Recombinant Rhodopirellula baltica 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (ispF)

What is the biological function of IspF in Rhodopirellula baltica?

IspF (2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase) in Rhodopirellula baltica serves as the fifth enzyme in the MEP pathway, which is responsible for the biosynthesis of isoprenoid precursors. Specifically, IspF catalyzes the cyclization and dephosphorylation reactions that convert 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate (CDP-ME2P) to 2C-methyl-D-erythritol 2,4-cyclodiphosphate (MECDP) while releasing CMP . This reaction represents a critical step in the non-mevalonate pathway for the synthesis of isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP), which are universal five-carbon building blocks for all isoprenoids . In Rhodopirellula baltica, this pathway supports the production of various essential cellular components, including membrane lipids and potentially pigments that contribute to the organism's pink-to-red coloration .

What cofactors are required for optimal IspF activity?

IspF requires divalent metal ions as essential cofactors for catalytic activity. Specifically, magnesium (Mg²⁺) or manganese (Mn²⁺) ions are necessary for the enzyme to function properly . Crystal structure analysis reveals that the metal ion adopts a tetrahedral coordination geometry at the base of the active site cleft, where it interacts with a phosphate oxygen of the product MECDP and the side chains of specific amino acid residues, namely Asp(8), His(10), and His(42) . The presence of these metal ions is critical for facilitating the cyclization reaction. Experimental research indicates that the enzyme has a pH optimum at pH 7.0, which should be maintained for optimal activity measurements . When designing experiments to assess IspF enzymatic activity, researchers should ensure that appropriate concentrations of either Mg²⁺ or Mn²⁺ are included in the reaction buffer.

How is Rhodopirellula baltica typically isolated and maintained in laboratory settings?

The recommended procedure includes:

• Collection of particles, plankton catches, or sediment grains from marine samples

• Direct spreading of these materials (containing attached-living bacteria) on plate surfaces

• Use of selective agents such as ampicillin plus either cycloserine or streptomycin

• Identification of Rhodopirellula colonies using specific PCR reactions targeting a portion of the 16S rRNA gene

This approach is essential because R. baltica primarily grows attached to surfaces rather than free-floating, and the combination of selective antibiotics significantly reduces the number of competing colony-forming bacteria (from around 10,365 cfu/ml with ampicillin alone to 262 or 107 cfu/ml when combined with cycloserine or streptomycin, respectively) . For long-term maintenance, cultures should be regularly subcultured on marine media containing appropriate selective agents.

What structural features of R. baltica IspF contribute to its catalytic mechanism?

R. baltica IspF, like other orthologs, adopts a homotrimeric quaternary structure that displays a bell-shaped architecture with three crucial characteristics that contribute to its catalytic mechanism :

• Trimeric Assembly: Each IspF forms a homotrimer with a central hydrophobic cavity and three externally facing active sites located at the interfaces between adjacent monomers, creating three catalytic centers per enzyme complex .

• Metal-Binding Site: A tetrahedrally arranged transition metal binding site occupied by Mn²⁺ is positioned at the base of each active site cleft. This metal ion is coordinated by a phosphate oxygen of the MECDP product and the side chains of specific conserved residues (Asp8, His10, and His42) .

• Substrate Recognition Elements: The active site contains specific structural elements that recognize and properly orient the substrate CDP-ME2P for the cyclization reaction.

The catalytic mechanism likely proceeds through the metal ion activating the substrate phosphate groups, facilitating nucleophilic attack that results in the formation of the cyclic diphosphate and release of CMP. The unique arrangement of the active sites at monomer interfaces suggests that proper trimerization is essential for catalytic activity, and disruption of this quaternary structure could serve as a potential strategy for enzyme inhibition .

The hydrophobic cavity at the center of the trimer may also play a regulatory role, as downstream products of the MEP pathway such as IPP/DMAPP, geranyl diphosphate (GDP), and farnesyl diphosphate (FDP) have been reported to bind to this hydrophobic region in IspF orthologs, suggesting potential feedback regulation mechanisms .

How does the expression and activity of IspF vary during different growth phases in R. baltica?

While specific data on IspF expression profiles in R. baltica throughout growth phases is limited in the provided search results, research on growth phase-dependent protein regulation in R. baltica provides valuable context. Quantitative proteomic analysis using two-dimensional difference gel electrophoresis (2D DIGE) has shown that the number of regulated proteins (with fold changes >2) increases dramatically from early stationary phase (10 proteins) to late stationary phase (179 proteins), with fold changes reaching maximal values of 40 .

The regulation pattern suggests opposing regulation of the tricarboxylic acid cycle and oxidative pentose phosphate cycle during the transition to stationary phase, alongside downregulation of several enzymes involved in amino acid biosynthesis and upregulation of the alternative sigma factor sigmaH . Of particular note, 26 proteins of unknown function were differentially regulated in the stationary phase.

For IspF specifically, researchers should consider examining:

• Changes in expression levels across growth phases using quantitative proteomics

• Correlation between IspF expression/activity and isoprenoid production

• Potential post-translational modifications affecting enzyme activity

• Regulatory mechanisms that might involve feedback from pathway intermediates or end products

Interestingly, recent research on IspF orthologs suggests this enzyme may be involved in metabolic regulation, as it is stabilized and activated by MEP (the upstream intermediate produced by DXR) – indicating a potential feedforward activation mechanism . Moreover, the product of IspF, MEcPP, appears to function as an antistressor signal in bacteria that accumulates under oxidative stress conditions . These findings suggest that IspF activity may be particularly important during stress responses, which often coincide with stationary phase growth.

What are the key differences between IspF from R. baltica and orthologs from pathogenic bacteria?

Although the crystal structure of R. baltica IspF has not been specifically described in the provided search results, comparisons can be made based on the general characteristics of IspF enzymes and the available information on IspF from other species:

The lipophilic character of the IspF active site in pathogenic bacteria like M. tuberculosis makes this enzyme particularly advantageous as a drug target, as highly lipophilic inhibitors have better potential to penetrate the lipid-rich cell walls of mycobacteria . Research on R. baltica IspF could provide valuable comparative data to highlight unique structural or functional aspects that might be exploited for selective inhibitor design against pathogenic homologs.

What is the optimal protocol for expressing and purifying recombinant R. baltica IspF?

Based on the methods described for IspF expression from other organisms, here is a recommended protocol for expressing and purifying recombinant R. baltica IspF:

Expression System:

• Clone the catalytic domain of the R. baltica ispF gene into a suitable expression vector (e.g., pET series) with an appropriate affinity tag.

• Transform the recombinant plasmid into an E. coli expression strain (e.g., BL21(DE3)).

• Culture transformed cells in rich medium (e.g., LB) with appropriate antibiotics at 37°C until mid-log phase (OD₆₀₀ ≈ 0.6-0.8).

• Induce protein expression with IPTG (typically 0.5-1.0 mM) at reduced temperature (18-30°C) to enhance protein solubility.

• Harvest cells after 4-18 hours by centrifugation.

Purification Strategy:

• Resuspend cell pellet in lysis buffer containing:

  • 50 mM Tris-HCl, pH 7.0 (optimal pH for enzyme activity)

  • 300 mM NaCl

  • 10 mM imidazole (for His-tagged proteins)

  • 5 mM MgCl₂ or MnCl₂ (required divalent cations for activity)

  • Protease inhibitors

• Lyse cells by sonication or high-pressure homogenization.

• Clear lysate by centrifugation (20,000 × g, 30 min, 4°C).

• Purify the protein using immobilized metal affinity chromatography (IMAC).

• Further purify by size exclusion chromatography to obtain the homotrimeric form.

Important Considerations:

• Experience with IspF from other organisms suggests expressing only the catalytic domain rather than the full-length protein may yield better results, as attempts to express the full-length protein have resulted in insoluble protein that could not be folded in vitro .

• Include divalent metal ions (Mg²⁺ or Mn²⁺) in all buffers throughout purification to maintain enzyme stability and activity .

• Monitor protein oligomerization state, as the functional enzyme exists as a homotrimer with active sites at monomer interfaces .

• Verify enzyme activity using a coupled assay system that can detect either the consumption of CDP-ME2P or the formation of MECDP.

This protocol should yield purified, active recombinant R. baltica IspF suitable for structural and functional studies.

What assays are available for measuring IspF enzymatic activity, and what are their limitations?

Several assay methods are available for measuring IspF enzymatic activity, each with specific advantages and limitations:

1. Direct Product Detection Assays:

2. Indirect Coupled Assays:

3. Binding Assays:

Key Challenges and Considerations:

• Substrate Instability: The substrate CDP-ME2P is unstable, complicating assay development and making commercial availability limited .

• Direct Measurement Limitations: There is a reported lack of assays for direct measurement of IspF substrate turnover , requiring researchers to develop creative approaches or rely on coupled systems.

• Enzyme Stability Issues: IspF can exhibit instability and insolubility , requiring careful handling during assay development and execution.

• Metal Dependency: Assays must include appropriate divalent metal ions (Mg²⁺ or Mn²⁺) for enzymatic activity .

• pH Considerations: The optimal pH for activity is around 7.0 , and assay buffers should be designed accordingly.

Researchers should select an assay method that best suits their specific experimental questions, available equipment, and throughput requirements while accounting for these potential limitations.

How can researchers develop effective inhibitors targeting R. baltica IspF?

Developing effective inhibitors targeting R. baltica IspF requires a multifaceted approach combining structural analysis, rational design, and screening methodologies:

1. Structure-Based Design Approach:

• Utilize homology modeling based on available IspF crystal structures (if R. baltica IspF structure is unavailable)

• Target the key structural features:

  • The active site located at monomer interfaces

  • The metal-binding site coordinated by Asp8, His10, and His42

  • The central hydrophobic cavity that may be involved in regulatory binding

2. Inhibitor Design Strategies:

• Substrate/Product Analogs: Design compounds that mimic CDP-ME2P or MECDP but cannot be processed

• Metal-Chelating Agents: Develop inhibitors that interfere with the essential metal cofactor

• Disruptors of Trimerization: Target the protein-protein interfaces critical for forming the active trimeric structure

• Non-cytidine-like Compounds: Explore thiazolopyrimidine derivatives which have shown promise against Mycobacterial IspF (IC₅₀ as low as 2.1 μM)

3. Physicochemical Considerations:

• Lipophilicity Optimization: Design compounds with appropriate lipophilicity (clogP) values to ensure cell penetration

• Metabolic Stability: Incorporate features that enhance stability against degradation

• Solubility Balance: Maintain sufficient aqueous solubility for testing while preserving membrane permeability

4. Screening and Validation Workflow: