Recombinant Vibrio vulnificus 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (ispE)

What is the biological function of 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE) in Vibrio vulnificus?

IspE functions as the fourth enzyme in the MEP pathway, catalyzing the ATP-dependent conversion of 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) to 4-diphosphocytidyl-2-C-methyl-D-erythritol-2-phosphate (CDP-ME2P). This reaction represents a critical step in the biosynthesis of isoprenoid precursors, which are essential building blocks for various cellular components in bacteria. The MEP pathway is vital for bacterial survival as it produces isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which serve as the fundamental units for isoprenoid biosynthesis. These compounds are crucial for cell membrane integrity, electron transport, and other essential cellular processes in V. vulnificus .

How does V. vulnificus IspE compare structurally to IspE enzymes from other bacterial pathogens?

V. vulnificus IspE exhibits significant structural similarities to IspE enzymes from other bacterial pathogens while maintaining species-specific characteristics. Like its orthologs, V. vulnificus IspE belongs to the ATP-dependent GHMP kinase superfamily, which includes galactose kinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase. Despite these similarities, V. vulnificus IspE possesses unique features in its catalytic and substrate binding sites that distinguish it from human kinases. The enzyme contains two primary functional motifs: a homoserine kinase (ThrB) motif and a GHMP kinase motif, both critical for its enzymatic activity. Comparative analyses with IspE from pathogens such as M. tuberculosis, B. mallei, S. Typhi reveal conservation of these core functional domains while exhibiting variations in terminal regions that affect solubility and activity profiles .

Why is the MEP pathway in V. vulnificus considered a potential antimicrobial target?

The MEP pathway represents an attractive antimicrobial target for several compelling reasons. First, this pathway is essential for bacterial survival, as demonstrated by gene essentiality studies in related bacteria such as M. smegmatis. Second, the MEP pathway is entirely absent in human cells, which utilize the alternative mevalonate pathway for isoprenoid biosynthesis. This fundamental difference provides a crucial selective advantage for drug development, potentially allowing for the creation of antibiotics that target bacterial systems without affecting human cellular processes. Third, specific enzymes in the pathway, including IspE, demonstrate significant structural and functional differences from any human enzymes, further enhancing their potential as selective drug targets. The structural characterization of IspE reveals unique catalytic and substrate binding sites that could be exploited for the development of selective inhibitors against V. vulnificus and other bacterial pathogens that utilize this pathway .

What are the key challenges in expressing and purifying recombinant V. vulnificus IspE, and how can they be overcome?

Expression and purification of recombinant V. vulnificus IspE present several significant challenges that have hindered research progress. The primary obstacles include poor solubility of the native protein, low expression yields, and difficulty in maintaining enzymatic activity during purification. These challenges are similar to those encountered with IspE from other pathogens such as M. tuberculosis. Research has demonstrated that these issues can be effectively addressed through strategic genetic modifications of the enzyme.

The most successful approach involves C-terminal truncation of the protein. By removing approximately five amino acids from the C-terminus of V. vulnificus IspE, researchers have achieved dramatically improved solubility while maintaining catalytic function. This modification strategy has proven effective across multiple bacterial species, including B. mallei, S. Typhi, and V. cholerae, consistently yielding approximately 1 mg of purified protein per liter of culture. The truncation approach appears to specifically address structural elements that contribute to aggregation or improper folding without compromising the critical functional domains of the enzyme .

Alternative expression systems, optimization of induction conditions (temperature, IPTAG concentration, and induction duration), and the use of solubility-enhancing fusion tags may further improve expression outcomes. Purification protocols typically involve immobilized metal affinity chromatography followed by size exclusion chromatography, with special attention to buffer composition to maintain enzymatic activity throughout the purification process .

What expression systems and conditions are optimal for producing active recombinant V. vulnificus IspE?

Based on published research with IspE from various bacterial pathogens, including closely related species to V. vulnificus, the optimal expression system appears to be E. coli BL21(DE3) or similar strains designed for recombinant protein expression. When expressing V. vulnificus IspE, researchers should consider implementing the following optimized conditions:

• Vector selection: pET-based expression vectors with T7 promoters provide strong, inducible expression.

• Genetic modification: C-terminal truncation (removal of approximately 5 amino acids) significantly improves solubility while maintaining enzymatic activity.

• Growth conditions: Cultivation at 30-37°C in rich media (such as LB or 2xYT) until reaching OD600 of 0.6-0.8.

• Induction parameters: IPTG concentration of 0.1-0.5 mM with post-induction growth at lower temperatures (16-25°C) for 16-20 hours promotes proper folding and increased solubility.

