Recombinant Photobacterium profundum 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (ispE)

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

IspE is the fourth enzyme in the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis. It 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-MEP). This reaction represents the only ATP-dependent step in the MEP pathway, which is essential for bacterial survival and absent in the human proteome . The MEP pathway ultimately leads to the production of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), which are precursors for various isoprenoids critical for bacterial cell function.

Why is Photobacterium profundum IspE of particular interest to researchers?

Photobacterium profundum is a deep-sea bacterium that has evolved to survive under high hydrostatic pressure conditions. Similar to other proteins from this organism, such as its cytochrome P450, P. profundum IspE is likely to possess unique structural adaptations that allow it to function optimally under pressure . These adaptations may include altered protein hydration, conformational changes, and modified active site accessibility. Studying P. profundum IspE provides insights into both enzymatic adaptations to extreme environments and potential novel antimicrobial targets, as the MEP pathway is absent in humans but essential in many pathogens .

How does the structure of IspE differ from other kinases in the bacterial proteome?

IspE belongs to the galactose/homoserine/mevalonate/phosphomevalonate (GHMP) kinase superfamily with a distinctive two-domain architecture and an α/β-fold . The enzyme contains two predicted functional motifs: a homoserine kinase (ThrB) motif and a GHMP kinase motif . Unlike many other bacterial kinases, IspE's catalytic core is located in a deep cleft at the interface of the cofactor and substrate-binding (CDP-ME) domains . Phylogenetic analysis shows that IspE enzymes form a distinct group from human mevalonate kinases, making them promising drug targets .

What strategies have proven most effective for expressing recombinant P. profundum IspE?

Based on approaches used for other IspE enzymes, expression of recombinant P. profundum IspE can be achieved using Escherichia coli expression systems. For optimal expression, consider the following methodology:

• Cloning the P. profundum ispE gene into an expression vector with an N-terminal or C-terminal His-tag

• Transforming E. coli BL21(DE3) or similar expression strains

• Growing cultures at 37°C until reaching OD600 of 0.6-0.8

• Inducing expression with IPTG (0.1-1.0 mM) at lower temperatures (16-20°C) for 16-20 hours to enhance protein solubility

This approach has been successful for related IspE enzymes from M. tuberculosis, B. mallei, S. Typhi, and V. cholerae .

What purification challenges are specific to P. profundum IspE and how can they be addressed?

Like other proteins from deep-sea bacteria, P. profundum IspE may present unique purification challenges due to its adaptation to high-pressure environments. Drawing from experiences with other bacterial IspE enzymes and pressure-adapted proteins, researchers should consider:

• Initial purification using Ni-NTA affinity chromatography with imidazole gradient elution

• Secondary purification via size exclusion chromatography to achieve high purity

• Including pressure stabilizers such as trimethylamine N-oxide (TMAO) or glycine betaine in purification buffers

• Testing purification at various pressures if specialized equipment is available

• Using truncation strategies if the full-length protein proves difficult to purify, as was effective for M. tuberculosis IspE

Researchers working with M. tuberculosis IspE found that generating truncated versions of the protein significantly improved solubility and yield, with approximately 1 mg of purified protein per liter of culture .

How can researchers verify the activity and purity of recombinant P. profundum IspE?

Activity and purity verification can be performed through multiple complementary approaches:

• SDS-PAGE analysis: To assess protein purity, with expected molecular weight verification

• Western blotting: Using anti-His antibodies or custom antibodies against IspE

• Enzyme activity assay: Typically involving a coupled assay system where IspE activity is linked to NADH oxidation through pyruvate kinase and lactate dehydrogenase (PK/LDH), with spectrophotometric monitoring at 340 nm

• Mass spectrometry: For precise molecular weight determination and detection of post-translational modifications

• Circular dichroism: To verify proper protein folding

When using coupled enzyme assays, it is critical to maintain a difference between PK/LDH and IspE enzymatic activities to ensure that observed effects are due to IspE inhibition rather than effects on the auxiliary enzymes .

What are the key structural features that enable P. profundum IspE to function under high-pressure conditions?

While no crystal structure of P. profundum IspE has been reported, insights can be drawn from studies of other P. profundum proteins and IspE enzymes from different organisms. P. profundum cytochrome P450 (P450-SS9) exhibits pressure-adaptive features that may be similar in P. profundum IspE:

• Pressure-induced transition to a state with reduced accessibility of the active site

• Stabilization of specific conformational states under pressure

• Substantial hydration of the protein with reaction volume changes (ΔV) around -100 to -200 mL/mol

• Pressure adaptation at P(1/2) of 300-800 bar, which corresponds to the natural habitat pressure of P. profundum

• Confined water accessibility in the active site, which may be a common feature serving to coordinate active site hydration with ligand binding

These adaptations likely represent evolutionary mechanisms that allow the protein to maintain functionality under high hydrostatic pressures.

How do the kinetic parameters of P. profundum IspE compare with those from non-piezophilic bacteria?

Based on data from other IspE enzymes, a comparative analysis of kinetic parameters might reveal:

Note: Actual values would need to be determined experimentally, as the predicted values are extrapolated from related enzymes and pressure adaptation principles.

What cofactors and metal ions are required for optimal P. profundum IspE activity?

Like other IspE enzymes, P. profundum IspE likely requires:

• Mg²⁺ ions: Essential for coordinating ATP and facilitating phosphoryl transfer

• K⁺ ions: May enhance catalytic activity

• Reducing environment: DTT or β-mercaptoethanol may be needed to maintain cysteine residues in a reduced state

The enzyme's requirement for these cofactors might be influenced by pressure, with potential changes in binding affinity under different pressure conditions. High pressure might alter the coordination geometry of metal ions, potentially requiring adjustments in ion concentrations for optimal activity compared to atmospheric pressure conditions.

How can pressure perturbation studies reveal insights into P. profundum IspE's adaptation mechanisms?

Pressure perturbation studies provide valuable insights into protein adaptation mechanisms. For P. profundum IspE, researchers can employ several approaches:

• Pressure perturbation spectroscopy: Monitoring changes in enzyme structure under varying pressures (0-1000 bar) using UV-Vis, fluorescence, or FTIR spectroscopy

• High-pressure enzyme kinetics: Measuring reaction rates at different pressures to determine if activity is maintained or enhanced under native habitat pressure conditions

• Pressure-jump experiments: Analyzing relaxation times after sudden pressure changes to understand conformational dynamics

• Volume profile analysis: Calculating reaction and activation volumes (ΔV and ΔV‡) to characterize the pressure dependence of enzyme catalysis

Studies on P. profundum cytochrome P450 showed that pressure failed to displace the spin equilibrium completely to the low-spin state, indicating a pressure-induced transition to a state with reduced active site accessibility . Similar studies on P. profundum IspE could reveal unique pressure adaptation mechanisms specific to this enzyme class.

What computational approaches can be used to model P. profundum IspE structure and predict pressure effects?

Several computational approaches can provide insights into P. profundum IspE structure and function:

• Homology modeling: Using known IspE structures as templates (potentially complemented with AlphaFold predictions as done for P. falciparum IspE )

• Molecular dynamics simulations: Simulating protein behavior under varying pressure conditions (1-1000 bar)

• Quantum mechanics/molecular mechanics (QM/MM): For detailed analysis of the catalytic mechanism under pressure

• Cavity analysis algorithms: To identify and characterize internal cavities that may be affected by pressure

• Free energy calculations: To quantify the effects of pressure on substrate binding and catalysis

Computational predictions can guide experimental design and help interpret results from functional studies, particularly when examining differences between P. profundum IspE and mesophilic homologs.

How can recombinant P. profundum IspE be used as a model system for studying enzyme adaptation to extreme environments?

P. profundum IspE represents an excellent model system for studying enzymatic adaptation to extreme environments for several reasons:

• Comparative studies: Comparing P. profundum IspE with homologs from mesophilic bacteria can reveal specific adaptations to high pressure

• Domain swapping experiments: Creating chimeric proteins with domains from piezophilic and mesophilic IspE can identify pressure-responsive regions

• Directed evolution under pressure: Using in vitro evolution to enhance or modify pressure adaptation mechanisms

• Structure-function relationship studies: Correlating structural features with pressure tolerance can inform protein engineering efforts

• Biophysical characterization across pressure ranges: Examining how pressure affects stability, flexibility, and catalytic efficiency

Such studies not only advance our understanding of pressure adaptation but may also inform the design of enzymes for industrial applications under non-standard conditions.

What strategies are most effective for screening potential inhibitors of P. profundum IspE?

Based on approaches used for P. falciparum IspE, effective inhibitor screening strategies include:

• High-throughput screening (HTS): Using spectrophotometric assays that couple IspE activity to NADH oxidation via pyruvate kinase and lactate dehydrogenase

• Fragment-based drug discovery: Screening small molecular fragments that bind to different regions of the enzyme

• Structure-based virtual screening: Using computational docking against homology models or AlphaFold predictions of P. profundum IspE

• Affinity-based proteome profiling (AfBPP): Using chemical probes containing reactive groups for covalent binding and reporter tags for detection, as demonstrated for P. falciparum IspE

• Competition assays: Testing whether candidate compounds compete with known ligands or substrates for binding

For P. falciparum IspE, researchers developed a probe (compound 23) containing a diazirine and an alkyne moiety that demonstrated binding to IspE in the presence of human cell lysate, providing evidence that both the probe and inhibitor competed for the same binding site .

How do inhibitors of IspE from other organisms perform against P. profundum IspE, and what structural modifications might improve their efficacy?

While specific data for P. profundum IspE inhibition is lacking, insights can be drawn from studies of other IspE enzymes:

• Cross-species activity: Inhibitors developed for P. falciparum IspE, such as compound 19 with an IC₅₀ of 53 ± 19 μM , could be tested against P. profundum IspE

• Pressure-dependent binding: Inhibitors effective at atmospheric pressure may show altered binding under high pressure due to conformational changes

• Structure-activity relationship (SAR) studies: Similar to those conducted for P. falciparum IspE inhibitors, SAR studies could identify optimal substitution patterns for P. profundum IspE inhibition

• Hydrophilic modifications: Since the MEP pathway enzymes have highly polar active sites , inhibitors may require specific hydrophilic features to achieve potent inhibition

• Pressure-stabilized binding pockets: Targeting unique binding pockets that emerge under pressure conditions could lead to selective inhibitors

Compounds that show activity against multiple IspE homologs could serve as starting points for developing specific P. profundum IspE inhibitors, with modifications guided by computational modeling and experimental feedback.

What technical challenges must be overcome when validating IspE inhibitors using whole-cell assays with P. profundum?

Validating IspE inhibitors in whole-cell assays with P. profundum presents several unique challenges:

• High-pressure cultivation requirements: P. profundum requires specialized equipment for growth under native pressure conditions (~300-800 bar)

• Membrane permeability under pressure: Inhibitor penetration into cells may differ under high pressure compared to atmospheric conditions

• Compound stability under pressure: Chemical stability of inhibitors may be affected by high hydrostatic pressure

• Distinguishing target-specific effects: Confirming that growth inhibition is due to IspE inhibition rather than off-target effects requires careful controls

• Correlation between enzymatic and cellular activity: Establishing clear relationships between in vitro IspE inhibition and whole-cell effects

These challenges necessitate specialized approaches, such as gene essentiality confirmation through allelic disruption (as demonstrated for IspE in M. smegmatis ), combined with target verification through affinity probes and proteomics.

How can structural comparisons between P. profundum IspE and pathogenic bacterial IspE inform drug development?

Structural comparisons between P. profundum IspE and pathogenic homologs can provide valuable insights for drug development:

• Conserved binding pockets: Identifying structural elements that are conserved across species can guide the development of broad-spectrum inhibitors

• Species-specific features: Recognizing unique structural features in pathogenic IspE can enable selective targeting

• Pressure-induced conformational changes: Understanding how pressure affects protein conformation may reveal transiently accessible binding sites

• Dynamic protein regions: Comparing flexibility and dynamics across homologs can identify targetable regions that undergo significant conformational changes

• Solvent accessibility patterns: Analyzing differences in solvent exposure of key residues can inform inhibitor design

For example, studies of M. tuberculosis IspE have shown that generating truncated versions of the protein (Rv1011 fragments) with varying degrees of the ThrB and GHMP kinase motifs resulted in different catalytic activities, with Rv1011 I (Δ301-305) showing the greatest activity . Similar structure-function relationships in P. profundum IspE could guide inhibitor development.

What methodological approaches are most effective for determining the essentiality of IspE in P. profundum compared to pathogenic bacteria?

To determine IspE essentiality in P. profundum, researchers can employ several approaches that have proven successful in other bacteria:

• Allelic disruption: Similar to methods used for M. smegmatis IspE , attempting gene knockout through homologous recombination

• Conditional expression systems: Creating strains where IspE expression is controlled by inducible promoters to observe effects of depletion

• CRISPR-Cas9 genome editing: Attempting targeted disruption of the ispE gene

• Chemical genetics: Using validated IspE inhibitors to assess effects on bacterial growth and survival

• Complementation studies: Testing whether IspE from other species can functionally replace P. profundum IspE

Each approach must be adapted for high-pressure conditions, potentially requiring specialized equipment for cultivation and analysis under native pressure environments.

How do environmental adaptations of P. profundum IspE compare with pathogenic bacterial adaptations to host environments?

The environmental adaptations of P. profundum IspE likely differ significantly from those of pathogenic bacteria:

Understanding these differences can inform both fundamental questions about protein evolution and applied research on drug development and enzyme engineering.

What new experimental approaches could advance our understanding of P. profundum IspE structure and function?

Several cutting-edge approaches could significantly advance our understanding of P. profundum IspE:

• Cryo-electron microscopy under pressure: Developing methodologies to visualize protein structures under native pressure conditions

• Single-molecule FRET under pressure: Measuring conformational dynamics of individual enzyme molecules under varying pressure conditions

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Characterizing pressure-dependent changes in protein dynamics and solvent accessibility

• Time-resolved X-ray crystallography: Capturing intermediate states during catalysis under different pressure conditions

• Neutron crystallography: Providing detailed information about hydrogen bonding networks and water organization under pressure

• Deep mutational scanning: Systematically assessing the effects of all possible single amino acid substitutions on pressure adaptation

These approaches would provide unprecedented insights into how P. profundum IspE has evolved to function optimally in high-pressure environments.

How might synthetic biology approaches utilize P. profundum IspE for novel applications?

P. profundum IspE offers several exciting possibilities for synthetic biology applications:

• Pressure-resistant biocatalysts: Engineering pressure-stable enzymes for industrial processes under high-pressure conditions

• Biosensors for pressure detection: Creating molecular sensors based on pressure-induced conformational changes in P. profundum IspE

• Deep-sea bioremediation: Developing engineered organisms with enhanced isoprenoid production for environmental applications

• Novel antimicrobial discovery platforms: Using P. profundum IspE as a model for screening compounds that could inhibit pathogenic IspE enzymes

• Heterologous expression systems for pressure-adapted proteins: Creating expression platforms optimized for producing other deep-sea bacterial proteins

These applications could leverage the unique properties of P. profundum IspE to address challenges in biotechnology, medicine, and environmental science.

What interdisciplinary collaborations would most benefit P. profundum IspE research?

Advancing P. profundum IspE research would benefit from several key interdisciplinary collaborations:

• Deep-sea microbiologists and high-pressure biophysicists: Providing expertise in cultivating piezophilic organisms and analyzing biomolecules under pressure

• Structural biologists and computational chemists: Combining experimental structure determination with advanced modeling of pressure effects

• Enzymologists and chemical biologists: Developing assays and probes for studying enzyme function under various conditions

• Medicinal chemists and pharmacologists: Designing and synthesizing potential inhibitors based on structural insights

• Synthetic biologists and protein engineers: Creating modified versions of the enzyme with enhanced or altered properties

• Materials scientists and nanotechnologists: Exploring applications of pressure-stable enzymes in material synthesis and nanofabrication

Such collaborations would accelerate progress in understanding this unique enzyme and translating findings into practical applications.