Recombinant Rhodopirellula baltica 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (ispG)

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 (also known as HMB-PP synthase or IspG) is an enzyme belonging to the oxidoreductase family that catalyzes a critical step in the MEP pathway (non-mevalonate pathway) of isoprenoid precursor biosynthesis . Specifically, the enzyme catalyzes the conversion of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MEcPP) to (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMB-PP) . The systematic name of this enzyme class is (E)-4-hydroxy-3-methylbut-2-en-1-yl-diphosphate:protein-disulfide oxidoreductase (hydrating) . The reaction requires electrons donated by two reduced ferredoxin proteins per reaction cycle .

The chemical reaction can be represented as:
2-C-methyl-D-erythritol 2,4-cyclodiphosphate + protein-dithiol → (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate + H₂O + protein-disulfide

What is the significance of studying Rhodopirellula baltica ispG specifically?

Rhodopirellula baltica is a marine planctomycete bacterium with unique biochemical properties. Studying the ispG enzyme from this organism provides valuable insights into the evolutionary diversity of the MEP pathway across different bacterial species. The enzyme from R. baltica (strain SH1) has distinctive characteristics that may offer advantages for certain research applications . Since the MEP pathway is absent in mammals but present in many pathogens, understanding variations in ispG function across different bacterial species can potentially lead to the development of new antimicrobial compounds that target this pathway without affecting human metabolism.

What expression systems are available for producing recombinant Rhodopirellula baltica ispG?

Based on the available information, recombinant Rhodopirellula baltica ispG can be expressed in multiple systems depending on research requirements:

E. coli with Avi-tag biotinylation offers the advantage of site-specific biotinylation, where E. coli biotin ligase (BirA) covalently attaches biotin to the 15 amino acid AviTag peptide with high specificity . This approach facilitates protein detection, immobilization, and interaction studies through the biotin-streptavidin system.

What are the methodological considerations for optimizing expression of functional ispG?

When expressing recombinant ispG, several factors must be considered to obtain properly folded and active enzyme:

• Iron-sulfur cluster incorporation: As ispG contains iron-sulfur clusters essential for its catalytic activity, expression conditions must support proper cluster assembly. This may require supplementation with iron and sulfur sources and co-expression of iron-sulfur cluster assembly proteins.

• Temperature optimization: Lower induction temperatures (16-25°C) often improve the solubility and proper folding of complex enzymes like ispG.

• Expression timing: Since ispG is a full-length protein of 382 amino acids , optimizing induction time and duration is critical to balance between protein yield and solubility.

• Purification considerations: When designing a purification strategy, the expected purity (>85% by SDS-PAGE ) should be considered alongside the requirements of downstream applications.

How does the catalytic mechanism of ispG differ from other enzymes in the MEP pathway?

The catalytic mechanism of ispG is distinctive within the MEP pathway due to its reliance on iron-sulfur clusters for electron transfer. While other enzymes in the pathway may utilize different cofactors like NADPH or ATP, ispG employs a unique mechanism involving redox chemistry with iron-sulfur clusters and protein-dithiol groups .

The reaction catalyzed by ispG involves:

• Binding of the substrate MEcPP

• Reductive ring opening of the cyclic diphosphate

• Electron transfer via iron-sulfur clusters

• Formation of the product HMB-PP with the introduction of a double bond

This mechanism represents a specialized case of CH or CH₂ group modification with protein-disulfide as an acceptor , classified under EC 1.17.7.1 (oxidoreductases acting on CH or CH₂ groups) .

What are the optimal conditions for measuring ispG enzyme activity in vitro?

When designing experiments to measure ispG activity, researchers should consider:

• Buffer composition: A buffer system that maintains optimal pH (typically 7.5-8.0) and includes components that stabilize the iron-sulfur clusters, such as dithiothreitol (DTT) or β-mercaptoethanol.

• Electron donor system: Since the enzyme requires electrons for catalysis, an appropriate electron donor system must be included. This typically involves reduced ferredoxin or an artificial electron donor system.

• Anaerobic conditions: To prevent oxidative damage to the iron-sulfur clusters, activity assays should be performed under anaerobic conditions whenever possible.

• Substrate preparation: Pure MEcPP substrate should be used, either synthesized chemically or enzymatically.

• Product detection methods: HPLC, LC-MS, or enzyme-coupled spectrophotometric assays can be used to monitor the formation of HMB-PP.

How can isotopic labeling be utilized to investigate the reaction mechanism of ispG?

Isotopic labeling provides powerful insights into reaction mechanisms by tracking atom movements during catalysis. For ispG research, consider these approaches:

• ¹³C-labeled substrates: Using MEcPP with specific carbon atoms labeled with ¹³C allows tracking of carbon rearrangements during catalysis. NMR spectroscopy can then be used to determine the position of labeled carbons in the product.

• ²H-labeled substrates: Deuterium labeling helps identify positions where hydrogen atoms are removed or added during the reaction, providing insights into the timing and mechanism of C-H bond cleavage.

• ¹⁸O-labeled water: To determine if the incorporated oxygen in HMB-PP comes from water or the substrate, experiments can be conducted in H₂¹⁸O.

• Kinetic isotope effects: Comparing reaction rates with isotopically labeled and unlabeled substrates can identify rate-limiting steps in the catalytic cycle.

These approaches should be integrated with experimental designs based on discrete-choice experiments (DCEs) methodology to effectively evaluate multiple variables simultaneously .

How can structural biology techniques be applied to understand ispG function?

Advanced structural biology techniques provide crucial insights into ispG function:

• X-ray crystallography: Determining the three-dimensional structure of ispG in different states (substrate-bound, product-bound, or intermediate-trapped) reveals the spatial arrangement of catalytic residues and conformational changes during catalysis.

• Cryo-electron microscopy: For challenging crystallization targets, cryo-EM can provide structural information about ispG, particularly when in complex with other proteins or in different functional states.

• EPR spectroscopy: Electron paramagnetic resonance spectroscopy is particularly valuable for studying the iron-sulfur clusters in ispG, providing information about their oxidation states during catalysis.

• NMR spectroscopy: For studying protein dynamics and substrate interactions, solution NMR can provide insights into flexible regions and conformational changes upon substrate binding.

• HDX-MS: Hydrogen-deuterium exchange mass spectrometry can map changes in protein solvent accessibility upon substrate binding or during catalysis.

These approaches should be integrated with computational methods like molecular dynamics simulations to develop a comprehensive understanding of the enzyme's function.

What strategies can be employed to develop inhibitors against ispG as potential antimicrobial agents?

Since the MEP pathway is absent in humans but present in many bacteria and some parasites, ispG represents a promising target for antimicrobial development. Advanced strategies include:

• Structure-based drug design: Using the three-dimensional structure of ispG to design molecules that bind to the active site or allosteric sites.

• Fragment-based screening: Identifying small molecular fragments that bind to different regions of ispG and then linking or growing these fragments to develop potent inhibitors.

• Natural product screening: Testing collections of natural products, especially those from organisms that may have evolved compounds targeting competitive microbes.

• Mechanism-based inhibitors: Designing compounds that mimic transition states or intermediates in the ispG reaction.

• Allosteric inhibitors: Targeting non-active site regions that still affect enzyme function, potentially offering higher selectivity.

When evaluating potential inhibitors, researchers should implement experimental designs based on the ISPOR Good Research Practices guidelines , which recommend systematic approaches to testing multiple variables and interpreting complex data sets.

What are common challenges in working with recombinant ispG and how can they be addressed?

Researchers frequently encounter these challenges when working with recombinant ispG:

• Low protein solubility: Address by optimizing expression conditions (lower temperature, use of solubility tags, co-expression with chaperones) or employing specialized solubilization methods.

• Iron-sulfur cluster instability: Prevent by working under anaerobic conditions, including reducing agents in buffers, and reconstituting iron-sulfur clusters in vitro if necessary.

• Variable activity levels: Standardize activity assays by carefully controlling substrate quality, enzyme concentration, and reaction conditions. Consider using internal standards for quantitative measurements.

• Protein aggregation during storage: Optimize buffer conditions (glycerol content, salt concentration) and storage temperature. Consider flash-freezing small aliquots to minimize freeze-thaw cycles.

• Inconsistent purification results: Develop a standardized purification protocol with quality control steps at each stage. Monitor protein purity using SDS-PAGE (aiming for >85% purity ) and activity assays.

How should contradictory experimental results with ispG be interpreted and resolved?

When faced with contradictory experimental results:

• Systematically evaluate variables: Identify all variables that might affect the experiment, including protein batch, substrate quality, buffer composition, and experimental conditions.

• Control experiments: Design appropriate positive and negative controls to validate assay performance and identify potential interfering factors.

• Statistical analysis: Apply appropriate statistical methods to determine if observed differences are significant. Consider using frameworks from the ISPOR guidelines for experimental design .

• Cross-validation: Employ multiple independent techniques to measure the same parameter. For example, confirm enzyme activity using both spectrophotometric assays and HPLC-based product detection.

• Collaborative verification: Consider engaging collaborators with expertise in different methods to independently verify critical results.

• Literature reconciliation: Carefully compare your experimental design with published protocols, noting any methodological differences that might explain contradictory results.