Recombinant Mycoplasma gallisepticum 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (ispF)

What is 2-C-Methyl-D-Erythritol 2,4-Cyclodiphosphate Synthase (IspF) and what pathway does it participate in?

IspF functions as a key enzyme in the 2-C-Methyl-D-erythritol-4-phosphate (MEP) pathway of isoprenoid biosynthesis. It specifically catalyzes the fifth step in this pathway, converting 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (CDPME2P) to 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MEcDP) with concomitant release of cytidine 5′-diphosphate (CMP) . This reaction represents a critical step in the biosynthesis of isoprenoid precursors, which are essential for numerous cellular functions including membrane maintenance, hormone production, and protein modification. The MEP pathway is present in many bacteria and some parasites but absent in humans, making it a potential target for antimicrobial development .

What are the major structural features of IspF enzymes?

IspF enzymes typically function as homotrimers with each monomer containing distinct domains for substrate binding and catalysis. High-resolution crystal structures have revealed that:

• Each monomer contains a binding pocket for the cytidine moiety of the substrate

• A zinc ion is coordinated at the active site, essential for enzymatic activity

• The enzyme displays a characteristic fold with a central β-sheet surrounded by α-helices

Structural comparisons between species reveal subtle differences in subunit packing. For example, Bacillus subtilis IspF shows looser packing of subunits compared to Escherichia coli, while having smaller solvent-accessible surface area in its active pockets . These structural variations may influence substrate specificity and catalytic efficiency across different species, offering important considerations for researchers studying this enzyme family.

What analytical methods are commonly used to assess IspF activity?

Several methodological approaches can be employed to assess IspF enzymatic activity:

When selecting an assay method, researchers should consider factors such as available equipment, required sensitivity, and potential interfering factors in their experimental system .

How do regulatory mechanisms control IspF activity in metabolic pathways?

IspF regulation involves sophisticated feed-forward and feedback mechanisms that fine-tune isoprenoid biosynthesis. Recent research has uncovered that 2-C-methyl-D-erythritol 4-phosphate (MEP), an earlier intermediate in the pathway, acts as an activator that enhances and sustains IspF activity . This creates a novel feed-forward regulatory mechanism whereby:

• MEP binds to IspF, forming an IspF-MEP complex with enhanced catalytic activity

• The methylerythritol scaffold unique to this pathway drives the activation and stabilization of active IspF

• The IspF-MEP complex appears to be the physiologically relevant form of the enzyme in vivo

Importantly, this activated complex can be inhibited by farnesyl diphosphate (FDP), suggesting a feedback inhibition mechanism to prevent overproduction of isoprenoids . This regulatory network demonstrates how metabolic intermediates coordinate pathway flux, a consideration essential for researchers designing experiments to characterize IspF function in cellular contexts or developing potential inhibitors targeting this enzyme.

What experimental approaches can be used to investigate potential protein-protein interactions between IspF and other MEP pathway enzymes?

The investigation of potential protein-protein interactions involving IspF requires multiple complementary approaches:

• Co-immunoprecipitation (Co-IP): Using antibodies against IspF to pull down potential interacting partners from cellular lysates, followed by Western blotting or mass spectrometry analysis.

• Bacterial two-hybrid systems: Modified for membrane-associated proteins if necessary, these can detect direct interactions between IspF and other MEP pathway enzymes.

• Fluorescence resonance energy transfer (FRET): By tagging IspF and potential partners with appropriate fluorophores, interactions can be detected in live cells.

• Surface plasmon resonance (SPR): This allows quantitative measurement of binding affinities between purified IspF and other pathway components.

• Analytical ultracentrifugation: Useful for detecting complex formation and determining stoichiometry of interactions.

The controversial question of whether multienzyme complexes form between IspD, IspE, and IspF remains unresolved . While bioinformatics analyses have indicated gene fusion between ispD and ispF in several bacterial species (creating bifunctional IspDF enzymes), conclusive evidence for physical interactions between the monofunctional enzymes is lacking. Further investigations are required to clarify these associations and their potential implications for metabolic channeling in isoprenoid biosynthesis .

What are the methodological challenges in crystallizing Mycoplasma gallisepticum IspF, and how might they be overcome?

Crystallizing Mycoplasma gallisepticum IspF presents several technical challenges that researchers should anticipate:

• Protein stability issues: Mycoplasma proteins often show reduced stability compared to those from other bacterial sources. Consider incorporating stability-enhancing mutations or using fusion tags that improve folding.

• Conformational heterogeneity: The active site flexibility required for catalysis can impede crystal formation. Co-crystallization with substrates, products, or inhibitors can stabilize specific conformations.

• Obtaining sufficient protein quantities: Mycoplasma expression systems may yield lower protein amounts. Optimization of codon usage for the expression host and culture conditions can improve yields.

• Crystal packing constraints: The homotrimeric nature of IspF can complicate crystal packing. Screening multiple crystallization conditions with varying precipitants, pH ranges, and additives is essential.

• Post-translational modifications: If present, these can introduce heterogeneity. Mass spectrometry analysis prior to crystallization attempts can identify potential modifications.

Successful approaches have included utilizing high-throughput crystallization screening methods, fragment-based approaches to identify stabilizing ligands, and surface entropy reduction mutations to create new crystal contacts . Researchers studying IspF from other species have successfully employed these techniques to obtain high-resolution structures.

How does the substrate specificity of Mycoplasma gallisepticum IspF compare with orthologs from other species?

Substrate specificity comparisons between IspF orthologs reveal both conserved mechanisms and species-specific variations:

*Values estimated based on related species as specific kinetic parameters for M. gallisepticum IspF are not directly reported in the literature

These differences in catalytic parameters may reflect evolutionary adaptations to different cellular environments and metabolic demands. The structural variations, particularly in active site architecture, suggest potential opportunities for developing species-selective inhibitors. When conducting comparative studies, researchers should carefully consider buffer conditions, metal ion concentrations, and temperature, as these factors can significantly impact measured kinetic parameters .

What approaches can be used to develop selective inhibitors of IspF for antimicrobial research?

Developing selective IspF inhibitors requires multifaceted approaches leveraging the enzyme's structural and mechanistic features:

• Structure-based design: Crystal structures of IspF from various species, including complexes with substrates and inhibitors, provide valuable templates for rational design. Focus should be placed on:

  • The zinc-binding site, essential for catalysis

  • The cytidine-binding pocket, which shows high conservation

  • Species-specific features in the active site that could enable selectivity

• Fragment-based screening: This approach has identified novel chemical scaffolds with binding affinity for IspF. The method involves:

  • Screening small molecular fragments (MW <300)

  • Using X-ray crystallography or NMR to confirm binding

  • Growing or linking fragments to improve potency

• High-throughput enzymatic assays: Enable rapid screening of compound libraries against recombinant IspF:

  • Primary screens using spectrophotometric methods

  • Secondary validation with orthogonal assays

  • Counter-screening against human enzymes to ensure selectivity

• Targeting the IspF-MEP complex: Recent research indicates that the IspF-MEP complex may be the physiologically relevant form of the enzyme. Inhibitors designed to disrupt this complex could represent a novel inhibitory mechanism .

Several compounds targeting IspF have shown antimicrobial activity, including against the malaria parasite Plasmodium falciparum with IC50 values in the low micromolar range (1.4-1.6 μM) . These findings validate IspF as a promising target for antimicrobial development, particularly for pathogens where this pathway is essential.

What are optimal expression conditions for producing high-yield recombinant Mycoplasma gallisepticum IspF?

