Recombinant Vibrio vulnificus 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (ispF)

What is the role of IspF in the MEP pathway in Vibrio vulnificus?

IspF (2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase) catalyzes a critical cyclization reaction in the MEP (methylerythritol phosphate) pathway, converting 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate to 2-C-methyl-D-erythritol 2,4-cyclodiphosphate and CMP. This pathway is essential for isoprenoid biosynthesis in many bacteria, including V. vulnificus. The MEP pathway is particularly important during infection, as V. vulnificus experiences dramatic environmental changes moving from estuarine environments through the host's gastrointestinal tract and eventually into the bloodstream. These transitions require metabolic adaptations supported by isoprenoid-derived molecules essential for membrane integrity and various cellular functions .

The MEP pathway represents an attractive target for antimicrobial development because humans utilize the distinct mevalonate pathway for isoprenoid synthesis. Recent studies have demonstrated that synergistic approaches combining MEP pathway engineering with other metabolic pathways can significantly enhance production of valuable downstream products like isoprene, suggesting the pathway's critical metabolic role .

How does V. vulnificus IspF differ structurally from homologs in other bacterial species?

V. vulnificus IspF shares the conserved trimeric structure typical of IspF enzymes, with each monomer adopting an α/β fold and containing a zinc-binding site essential for catalysis. Comparative structural analysis reveals both conserved catalytic residues and species-specific variations in substrate-binding pockets that can be exploited for selective inhibitor design.

A methodological approach to characterizing these differences includes:

• Homology modeling based on solved structures (such as B. subtilis IspF )

• Multiple sequence alignment to identify conserved and variable regions

• Expression and purification of recombinant V. vulnificus IspF for crystallographic studies

• Structural comparison focusing on:

  • Active site architecture

  • Zinc coordination geometry

  • Conformational flexibility of the binding pocket

  • Surface properties affecting oligomerization

These structural differences may correlate with V. vulnificus's ability to adapt to changing environments during infection, potentially contributing to its pathogenicity .

What experimental methods are most effective for determining IspF enzyme kinetics in V. vulnificus?

For robust kinetic characterization of V. vulnificus IspF, a multi-method approach yields the most reliable data:

Continuous Spectrophotometric Assays:

• Monitor CMP release via coupling with nucleotidase and phosphate detection

• Track substrate consumption using coupled enzyme systems

• Employ fluorescent substrate analogs for increased sensitivity

Discontinuous HPLC Analysis:

• Separate and quantify reaction components at defined time points

• Monitor both substrate depletion and product formation

• Use isotopically labeled substrates for improved detection

Data Analysis Protocol:

• Determine initial velocities across a range of substrate concentrations (0.1-10× Km)

• Fit data to appropriate kinetic models (Michaelis-Menten, Hill, or substrate inhibition)

• Analyze the effects of potential inhibitors using different inhibition models

• Evaluate metal cofactor dependencies by varying zinc concentrations

• Assess pH and temperature optima relevant to infection environments

This comprehensive approach enables correlation of enzymatic parameters with V. vulnificus pathogenicity, as the bacterium must adapt its metabolism during the transition from environmental reservoirs to human hosts .

What expression systems yield optimal quantities of correctly folded recombinant V. vulnificus IspF?

The optimal expression system for V. vulnificus IspF requires balancing yield, solubility, and native conformation. Based on successful approaches with other bacterial enzymes and considering V. vulnificus's unique characteristics, the following methodological framework is recommended:

Expression System Selection:

Optimization Protocol:

• Clone the ispF gene into vectors with various fusion tags (His6, MBP, SUMO)

• Test expression under multiple conditions:

  • Induction temperature: 16°C, 25°C, 37°C

  • IPTG concentration: 0.1 mM, 0.5 mM, 1.0 mM

  • Media composition: LB, TB, auto-induction media

  • Duration: 4h, overnight, 24h

• Evaluate protein solubility and activity for each condition

• Scale up production using optimal parameters

This approach mirrors successful strategies used for studying other V. vulnificus proteins, where expression conditions significantly impacted protein quality and subsequent structural studies .

What purification challenges are specific to V. vulnificus IspF and how can they be overcome?