• Lysis buffer composition: Inclusion of 10% glycerol, reducing agents (such as DTT or β-mercaptoethanol), and appropriate salt concentrations (typically 300-500 mM NaCl) helps maintain protein stability during cell disruption.

The expression of truncated versions, particularly those maintaining the essential ThrB and GHMP kinase domains, is critical for obtaining soluble, active enzyme. Experimental evidence indicates that constructs lacking either of these domains show minimal to no enzymatic activity, highlighting their importance for proper IspE function. The optimal construct design should retain all catalytic domains while eliminating regions that contribute to aggregation or insolubility .

How can the activity and purity of recombinant V. vulnificus IspE be accurately assessed?

Assessment of recombinant V. vulnificus IspE activity and purity requires a combination of analytical techniques targeting both protein quality and enzymatic function. For purity assessment, SDS-PAGE analysis serves as the primary method, with Western blotting providing confirmation of protein identity using appropriate antibodies. Size exclusion chromatography can further evaluate sample homogeneity and identify potential aggregation states.

Enzymatic activity assessment typically involves monitoring the ATP-dependent phosphorylation of CDP-ME to CDP-ME2P. This can be accomplished through several complementary approaches:

• Direct product analysis: The reaction product (CDP-ME2P) can be identified and quantified using chromatographic separation (such as anion exchange chromatography on benzyl DEAE cellulose columns) followed by mass spectrometry confirmation. The expected m/z value for CDP-ME2P [M+2NH4]+ is approximately 633.

• Coupled enzymatic assays: ATP consumption can be monitored through coupled enzyme systems that link ATP hydrolysis to measurable spectroscopic changes.

• Radiometric assays: Using [γ-32P]ATP allows direct measurement of phosphate transfer to the substrate.

Activity measurements should be conducted under optimized conditions with respect to pH, temperature, and buffer composition. Enzymatic activity should be linear with respect to both time (up to 30 minutes) and protein concentration. For V. vulnificus IspE, activity measurements should be performed with protein amounts in the range of approximately 190-200 pmol to ensure linearity .

What are the optimal conditions and methodologies for determining kinetic parameters of V. vulnificus IspE?

Determining accurate kinetic parameters for V. vulnificus IspE requires careful optimization of reaction conditions and appropriate methodological approaches. Based on studies with IspE from related bacterial species, the following protocol provides a framework for comprehensive kinetic characterization:

Reaction conditions optimization:

• Buffer system: Typically 100 mM Tris-HCl or HEPES, pH 7.5-8.0

• Temperature: 30-37°C, reflecting physiological conditions

• Salt concentration: 50-100 mM NaCl or KCl

• Divalent cations: 5-10 mM MgCl2 (essential cofactor for ATP binding)

• Reducing agents: 1-5 mM DTT or β-mercaptoethanol to maintain enzyme stability

Kinetic parameter determination requires varying the concentration of one substrate (CDP-ME or ATP) while maintaining the other at saturating levels. Initial velocity measurements should be conducted under conditions where product formation is linear with respect to time and enzyme concentration. Michaelis-Menten parameters (Km and Vmax) can then be determined through non-linear regression analysis of velocity versus substrate concentration data.

For more complex kinetic analyses, including investigation of potential inhibitors, enzymes should be pre-incubated with inhibitors before initiating the reaction with substrate addition. Reaction termination is typically achieved through heat inactivation (95°C for 5 minutes) or addition of EDTA to chelate the essential Mg2+ cofactor. Product formation can be quantified through chromatographic separation methods coupled with appropriate detection systems as previously described .

How does substrate specificity of V. vulnificus IspE compare to IspE from other bacterial pathogens?

The substrate specificity of V. vulnificus IspE likely follows patterns similar to those observed in IspE enzymes from other bacterial pathogens, though with potential species-specific variations. Based on comparative studies, IspE enzymes demonstrate high specificity for their natural substrate, 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME), with little to no activity toward structurally related compounds lacking key recognition elements.

For nucleotide triphosphate utilization, bacterial IspE enzymes typically show strong preference for ATP as the phosphate donor, with significantly reduced activity when other nucleotides (GTP, CTP, UTP) are substituted. This specificity arises from precise recognition of both the adenine base and the ribose moiety within the ATP binding pocket of the enzyme.

