Recombinant Chlamydophila caviae 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (ispG), partial

What is 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (IspG) and what reaction does it catalyze?

4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (IspG) is an enzyme that catalyzes the penultimate step in the methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis. Specifically, IspG converts 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MEcPP) into (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMB-PP) . The reaction involves a reduction and ring-opening process that requires electron transfer, typically provided by reduced ferredoxin proteins. The systematic name for this enzyme is (E)-4-hydroxy-3-methylbut-2-en-1-yl-diphosphate:protein-disulfide oxidoreductase (hydrating) .

What is the domain structure of IspG in bacteria versus eukaryotic organisms?

IspG enzymes exhibit distinct structural organizations depending on their biological source:

• Bacterial IspGs (e.g., from Aquifex aeolicus): Contain two domains that function as (AB)₂ dimers in solution. The N-terminus (A) has a TIM barrel structure with homology to dihydropteroate synthase, while the C-terminus (B) contains a 4Fe-4S cluster domain with homology to sulfite/nitrite reductase .

• Plant, algae, and apicomplexan parasite IspGs (e.g., from Arabidopsis thaliana): Feature three domains (AAB) that function as monomers. The additional insert domain (A) appears to be a second TIM barrel that plays a structural role, enabling the A and B domains to interact during catalysis in a "cup and ball" arrangement similar to bacterial systems .

This structural diversity is significant for understanding enzyme evolution and developing targeted inhibitors for pathogen-specific IspG enzymes .

What are the optimal conditions for expressing recombinant Chlamydophila caviae IspG in E. coli?

For optimal expression of recombinant Chlamydophila caviae IspG in E. coli, researchers should consider the following methodology:

• Vector selection: Use a broad host range, IPTG-inducible expression vector such as pRL814 for controlled expression .

• Ribosomal binding site optimization: Employ the Salis algorithm to optimize the ribosomal binding site sequence for improved translation efficiency in E. coli .

• Expression conditions: Culture at lower temperatures (16-20°C) after induction to enhance proper folding of the iron-sulfur cluster-containing protein.

• Media supplementation: Add iron (Fe²⁺) and sulfur sources to the growth medium to support proper formation of the [4Fe-4S] cluster essential for enzyme activity.

• Anaerobic conditions: Maintain low oxygen conditions during later stages of expression and throughout purification to preserve the integrity of the oxygen-sensitive [4Fe-4S] cluster .

These conditions have been successfully applied to express various IspG proteins and can be adapted specifically for Chlamydophila caviae IspG expression.

What are the challenges in maintaining IspG activity during purification, and how can they be addressed?

Maintaining IspG activity during purification presents several challenges due to the oxygen-sensitive [4Fe-4S] cluster. Methodological approaches to address these challenges include:

• Anaerobic purification: Perform all purification steps in an anaerobic chamber or using degassed buffers containing reducing agents to prevent oxidative damage to the [4Fe-4S] cluster .

• Reducing agents: Include dithiothreitol (DTT) or β-mercaptoethanol in all buffers to maintain a reducing environment .

• Metal chelators: Avoid EDTA and other strong metal chelators that might strip iron from the [4Fe-4S] cluster.

• Reconstitution of Fe-S clusters: If activity is compromised during purification, implement in vitro reconstitution of the [4Fe-4S] cluster using iron salts, sulfide sources, and reducing agents under anaerobic conditions .

• Activity testing: Monitor enzyme activity throughout purification using spectrophotometric assays or HPLC-based methods to track conversion of MEcPP to HMB-PP .

Even with optimal purification conditions, IspG activity may be lower than expected. The observed low rate of 1 nmol⋅mg⁻¹⋅min⁻¹ in some studies may reflect partial aerobic disruption of the holoenzyme during isolation procedures .

How can researchers effectively assay IspG activity in vitro?

Effective in vitro assays for IspG activity include:

• Spectrophotometric assays:

  • Monitor the decrease in absorbance at 340 nm due to the oxidation of reduced ferredoxin or artificial electron donors.

  • This approach allows real-time kinetic analysis but may be less specific.