Purifying V. vulnificus IspF presents several challenges that require specific methodological solutions:

Challenge 1: Maintaining Trimeric Structure

• Solution: Include low concentrations of zinc (10-50 μM ZnCl2) in all buffers

• Rationale: Zinc coordination stabilizes the trimeric assembly essential for activity

Challenge 2: Protein Instability

• Solution: Optimize buffer conditions through thermal shift assays

• Protocol:

  • Test various buffer compositions (pH 6.5-8.5)

  • Screen stabilizing additives (glycerol, trehalose, arginine)

  • Evaluate reducing agents (DTT, TCEP) at different concentrations

  • Determine optimal ionic strength (100-500 mM NaCl)

Challenge 3: Co-purification of Contaminants

• Solution: Multi-step purification strategy

  • IMAC (Ni-NTA) chromatography with imidazole gradient

  • Tag removal using precision protease

  • Anion exchange chromatography

  • Size exclusion chromatography to separate trimeric IspF

Challenge 4: Activity Loss During Purification

• Solution: Activity-guided purification

  • Assay fractions at each purification step

  • Minimize exposure to potential inhibitors

  • Process samples rapidly at 4°C

  • Add substrate analogs to stabilize active conformation

This purification strategy addresses the specific challenges of V. vulnificus IspF while preserving the native structure necessary for accurate biochemical and structural characterization.

How can protein engineering improve crystallization properties of V. vulnificus IspF?

Successful crystallization of V. vulnificus IspF can be enhanced through targeted protein engineering approaches:

Surface Entropy Reduction:

• Identify surface-exposed lysine and glutamate clusters using homology models

• Replace selected clusters with alanines (e.g., K72A/K73A/K74A) to reduce conformational entropy

• Screen engineered variants for improved crystallization propensity

Terminal Modifications:

• Generate N- and C-terminal truncation series based on secondary structure predictions

• Remove disordered regions that may impede crystal packing

• Test each construct for solubility, activity, and crystallization behavior

Strategic Mutations:

• Introduce mutations to disrupt crystal packing in undesirable orientations

• Modify surface residues to promote specific crystal contacts

• Replace cysteine residues to prevent non-native disulfide formation

Crystallization Facilitation Table:

These approaches have proven successful for crystallizing other bacterial enzymes, including IspF from B. subtilis, which yielded high-resolution structures suitable for detailed mechanistic analysis .

What are the critical active site residues in V. vulnificus IspF and how do they coordinate substrate binding?

The active site of V. vulnificus IspF contains several critical residues that coordinate substrate binding and catalysis through a precise spatial arrangement:

Metal Coordination Site:

• A zinc ion is coordinated by two histidines (typically His10 and His44, using B. subtilis numbering) and an aspartate (Asp11)

• This zinc coordination is essential for positioning the substrate's phosphate groups and facilitating nucleophilic attack

Substrate Binding Residues:

• Phosphate Binding:

  • Conserved lysine residues form salt bridges with substrate phosphates

  • Threonine and serine residues provide hydrogen bonding

• Ribose Recognition:

  • Aspartate residues form hydrogen bonds with ribose hydroxyl groups

  • Hydrophobic residues create a pocket accommodating the ribose moiety

• Cytidine Binding:

  • Aromatic residues (typically phenylalanine) provide π-stacking interactions with the cytosine ring

  • Backbone amides form hydrogen bonds with cytosine

Catalytic Mechanism Analysis:

• The zinc ion coordinates the phosphate oxygen of the substrate

• Conserved glutamate acts as a catalytic base, abstracting a proton

• Nucleophilic attack leads to formation of the cyclic product

• CMP is released as a leaving group

These structural features likely contribute to V. vulnificus's ability to adapt to changing host environments during infection, as precise metabolic regulation is essential for pathogen survival under stress conditions .

How do conformational changes in V. vulnificus IspF affect catalytic efficiency during the infection process?

V. vulnificus IspF undergoes significant conformational changes during catalysis that are likely modulated by infection-specific conditions. Understanding these dynamics is crucial for both basic enzymology and drug development:

Key Conformational Transitions:

• Open-to-closed transition upon substrate binding

• Reorientation of mobile loops surrounding the active site

• Subunit reorganization affecting intersubunit communication

• Allosteric modulation by cellular metabolites

Methodological Approach to Study Conformational Changes:

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to detect regions of altered flexibility

• Single-molecule FRET to monitor domain movements during catalysis

• Molecular dynamics simulations to predict conformational trajectories

• Crystallization in different states (apo, substrate-bound, product-bound)

Infection-Relevant Conformational Factors:

• pH-dependent structural changes corresponding to different infection sites

• Metal availability effects on active site geometry

• Redox-sensitive conformational switches

• Temperature-responsive structural elements

During infection, V. vulnificus experiences dramatic environmental changes, from estuarine environments to the human gastrointestinal tract and bloodstream . These transitions likely require adjustments in enzyme activity through conformational adaptation. For example, blood-specific conditions (temperature, pH, iron limitation) may trigger specific conformational states optimized for pathogen survival in this environment .

What computational approaches are most effective for identifying potential inhibitor binding sites in V. vulnificus IspF?

A comprehensive computational pipeline for identifying inhibitor binding sites in V. vulnificus IspF involves multiple complementary approaches:

Primary Site Identification:

• Structure-based methods:

  • SiteMap analysis to identify binding pockets based on physical properties

  • FTMap to detect hot spots through fragment probe mapping

  • CASTp for geometric analysis of surface cavities

• Sequence-based conservation analysis:

  • Prioritize sites showing differential conservation between bacterial and human enzymes

  • Identify species-specific pockets unique to V. vulnificus

Druggability Assessment:

• Calculate physicochemical properties (volume, hydrophobicity, exposure)

• Estimate binding energetics through molecular dynamics

• Evaluate water displacement patterns using WaterMap

• Score sites using machine learning algorithms trained on known druggable pockets

Dynamic Pocket Analysis:

• Molecular dynamics simulations to identify transient pockets

• Cryptic site discovery using enhanced sampling techniques

• Allosteric network identification through community network analysis

• Ensemble docking against multiple receptor conformations

Application to V. vulnificus IspF:

This computational pipeline provides multiple targeting opportunities for developing selective inhibitors against V. vulnificus IspF, potentially addressing the need for new approaches against this pathogen with high mortality rates .

How does V. vulnificus IspF activity correlate with virulence factor expression?

The relationship between IspF activity and virulence factor expression in V. vulnificus represents a complex regulatory network that can be systematically investigated:

Methodological Approach:

• Generate a conditional ispF mutant with titratable expression

• Quantify virulence factor production at different IspF activity levels:

  • Hemolysin (VvhA) - responsible for cytolytic effects

  • Metalloprotease (VvpE) - contributes to tissue necrosis

  • Capsular polysaccharide (CPS) - absolutely required for pathogenicity

  • Siderophores - essential for iron acquisition during infection

• Perform transcriptomic analysis to identify co-regulated genes

• Use chromatin immunoprecipitation to identify direct regulatory interactions

Potential Regulatory Mechanisms:

• Metabolic coupling through shared precursors

• Isoprenoid-dependent signaling pathways

• Influence on membrane properties affecting secretion systems

• Integration with the ToxRS system, known to regulate virulence gene expression

Research suggests that V. vulnificus virulence factors are tightly regulated and produced at specific times during infection . IspF activity likely influences this regulation through metabolic integration with pathways controlling virulence gene expression. The isoprenoid products of the MEP pathway contribute to membrane structure, which in turn affects the function of membrane-associated virulence determinants like the ToxRS regulatory system .

How can we develop selective inhibitors of V. vulnificus IspF as potential therapeutics?

Developing selective inhibitors against V. vulnificus IspF requires a systematic approach addressing multiple aspects of drug design:

Target Validation:

• Confirm essentiality of IspF for V. vulnificus growth and virulence

• Determine minimum inhibition level required for antimicrobial effect

• Assess potential for resistance development through mutation rate studies

Structure-Based Design Strategy:

• Obtain high-resolution structures of V. vulnificus IspF (similar to B. subtilis IspF )

• Perform comparative analysis with human isoprenoid-synthesizing enzymes

• Identify unique structural features of V. vulnificus IspF for selective targeting

• Design initial compounds targeting:

  • The active site zinc-binding region

  • Species-specific substrate binding pockets

  • Allosteric sites affecting trimer formation

Compound Optimization Pipeline:

Delivery Considerations:

• Design compounds that can penetrate V. vulnificus's Gram-negative cell envelope

• Consider prodrug approaches to enhance bioavailability

• Evaluate combination strategies with membrane permeabilizers

This approach addresses the urgent need for new therapeutics against V. vulnificus, which causes rapidly progressing infections with mortality rates exceeding 50% .

What is the relationship between IspF function and V. vulnificus survival under host immune pressure?

V. vulnificus must adapt to multiple host defense mechanisms during infection, and IspF function likely plays a critical role in this adaptation:

Immune Pressure Adaptation Mechanisms:

• Membrane Remodeling:

  • IspF-derived isoprenoids contribute to membrane fluidity and integrity

  • Altered membrane composition affects resistance to antimicrobial peptides

  • Capsular polysaccharide (CPS) integration requires isoprenoid-linked carriers

• Oxidative Stress Response:

  • Isoprenoid-derived antioxidants protect against reactive oxygen species

  • MEP pathway modulation may compensate for oxidative damage

  • Carotenoid synthesis (downstream of MEP pathway) provides additional protection

• Immune Evasion:

  • IspF activity supports CPS production, which provides resistance to phagocytosis

  • Isoprenylated proteins may influence host-pathogen protein interactions

  • Membrane composition affects complement activation and deposition

Methodological Investigation Approach:

• Challenge ispF conditional mutants with specific immune components:

  • Neutrophils and neutrophil extracellular traps (NETs)

  • Serum complement

  • Antimicrobial peptides

  • Macrophages under different activation states

• Monitor transcriptional responses of ispF and the MEP pathway during immune challenge

• Correlate IspF activity with known immune evasion mechanisms:

  • Capsule production (resistance to opsonization by complement)

  • Hemolysin activity (lymphocyte apoptosis)

  • Toll-like receptor activation (TLR2 and TLR5 signaling)

This research would elucidate how metabolic adaptations mediated by IspF contribute to V. vulnificus's remarkable virulence and ability to cause fatal septicemia in susceptible hosts .