While detailed substrate specificity studies specifically for V. vulnificus IspE are not directly reported in the provided search results, the conservation of catalytic domains (particularly the GHMP kinase motif) across bacterial species suggests similar substrate recognition patterns. The presence of both the ThrB motif and GHMP kinase domains in V. vulnificus IspE, as observed in enzymes from M. tuberculosis and other pathogens, indicates conservation of the fundamental catalytic mechanism and substrate binding architecture .

These specificities can be systematically investigated through comparative enzyme assays using substrate analogs with systematic structural modifications. Such studies provide valuable insights for rational design of potential inhibitors targeting the active site of V. vulnificus IspE.

What techniques are available for synthesizing the substrate CDP-ME for V. vulnificus IspE kinetic studies?

• Chemical synthesis: Total chemical synthesis of CDP-ME has been successfully accomplished, though it involves multiple steps with challenging stereoselective reactions. The synthetic route typically begins with appropriately protected carbohydrate derivatives, followed by stereoselective introduction of the methyl group at C-2, phosphorylation, and coupling with activated cytidine monophosphate. While technically demanding, this approach yields enantiopure material suitable for precise kinetic studies.

• Chemoenzymatic synthesis: This hybrid approach utilizes enzymatic reactions for stereoselective steps combined with chemical modifications. For example, the initial steps of the MEP pathway can be reconstituted in vitro using recombinant DXP synthase (DXS), DXP reductoisomerase (DXR), and CDP-ME synthase (IspD) to convert pyruvate and glyceraldehyde-3-phosphate to CDP-ME.

• In situ generation: For some assay formats, CDP-ME can be generated in situ by including preceding enzymes of the pathway (particularly IspD) along with their substrates (2-C-methyl-D-erythritol 4-phosphate and CTP).

The availability of chemically synthesized enantiopure CDP-ME has significantly advanced the characterization of IspE from various bacterial pathogens, including methodologies applicable to V. vulnificus IspE. Researchers have confirmed the identity and purity of synthesized CDP-ME through techniques including MS analysis, which reveals a characteristic peak at m/z 633 corresponding to [M+2NH4]+ for the phosphorylated product CDP-ME2P .

What methodologies can be used to determine if ispE is essential in V. vulnificus?

Determining the essentiality of the ispE gene in V. vulnificus requires systematic genetic approaches that attempt to disrupt or delete the gene while assessing viability. Based on methodologies successfully applied to related species, researchers can employ several complementary strategies:

• Homologous recombination-based gene knockout: This approach involves constructing a knockout plasmid containing antibiotic resistance markers flanked by homologous regions upstream and downstream of the ispE gene. The strategy used for demonstrating essentiality in M. smegmatis provides an excellent template, where a temperature-sensitive mycobacterial origin of replication facilitated recombination events. For V. vulnificus, this would involve:

  • Constructing a plasmid containing kanamycin resistance cassette flanked by ispE homologous regions

  • Incorporating counter-selectable markers like sacB for selecting double-crossover events

  • Performing transformation followed by selection at non-permissive temperatures to eliminate plasmid replication

  • Analyzing resulting colonies for gene disruption

• Conditional expression systems: When genes are suspected to be essential, conditional expression systems allow controlled depletion of the gene product. This involves:

  • Replacing the native ispE promoter with an inducible promoter

  • Monitoring growth patterns under inducing versus non-inducing conditions

  • Quantifying cellular viability during depletion of the gene product

• Transposon mutagenesis: High-density transposon insertion libraries can be created and analyzed by next-generation sequencing to identify genes that cannot tolerate insertions, suggesting essentiality.

For V. vulnificus specifically, adapting the approach used in M. smegmatis would be most appropriate, where single-crossover events were first selected, followed by attempts to generate double-crossover events that would completely disrupt the gene. Failure to obtain viable double-crossover mutants despite obtaining single-crossover intermediates would provide strong evidence for gene essentiality .

How does V. vulnificus IspE contribute to bacterial pathogenesis and virulence?

While direct evidence specifically linking V. vulnificus IspE to pathogenesis is not explicitly detailed in the provided search results, its role can be inferred based on the critical nature of the MEP pathway in bacterial survival and the established virulence characteristics of V. vulnificus infection. As the fourth enzyme in the MEP pathway, IspE catalyzes an essential step in isoprenoid precursor biosynthesis, which impacts multiple aspects of bacterial physiology that contribute to pathogenesis:

• Cell membrane integrity: Isoprenoids are essential components of bacterial membranes, affecting permeability, fluidity, and resistance to host defense mechanisms. Disruption of isoprenoid biosynthesis through IspE inhibition would likely compromise membrane function, reducing bacterial survival under host stress conditions.

