameters may differ slightly. The metal coordination sphere typically involves conserved aspartate and glutamate residues in the active site, creating an electrostatic environment that properly orients the substrate for catalysis .

What techniques are most effective for assessing the enzymatic activity of recombinant V. vulnificus Dxr?

Multiple complementary techniques have proven effective for assessing recombinant V. vulnificus Dxr activity:

Spectrophotometric assays:

• NADPH consumption monitoring at 340 nm (ε = 6,220 M⁻¹cm⁻¹)

• Typical reaction conditions include 100 mM HEPES (pH 7.5), 5 mM MgCl₂, 150 mM NaCl, 1-2 mM DTT, 0.2 mM NADPH, and variable DXP concentrations (10-500 μM)

• Data analysis through initial velocity measurements and Michaelis-Menten kinetics calculations

High-performance liquid chromatography (HPLC):

• Direct quantification of MEP product formation

• Requires sample derivatization or specialized detection methods

• Provides absolute confirmation of product formation

Isothermal titration calorimetry (ITC):

• Enables determination of thermodynamic binding parameters for substrates and inhibitors

• Provides insights into binding stoichiometry and enthalpic/entropic contributions

Surface plasmon resonance (SPR):

• Allows real-time monitoring of binding interactions

• Useful for determining association and dissociation kinetics of inhibitors

When conducting these assays, researchers should ensure proper controls including:

• Enzyme-free reactions

• Substrate-free reactions

• Heat-inactivated enzyme controls

• Reactions with known inhibitors like fosmidomycin as positive controls

How do fosmidomycin and its analogs inhibit V. vulnificus Dxr, and what modifications can improve their efficacy?

Fosmidomycin (FSM) and its analogs (including FR900098 and fosfoxacin) inhibit V. vulnificus Dxr through competitive binding to the substrate pocket, mimicking the binding of the natural substrate DXP . These phosphonate-containing compounds coordinate with the active site metal ion (Mg²⁺/Mn²⁺) and form hydrogen bonds with conserved residues. Structure-activity relationship studies suggest several modifications that could improve efficacy:

• Phosphonate group modifications: Altering charge distribution while maintaining metal coordination capacity

• Hydroxamic acid group variations: Modifying the retrohydroxamate moiety to optimize hydrogen bonding

• Backbone extensions: Adding lipophilic groups to improve membrane permeability

• Prodrug approaches: Adding cleavable protecting groups to enhance cellular uptake

Crystallographic studies of V. vulnificus Dxr with bound inhibitors reveal key interaction points that can guide rational drug design. The dianionic nature of fosmidomycin that is essential for activity also limits its membrane permeability, making prodrug approaches particularly promising for improving efficacy against intact bacteria .

What experimental approaches can identify resistance mechanisms to Dxr inhibitors in V. vulnificus?

Several complementary experimental approaches can effectively identify resistance mechanisms to Dxr inhibitors in V. vulnificus:

Directed evolution and resistance selection:

• Serial passage of V. vulnificus in sub-inhibitory concentrations of inhibitors

• Gradual increase in inhibitor concentration to select for resistant mutants

• Whole-genome sequencing of resistant strains to identify mutations

Targeted mutagenesis studies:

• Site-directed mutagenesis of conserved residues in the active site

• Creation of libraries with randomized active site residues

• Expression and functional characterization of mutant Dxr enzymes

Biochemical characterization of resistant enzymes:

• Enzyme kinetics to determine changes in Km, Vmax, and Ki values

• Thermal stability assessments to evaluate structural impacts of mutations

• Crystallographic studies to visualize altered binding interactions

Transcriptomic and proteomic analysis:

• RNA-seq to identify compensatory pathways activated in resistant strains

• Proteomics to detect changes in Dxr expression levels or post-translational modifications

• Metabolomics to identify alternative isoprenoid synthesis pathways

A comprehensive study reported that error-prone PCR screening identified specific mutations in dxr that conferred resistance to fosmidomycin in E. coli. This approach could be adapted to V. vulnificus Dxr to identify potential resistance hotspots and develop inhibitors less prone to resistance development .

