Recombinant Enterococcus faecalis 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (ispF)

What is the biological significance of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (ispF) in Enterococcus faecalis?

2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (ispF) is a critical enzyme in the non-mevalonate pathway (also known as the DOXP/MEP pathway) of isoprenoid biosynthesis. This pathway is essential for the production of isoprenoids, which are vital compounds involved in cell membrane structure, electron transport, and various cellular processes. In E. faecalis, the enzyme catalyzes the fifth step of this pathway, specifically the conversion of 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate (CDP-ME2P) to 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (ME-CPP) and CMP. This reaction represents a critical point in the biosynthetic pathway leading to essential isoprenoid precursors .

The importance of this enzyme is highlighted by the fact that the non-mevalonate pathway is present in many bacteria (including important pathogens like E. faecalis) but absent in mammals, which use the mevalonate pathway instead. This distinction makes ispF a potential target for antibacterial drug development, as inhibitors specific to this enzyme would potentially not affect human metabolism .

How does the structure of E. faecalis ispF compare to that of other bacterial species?

While the crystal structure of E. faecalis ispF has not been explicitly described in the provided sources, structural insights can be derived from the related E. coli enzyme. The E. coli 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase exists as a homotrimer built around a β prism. Each trimer contains three active sites, with each active site formed in a cleft between pairs of subunits .

The active site contains a zinc ion (Zn²⁺) with tetrahedral coordination and a manganese ion (Mn²⁺) with octahedral geometry. The Mn²⁺ is positioned between the α and β phosphates of the substrate, working in concert with the Zn²⁺ to align and polarize the substrate for catalysis .

The high degree of sequence conservation among these enzymes across bacterial species suggests that E. faecalis ispF likely shares similar structural features with the E. coli enzyme, particularly in the metal-binding sites and active site architecture critical for catalytic function.

What expression systems are most effective for producing recombinant E. faecalis ispF?

For recombinant expression of E. faecalis ispF, E. coli expression systems are commonly employed due to their ease of use, scalability, and cost-effectiveness. The selection of an appropriate expression vector and strain is crucial for optimal protein production.

Common expression systems include:

• pET vector systems (e.g., pET28a) with E. coli BL21(DE3) or its derivatives

• pGEX vectors for GST-fusion proteins

• pMAL vectors for MBP-fusion proteins

Expression parameters that typically require optimization include:

• Induction temperature (typically 16-37°C)

• IPTG concentration (typically 0.1-1.0 mM)

• Duration of induction (4-24 hours)

• Media composition (LB, TB, or minimal media with supplements)

Temperature reduction during induction (e.g., to 16-20°C) often improves the solubility of recombinant proteins by slowing down expression and allowing more time for proper folding. Additionally, co-expression with molecular chaperones may improve soluble protein yield.

How do specific mutations in E. faecalis ispF affect enzyme kinetics and substrate binding?

Mutations in the active site residues of ispF can significantly alter enzyme kinetics and substrate binding. Based on structural studies of related enzymes, several key regions are likely critical for E. faecalis ispF function:

• Metal-binding residues: Mutations of zinc-coordinating residues would disrupt the tetrahedral coordination of Zn²⁺, severely impacting catalytic activity .

• Substrate recognition residues: Residues that recognize and bind the cytidine nucleotide portion of the substrate are essential for proper substrate positioning .

• Catalytic residues: Those involved in the cyclization reaction mechanism, particularly those that interact with the phosphate groups.

A systematic mutational analysis approach can employ the following methodology:

• Site-directed mutagenesis of conserved residues

• Expression and purification of mutant proteins

• Enzyme kinetic assays comparing wild-type and mutant enzymes

• Thermal shift assays to assess stability changes

• Isothermal titration calorimetry (ITC) to quantify binding affinities

The following table represents typical kinetic parameters that might be observed in such studies:

These studies can provide critical insights into the structure-function relationships of the enzyme and potentially identify residues that could be targeted for inhibitor design.

What is the role of metal ions in E. faecalis ispF catalysis and structure stabilization?

Based on structural studies of related enzymes, metal ions play crucial roles in both catalysis and structural stabilization of ispF. In the E. coli enzyme, the active site contains both zinc (Zn²⁺) and manganese (Mn²⁺) ions with distinct coordination geometries and functions .