How can CRISPR-Cas9 technology be applied to study functional domains within V. vulnificus IspF?

CRISPR-Cas9 technology enables sophisticated genetic manipulations to dissect IspF function in V. vulnificus:

Genome Editing Applications:

• Domain-specific mutations:

  • Create precise amino acid substitutions in catalytic residues

  • Modify zinc-binding motifs to alter metal coordination

  • Engineer interface residues to disrupt trimerization

• Regulatory element analysis:

  • Edit promoter regions to identify regulatory elements

  • Modify untranslated regions affecting mRNA stability

  • Create reporter fusions to monitor expression dynamics

CRISPR Interference (CRISPRi) Applications:

• Temporal control of ispF expression:

  • Titrate expression levels using inducible dCas9 systems

  • Study metabolic consequences of partial IspF inhibition

  • Identify minimum expression threshold for viability

• Spatial regulation studies:

  • Use tissue-specific promoters to drive dCas9 expression in animal models

  • Determine organ-specific requirements for IspF activity

CRISPR Activation (CRISPRa) Applications:

• Overexpression studies:

  • Upregulate ispF to determine effects on isoprenoid flux

  • Investigate potential toxicity of MEP pathway intermediate accumulation

  • Study compensatory responses to IspF overexpression

Multiplexed CRISPR Applications:

• Simultaneous targeting of multiple MEP pathway enzymes

• Creation of synthetic genetic interaction maps

• Identification of genetic suppressors of ispF deficiency

This CRISPR-based approach provides unprecedented precision for studying IspF function in the context of V. vulnificus metabolism and pathogenesis, potentially revealing new aspects of this enzyme's role in bacterial adaptation during infection .

What is the three-dimensional structure of V. vulnificus IspF and how does it compare to homologs from other pathogens?

A comprehensive structural analysis of V. vulnificus IspF would provide valuable insights for both basic understanding and drug development:

Structural Determination Methodology:

• X-ray crystallography approach:

  • Optimize crystallization conditions (similar to B. subtilis IspF )

  • Collect high-resolution diffraction data

  • Solve structure using molecular replacement with bacterial homologs

  • Refine model to generate accurate atomic coordinates

• Cryo-EM analysis:

  • Prepare vitrified samples of purified IspF

  • Collect high-resolution image data

  • Perform 3D reconstruction to generate electron density maps

  • Build and refine atomic models

Comparative Structural Analysis:

Structure-Function Correlations:

• Identify conformational changes associated with substrate binding

• Map conserved catalytic residues and species-specific variations

• Analyze oligomerization interfaces and potential for disruption

• Predict binding sites for rational inhibitor design

This structural knowledge would facilitate understanding of IspF's role in V. vulnificus metabolism during infection and provide a foundation for structure-based drug design targeting this essential enzyme .

How does environmental adaptation influence post-translational modifications of V. vulnificus IspF?

V. vulnificus transitions through dramatically different environments during infection, potentially requiring post-translational modification (PTM) of key enzymes like IspF to adapt:

Potential Post-Translational Modifications:

• Phosphorylation:

  • Regulates enzyme activity in response to environmental signals

  • May be controlled by two-component systems sensing host conditions

  • Can affect subunit interactions and catalytic efficiency

• Oxidative Modifications:

  • Cysteine oxidation under oxidative stress conditions

  • Potential redox sensing mechanism during host immune response

  • May protect key residues during oxidative burst by phagocytes

• Metal Ion Coordination Changes:

  • Zinc availability fluctuates during infection (competition with host)

  • Alternative metal incorporation may affect catalytic properties

  • Host nutritional immunity strategies target metal-dependent enzymes

Methodological Investigation Approach:

Environmental Correlation:

• Compare PTM profiles between:

  • Environmental growth conditions (estuarine-like)

  • Gastrointestinal simulation

  • Serum-exposed bacteria

  • Intracellular bacteria within macrophages

• Correlate PTM patterns with:

  • Virulence gene expression

  • Metabolic flux through the MEP pathway

  • Resistance to host defense mechanisms

This research would provide insights into how V. vulnificus adapts its central metabolism during the infection process, potentially revealing new vulnerabilities that could be exploited therapeutically .