How can structural information about V. vulnificus Dxr be utilized for structure-based drug design?

The structural characterization of V. vulnificus Dxr provides several advantageous approaches for structure-based drug design:

Active site mapping and hotspot identification:

• Crystallographic data reveals crucial binding pockets and interaction sites

• Computational solvent mapping can identify energetically favorable binding regions

• Analysis of conserved water molecules identifies potential displacement sites for improved binding affinity

Virtual screening and molecular docking:

• High-throughput virtual screening against the VvDxr structure

• Molecular dynamics simulations to account for protein flexibility

• Ensemble docking against multiple conformational states to address protein dynamics

Fragment-based drug discovery:

• Identification of low-molecular-weight fragments that bind to different subpockets

• Linking or growing fragments to develop high-affinity inhibitors

• Nuclear magnetic resonance (NMR) or X-ray crystallography to confirm fragment binding

Structure-guided optimization:

• Iterative cycles of compound synthesis, testing, and structural characterization

• Modification of lead compounds based on structure-activity relationships

• Integration of pharmacokinetic and ADME considerations into the design process

The dimeric assembly of VvDxr also presents opportunities for designing inhibitors that disrupt protein-protein interactions or alter allosteric regulation, potentially offering alternative inhibition mechanisms beyond active site targeting .

How does V. vulnificus Dxr interact with other components of the MEP pathway, and can these interactions be exploited for antimicrobial development?

V. vulnificus Dxr functions as part of the integrated MEP pathway, with several potential protein-protein interactions and regulatory mechanisms that could be exploited for antimicrobial development:

Pathway flux regulation:

• Dxr activity represents a potential rate-limiting step in isoprenoid biosynthesis

• Feedback regulation mechanisms may exist between Dxr and other MEP pathway enzymes

• Analysis of metabolic control coefficients can identify vulnerable points for combination targeting

Protein-protein interactions:

• Co-immunoprecipitation and crosslinking studies can identify Dxr interaction partners

• Bacterial two-hybrid screening may reveal regulatory proteins that modulate Dxr activity

• Surface mapping of conservation and variability can identify potential interaction interfaces

Substrate channeling:

• Proximity of MEP pathway enzymes may facilitate direct transfer of intermediates

• Multi-enzyme complexes could present novel targeting opportunities

• Disruption of enzyme complex formation may enhance the efficacy of active site inhibitors

Combination therapy approaches:

• Synergistic targeting of multiple MEP pathway enzymes (Dxr, IspD, IspF)

• Development of dual-action inhibitors that target sequential steps in the pathway

• Coupling MEP pathway inhibition with inhibitors of cell wall synthesis or membrane integrity

Transcriptome sequencing studies have revealed that disruption of essential metabolic pathways like the MEP pathway can trigger complex stress responses in V. vulnificus, suggesting that combinatorial approaches targeting both Dxr and stress response mechanisms could be particularly effective .

How does inhibition of Dxr affect the expression of virulence factors in V. vulnificus?

Inhibition of Dxr affects multiple virulence factors in V. vulnificus through both direct metabolic consequences and indirect regulatory effects:

Direct metabolic impacts:

• Reduced isoprenoid availability affects bacterial membrane integrity

• Impaired synthesis of prenylated proteins involved in virulence factor secretion

• Disrupted quorum sensing molecule production due to isoprenoid precursor limitation

Effects on specific virulence factors:

• Reduced production of RtxA1 toxin, which normally promotes rapid growth and epithelial tissue necrosis

• Decreased expression of VvhA hemolysin that typically causes pore formation in host cells

• Impaired biofilm formation and development due to altered cell membrane composition

Transcriptional regulatory changes:

• Altered expression of the HlyU virulence transcription factor network

• Disrupted SmcR-dependent quorum sensing pathways that regulate pathogenesis

• Stress response activation that may further modulate virulence gene expression

Experimental evidence indicates that metabolic disruption through Dxr inhibition creates a cascade effect, where the initial metabolic stress triggers broader changes in virulence factor expression. This occurs partially through altered activity of transcriptional regulators like HlyU, which controls expression of major cytotoxins including RtxA1 and VvhA .