Optimizing expression conditions for Mycoplasma gallisepticum IspF requires careful consideration of several parameters:

Expression system selection:

• Yeast systems (particularly Pichia pastoris) have proven effective for producing recombinant M. gallisepticum IspF with His-tag conjugation

• E. coli systems may offer higher yields but can present challenges with proper folding

• Mammalian expression systems might be considered for specialized applications requiring specific post-translational modifications

Critical optimization parameters:

• Temperature: Lower expression temperatures (16-20°C) often improve protein folding and solubility

• Induction timing and concentration: For inducible systems, induction at mid-log phase typically yields optimal results

• Media composition: Enriched media can increase biomass but may reduce specific protein expression

• Codon optimization: Adapting codons to the expression host can significantly improve translation efficiency

• Fusion tags: Beyond the His-tag for purification, solubility-enhancing tags (MBP, SUMO) may improve yields

Purification considerations:

• Multi-step purification protocols typically achieve >90% purity

• Inclusion of zinc in purification buffers helps maintain enzyme structure and activity

• Size exclusion chromatography as a final step ensures isolation of properly folded trimeric enzyme

Researchers should conduct small-scale expression trials varying these parameters to determine optimal conditions before scaling up production .

How can researchers accurately assess the structural integrity and functional activity of purified recombinant IspF?

A comprehensive approach to quality assessment combines structural and functional analyses:

Structural integrity assessment:

Functional activity assessment:

• Enzymatic assay: Measure conversion of CDPME2P to MEcDP and CMP using:

  • HPLC-based detection of products

  • Coupled enzyme assays linking CMP production to a spectroscopic readout

  • Mass spectrometry to directly detect MEcDP formation

• Zinc content analysis: Since zinc is essential for catalytic activity, atomic absorption spectroscopy or colorimetric assays can verify zinc incorporation

• Differential Scanning Fluorimetry (DSF): Assess thermal stability shifts upon substrate binding, which correlate with functional integrity

• Isothermal Titration Calorimetry (ITC): Determine binding constants for substrates and inhibitors to verify active site functionality

By combining multiple orthogonal methods, researchers can confidently establish both the structural and functional quality of their purified IspF preparations before proceeding with more complex experiments .

What considerations are important when designing site-directed mutagenesis experiments for IspF structure-function studies?

Effective site-directed mutagenesis studies of IspF should consider:

Key residue selection:

• Catalytic residues: Those directly involved in zinc coordination and substrate binding

• Substrate recognition residues: Those that interact with the cytidine moiety or the methylerythritol portion

• Oligomerization interface residues: Those maintaining the trimeric structure

• Regulatory sites: Residues involved in binding MEP or other regulatory molecules

Mutation strategy:

• Conservative substitutions: Replacing with chemically similar amino acids to probe the importance of specific interactions

• Charge reversals: To test electrostatic interactions

• Alanine scanning: Systematic replacement with alanine to identify critical residues

• Introduction of reporter groups: Such as cysteine for subsequent chemical modification

Special considerations for IspF:

• Mutations near the zinc-binding site may disrupt metal coordination, leading to complete loss of activity

• The trimeric structure is essential for function; mutations disrupting oligomerization will affect activity indirectly

• Some residues may play dual roles in substrate binding and maintaining structural integrity

Control experiments:

• Verify expression and purification yield of mutants compared to wild-type

• Assess structural integrity through CD spectroscopy or thermal stability assays

• Use multiple assay methods to verify activity changes

• Consider generating a homology model based on existing crystal structures to predict mutational effects

By carefully designing mutagenesis experiments with these considerations in mind, researchers can generate valuable insights into structure-function relationships in IspF enzymes .

How can IspF be utilized as a target for developing new antimicrobial agents against Mycoplasma infections?

IspF presents several advantageous characteristics as an antimicrobial target against Mycoplasma infections:

• Essential pathway: The MEP pathway, including IspF, is essential for bacterial survival in multiple studied species . Genetic validation has established that the ispF gene is necessary for growth in several bacterial models.

• Absence in humans: The MEP pathway is absent in humans, who exclusively utilize the alternative mevalonate pathway for isoprenoid biosynthesis . This difference provides a theoretical basis for selective toxicity.

• Structural knowledge: High-resolution structural data from related bacterial IspF enzymes provides templates for structure-based drug design approaches.

Target-based screening approaches:

• Biochemical assays using purified recombinant M. gallisepticum IspF

• Fragment-based screening followed by structure-guided optimization

• Virtual screening against the IspF active site

• Design of transition state analogs or substrate mimics

Potential challenges to address:

• Ensuring sufficient penetration of inhibitors through the Mycoplasma cell membrane

• Achieving specificity for Mycoplasma IspF over other bacterial orthologs when treating mixed infections

• Preventing resistance development through rational design or combination approaches

Recent studies have shown promising results with IspF inhibitors against other pathogens, with some compounds demonstrating IC50 values in the low micromolar range against Plasmodium falciparum within infected erythrocytes . These precedents suggest that similar approaches could be successful against Mycoplasma species.

What methods can be employed to study the potential role of IspF in multienzyme complexes within the MEP pathway?

Investigating potential multienzyme complexes involving IspF requires multiple complementary approaches:

In vitro approaches:

• Protein-protein interaction assays:

  • Pull-down assays with purified components (IspD, IspE, IspF)

  • Surface plasmon resonance to quantify binding kinetics

  • Isothermal titration calorimetry for thermodynamic parameters

  • Native mass spectrometry to detect intact complexes

• Structural studies:

  • Cryo-electron microscopy for large complexes

  • X-ray crystallography of co-purified components

  • Small-angle X-ray scattering for solution structure analysis

In vivo approaches:

• Proximity labeling techniques:

  • BioID or TurboID fusions to IspF expressed in native hosts

  • APEX2-based proximity labeling

  • These methods identify proteins in close proximity in living cells

• Imaging techniques:

  • Fluorescence microscopy with differentially labeled pathway enzymes

  • Förster resonance energy transfer (FRET) to detect direct interactions

  • Bimolecular fluorescence complementation to visualize interactions

Functional evidence:

• Substrate channeling experiments:

  • Compare kinetics of individual enzymes versus combined enzymes

  • Isotope dilution experiments to detect channeled intermediates

Current evidence suggests variable organization across species - some organisms (like Campylobacter jejuni) have gene fusions creating bifunctional IspDF enzymes, while in other species like B. subtilis and E. coli, there's ongoing debate about whether monofunctional IspD, IspE, and IspF form multienzyme complexes . The existence and physiological relevance of such complexes remain controversial and require further investigation.

What approaches can be used to investigate differences in IspF regulation between Mycoplasma gallisepticum and other bacterial species?

Investigating regulatory differences in IspF across species requires systematic comparative analysis:

Regulatory mechanism comparison:

• Feed-forward activation: Test whether MEP activates M. gallisepticum IspF similar to E. coli IspF

  • Enzymatic assays with and without MEP

  • Binding studies (ITC, fluorescence) to quantify MEP interaction

  • Crystal structures of the enzyme with MEP

• Feedback inhibition: Determine if downstream products (IDP, DMADP, FDP) inhibit the enzyme

  • Concentration-dependent inhibition assays

  • Competition studies to determine inhibition mechanisms

Structural basis for regulatory differences:

• Sequence alignment analysis: Identify conservation or divergence in regulatory sites

• Homology modeling: If M. gallisepticum IspF structure is unavailable

• Chimeric proteins: Create domain-swapped variants between species to isolate regulatory elements

Experimental design considerations:

• Standardized conditions: Use identical buffer systems, temperature, and substrate concentrations when comparing across species

• Multiple orthogonal methods: Combine kinetic, thermodynamic, and structural approaches

• Physiological context: Consider the cellular environment of Mycoplasma (pH, ion concentrations, metabolite levels)

A comparative study of IspF regulation across multiple bacterial species could reveal evolutionary adaptations in isoprenoid biosynthesis regulation. This information would be valuable for understanding the metabolic adaptation of Mycoplasma gallisepticum and could potentially reveal species-specific regulatory mechanisms that might be exploited for selective targeting .

What are common challenges in expressing and purifying functional recombinant IspF, and how can they be addressed?

Researchers frequently encounter several challenges when working with recombinant IspF:

Expression challenges:

Purification challenges:

• Aggregation issues:

  • Include reducing agents (DTT, β-mercaptoethanol) in buffers

  • Optimize ionic strength (typically 150-300 mM NaCl)

  • Add stabilizing agents (glycerol 5-10%, specific amino acids)

• Loss of activity during purification:

  • Minimize purification steps and processing time

  • Maintain constant zinc concentration in buffers

  • Verify trimeric assembly by size exclusion chromatography

• Co-purifying contaminants:

  • Implement additional chromatography steps (ion exchange, hydrophobic interaction)

  • Consider on-column refolding protocols if necessary

  • Use high-resolution size exclusion as final polishing step

• Establishing proper quality control:

  • Verify zinc content by atomic absorption spectroscopy

  • Confirm trimeric assembly by native PAGE or analytical SEC

  • Validate activity with established enzymatic assays

Researchers have successfully overcome these challenges by implementing systematic optimization of expression and purification protocols, with recombinant IspF routinely achievable at >90% purity .

How can researchers address data inconsistencies when comparing IspF activity measurements across different studies?

When encountering data inconsistencies in IspF literature, researchers should consider several potential sources of variation:

Methodological variations:

• Different assay methods: Direct vs. coupled assays may yield different apparent kinetic values

• Buffer composition: pH, ionic strength, and specific buffer components significantly affect activity

• Metal content: Zinc occupancy varies with purification methods and affects catalytic performance

• Temperature: Assays performed at different temperatures (25°C vs. 37°C) are not directly comparable

• Substrate quality: Commercial vs. synthesized substrates may contain different impurities

Standardization approaches:

• Include reference standards: When possible, obtain and test a well-characterized IspF preparation alongside new samples

• Detailed methods reporting: Document all assay conditions comprehensively

• Multiple assay methods: Validate results using orthogonal activity measurement techniques

• Molecular quality control: Verify protein integrity through methods such as mass spectrometry prior to activity measurements

Data interpretation strategies:

• Focus on trends rather than absolute values: When comparing across studies, relative changes may be more reliable than absolute measurements

• Consider physiological relevance: Evaluate activity under conditions that approximate the cellular environment

• Meta-analysis approaches: Systematically compare multiple studies while accounting for methodological differences

A standardized approach to measuring and reporting IspF activity would facilitate more meaningful comparisons across studies. When integrating data from multiple sources, researchers should carefully consider methodological differences and their potential impact on the reported values .

What considerations are important when designing inhibitor screening assays for IspF?

Designing effective inhibitor screening assays for IspF requires careful optimization of multiple parameters:

Assay design considerations:

• Assay format selection:

  • Primary screening: Higher throughput methods (fluorescence, absorbance-based)

  • Secondary confirmation: More direct but lower throughput methods (HPLC, mass spectrometry)

• Critical parameters to optimize:

  • Signal-to-background ratio: Aim for >3:1 for reliable screening

  • Z'-factor: Values >0.5 indicate suitable assay quality for screening

  • DMSO tolerance: Essential for compound solubility, typically up to 1-5%

  • Stability over time: Minimal drift during the screening timeframe

• Controls and counter-screens:

  • Positive controls: Known inhibitors or heat-inactivated enzyme

  • Negative controls: Full reaction with DMSO vehicle

  • Interference counter-screens: For compounds showing fluorescence or absorbance

  • Selectivity counter-screens: Against human enzymes or unrelated bacterial targets

IspF-specific considerations:

• Enzyme preparation:

  • Ensure consistent zinc content across preparations

  • Verify trimeric assembly for each batch

  • Consider using the IspF-MEP complex for physiological relevance

• Substrate concentrations:

  • For identifying competitive inhibitors: Use substrate at or below Km

  • For identifying any mode of inhibition: Use substrate at 2× Km

• Mechanistic insights:

  • Design assays capable of distinguishing competitive, uncompetitive, and non-competitive inhibition

  • Consider time-dependence to identify slow-binding inhibitors

• Detection methods optimized for IspF:

By carefully addressing these considerations, researchers can develop robust screening assays for identifying novel IspF inhibitors with potential antimicrobial applications .