• HPLC or LC-MS based detection:

  • Separate and quantify substrate (MEcPP) and product (HMB-PP) using chromatographic methods.

  • This provides direct measurement of product formation but requires specialized equipment.

• Radiometric assays:

  • Use ¹⁴C-labeled or ³H-labeled substrates to track product formation.

  • Provide high sensitivity but require radiation safety precautions .

• Coupled enzymatic assays:

  • Link IspG activity to subsequent enzymes in the pathway (e.g., IspH) and detect final products.

  • This approach can amplify the signal but may introduce additional variables .

• Photoreduced deazaflavin activation method:

  • For challenging samples, IspG can be activated using photoreduced deazaflavin as an electron donor.

  • This technique has been successful in cases where traditional electron donors are ineffective .

When developing these assays, researchers should consider the sensitivity to oxygen and the requirement for adequate electron donors to support the redox chemistry.

What experimental approaches can be used to investigate the reaction intermediates of IspG?

Investigating reaction intermediates in the IspG-catalyzed reaction requires sophisticated methodological approaches:

• EPR/ENDOR/HYSCORE spectroscopy:

  • These techniques can detect paramagnetic intermediates that form during catalysis.

  • They have successfully identified radical species coordinated to the [4Fe-4S] cluster .

  • HYSCORE (Hyperfine Sublevel Correlation) spectroscopy is particularly useful for characterizing the electronic structure of intermediates bound to the 4Fe-4S cluster .

• Trapping reaction intermediates:

  • Use of substrate analogs or inhibitors that stabilize reaction intermediates.

  • Rapid freeze-quench techniques to capture short-lived intermediates for spectroscopic analysis .

• Crystallographic approaches:

  • X-ray crystallography of IspG in complex with substrate analogs or inhibitors.

  • Structures of discrete stages on the reaction pathway have been captured, revealing epoxide, ferraoxetane, radical, carbocation, and carbanion structures .

• Isotope labeling experiments:

  • Use of ¹³C, ²H, or other isotopically labeled substrates to track atom movements during catalysis.

  • Combined with NMR or mass spectrometry to identify reaction intermediates .

These approaches have helped resolve the controversial nature of IspG reaction intermediates and provided insights into the catalytic mechanism .

How does the oligomeric state of IspG affect its function, and how can researchers verify the biologically relevant quaternary structure?

The oligomeric state of IspG is crucial for its function, with different structures observed across organisms:

• Bacterial IspGs: Function as (AB)₂ dimers where the A domain (TIM barrel) of one monomer interacts with the B domain (4Fe-4S cluster) of the second monomer to form the active site. This arrangement creates a "cup and ball" structure with the substrate trapped between domains .

• Plant and parasite IspGs: Function as AAB monomers where the additional A domain (also a TIM barrel) interacts with the A domain, allowing proper positioning of the A and B domains to maintain the "cup and ball" arrangement despite being on a single polypeptide chain .

To verify the biologically relevant quaternary structure, researchers can employ:

• Gel filtration chromatography: Determine the apparent molecular weight of the native protein complex under non-denaturing conditions .

• Cross-linking studies: Use chemical cross-linkers to capture transient protein-protein interactions .

• Analytical ultracentrifugation: Provide accurate molecular weight and shape information.

• Electron microscopy: Directly visualize protein complexes, as demonstrated for Arabidopsis thaliana IspG .

• Functional studies with chimeras: Create chimeric proteins with mutations in different domains to assess how domain interactions affect activity. For example, bacterial IspG chimeras with A-domain mutations in one chain and B-domain mutations in the other retain 50% activity, confirming the functional importance of the dimer structure .

What structural features of IspG should be considered when designing targeted inhibitors?

When designing targeted inhibitors for IspG, researchers should consider several key structural features:

The most successful inhibitor design strategies have focused on compounds that mimic the substrate or transition states and interact with both domains of the enzyme.

How does Chlamydophila caviae IspG compare to IspG enzymes from other organisms in terms of structure and function?