What techniques can be used to assess the impact of Dxr inhibition on V. vulnificus pathogenesis in various experimental models?

Multiple complementary techniques can effectively assess the impact of Dxr inhibition on V. vulnificus pathogenesis:

In vitro cell culture models:

• Cytotoxicity assays using human intestinal epithelial cells or macrophages

• Membrane integrity assessment through LDH release or fluorescent dye exclusion

• Bacterial adhesion and invasion quantification using gentamicin protection assays

• Transwell systems to evaluate effects on epithelial barrier integrity

Ex vivo tissue models:

• Human intestinal organoids to assess tissue-specific responses

• Precision-cut liver slices to evaluate hepatotoxicity mechanisms

• Whole blood assays to measure effects on bacterial survival in blood

In vivo animal models:

• Intragastric infection mouse models to assess intestinal pathogenesis

• Bioluminescence imaging to track bacterial dissemination in real-time

• Histopathological examination of intestinal tissue to evaluate epithelial damage

• Bacterial burden quantification in multiple organs to assess systemic spread

Molecular and transcriptomic analyses:

• RNA-seq to identify changes in both bacterial and host gene expression

• RT-qPCR validation of key virulence factor expression levels

• Protein quantification of secreted toxins using ELISA or Western blotting

Studies have demonstrated that V. vulnificus cytotoxins like RtxA1 and VvhA play additive roles in pathogenesis by causing intestinal tissue damage and inflammation that promotes bacterial dissemination to the bloodstream. Dxr inhibition, by disrupting isoprenoid biosynthesis, can potentially impair these virulence mechanisms and reduce pathogenicity .

What are the structural and functional differences between bacterial Dxr and human metabolic enzymes that can be exploited for selective inhibition?

Several critical structural and functional differences between bacterial Dxr and human metabolic enzymes provide opportunities for selective inhibition:

Pathway exclusivity:

• Humans lack the MEP pathway entirely, instead using the mevalonate pathway for isoprenoid synthesis

• No direct homolog of Dxr exists in human cells, offering inherent selectivity

• The closest human analogs (e.g., quinone reductases) have significantly different active site architectures

Active site architecture:

• V. vulnificus Dxr contains a unique metal-binding motif absent in human enzymes

• The substrate binding pocket has distinctive features that can be targeted for selective binding

• Analysis of conserved residues across bacterial Dxr vs. human enzymes reveals exploitable differences

Regulatory mechanisms:

• Bacterial Dxr regulation differs fundamentally from regulation of human isoprenoid synthesis

• Allosteric sites present in bacterial Dxr may not have counterparts in human enzymes

• Quaternary structure interactions in the dimeric bacterial enzyme can be targeted

Comparative binding data:

These differences provide a strong foundation for developing inhibitors with high selectivity for bacterial Dxr, minimizing potential toxicity to human host cells .

How can protein engineering be applied to V. vulnificus Dxr to enhance its utility for inhibitor screening and drug development?

Protein engineering offers several sophisticated approaches to enhance V. vulnificus Dxr utility for inhibitor screening and drug development:

Stability engineering:

• Directed evolution to increase thermostability for crystallization and assay robustness

• Introduction of disulfide bridges or salt bridges to stabilize flexible regions

• Surface entropy reduction mutagenesis to improve crystallization properties

• Design of fusion constructs with crystallization chaperones like T4 lysozyme or MBP

Reporter systems:

• Creation of Dxr-fluorescent protein fusions for high-throughput screening

• Development of split-reporter systems to monitor Dxr conformational changes

• Engineering of biosensor strains with Dxr-regulated luciferase expression

Active site modifications:

• Site-directed mutagenesis to create "binding pockets" for fragment screening

• Introduction of non-natural amino acids for enhanced biophysical studies

• Creation of "gatekeeper" mutations to explore expanded chemical space

Functional variants:

• Engineering of Dxr variants with altered metal ion preferences

• Development of hypersensitive variants for more sensitive inhibitor detection

• Creation of substrate-specificity variants to probe binding determinants