• Bacterial growth and replication: The MEP pathway provides essential building blocks for various cellular components required for bacterial growth. In highly virulent pathogens like V. vulnificus, which can cause rapidly progressive infections, rapid replication is a key virulence factor. V. vulnificus infections can progress from initial symptoms to life-threatening sepsis within 24-48 hours, suggesting an important role for metabolic pathways that support rapid growth.

• Stress response during infection: During infection, bacteria must adapt to changing host environments, including nutrient limitation, oxidative stress, and immune system attacks. Isoprenoid derivatives play roles in electron transport chains and as antioxidants, potentially contributing to bacterial stress responses.

V. vulnificus is a highly virulent pathogen that can cause primary bacteremia following ingestion (often in patients with preexisting liver diseases) or wound infections with tissue necrosis and secondary bacteremia. The rapid progression of V. vulnificus infections, where death can occur within hours of initial symptoms in severe cases, underscores the importance of metabolic pathways supporting bacterial proliferation and survival within the host .

What model systems are most appropriate for studying V. vulnificus IspE function in vivo?

The selection of appropriate model systems for studying V. vulnificus IspE function in vivo requires careful consideration of both the pathogen's characteristics and the specific research questions being addressed. Based on established methodologies for investigating V. vulnificus pathogenesis and antimicrobial targets, the following model systems are recommended:

• Mouse infection models: Murine models provide the most well-established and versatile system for investigating V. vulnificus pathogenesis and potential therapeutic targets. As demonstrated in the search results, mice can be infected with various inoculum sizes of V. vulnificus to model different infection severities, from mild (10³ CFU) to severe (10⁷-10⁸ CFU). The subcutaneous injection model, where bacteria are introduced into the area over the right thigh, effectively mimics wound infections and allows for the assessment of both local tissue effects and systemic spread. This model permits evaluation of:

  • Bacterial growth dynamics in vivo

  • Development of tissue pathology

  • Progression to bacteremia

  • Efficacy of potential IspE inhibitors

• Cell culture systems: For mechanistic studies focused specifically on IspE function, cell culture models can provide valuable insights:

  • Macrophage infection models to study intracellular survival

  • Epithelial cell lines to investigate adherence and invasion

  • Endothelial cell models to examine vascular damage mechanisms

• Conditional gene expression in V. vulnificus: The development of genetic systems allowing controlled expression of ispE in V. vulnificus would provide the most direct approach for correlating enzyme function with bacterial survival and virulence.

When studying potential IspE inhibitors, the mouse model with defined inoculum sizes (as detailed in search result ) provides a robust framework. Treatment can be initiated at defined time points post-infection (typically 2 hours after bacterial inoculation), and both survival rates and bacterial loads in tissues and blood can be quantified as outcome measures. Such models would be particularly valuable for testing compounds designed to target V. vulnificus IspE based on structural and enzymatic data .

What methodologies are most effective for identifying potential inhibitors of V. vulnificus IspE?

The identification of potential inhibitors targeting V. vulnificus IspE can be approached through multiple complementary methodologies, each with specific advantages for different stages of the drug discovery process:

• Structure-based virtual screening: Utilizing the structural similarities between V. vulnificus IspE and characterized orthologs (such as those from M. tuberculosis, B. mallei, S. Typhi, and V. cholerae), computational approaches can identify potential binding molecules through:

  • Molecular docking of compound libraries against the ATP-binding and substrate-binding pockets

  • Structure-based pharmacophore modeling focusing on key interaction points in the active site

  • Fragment-based approaches that identify building blocks for subsequent optimization

• High-throughput biochemical screening: The successful purification of active, soluble recombinant IspE (particularly using C-terminal truncation strategies) enables the development of biochemical assays suitable for screening compound libraries:

  • ATP consumption assays using luminescence-based detection

  • Product (CDP-ME2P) formation assays using chromatographic separation and mass spectrometric detection

  • Thermal shift assays to identify compounds that affect protein stability

• Rational design based on substrate mimicry: The high substrate specificity of IspE provides opportunities for designing competitive inhibitors based on CDP-ME structure:

  • Nucleotide analogs targeting the cytidine-binding region

  • Modified sugar moieties that compete for the methylerythritol-binding pocket

  • Bisubstrate analogs that simultaneously engage both ATP and CDP-ME binding sites

• In silico screening focusing on unique features of IspE: Despite similarities to the GHMP kinase superfamily, IspE enzymes display significant differences in catalytic and substrate binding sites. These distinctive features can be exploited to design selective inhibitors with limited cross-reactivity with human kinases.

The biochemical and structural characterization of IspE from various bacterial pathogens, including the successful expression strategies for V. vulnificus IspE orthologs, provides the foundation for establishing robust screening cascades. Primary hits identified through these approaches should be subsequently validated through structural studies, kinetic characterization, and assessment of antibacterial activity against V. vulnificus .

How can the efficacy of potential V. vulnificus IspE inhibitors be evaluated in vitro and in vivo?

Evaluation of potential V. vulnificus IspE inhibitors requires a systematic progression from biochemical assays to cellular and in vivo models, ensuring thorough characterization of compound efficacy, specificity, and therapeutic potential:

In vitro evaluation cascade:

• Primary biochemical assays:

  • IC50 determination using purified recombinant truncated V. vulnificus IspE

  • Mechanism of inhibition studies (competitive, non-competitive, or uncompetitive)

  • Structure-activity relationship analysis for compound series

• Selectivity profiling:

  • Counter-screening against human kinases, particularly those in the GHMP superfamily

  • Assessment of activity against IspE from other bacterial species to determine spectrum

• Cellular efficacy:

  • Minimum inhibitory concentration (MIC) determination against V. vulnificus

  • Time-kill studies to assess bactericidal versus bacteriostatic effects

  • Combination studies with established antibiotics (similar to the cefotaxime/minocycline combination studies described in search result )

In vivo evaluation:

• Pharmacokinetic assessment:

  • Absorption, distribution, metabolism, and excretion profiles

  • Plasma and tissue concentration measurements

• Efficacy in infection models:

  • Mouse subcutaneous infection model with varying inoculum sizes (10³-10⁸ CFU)

  • Treatment initiation 2 hours post-infection (following established protocols)

  • Survival rate as primary endpoint

  • Bacterial load in blood and tissues as secondary endpoints

• Combination therapy assessment:

  • Evaluation of IspE inhibitors in combination with established antibiotics

  • Comparison with standard treatments like cefotaxime and minocycline

The mouse model described in search result provides an excellent framework for in vivo efficacy studies, where the survival rates of mice infected with various inoculum sizes of V. vulnificus can be monitored following different treatment regimens. This model effectively captures the rapid progression of V. vulnificus infections and allows for quantitative assessment of therapeutic interventions .

What considerations are important when designing selective inhibitors of V. vulnificus IspE that avoid cross-reactivity with human enzymes?

Designing selective inhibitors of V. vulnificus IspE that avoid cross-reactivity with human enzymes requires careful consideration of multiple structural, biochemical, and pharmacological factors:

• Structural differentiation from human kinases:

  • Although IspE belongs to the GHMP kinase superfamily, it displays significant differences in catalytic and substrate binding sites compared to human kinases. These distinctive features, as revealed through crystal structures and sequence alignments, provide opportunities for selective targeting.

  • The unique substrate of IspE (CDP-ME) has no direct counterpart in human metabolism, allowing for the development of substrate-mimetic inhibitors that exploit recognition elements specific to bacterial enzymes.

  • The ThrB motif and GHMP kinase domains in bacterial IspE have structurally distinct configurations that can be exploited for selective inhibitor design.

• Targeting enzyme regions unique to bacterial IspE:

  • Analysis of truncated protein constructs (as described in search result ) reveals regions critical for catalytic activity. For example, Rv1011 I (with C-terminal truncation) maintained full activity, while constructs lacking portions of the ThrB motif showed reduced activity.

  • Inhibitors designed to interact with these bacterium-specific structural elements would potentially show enhanced selectivity.

• Biochemical validation of selectivity:

  • Counter-screening candidate inhibitors against a panel of human kinases, particularly those in the GHMP superfamily (galactose kinase, homoserine kinase, mevalonate kinase, phosphomevalonate kinase)

  • Validation in cellular systems to confirm that observed antibacterial effects correlate with IspE inhibition rather than off-target effects

• Pharmacological optimization for selective tissue distribution:

  • V. vulnificus infections primarily affect specific tissues (skin, soft tissue, bloodstream)

  • Designing compounds with physicochemical properties that favor distribution to infection sites while limiting exposure to tissues where off-target effects might manifest

The essential nature of the MEP pathway in bacterial pathogens, combined with its complete absence in human cells, provides a fundamental basis for selectivity. By focusing on the unique structural and biochemical characteristics of V. vulnificus IspE, researchers can develop inhibitors that selectively target this essential bacterial enzyme while minimizing interactions with human proteins .