Recombinant Bacillus thuringiensis subsp. konkukian 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (ispH)

What is 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (ispH) and what is its role in bacterial metabolism?

4-Hydroxy-3-methylbut-2-enyl diphosphate reductase (ispH, also known as LytB) is a crucial enzyme that catalyzes the terminal step of the MEP/DOXP (2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate) pathway. In this reaction, ispH converts (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) into two essential isoprenoid precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) . The reaction mechanism involves the reductive elimination of the C4 hydroxyl group from HMBPP, utilizing two electrons in the process .

The active form of ispH contains a [4Fe-4S] cluster that is essential for its catalytic activity . This iron-sulfur cluster plays a critical role in the electron transfer process during catalysis, distinguishing ispH from many other reductases.

In Bacillus thuringiensis, as in other bacteria that utilize the MEP/DOXP pathway, ispH is essential for the biosynthesis of isoprenoids, which are required for various cellular processes including cell wall biosynthesis, electron transport, and hormone production.

What genomic characteristics of Bacillus thuringiensis subsp. konkukian influence ispH expression and function?

Bacillus thuringiensis subsp. konkukian strain 97-27 has several notable genomic features that may influence ispH expression and function:

• Phylogenetic position: This strain is very closely related to Bacillus anthracis based on phylogenetic analysis, which suggests potential similarities in metabolic pathways including isoprenoid biosynthesis .

• Pathogenic potential: Unlike most B. thuringiensis strains which are primarily insect pathogens, strain 97-27 was isolated from a case of severe human tissue necrosis, indicating unique adaptations that may affect metabolic enzyme function .

• Gene regulation: The regulatory elements controlling ispH expression likely reflect the dual lifestyle of B. thuringiensis as both a soil saprophyte and an opportunistic pathogen .

• Genomic context: Analysis of the genomic context of the ispH gene in strain 97-27 reveals its integration within the MEP/DOXP pathway operon, suggesting coordinated expression with other pathway enzymes.

How does the MEP/DOXP pathway in B. thuringiensis differ from other isoprenoid biosynthesis pathways?

The MEP/DOXP pathway in B. thuringiensis represents one of two known natural pathways for isoprenoid biosynthesis, with several distinguishing characteristics:

The MEP/DOXP pathway is unique to many bacteria, including B. thuringiensis, as well as plastids in plants, making enzymes like ispH potential targets for antimicrobial or herbicidal development.

What structural features of recombinant B. thuringiensis subsp. konkukian ispH contribute to its catalytic mechanism?

The catalytic mechanism of B. thuringiensis ispH hinges on several critical structural features:

• [4Fe-4S] cluster coordination: The enzyme contains a unique [4Fe-4S] cluster rather than the [3Fe-4S] cluster initially proposed in earlier studies . This cluster is coordinated by three conserved cysteine residues, creating an unusual coordination environment with one "free" iron site that participates directly in substrate binding.

• Substrate binding pocket: The active site forms a hydrophobic pocket that positions HMBPP optimally for interaction with the [4Fe-4S] cluster. Key residues in this pocket form hydrogen bonds with the substrate's diphosphate group and hydroxyl moiety.

• Proton donation network: A network of conserved residues facilitates proton donation during the reduction of HMBPP. This includes acidic amino acids that act as proton donors in conjunction with electron transfer from the [4Fe-4S] cluster.

• Conformational changes: Substantial evidence indicates that ispH undergoes conformational changes upon substrate binding, bringing catalytic residues into optimal positions for reaction.

• Domain organization: The enzyme typically consists of three domains that form a trefoil-like structure, creating a central cavity where the [4Fe-4S] cluster and substrate binding occur.

What are the challenges in expressing and purifying functional recombinant B. thuringiensis subsp. konkukian ispH?

Researchers face several significant challenges when working with recombinant ispH from B. thuringiensis:

• Oxygen sensitivity: The [4Fe-4S] cluster is highly sensitive to oxidation, requiring strictly anaerobic conditions during purification and handling to maintain enzyme activity.

• Iron-sulfur cluster assembly: Heterologous expression systems often struggle to properly incorporate the [4Fe-4S] cluster, necessitating co-expression with iron-sulfur cluster assembly machinery or in vitro reconstitution.

• Protein solubility: IspH often forms inclusion bodies when overexpressed in E. coli, requiring optimization of expression conditions (temperature, induction parameters) and potentially the use of solubility tags.

• Maintaining stability: Even after successful purification, the enzyme can rapidly lose activity due to cluster degradation, requiring stabilizing agents and precise buffer conditions.

• Assay limitations: Traditional spectrophotometric assays for ispH activity can be complicated by the oxygen sensitivity of the enzyme and potential side reactions.

An optimized protocol for functional expression typically includes:

• Expression in E. coli strains with enhanced capacity for iron-sulfur protein production

• Growth under microaerobic conditions with iron and sulfur supplementation

• Rapid purification under strict anaerobic conditions

• Addition of reducing agents like DTT or β-mercaptoethanol throughout the purification

How can hybrid protein technology be applied to enhance ispH function in B. thuringiensis?

Drawing from successful examples of hybrid protein engineering in B. thuringiensis crystal proteins , several approaches can be applied to ispH:

• Domain swapping: Exchanging domains between ispH enzymes from different bacterial species may create variants with enhanced catalytic properties or stability. Similar to how domain III of CryIC was transferred to CryIE to create a protein with broader insecticidal activity , domains from thermophilic bacteria could be introduced to increase thermostability.

• Active site engineering: Targeted modifications of the substrate binding pocket can be made to alter substrate specificity or improve catalytic efficiency.

• Surface modification: Alterations to surface residues distant from the active site can enhance solubility and stability without compromising catalytic function.

• [4Fe-4S] cluster coordination optimization: Modifications to the microenvironment around the iron-sulfur cluster may improve oxygen tolerance and stability.

• Fusion protein strategies: Creating fusion proteins with redox partners or other functional domains may enhance electron transfer efficiency or create bifunctional enzymes.

Implementation of these strategies requires:

• Detailed structural knowledge of ispH

• Careful boundary selection for domain swapping

• High-throughput screening methods to identify improved variants

• Rigorous characterization of hybrid proteins to verify preserved or enhanced function

What experimental approaches can be used to characterize the kinetic properties of recombinant ispH?

A comprehensive kinetic characterization of recombinant ispH requires multiple complementary approaches:

• Steady-state kinetics:

  • Spectrophotometric assays coupling NADPH oxidation to ispH activity

  • HPLC-based product formation analysis

  • Real-time monitoring using fluorescent substrate analogs

• Pre-steady-state kinetics:

  • Stopped-flow spectroscopy to monitor rapid changes in [4Fe-4S] cluster during catalysis

  • Rapid-quench techniques to identify reaction intermediates

  • EPR spectroscopy to characterize paramagnetic intermediates

• pH and temperature dependence studies:

  • Determination of optimal pH and temperature ranges

  • van't Hoff and Arrhenius analysis for thermodynamic parameters

  • pKa determination of catalytically important residues

• Inhibition studies:

  • Competitive vs. non-competitive inhibition patterns

  • Time-dependent inhibition for mechanism-based inhibitors

  • Dissociation constant determination using isothermal titration calorimetry

A typical experimental design for steady-state kinetic analysis includes:

From such data, researchers can determine KM, Vmax, kcat, and the catalytic efficiency (kcat/KM) under various conditions.

How can site-directed mutagenesis be used to investigate the catalytic mechanism of B. thuringiensis ispH?

Site-directed mutagenesis represents a powerful approach for elucidating the catalytic mechanism of ispH through systematic modification of key residues:

• Target selection strategy:

  • Conserved residues identified through multiple sequence alignment

  • Residues predicted to interact with substrate based on homology models

  • Residues near the [4Fe-4S] cluster that may participate in electron transfer

  • Residues implicated in proton donation/abstraction

• Mutation design considerations:

  • Conservative mutations (e.g., Asp→Glu) to probe size effects

  • Charge neutralization (e.g., Asp→Asn) to assess electrostatic contributions

  • Charge reversal (e.g., Asp→Lys) to detect charge-dependent interactions

  • Removal of functional groups (e.g., Ser→Ala) to identify hydrogen bonding partners

• Methodological approaches:

  • QuikChange mutagenesis for single mutations

  • Gibson Assembly for multiple mutations or domain swapping

  • Golden Gate Assembly for combinatorial mutagenesis libraries

• Functional characterization of mutants:

  • Steady-state kinetic parameters (kcat, KM)

  • Substrate binding affinity (Kd)

  • [4Fe-4S] cluster integrity (UV-vis and EPR spectroscopy)

  • pH-activity profiles to identify shifts in optimal pH

• Interpretation framework:

  • Correlation of structural location with functional impact

  • Energy diagram modifications based on rate-limiting step changes

  • Integration with computational modeling to explain observed effects

What spectroscopic techniques are most valuable for studying the [4Fe-4S] cluster in ispH?

A multi-spectroscopic approach is essential for comprehensive characterization of the [4Fe-4S] cluster in ispH:

• UV-visible spectroscopy:

  • Monitors the characteristic absorption bands of [4Fe-4S] clusters (~390-420 nm)

  • Provides quick assessment of cluster integrity during purification

  • Can track redox state changes during catalysis

  • Limitations: Low structural resolution and potential interference from other chromophores

• Electron Paramagnetic Resonance (EPR) spectroscopy:

  • Directly observes paramagnetic species (e.g., [4Fe-4S]+ state)

  • Provides information about the electronic structure and coordination environment

  • Can detect substrate-cluster interactions

  • Requires specialized equipment and cryogenic temperatures

• Mössbauer spectroscopy:

  • Provides detailed information about oxidation states of individual iron atoms

  • Can distinguish different types of Fe-S clusters

  • Allows monitoring of all iron sites regardless of paramagnetism

  • Requires 57Fe enrichment and specialized instrumentation

• X-ray Absorption Spectroscopy (XAS):

  • XANES provides information about oxidation states

  • EXAFS reveals bond distances and coordination geometries

  • Can be performed on frozen solutions

  • Requires synchrotron radiation source

• Resonance Raman spectroscopy:

  • Identifies vibrational modes of the Fe-S cluster

  • Can detect subtle changes in cluster geometry upon substrate binding

  • Provides fingerprint for cluster type and integrity

  • May require resonance enhancement for sufficient sensitivity

A comprehensive characterization typically combines these approaches, as illustrated in this example workflow:

How can computational methods enhance our understanding of B. thuringiensis ispH structure and function?

Computational approaches offer valuable insights into ispH structure and function that complement experimental data:

• Homology modeling:

  • Generates structural models based on known structures of ispH from other organisms

  • Identifies conserved structural elements and species-specific variations

  • Predicts substrate binding modes and active site architecture

  • Implementation: SWISS-MODEL, I-TASSER, or Rosetta can be used with ispH sequences from closely related species as templates

• Molecular dynamics simulations:

  • Reveals protein flexibility and conformational changes

  • Identifies water networks and proton transfer pathways

  • Simulates substrate binding and product release

  • Captures dynamics of the [4Fe-4S] cluster environment

  • Implementation: GROMACS or AMBER with specialized force fields for metal centers

• Quantum mechanics/molecular mechanics (QM/MM):

  • Models electronic structure of the [4Fe-4S] cluster and substrate

  • Calculates reaction energetics and transition states

  • Predicts effects of mutations on catalysis

  • Implementation: QM region (cluster, substrate, key residues) treated with DFT methods; MM region with classical force fields

• Bioinformatic analyses:

  • Multiple sequence alignments identify conserved residues across bacterial species

  • Ancestral sequence reconstruction traces evolutionary history of ispH

  • Coevolution analysis reveals functionally coupled residues

  • Implementation: MAFFT for alignments, followed by ConSurf for conservation mapping

• Docking and virtual screening:

  • Predicts binding modes of substrates, products, and potential inhibitors

  • Ranks compounds by predicted binding affinity

  • Guides rational design of ispH inhibitors as potential antimicrobials

  • Implementation: AutoDock Vina with careful parameterization for the [4Fe-4S] cluster

What are the best practices for interpreting inconsistent results from different ispH activity assays?

Researchers frequently encounter inconsistencies when using multiple assays to measure ispH activity. A systematic approach to resolving these discrepancies includes:

• Assay principle comparison:

  • Direct vs. coupled assays may yield different results due to coupling enzyme limitations

  • Endpoint vs. continuous assays differ in their ability to detect initial rates

  • Product formation vs. substrate consumption measurements may be affected differently by side reactions

• Analytical validation approach:

  • Determine linear range for each assay

  • Validate with known positive and negative controls

  • Establish minimum detectable activity levels

  • Verify absence of interfering substances

• Reconciliation strategies:

  • Standardize enzyme preparations across all assays

  • Perform assays under identical conditions (buffer, pH, temperature)

  • Use multiple batch preparations to identify preparation-specific artifacts

  • Apply statistical methods to determine significant differences

• Decision framework for selecting primary assay:

  • Consider physiological relevance of assay conditions

  • Evaluate precision, accuracy, and reproducibility

  • Assess compatibility with inhibitor screening if relevant

  • Consider practical factors (throughput, cost, equipment requirements)

• Integrated data analysis:

  • Use Bland-Altman plots to visualize systematic differences between assays

  • Apply correction factors based on careful calibration

  • Report results from multiple assays when publishing

  • Clearly state limitations of each assay method

How can hybrid protein approaches from crystal toxin research inform ispH engineering?

The successful engineering of hybrid B. thuringiensis crystal proteins provides valuable lessons for ispH engineering:

• Domain identification principles:

  • Crystal proteins like CryIC have well-defined functional domains that can be swapped to create proteins with new properties

  • Similarly, ispH can be analyzed to identify discrete functional domains (substrate binding, [4Fe-4S] coordination, etc.)

  • Domain boundaries must be carefully selected to maintain proper protein folding

• Selection of recombination partners:

  • Selection of CryIC and CryIE for hybridization was based on their different insecticidal specificities

  • For ispH, potential partners include enzymes with enhanced stability, altered substrate specificity, or improved catalytic efficiency

  • Partners should have sufficient sequence similarity to allow proper folding of hybrid structures

• Structure-function relationship analysis:

  • The hybrid crystal protein research revealed that domain III of CryIC is involved in toxicity towards specific insects

  • Similar approaches can map specific properties of ispH to discrete regions

  • Systematic domain swapping can create a functional map of the enzyme

• Receptor interaction considerations:

  • Hybrid crystal proteins bind to different receptors than the parent proteins

  • For ispH, altered interactions with electron donors or redox partners could be engineered

  • Changes in substrate binding specificity could lead to novel catalytic activities

• Experimental design for hybrid screening:

  • High-throughput screening methods are essential for identifying functional hybrids

  • Complementation assays in auxotrophic strains can rapidly identify functional ispH variants

  • In vitro activity assays must be designed to detect potentially altered substrate specificities

How can genomic and proteomic approaches improve our understanding of ispH variation across B. thuringiensis strains?

Genomic and proteomic strategies offer powerful tools for comparative analysis of ispH across B. thuringiensis strains:

• Comparative genomics approach:

  • Whole genome sequencing of multiple B. thuringiensis strains, including subsp. konkukian (strain 97-27)

  • Analysis of ispH gene context within the genome

  • Identification of regulatory elements and potential horizontal gene transfer events

  • Correlation of ispH sequence variations with strain-specific phenotypes

• Transcriptomic analysis:

  • RNA-Seq under various growth conditions reveals regulation patterns

  • Comparison of expression levels between pathogenic and non-pathogenic strains

  • Co-expression network analysis identifies functionally related genes

  • Identification of small RNAs potentially regulating ispH expression

• Proteomic characterization:

  • Quantitative proteomics to determine relative abundance of ispH

  • Post-translational modification analysis (phosphorylation, etc.)

  • Protein-protein interaction networks via co-immunoprecipitation or crosslinking

  • Structural proteomics (hydrogen-deuterium exchange MS) for conformational analysis

• Evolutionary analysis framework:

  • Phylogenetic analysis of ispH sequences across Bacillus species

  • Calculation of selection pressures (dN/dS ratios) to identify conserved vs. variable regions

  • Ancestral sequence reconstruction to trace evolutionary history

  • Correlation of sequence changes with ecological adaptations

• Integration of multi-omics data:

  • Correlation of genomic variants with transcriptomic and proteomic differences

  • Pathway analysis incorporating ispH and related enzymes

  • Machine learning approaches to identify patterns across datasets

  • Visualization tools for complex multi-omic data interpretation