Recombinant 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase 1 (ispG1)

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, EC 1.17.7.1) is an enzyme that catalyzes a critical reaction in the MEP pathway (non-mevalonate pathway) of isoprenoid precursor biosynthesis. 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), with electrons donated by two reduced ferredoxin proteins per reaction .

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

Where does IspG fit within the MEP pathway and why is it significant?

IspG catalyzes a late-stage reaction in the MEP pathway, which is responsible for the biosynthesis of isoprenoid precursors IPP (isopentenyl diphosphate) and DMAPP (dimethylallyl diphosphate). The MEP pathway operates in many bacteria and plants but is absent in mammals, which exclusively use the mevalonate pathway for isoprenoid biosynthesis .

The MEP pathway contains several enzymatic steps:

• DXP synthase (EC 2.2.1.7) - Formation of 1-deoxy-d-xylulose 5-phosphate

• DXP reductoisomerase (EC 1.1.1.267) - Conversion to 2-C-methylerythritol 4-phosphate (MEP)

• MEP cytidylyltransferase (EC 2.7.7.60) - Conversion of MEP to CDP-ME

• CDP-ME kinase (EC 2.7.1.148) - Phosphorylation to CDP-ME2P

• Later steps involving IspG (conversion of MEcPP to HMB-PP)

IspG's significance lies in its essential role in this pathway and its potential as a target for antimicrobial development, as the MEP pathway is absent in mammals but present in many pathogenic bacteria.

How is IspG classified in enzyme nomenclature?

IspG belongs to the family of oxidoreductases (EC 1), specifically those acting on CH or CH2 groups (EC 1.17) with a disulfide as acceptor (EC 1.17.7). Its complete EC number is 1.17.7.1 .

The systematic name of this enzyme class is:
(E)-4-hydroxy-3-methylbut-2-en-1-yl-diphosphate:protein-disulfide oxidoreductase (hydrating)

This classification places IspG among enzymes that catalyze redox reactions involving carbon-hydrogen bonds, with specific electron acceptor requirements.

What database resources are available for researching IspG?

Researchers can access information about IspG through several specialized databases:

These resources provide valuable information on enzyme characteristics, metabolic context, and structural features of IspG .

What methodologies can be employed to express and purify recombinant IspG?

To express and purify recombinant IspG, researchers should consider the following methodological approach:

• Expression System Selection: E. coli has proven effective for expression of recombinant IspG from various bacterial sources, including Haemophilus somnus .

• Vector Design:

  • Construct plasmids with strong inducible promoters (T7, tac)

  • Include affinity tags (His-tag, GST) for purification

  • Consider codon optimization for the expression host

• Expression Conditions:

  • Test multiple induction temperatures (16°C, 25°C, 37°C)

  • Optimize inducer concentration (IPTG typically 0.1-1.0 mM)

  • Evaluate expression duration (4-24 hours)

• Purification Strategy:

  • Initial capture: Affinity chromatography using the engineered tag

  • Intermediate purification: Ion exchange chromatography

  • Polishing: Size exclusion chromatography

  • Consider including reducing agents throughout purification to maintain enzymatic activity

• Activity Verification:

  • Employ DTNB assay to verify enzymatic activity of the purified protein

  • Assess product formation using mass spectrometry (MALDI-TOF)

The purification protocol must be developed with consideration for preserving the redox-sensitive activity of IspG, as it participates in electron transfer reactions.

How can researchers experimentally determine the kinetic parameters of IspG?

To determine kinetic parameters of IspG enzymatic activity, researchers should implement the following methodological approach:

• Spectrophotometric Assays:

  • Monitor NADPH oxidation at 340 nm if coupling the reaction with ferredoxin/ferredoxin reductase

  • Use 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) assay to measure thiol formation

• Experimental Design for Kinetic Studies:

  • Vary substrate (MEcPP) concentration while maintaining constant enzyme concentration

  • Maintain appropriate temperature and pH for optimal enzyme activity

  • Include appropriate electron donors (ferredoxin)

  • Account for potential cooperative binding effects using Hill plots

  • Consider product inhibition effects

• Product Analysis:

  • Quantify HMB-PP formation using MALDI-TOF mass spectrometry

  • HPLC analysis with UV detection

  • LC-MS/MS for sensitive detection of products

• Data Analysis:

  • Generate Michaelis-Menten or Hill plots depending on binding behavior

  • Calculate Km, Vmax, and catalytic efficiency (kcat/Km)

  • Analyze enzyme cooperativity using Hill coefficients (if applicable)

• Method Validation:

  • Include positive controls with characterized enzymes

  • Perform replicate measurements for statistical reliability

  • Consider varying enzyme concentration to confirm proportional response

This multi-faceted approach allows for comprehensive characterization of IspG kinetic behavior.