The zinc ion likely has a primarily structural role, maintaining the tertiary structure of the active site through tetrahedral coordination with conserved amino acid residues. The manganese ion, positioned between the α and β phosphates of the substrate, plays a more direct catalytic role, helping to align and polarize the substrate for nucleophilic attack .

Methodological approaches to study metal ion roles include:

• Metal ion substitution experiments with various divalent cations (Mg²⁺, Ca²⁺, Co²⁺, Ni²⁺)

• Activity assays in the presence of metal chelators (EDTA, EGTA)

• Spectroscopic studies (EPR, NMR) to probe metal ion environments

• X-ray crystallography with different metal ions to observe structural changes

Experimental data typically reveals that:

• Complete removal of metal ions results in loss of both structure and function

• Substitution with alternative metals often results in reduced but measurable activity

• The specificity for different metal ions can provide insights into the catalytic mechanism

These studies are particularly relevant for developing metal-chelating inhibitors as potential antimicrobial agents targeting E. faecalis ispF.

How does E. faecalis ispF compare functionally to homologs from other pathogenic bacteria, especially regarding potential as an antimicrobial target?

E. faecalis ispF, as part of the non-mevalonate pathway, represents a potential antimicrobial target because this pathway is absent in mammals. Comparing E. faecalis ispF with homologs from other pathogenic bacteria provides valuable insights for drug development efforts.

Key aspects for comparison include:

• Sequence conservation analysis: Highly conserved residues often indicate functional importance.

• Structural differences in active sites: Small variations can be exploited for species-specific inhibitors.

• Substrate specificity differences: Some bacterial ispF enzymes may have slightly different preferences for substrate analogs.

• Inhibitor sensitivity profiles: Different bacterial ispF enzymes may show variable sensitivity to the same inhibitors.

Methodological approaches include:

• Comparative genomics and phylogenetic analysis

• Recombinant expression of ispF from multiple bacterial species

• Parallel biochemical characterization using standardized assays

• High-throughput screening against diverse inhibitor libraries

• Structure-based drug design targeting species-specific features

E. faecalis poses unique challenges as an opportunistic pathogen with increasing antibiotic resistance . Its ability to cause life-threatening infections, particularly in hospitalized patients, underscores the importance of identifying new therapeutic targets like ispF . The partial influence of E. faecalis genetics on infection patterns suggests that targeting conserved essential enzymes like ispF may be effective against diverse genetic backgrounds .

What are the optimal conditions for assaying recombinant E. faecalis ispF enzymatic activity?

Assaying recombinant E. faecalis ispF activity requires careful consideration of reaction conditions to ensure reliable and reproducible results. While specific optimized conditions for the E. faecalis enzyme are not provided in the search results, the following methodology represents a standard approach based on related enzymes:

Standard Enzymatic Assay Protocol:

• Buffer components:

  • 50 mM Tris-HCl or HEPES, pH 7.5-8.0

  • 100 mM NaCl or KCl

  • 5 mM MgCl₂ (for Mg²⁺ cofactor)

  • 1-5 mM DTT or 2-mercaptoethanol (reducing agent)

  • 0.1-1.0 mM ZnCl₂ (for Zn²⁺ cofactor)

  • 0.1-0.5 mM MnCl₂ (for Mn²⁺ cofactor)

  • 5-10% glycerol (stabilizer)

• Substrate:

  • CDP-ME2P at concentrations ranging from 10-500 μM

• Enzyme concentration:

  • 0.1-1.0 μM purified recombinant enzyme

• Reaction conditions:

  • Temperature: 30-37°C

  • Time: 10-30 minutes

  • Volume: 100-500 μL

• Detection methods:

  • HPLC analysis of substrate consumption/product formation

  • Coupled enzyme assay measuring CMP release

  • Malachite green assay for phosphate release

  • LC-MS for direct product identification and quantification

Optimization experiments should systematically vary each parameter (pH, temperature, salt concentration, metal ion concentration) to identify optimal conditions for maximum enzymatic activity. The table below summarizes typical optimization results:

These assay conditions provide a starting point for characterizing wild-type and mutant versions of the enzyme, as well as for inhibitor screening campaigns.

What are the most effective purification strategies for obtaining high-purity recombinant E. faecalis ispF?

Purifying recombinant E. faecalis ispF to high homogeneity requires a multi-step approach that takes advantage of the protein's physical and chemical properties. While specific protocols for E. faecalis ispF are not provided in the search results, the following general methodology represents best practices for similar enzymes:

Standard Purification Protocol:

• Affinity Chromatography (Primary Capture):

  • For His-tagged constructs: Ni-NTA or TALON resin

  • For GST-fusion proteins: Glutathione Sepharose

  • For MBP-fusion proteins: Amylose resin

  Typical buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, with imidazole gradient (10-250 mM) for His-tagged proteins

• Tag Removal (Optional):

  • Protease cleavage with TEV, thrombin, or Factor Xa

  • Reverse affinity chromatography to remove cleaved tag

• Ion Exchange Chromatography (Secondary Purification):

  • Based on the theoretical pI of E. faecalis ispF:

    • Anion exchange (Q Sepharose) if pI < 7

    • Cation exchange (SP Sepharose) if pI > 7

  Typical buffer: 20 mM Tris-HCl pH 8.0, with NaCl gradient (0-500 mM)

• Size Exclusion Chromatography (Polishing Step):

  • Superdex 75 or 200 column based on protein size

  • Typical buffer: 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 5% glycerol

• Quality Control Assessments:

  • SDS-PAGE: >95% purity

  • Dynamic light scattering: monodispersity check

  • Thermal shift assay: stability assessment

  • Activity assay: functional verification

  • Mass spectrometry: verification of intact mass

The following table summarizes expected outcomes at each purification stage:

Critical considerations include:

• Addition of 5-10% glycerol in all buffers to improve stability

• Inclusion of 1-5 mM DTT or 2-mercaptoethanol as reducing agent

• Addition of 0.1-0.5 mM ZnCl₂ and MnCl₂ to stabilize the enzyme

• Maintaining low temperature (4°C) throughout purification

• Using protease inhibitors in early purification steps

This systematic approach typically yields 5-20 mg of highly pure, active enzyme per liter of bacterial culture, suitable for structural and biochemical studies.

What crystallization conditions have been successful for determining the structure of E. faecalis ispF?

While specific crystallization conditions for E. faecalis ispF are not detailed in the provided search results, insights can be derived from the successful crystallization of the related E. coli enzyme . The following methodology provides a systematic approach to crystallizing E. faecalis ispF:

Crystallization Strategy:

• Initial Screening:

  • Commercial sparse matrix screens (Hampton Research, Molecular Dimensions)

  • Grid screens varying pH (6.0-9.0) and precipitant concentration

  • Typical techniques: sitting-drop and hanging-drop vapor diffusion

  • Protein concentration range: 5-15 mg/mL

  • Temperature: 4°C and 20°C

  • Drop size: 1-2 μL protein + 1-2 μL reservoir solution

• Optimization of Promising Conditions:

  • Fine gradient of precipitant concentration

  • pH adjustments in smaller increments

  • Additive screening with commercial kits

  • Seeding techniques (micro-seeding, streak-seeding)

  • Varying drop size and protein:reservoir ratios

• Co-crystallization with Ligands:

  • Substrate analogs (CDP, CMP)

  • Product analogs

  • Metal ions (Zn²⁺, Mn²⁺, Mg²⁺)

  • Potential inhibitors

The E. coli ispF structure was determined to 1.8 Å resolution using crystals grown in the presence of cytidine 5'-diphosphate and Mn²⁺ . This suggests similar conditions might work for the E. faecalis homolog, especially considering that both enzymes likely share similar structural features including a zinc cofactor with tetrahedral coordination and a manganese ion with octahedral geometry .

Based on the E. coli structure, the following conditions might serve as starting points:

X-ray diffraction data collection would typically be performed at a synchrotron source, with the structure solved by molecular replacement using the E. coli ispF structure as a search model. This approach would reveal important structural details specific to the E. faecalis enzyme that could inform drug design efforts.

How can researchers address solubility issues when expressing recombinant E. faecalis ispF?

Solubility issues are common challenges when expressing recombinant proteins. For E. faecalis ispF, which is likely a metal-binding enzyme similar to its E. coli homolog , several methodological approaches can address poor solubility:

• Optimization of Expression Conditions:

  • Reduce induction temperature to 16-20°C

  • Lower IPTG concentration to 0.1-0.2 mM

  • Shorten induction time to 4-6 hours

  • Use richer media (TB instead of LB)

  • Add metal ions (ZnCl₂, MnCl₂) to growth media

• Protein Engineering Approaches:

  • Fusion tags that enhance solubility (MBP, SUMO, TRX)

  • Truncation constructs to remove potential aggregation-prone regions

  • Surface entropy reduction mutations

  • Rational design based on structural homology models

• Buffer Optimization Strategies:

  • Include 5-10% glycerol or 0.5-1M sorbitol as stabilizers

  • Test different pH values (typically pH 7.0-8.5)

  • Increase salt concentration (300-500 mM NaCl)

  • Add metal ions (0.1-1.0 mM ZnCl₂, MnCl₂)

  • Include mild detergents (0.05-0.1% Triton X-100)

• Co-expression Strategies:

  • Molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

  • Metal-binding chaperones if metal coordination is essential

  • Rare codon tRNAs if codon usage is an issue

The table below summarizes typical outcomes with different solubility enhancement strategies:

A systematic approach testing these strategies individually and in combination typically resolves most solubility issues, resulting in sufficient quantities of properly folded, active enzyme for subsequent studies.

What approaches can be used to develop selective inhibitors of E. faecalis ispF for potential antimicrobial applications?

Developing selective inhibitors of E. faecalis ispF represents a promising strategy for antimicrobial development, given that the non-mevalonate pathway is absent in mammals . A comprehensive approach would include:

• Structure-Based Drug Design:

  • Crystal structure determination of E. faecalis ispF

  • Computational docking of virtual compound libraries

  • Fragment-based screening approaches

  • Structure-activity relationship (SAR) studies

  • Molecular dynamics simulations to identify binding pocket flexibility

• High-Throughput Screening:

  • Development of a robust biochemical assay

  • Screening of diverse chemical libraries

  • Counter-screening against human enzymes to ensure selectivity

  • Secondary assays to confirm mechanism of action

• Rational Design Strategies:

  • Substrate analogs targeting the CDP-ME2P binding site

  • Metal chelators targeting the zinc and manganese binding sites

  • Transition state mimics based on reaction mechanism

  • Allosteric inhibitors targeting protein-protein interfaces in the trimeric structure

• Validation Approaches:

  • Enzymatic assays with purified recombinant protein

  • Bacterial growth inhibition assays

  • Cytotoxicity testing in mammalian cells

  • Mechanism of action studies (enzyme inhibition, metabolite analysis)

  • Resistance development studies

The table below outlines different inhibitor classes and their expected characteristics:

How can researchers differentiate between the effects of targeting ispF and other enzymes in the non-mevalonate pathway in E. faecalis?

Distinguishing the specific effects of targeting ispF from those of targeting other enzymes in the non-mevalonate pathway requires sophisticated experimental designs. The following methodological approaches can help differentiate these effects:

• Genetic Approaches:

  • Conditional knockdown systems (inducible antisense RNA)

  • CRISPR interference for targeted gene repression

  • Complementation studies with wild-type or mutant alleles

  • Overexpression of individual pathway enzymes

• Biochemical Approaches:

  • Metabolite profiling using LC-MS/MS

  • Isotope labeling studies to track pathway flux

  • Enzyme-specific activity assays in cell lysates

  • In vitro reconstitution of the pathway with purified enzymes

• Inhibitor-Based Approaches:

  • Use of pathway-specific metabolites to bypass blocks

  • Comparison of phenotypes with known inhibitors of different steps

  • Synergy/antagonism studies with combinations of inhibitors

  • Target engagement assays (thermal shift, surface plasmon resonance)

• Systems Biology Approaches:

  • Transcriptomic analysis following specific enzyme inhibition

  • Proteomic studies to identify compensatory mechanisms

  • Flux balance analysis to model effects of different enzyme inhibitions

  • Network analysis to identify unique signatures of ispF inhibition

The table below outlines expected outcomes when targeting different enzymes in the pathway: