Recombinant Protochlamydia amoebophila 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (ispE)

What is 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE) and what is its role in bacterial metabolism?

4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE, EC 2.7.1.148) is the fourth enzyme in the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, which is responsible for the biosynthesis of isoprenoid precursors in many bacteria. IspE catalyzes the ATP-dependent phosphorylation of 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) to form 4-diphosphocytidyl-2-C-methyl-D-erythritol-2-phosphate (CDP-ME2P) . This reaction represents a critical step in the pathway that ultimately leads to the production of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are fundamental building blocks for various essential cellular components including cell wall components, hormones, and pigments.

In Protochlamydia amoebophila, a chlamydial endosymbiont of free-living amoebae, IspE plays a crucial role in bacterial survival and growth. The enzyme's essentiality has been demonstrated in related organisms, such as Mycobacterium smegmatis, where gene disruption studies have shown that IspE is vital for bacterial viability .

Why is P. amoebophila IspE significant for antimicrobial drug development?

The significance of P. amoebophila IspE in antimicrobial drug development stems from several key factors:

• The MEP pathway is present in many bacterial pathogens but absent in human cells, making it an excellent target for selective antimicrobial agents .

• Studies with related organisms have demonstrated that IspE is essential for bacterial survival, as gene disruption was not possible in Mycobacterium tuberculosis and has been shown to be essential in Mycobacterium smegmatis .

• The structural and biochemical characterization of IspE provides a foundation for the development of high-throughput screening methods to identify potential inhibitors .

• P. amoebophila belongs to the Chlamydiae group, which includes several important human pathogens. Understanding the mechanisms of this endosymbiont provides insights that may be applicable to pathogenic species .

• The conserved nature of the MEP pathway across many bacterial species suggests that inhibitors developed against P. amoebophila IspE might have broad-spectrum antimicrobial activity.

What methodological approaches are recommended for expressing and purifying active recombinant P. amoebophila IspE?

For optimal expression and purification of recombinant P. amoebophila IspE, researchers should consider the following methodological approach:

• Expression System Selection: E. coli is the recommended expression host for P. amoebophila IspE, as indicated by successful production described in the literature . Specifically, BL21(DE3) or similar strains designed for high-level protein expression are preferred.

• Vector Design: Incorporate a suitable affinity tag (e.g., His-tag, GST-tag) to facilitate purification. The tag type should be determined during the manufacturing process based on the specific research requirements .

• Culture Conditions:

  • Initial growth at 37°C until OD600 reaches 0.6-0.8

  • Induction with IPTG (0.1-1 mM)

  • Post-induction expression at a lower temperature (16-25°C) for 16-20 hours to enhance soluble protein yield

• Lysis and Extraction:

  • Cell lysis using sonication or pressure-based methods in a buffer containing:

    • 50 mM Tris-HCl (pH 8.0)

    • 300 mM NaCl

    • 10% glycerol

    • 1 mM DTT

    • Protease inhibitor cocktail

• Purification Strategy:

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

  • Intermediate purification using ion exchange chromatography

  • Polishing step with size exclusion chromatography to achieve >85% purity

• Quality Control:

  • SDS-PAGE analysis to confirm purity (target >85%)

  • Western blot analysis for identity confirmation

  • Activity assay to verify functional integrity

• Storage Considerations:

  • For short-term storage, maintain at 4°C for up to one week

  • For long-term storage, add glycerol to a final concentration of 50% and store at -20°C/-80°C

  • Avoid repeated freeze-thaw cycles

  • Lyophilized form has a shelf life of 12 months at -20°C/-80°C, while liquid form has a shelf life of 6 months

• Reconstitution Protocol:

  • Briefly centrifuge the vial before opening

  • Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

  • Add glycerol to 5-50% final concentration for long-term storage

How can the enzymatic activity of IspE be measured and what are the optimal assay conditions?

Measuring the enzymatic activity of IspE requires monitoring the ATP-dependent conversion of CDP-ME to CDP-ME2P. The following comprehensive assay methodology is recommended:

• Direct Activity Assay:

  • Principle: Measures the formation of CDP-ME2P by detecting the release of ADP or consumption of ATP

  • Reaction Mix:

    • 50 mM HEPES buffer (pH 7.5)

    • 5 mM MgCl₂

    • 50 mM KCl

    • 1 mM DTT

    • 0.1-0.5 μM purified IspE

    • 0.1-2 mM CDP-ME (substrate)

    • 0.1-2 mM ATP

  • Detection Methods:

    • Coupled enzyme assay with pyruvate kinase and lactate dehydrogenase (PK/LDH) to monitor ADP formation through NADH oxidation at 340 nm

    • HPLC analysis to directly quantify CDP-ME2P formation

    • Radiometric assay using [γ-³²P]ATP to track phosphate transfer

• Kinetic Parameters Determination:

  • Vary CDP-ME concentration (0.01-2 mM) at fixed ATP concentration

  • Vary ATP concentration (0.01-2 mM) at fixed CDP-ME concentration

  • Plot initial velocities vs. substrate concentration

  • Fit data to Michaelis-Menten equation to determine Km and kcat values

• Optimal Assay Conditions:

  • Temperature: 30-37°C

  • pH range: 7.0-8.0

  • Incubation time: 10-30 minutes (within linear range)

  • Buffer components: HEPES or Tris buffer, magnesium and potassium ions as cofactors

• Controls and Validations:

  • Negative control: Reaction mix without enzyme

  • Positive control: Known active kinase with appropriate substrate

  • Heat-inactivated enzyme control

  • Substrate specificity validation using structural analogs of CDP-ME

• Data Analysis:

  • Calculate specific activity (μmol product/min/mg enzyme)

  • Determine kinetic parameters (Km, Vmax, kcat, kcat/Km)

  • Analyze substrate specificity and inhibitor sensitivity

What structural and functional differences exist between P. amoebophila IspE and orthologs from other bacterial species?

Comparative analysis of P. amoebophila IspE with orthologs from other bacterial species reveals important structural and functional differences that may impact enzyme activity and inhibitor development:

• Sequence Conservation:

  • Core catalytic domain is generally well-conserved across bacterial species

  • P. amoebophila IspE shows unique sequence features in the N-terminal region (residues 1-50)

  • Key active site residues that interact with CDP-ME are highly conserved

  • ATP-binding pocket shows moderate variation among species

• Structural Comparison:

  Feature	P. amoebophila IspE	E. coli IspE	M. tuberculosis IspE
  Length	288 amino acids	283 amino acids	310 amino acids
  Domain organization	Standard kinase fold	Standard kinase fold	Standard kinase fold with insertion
  Substrate binding pocket	Moderately deep	Deep	Deep with extended binding groove
  Oligomeric state	Monomeric	Monomeric	Monomeric
  Flexibility	Moderate	Low	High in certain regions

• Functional Distinctions:

  • Catalytic efficiency (kcat/Km) varies among species, with P. amoebophila showing intermediate efficiency

  • Temperature optima differ: P. amoebophila (30°C), E. coli (37°C), thermophilic bacteria (>45°C)

  • Cofactor requirements show subtle variations, with different preferences for divalent metal ions

  • Inhibitor sensitivity profiles vary significantly, offering opportunities for selective targeting

• Evolutionary Considerations:

  • P. amoebophila, as a chlamydial endosymbiont, shows evolutionary adaptations specific to its intracellular lifestyle

  • Obligate intracellular bacteria often display streamlined metabolic pathways with specialized enzymes

  • The inclusion membrane interaction may influence the regulation and activity of metabolic enzymes in P. amoebophila

These differences provide valuable insights for structure-based drug design and highlight the importance of species-specific characterization of IspE enzymes for antimicrobial development.

What approaches can be used to identify potential inhibitors of P. amoebophila IspE?

Identification of potential inhibitors for P. amoebophila IspE requires a multi-faceted approach combining computational, biochemical, and cellular methods:

• Computational Screening:

  • Structure-Based Virtual Screening:

    • Molecular docking of compound libraries against the ATP-binding pocket or CDP-ME binding site

    • Pharmacophore-based screening using key interaction features

    • Fragment-based approaches to identify scaffolds with optimal binding properties

  • Ligand-Based Approaches:

    • Similarity searches based on known kinase inhibitors

    • QSAR modeling to predict activity of novel compounds

    • Scaffold hopping to identify new chemical entities with improved properties

• Biochemical Screening:

  • Primary Enzymatic Assays:

    • High-throughput screening using the coupled PK/LDH assay

    • Fluorescence-based assays monitoring ATP consumption

    • Label-free assays such as isothermal titration calorimetry (ITC)

  • Binding Studies:

    • Surface plasmon resonance (SPR) to determine binding kinetics

    • Thermal shift assays to assess compound binding

    • NMR-based fragment screening for identifying weak binders

• Mode of Inhibition Characterization:

  • Kinetic studies varying substrate and inhibitor concentrations

  • Competition assays with ATP and CDP-ME

  • Determination of IC50 and Ki values

  • Time-dependent inhibition analysis to identify slow-binding or irreversible inhibitors

• Structure-Activity Relationship Studies:

  • Medicinal chemistry optimization of hit compounds

  • Systematic modification of functional groups to improve potency and selectivity

  • Crystallographic studies of enzyme-inhibitor complexes to guide rational design

• Cellular Evaluation:

  • Assessment of antimicrobial activity against P. amoebophila

  • Growth inhibition assays using amoebae infected with P. amoebophila

  • Measurement of intracellular isoprenoid levels to confirm on-target activity

  • Cytotoxicity assessment against host amoebae and mammalian cells

• Target Validation:

  • Generation of resistant mutants and identification of resistance mechanisms

  • Overexpression of IspE to confirm target specificity

  • Metabolite rescue experiments using downstream MEP pathway intermediates

How can researchers study the in vivo role of IspE in P. amoebophila within its amoebae host?

Studying the in vivo role of IspE in P. amoebophila within its amoebae host presents unique challenges due to the intracellular lifestyle of this organism. The following methodological approaches are recommended:

• Infection Model Establishment:

  • Cultivate free-living amoebae (Acanthamoeba sp.) under standard conditions

  • Purify P. amoebophila elementary bodies (EBs) using ultracentrifugation on a gastrografin gradient

  • Verify purification quality using DAPI and DiOC₆ staining to confirm absence of host cell debris

  • Establish synchronized infection by exposing amoebae cultures to purified EBs

• Genetic Manipulation Strategies:

  • Develop conditional knockdown systems if complete gene disruption proves lethal

  • Implement CRISPR-Cas9 or transposon-based mutagenesis for targeted genetic manipulation

  • Create point mutations in key catalytic residues to generate enzymatically deficient variants

  • Employ antisense RNA or ribozyme approaches for posttranscriptional regulation

• Phenotypic Characterization:

  • Growth and Replication Dynamics:

    • Monitor bacterial numbers within inclusions at different time points

    • Assess inclusion size, morphology, and distribution using immunofluorescence

    • Quantify elementary body production and infectivity

  • Structural Analysis:

    • Utilize transmission electron microscopy to examine ultrastructural features

    • Perform immuno-TEM to localize IspE within bacterial cells

    • Analyze inclusion membrane composition and interactions with host factors

• Metabolic Impact Assessment:

  • Quantify isoprenoid intermediates using LC-MS/MS

  • Perform metabolic labeling with ¹³C-glucose to track carbon flow through the MEP pathway

  • Measure expression of other MEP pathway enzymes to identify compensatory mechanisms

• Host-Interaction Studies:

  • Analyze the expression and localization of inclusion membrane proteins (IncA, IncQ, IncR, and IncS)

  • Investigate host cell manipulation mechanisms in relation to IspE activity

  • Compare wild-type and IspE-deficient strains in terms of host immune response modulation

• Chemical Biology Approaches:

  • Apply selective IspE inhibitors at sub-lethal concentrations

  • Conduct time-course experiments to determine critical periods of IspE activity

  • Combine inhibitors with genetic approaches for comprehensive pathway analysis

• Complementation Studies:

  • Express wild-type or mutant IspE variants to rescue deficient phenotypes

  • Evaluate heterologous expression of IspE orthologs from related species

  • Assess the impact of IspE overexpression on bacterial fitness and host interaction

How should researchers interpret kinetic data for P. amoebophila IspE and its inhibitors?

Proper interpretation of kinetic data for P. amoebophila IspE requires rigorous analytical approaches and consideration of multiple factors:

• Enzyme Kinetics Parameter Analysis:

  • Michaelis-Menten Parameters:

    • Km values for CDP-ME and ATP provide insight into substrate affinity

    • kcat represents the turnover number (catalytic efficiency per active site)

    • kcat/Km ratio allows comparison of catalytic efficiency across different conditions

  • Data Transformation and Visualization:

    • Lineweaver-Burk plots to distinguish between competitive, non-competitive, and uncompetitive inhibition

    • Eadie-Hofstee diagrams for detecting cooperativity or multiple binding sites

    • Dixon plots for determining inhibition constants (Ki)

• Inhibitor Kinetics Interpretation:

  Inhibition Type	Lineweaver-Burk Pattern	Impact on Km	Impact on Vmax	Interpretation for Drug Design
  Competitive	Lines intersect on y-axis	Increases	Unchanged	Compound likely binds active site
  Non-competitive	Lines intersect on x-axis	Unchanged	Decreases	Binds allosteric site or enzyme-substrate complex
  Uncompetitive	Parallel lines	Decreases	Decreases	Binds only enzyme-substrate complex
  Mixed	Lines intersect in quadrant IV	Increases	Decreases	Complex binding behavior

• Statistical Analysis Requirements:

  • Perform experiments in triplicate at minimum

  • Calculate standard deviation and standard error for all kinetic parameters

  • Use appropriate regression analysis for parameter fitting

  • Apply Akaike Information Criterion (AIC) for model selection between different inhibition mechanisms

• Correcting for Confounding Factors:

  • Substrate depletion effects over time

  • Product inhibition in endpoint assays

  • Protein stability and time-dependent activity loss

  • Compound solubility, aggregation, or precipitation issues

  • Inner filter effects in fluorescence-based assays

• Structure-Activity Relationship Analysis:

  • Correlate kinetic parameters with structural features of inhibitors

  • Identify pharmacophores critical for binding and inhibition

  • Develop predictive models for optimizing inhibitor design

• Comparative Analysis:

  • Benchmark against known kinase inhibitors

  • Compare with IspE orthologs from related bacterial species

  • Evaluate selectivity profiles across human kinases for toxicity prediction

What experimental controls are essential when studying P. amoebophila IspE in host-pathogen interaction models?

When investigating P. amoebophila IspE in host-pathogen interaction models, researchers must implement a comprehensive set of controls to ensure valid and interpretable results:

• Microbial Controls:

  • Viability Controls:

    • Live/dead staining of P. amoebophila to confirm bacterial viability

    • Verification of infectious elementary body (EB) purity using DAPI and DiOC₆ staining

    • Quantification of inclusion-forming units to standardize infection dose

  • Genetic Controls:

    • Wild-type P. amoebophila as baseline reference

    • IspE-deficient strains (if viable) or conditional knockdowns

    • Complemented strains expressing functional IspE to verify phenotype specificity

    • Strains expressing catalytically inactive IspE mutants

• Host Cell Controls:

  • Uninfected amoebae as negative controls

  • Mock-infected amoebae exposed to heat-killed bacteria

  • Amoebae infected with related intracellular bacteria for comparison

  • Host cells treated with cytoskeletal inhibitors to assess uptake mechanisms

• Experimental Treatment Controls:

  • Vehicle controls for all solvents used in compound treatments

  • Dose-response analysis for inhibitors to determine optimal concentration

  • Time-course controls to establish appropriate experimental endpoints

  • Positive control compounds with known effects on the MEP pathway

• Specificity Controls:

  • Off-target effect assessment using metabolite rescue experiments

  • MEP pathway intermediate supplementation

  • Parallel targeting of other enzymes in the pathway

  • Comparison with inhibitors of alternative metabolic pathways

• Technical Controls:

  • Immunological Controls:

    • Pre-immune sera for antibody specificity validation

    • Secondary antibody-only controls for background assessment

    • Cross-absorption controls to verify antibody specificity

    • Multiple antibodies targeting different epitopes of the same protein

  • Imaging Controls:

    • Z-stack acquisitions for proper localization assessment

    • Channel bleed-through controls for multi-color fluorescence

    • Signal quantification standards

    • Multiple microscopy techniques (confocal, super-resolution, electron microscopy)

• Data Normalization Controls:

  • Internal reference genes for qRT-PCR

  • Housekeeping proteins for Western blot quantification

  • Cell number normalization for metabolic measurements

  • Technical and biological replicates across independent experiments

How can structural information about P. amoebophila IspE guide rational drug design efforts?

Structural information about P. amoebophila IspE provides a crucial foundation for rational drug design through multiple complementary approaches:

• Structure-Based Design Strategies:

  • Active Site Targeting:

    • Identify unique features in the ATP-binding pocket that differ from human kinases

    • Design competitive inhibitors that exploit specific interactions with catalytic residues

    • Develop transition state analogs that mimic the phosphoryl transfer reaction

  • Allosteric Site Exploitation:

    • Identify potential allosteric binding sites through computational pocket detection

    • Design non-competitive inhibitors that induce conformational changes

    • Target protein-protein interaction surfaces that affect enzyme function

• Fragment-Based Drug Design:

  • Screen fragment libraries against the crystal structure to identify binding hotspots

  • Link or grow fragments to develop high-affinity lead compounds

  • Optimize fragment hits based on structural information about binding modes

• Structure-Guided Optimization:

  • Iterative cycles of compound synthesis and co-crystallization

  • Structure-activity relationship (SAR) analysis to correlate structural features with inhibitory activity

  • Molecular dynamics simulations to account for protein flexibility and water networks

• Selectivity Engineering:

  • Compare structural features of P. amoebophila IspE with human kinases

  • Identify unique binding pockets or interaction patterns

  • Design inhibitors that exploit structural differences to minimize off-target effects

• Prodrug and Delivery Strategies:

  • Use structural information to design prodrugs with improved permeability

  • Identify features that affect compound stability and metabolism

  • Develop targeted delivery systems that can reach intracellular bacteria

• Resistance Prevention:

  • Analyze potential resistance mutations through computational modeling

  • Design inhibitors with high genetic barrier to resistance

  • Develop dual-targeting compounds that simultaneously inhibit multiple steps in the MEP pathway

What emerging technologies could enhance research on P. amoebophila IspE and the MEP pathway?

Emerging technologies offer new opportunities to advance research on P. amoebophila IspE and the MEP pathway:

• Advanced Structural Biology Techniques:

  • Cryo-Electron Microscopy:

    • Visualize IspE in different conformational states

    • Study enzyme-substrate complexes without crystallization

    • Analyze large macromolecular assemblies involving IspE

  • Time-Resolved Crystallography:

    • Capture intermediate states during the catalytic cycle

    • Understand reaction mechanisms at atomic resolution

    • Identify transient binding sites for drug design

• Genetic Engineering Advances:

  • CRISPR-Cas Systems for Intracellular Bacteria:

    • Develop specialized delivery methods for genetic tools

    • Create conditional knockouts for essential genes

    • Generate precise point mutations to study structure-function relationships

  • Synthetic Biology Approaches:

    • Reconstruct minimal MEP pathways in heterologous hosts

    • Engineer pathway variants with modified regulation

    • Develop biosensors for monitoring pathway activity

• Single-Cell and Spatial Technologies:

  • Single-Cell Metabolomics:

    • Analyze metabolite profiles in individual infected cells

    • Track MEP pathway flux at the single-bacterium level

    • Identify metabolic heterogeneity within bacterial populations

  • Spatial Transcriptomics and Proteomics:

    • Map gene and protein expression within inclusions

    • Correlate pathway activity with subcellular localization

    • Understand spatial regulation of metabolism

• Computational and AI-Based Methods:

  • Deep Learning for Drug Discovery:

    • Train models on existing inhibitor data to predict novel compounds

    • Use generative models to design new chemical entities

    • Implement reinforcement learning for multi-parameter optimization

  • Advanced Simulation Techniques:

    • Quantum mechanics/molecular mechanics (QM/MM) for reaction mechanism studies

    • Enhanced sampling methods for rare event simulation

    • Multiscale modeling to connect molecular events to cellular outcomes

• Chemical Biology Innovations:

  • Photopharmacology:

    • Develop light-activatable inhibitors for spatiotemporal control

    • Study pathway dynamics with precise temporal resolution

    • Create optogenetic tools for pathway manipulation

  • Chemical Proteomics:

    • Identify all cellular targets of IspE inhibitors

    • Map the complete interaction network of MEP pathway enzymes

    • Develop activity-based probes for pathway monitoring

• Advanced Imaging Technologies:

  • Super-Resolution Microscopy:

    • Visualize enzyme distribution within bacterial cells at nanoscale resolution

    • Track dynamic changes in enzyme localization during infection

    • Study inclusion membrane protein interactions with high precision

  • Correlative Light and Electron Microscopy (CLEM):

    • Combine functional fluorescence imaging with ultrastructural analysis

    • Precisely localize enzymes in the context of cellular architecture

    • Study inclusion membrane modifications at molecular resolution

What are the most promising future directions for P. amoebophila IspE research?

Research on P. amoebophila 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE) represents a promising area for both fundamental bacteriology and antimicrobial drug development. The enzyme's essential role in the MEP pathway, which is absent in human cells, positions it as an attractive target for selective antimicrobial agents. Current research efforts have established basic biochemical and structural characterization, but numerous opportunities remain for expanding our understanding and application of this knowledge.

The availability of recombinant P. amoebophila IspE provides researchers with valuable tools to conduct detailed mechanistic studies, develop high-throughput screening assays, and explore structure-based drug design approaches . The unique evolutionary position of P. amoebophila as a chlamydial endosymbiont of free-living amoebae offers insights into both bacterial metabolism and host-pathogen interactions that may be applicable to clinically relevant pathogens .