What experimental design principles should be applied when studying IspG inhibition?

When investigating potential inhibitors of IspG, researchers should follow these experimental design principles:

These approaches create a robust framework for identifying and characterizing IspG inhibitors with potential antimicrobial applications.

What techniques are most effective for studying IspG structure-function relationships?

To elucidate structure-function relationships of IspG, researchers should employ these methodological approaches:

• Structural Characterization:

  • X-ray crystallography for high-resolution structures

  • Cryo-EM for visualization of conformational states

  • NMR for dynamic studies of smaller domains

  • Small-angle X-ray scattering (SAXS) for solution structure

• Functional Site Mapping:

  • Site-directed mutagenesis of conserved residues

  • Chemical modification studies

  • Hydrogen-deuterium exchange mass spectrometry

  • Crosslinking studies to identify interaction surfaces

• Computational Analysis:

  • Homology modeling using related structures

  • Molecular dynamics simulations to study conformational changes

  • Quantum mechanical calculations for reaction mechanism studies

  • Evolutionary analysis of conserved residues across species

• Ligand Binding Studies:

  • Isothermal titration calorimetry for thermodynamic parameters

  • Surface plasmon resonance for binding kinetics

  • Fluorescence-based binding assays

  • Differential scanning fluorimetry for thermal stability shifts upon binding

• Enzyme Variant Analysis:

  • Compare kinetic parameters of wild-type and mutant enzymes

  • Analyze effects of mutations on substrate specificity

  • Study pH and temperature dependencies of variants

This integrated approach provides comprehensive insights into how IspG structure relates to its catalytic function.

How does genomic context analysis contribute to understanding IspG function across bacterial species?

Analyzing the genomic context of ispG genes provides valuable insights into function, regulation, and evolution:

• Comparative Genomic Approaches:

  • Identify gene organization patterns across species

  • Map operon structures containing ispG

  • Detect horizontally transferred regions through GC content analysis

  • Analyze the presence of mobile genetic elements near ispG

• Case Study - Porphyromonas gingivalis:
  The extensive genome rearrangements observed in P. gingivalis strains demonstrate how mobile genetic elements influence genomic architecture, potentially affecting enzyme function and regulation :

  Element Type	W83 Strain	ATCC 33277 Strain	Impact
  IS elements	93 (51 partial)	93 (38 partial)	Mediates genomic rearrangements
  MITEs	35 (9 partial)	48 (18 partial)	Contributes to strain diversity
  IS types	ISPg1-ISPg6	Expansion of ISPg1, ISPg3	Strain-specific patterns

• Functional Correlation Methodologies:

  • Analyze co-expression patterns with other MEP pathway genes

  • Identify potential regulatory elements in the promoter region

  • Study transcription factor binding sites using ChIP-seq

  • Compare expression under different growth conditions

• Evolutionary Analysis Techniques:

  • Construct phylogenetic trees based on IspG sequences

  • Calculate selection pressure (dN/dS ratio) across domains

  • Detect recombination events using specialized algorithms

  • Map functional divergence across bacterial lineages

This multi-layered analysis provides a comprehensive understanding of how IspG functions within different genomic contexts and evolutionary trajectories.

What considerations are important when designing experiments to study IspG in different bacterial contexts?

When investigating IspG across different bacterial species, researchers should consider:

• Experimental Design Framework:

  • Follow systematic scientific method approaches with clear research questions

  • Design controlled experiments with appropriate variables

  • Include replicate measurements for statistical validity

  • Consider both within-subjects and between-subjects experimental designs when applicable

• Species Selection Criteria:

  • Include diverse bacterial species representing different phyla

  • Consider pathogenic and non-pathogenic representatives

  • Select model organisms with genetic manipulation tools available

  • Include species with variations in MEP pathway organization

• Control Considerations:

  • Account for differences in optimal growth conditions

  • Normalize enzyme activity to protein concentration

  • Include species-specific positive and negative controls

  • Control for differences in gene expression systems

• Comparative Analysis Approach:

  • Standardize assay conditions across species when possible

  • Develop species-specific modifications when necessary

  • Use recombinant systems with standardized tags for fair comparison

  • Account for differences in codon usage and protein folding machinery

• Data Integration Methods:

  • Implement bioinformatic pipelines for cross-species comparisons

  • Use statistical methods appropriate for comparative analysis

  • Develop normalization strategies for cross-species data

  • Create visualization tools for multi-species datasets

This methodological framework enables rigorous comparative studies of IspG across different bacterial contexts, providing insights into functional conservation and specialization.

How can researchers utilize transposon mutagenesis to study IspG function in bacterial pathogens?

Transposon mutagenesis offers powerful approaches for studying IspG function in pathogenic bacteria:

• Transposon Sequencing (Tn-Seq) Methodology:

  • Generate saturated transposon libraries in the bacterial species of interest

  • Sequence transposon insertion sites to identify essential genes

  • Compare growth in different conditions to identify condition-specific roles

  • Use statistical analyses to determine fitness effects of gene disruption

• Application Example - P. gingivalis:
  While not specifically studying IspG, the approach used for tyrosine kinase Ptk1 demonstrates the methodology applicable to IspG research :

  "In this study, we used transposon sequencing (Tn-Seq) to identify P. gingivalis genes that confer fitness during cooperative growth with S. gordonii."

• Data Analysis Strategy:

  • Calculate fitness scores based on relative abundance of mutants

  • Define significant fitness effects (typically score >0.5 with adjusted P-value <0.05)

  • Analyze negative fitness scores as indicators of essential functions

  • Integrate with transcriptomic and proteomic data for comprehensive insights

• Validation Approaches:

  • Create targeted deletion mutants of identified genes

  • Perform complementation studies to confirm phenotypes

  • Conduct biochemical assays to characterize effects on specific pathways

  • Evaluate changes in virulence or fitness in infection models

• Biological Context Interpretation:

  • Analyze the impact on downstream isoprenoid-dependent processes

  • Evaluate effects on cell envelope properties and stress responses

  • Assess changes in virulence factor expression

  • Determine effects on antibiotic susceptibility

This transposon-based approach provides a powerful method for understanding IspG function in the context of bacterial pathogenesis and identifying potential antimicrobial targets.

What role does IspG play in bacterial pathogenesis and how can this be experimentally demonstrated?

While the search results don't directly address IspG's role in pathogenesis, we can outline methodological approaches to investigate this question:

• Genetic Manipulation Strategies:

  • Generate ispG deletion or conditional mutants in bacterial pathogens

  • Create point mutations affecting catalytic activity

  • Develop complementation strains expressing wild-type or mutant variants

  • Use inducible systems to control expression levels

• Pathogenesis Model Systems:

  • Employ cell culture infection models with appropriate host cells

  • Use animal infection models suited to the pathogen being studied

  • Develop ex vivo tissue models for organ-specific infections

  • Consider alternative infection models (e.g., Caenorhabditis elegans, Galleria mellonella)

• Virulence Assessment Methodologies:

  • Measure bacterial survival and replication within host cells

  • Quantify virulence factor production in wild-type vs. ispG mutants

  • Assess host immune response parameters (cytokine production, immune cell recruitment)

  • Determine changes in biofilm formation capacity

• Analytical Approaches:

  • Conduct transcriptomic analysis of ispG mutants vs. wild-type during infection

  • Perform metabolomic profiling focusing on isoprenoid-dependent molecules

  • Use imaging techniques to visualize pathogen-host interactions

  • Measure stress response activation in mutant strains

• Potential Applications:

  • Develop IspG inhibitors as potential antimicrobials

  • Identify biomarkers associated with IspG activity during infection

  • Create attenuated strains for vaccine development

  • Design diagnostic tools based on IspG activity or products

These methodological approaches provide a comprehensive framework for investigating IspG's contribution to bacterial pathogenesis.