Chlamydophila caviae IspG shares fundamental characteristics with other bacterial IspGs while exhibiting some distinct features:

Key comparative observations:

• Sequence conservation: The protein is highly conserved across apicomplexans and prokaryotes, particularly in residues involved in catalysis .

• Structural adaptations: While C. caviae IspG maintains the two-domain bacterial architecture, specific amino acid variations may reflect adaptation to its intracellular lifestyle.

• Catalytic mechanism: Despite structural differences, EPR/ENDOR/HYSCORE spectra indicate that the same reactive intermediates form with both 2- and 3-domain enzymes, suggesting a conserved catalytic mechanism .

• Inhibitor sensitivity: Both two-domain (bacterial) and three-domain (plant/parasite) IspGs show similar patterns of inhibition by alkyne diphosphates, with inhibitors binding to both A and B domains .

These comparative insights are valuable for understanding the evolution of isoprenoid biosynthesis pathways and for developing targeted antimicrobial strategies.

What is the significance of IspG in the context of Chlamydophila caviae pathogenesis?

The significance of IspG in Chlamydophila caviae pathogenesis stems from several factors:

• Essential metabolic pathway:

  • IspG catalyzes a critical step in the MEP pathway, which is the sole route for isoprenoid biosynthesis in many bacteria.

  • Isoprenoids are essential for membrane integrity, electron transport, and other vital cellular functions .

• Intracellular lifestyle adaptation:

  • C. caviae, as an obligate intracellular bacterium, relies on optimized metabolic pathways.

  • The MEP pathway may be particularly important during replicative phases of the Chlamydial developmental cycle.

• Phage interactions:

  • C. caviae is susceptible to phiCPG1 bacteriophage infection, which can modify the course of infection in animal models.

  • In guinea pig conjunctival infection models, phage presence delayed the peak level of chlamydiae and decreased the pathological response .

  • The relationship between phage infection and metabolic enzymes like IspG remains an area for investigation.

• Potential therapeutic target:

  • The absence of the MEP pathway in mammals makes IspG an attractive target for selective inhibition.

  • Targeting IspG could potentially reduce virulence without directly affecting host metabolism .

• Disease relevance:

  • C. caviae is a specific pathogen of guinea pigs causing conjunctivitis .

  • Understanding IspG function may provide insights into ocular infections caused by related Chlamydial species in humans.

The strategic targeting of IspG could potentially disrupt C. caviae's ability to establish productive infection, making it a promising avenue for therapeutic intervention.

How can recombinant Chlamydophila caviae IspG be utilized in drug discovery efforts targeting the MEP pathway?

Recombinant C. caviae IspG can serve as a valuable tool in drug discovery through several methodological approaches:

• High-throughput screening platforms:

  • Develop activity-based assays using purified recombinant IspG for screening chemical libraries.

  • Design fluorescence or luminescence-based readouts for rapid assessment of inhibitor efficacy.

• Structure-based drug design:

  • Use crystallographic or computational models of C. caviae IspG to identify potential binding pockets.

  • Employ in silico docking studies to predict binding modes of potential inhibitors.

  • The [4Fe-4S] cluster environment offers a unique target for metal-coordinating inhibitors .

• Fragment-based drug discovery:

  • Screen small molecular fragments for binding to specific regions of IspG.

  • Link or grow fragments that bind to different parts of the protein to develop high-affinity inhibitors.

• Mechanism-based inhibitor design:

  • Develop compounds that mimic reaction intermediates or transition states.

  • Alkyne diphosphate analogs have shown promise as potent inhibitors that bind near the 4Fe-4S cluster .

• Comparative inhibition studies:

  • Test inhibitors against IspG enzymes from multiple species to identify compounds with specificity for bacterial versus human pathways.

  • In-silico screening of different drug entities has suggested the inhibitory role of alkyne diphosphate analogues and fosmidomycin against IspG enzymes .

• Whole-cell validation:

  • Confirm that compounds targeting IspG in biochemical assays also inhibit bacterial growth in cellular models.

  • Assess the ability of inhibitors to reduce virulence in infection models.

The unique properties of the MEP pathway as a prokaryote-specific metabolic route make IspG an attractive target for developing novel antibiotics against Chlamydial infections.

What approaches can be used to study the interaction between IspG and IspH in the MEP pathway of Chlamydophila caviae?

Investigating the functional relationship between IspG and IspH enzymes in the MEP pathway requires sophisticated experimental approaches:

• Co-expression and co-purification studies:

  • Clone and co-express ispG and ispH genes in expression systems.

  • Assess whether co-expression improves stability or activity of either enzyme.

  • Recent studies have revealed the surprising finding that IspG and IspH need to be co-optimized to improve flux via the methyl erythritol phosphate pathway .

• Coupled enzyme assays:

  • Develop assays where the product of IspG (HMB-PP) serves as the substrate for IspH.

  • Monitor the conversion efficiency of MEcPP to IPP/DMAPP through the sequential action of both enzymes.

  • Previous studies have shown that mixtures containing IspG protein, IspH protein, 14C-labeled substrates, and photoreduced deazaflavin can demonstrate the sequential pathway operation .

• Protein-protein interaction studies:

  • Employ techniques like pull-down assays, surface plasmon resonance, or fluorescence resonance energy transfer to detect direct interactions.

  • Investigate whether these enzymes form a metabolic complex in vivo.

• Metabolic flux analysis:

  • Use isotope-labeled precursors to trace the flow of metabolites through the pathway.

  • Quantify the relative rates of the IspG and IspH reactions under various conditions.

  • Previous studies indicate that IspH protein operates at a higher rate than the IspG protein, suggesting that IspG may be rate-limiting in the pathway .

• Co-localization studies:

  • Investigate the subcellular localization of both enzymes in Chlamydophila cells.

  • Determine whether they co-localize to specific cellular compartments.

• Genetic approaches:

  • Create partial knockdowns or conditional mutants to assess how altering the ratio of IspG to IspH affects pathway flux.

  • Implement ribosomal binding site optimization for both genes as demonstrated in other bacteria .

Understanding the interplay between IspG and IspH can provide insights into metabolic regulation in Chlamydophila caviae and identify potential vulnerabilities for therapeutic targeting.

What are the main technical challenges in working with recombinant Chlamydophila caviae IspG, and how can they be overcome?

Working with recombinant C. caviae IspG presents several technical challenges that require specialized methodological solutions:

• Challenge: Oxygen sensitivity of the [4Fe-4S] cluster

  Solutions:

  • Perform all work in an anaerobic chamber or using Schlenk techniques.

  • Include oxygen scavengers (glucose oxidase/catalase) in buffer systems.

  • Use reducing agents such as dithionite or dithiothreitol to maintain cluster integrity.

• Challenge: Low expression levels in heterologous systems

  Solutions:

  • Optimize codon usage for the expression host.

  • Test multiple expression vectors and promoter systems.

  • Co-express with iron-sulfur cluster assembly proteins (ISC or SUF systems).

  • Optimize ribosomal binding sites using algorithms like the Salis calculator .

• Challenge: Incomplete [4Fe-4S] cluster incorporation

  Solutions:

  • Supplement growth media with iron and cysteine.

  • Add cluster precursors during induction.

  • Perform in vitro reconstitution of the [4Fe-4S] cluster after purification.

  • Biochemical reconstitution and in silico docking of [4Fe-4S] clusters have demonstrated their importance for enzyme activity .

• Challenge: Low enzymatic activity

  Solutions:

  • Activate enzyme with photoreduced deazaflavin as demonstrated for other IspG enzymes .

  • Optimize electron donor systems (ferredoxin/ferredoxin reductase pairs).

  • Consider co-expression with IspH to improve pathway flux .

• Challenge: Difficulty in assaying activity

  Solutions:

  • Employ multiple detection methods (spectrophotometric, HPLC, radiometric).

  • Develop coupled assays with downstream enzymes.

  • Use isotope-labeled substrates for enhanced detection sensitivity.

• Challenge: Protein instability during purification

  Solutions:

  • Include glycerol and reducing agents in all buffers.

  • Minimize purification steps and handling time.

  • Consider fusion tags that enhance solubility (MBP, SUMO).

  • Perform activity assays at each purification stage to track activity loss.

These methodological approaches have been successful for working with related IspG enzymes and can be adapted specifically for C. caviae IspG.

How can researchers effectively model and predict the three-dimensional structure of Chlamydophila caviae IspG for structure-based studies?

Effective modeling and prediction of C. caviae IspG structure involves multi-faceted computational and experimental approaches:

These approaches provide a robust framework for developing reliable structural models of C. caviae IspG that can inform drug discovery and mechanistic studies.

What are the current gaps in understanding Chlamydophila caviae IspG function, and what research directions should be prioritized?

Current knowledge gaps and priority research directions for C. caviae IspG include:

• Structural characterization:

  • No high-resolution structure of C. caviae IspG is currently available.

  • Priority: Determine crystal or cryo-EM structure to understand specific adaptations related to its intracellular lifestyle.

• Catalytic mechanism nuances:

  • While the general IspG mechanism is known, organism-specific variations remain unclear.

  • Priority: Characterize reaction intermediates using spectroscopic methods to identify any unique features of C. caviae IspG catalysis.

• Regulatory mechanisms:

  • Little is known about how IspG activity is regulated during the Chlamydial developmental cycle.

  • Priority: Investigate expression patterns and post-translational modifications under different growth conditions.

• Phage interactions:

  • The phiCPG1 phage affects C. caviae infection dynamics, but impacts on metabolism are unknown.

  • Priority: Study how phage infection affects IspG expression and activity, potentially linking metabolism to virulence .

• Drug development:

  • Few inhibitors have been specifically tested against C. caviae IspG.

  • Priority: Screen compound libraries against recombinant enzyme and assess effects on bacterial growth and pathogenesis.

• Pathway integration:

  • The coordination between IspG and other MEP pathway enzymes in C. caviae remains unexplored.

  • Priority: Study whether co-optimization of IspG and IspH improves pathway flux, as observed in other systems .

• Host-pathogen interactions:

  • The role of IspG-derived metabolites in modulating host immune responses is poorly understood.

  • Priority: Investigate whether HMB-PP from C. caviae activates γδ T cells as observed with other bacterial pathogens.

Addressing these research priorities would significantly advance our understanding of both basic Chlamydial biology and potential therapeutic approaches.

How might advances in studying Chlamydophila caviae IspG contribute to broader understanding of bacterial metabolism and pathogenesis?

Research on C. caviae IspG has significant potential to contribute to broader scientific understanding through several avenues:

• Evolution of metabolic pathways:

  • Comparing IspG across diverse organisms provides insights into the evolution of isoprenoid biosynthesis.

  • The adaptation of metabolic enzymes in obligate intracellular bacteria represents a unique evolutionary scenario.

• Drug resistance mechanisms:

  • Understanding how variations in IspG structure affect inhibitor binding could explain differential antibiotic susceptibility.

  • This knowledge may inform strategies to combat emerging antimicrobial resistance.

• Bacterial adaptation to intracellular environments:

  • C. caviae IspG adaptations may reveal general principles about how metabolic enzymes evolve for intracellular survival.

  • These insights could apply to other intracellular pathogens with similar lifestyles.

• Novel antimicrobial strategies:

  • Targeting essential bacterial metabolic pathways absent in humans represents a promising approach for antibiotic development.

  • Insights from C. caviae IspG could lead to broad-spectrum agents effective against multiple pathogens .

• Understanding phage-bacteria interactions:

  • The relationship between phage infection and bacterial metabolism (including the MEP pathway) remains poorly understood.

  • Studies on how phiCPG1 affects C. caviae metabolism could reveal general principles applicable to other phage-bacteria systems .

• Metabolic network modeling:

  • Detailed characterization of IspG within the context of C. caviae metabolism contributes to more accurate metabolic models.

  • These models have applications in synthetic biology and metabolic engineering.

• Animal model development:

  • Understanding C. caviae IspG function could improve guinea pig models of chlamydial infection.

  • This may lead to better models for studying human chlamydial infections and therapeutic interventions .