Recombinant Pseudomonas syringae pv. tomato 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (ispG)

What is the function of 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (ispG) in the MEP pathway?

IspG (also known as GcpE) is a crucial enzyme in the methylerythritol phosphate (MEP) pathway that catalyzes the penultimate step in isoprenoid precursor biosynthesis. Specifically, ispG converts 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcPP) into (E)-4-hydroxy-3-methylbut-2-enyl pyrophosphate (HMBPP). This conversion involves the reduction and opening of the cyclic diphosphate intermediate . The MEP pathway is essential for isoprenoid biosynthesis in bacteria and plastids of plants, making ispG a critical enzyme for the survival of organisms like Pseudomonas syringae.

How does the structure of ispG relate to its catalytic function?

IspG contains a [4Fe-4S] cluster at its active site, which is essential for its catalytic activity. The enzyme requires this iron-sulfur cluster for electron transfer during the reduction reaction. The active site structure creates a specific binding pocket for MEcPP, allowing for the coordination of the substrate with the iron-sulfur cluster. In its active form, the enzyme contains a [4Fe-4S] cluster, not the [3Fe-4S] cluster sometimes reported in earlier literature . This structural arrangement is crucial for the enzyme's ability to perform the reductive transformation of MEcPP to HMBPP through a series of electron transfer steps.

What expression systems are most effective for producing active recombinant P. syringae ispG?

For recombinant expression of ispG from P. syringae, the following methodology has proven effective:

• Bacterial expression system: E. coli BL21(DE3) strains with pET-based vectors under T7 promoter control.

• Expression conditions: Induction with 0.1-0.5 mM IPTG at lower temperatures (16-20°C) for 16-20 hours to enhance protein folding and [4Fe-4S] cluster incorporation.

• Media supplementation: Addition of iron (FeCl₃, 50-100 μM) and cysteine (0.5-1 mM) to enhance iron-sulfur cluster formation.

• Anaerobic conditions: Expression under microaerobic or anaerobic conditions to prevent oxidative damage to the [4Fe-4S] cluster.

Due to the oxygen sensitivity of the [4Fe-4S] cluster, all purification steps should be performed under anaerobic conditions using glove boxes or Schlenk techniques to maintain enzyme activity .

What are the most effective purification methods for obtaining active recombinant ispG?

The purification of recombinant ispG requires specific techniques to preserve the oxygen-sensitive [4Fe-4S] cluster:

• Anaerobic purification: All steps must be conducted in an anaerobic chamber or using Schlenk techniques.

• Buffer composition: Standard buffers include 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5-10% glycerol, and 1-5 mM DTT or 2-mercaptoethanol as reducing agents.

• Purification steps:

  • Immobilized metal affinity chromatography (IMAC) using His-tagged protein

  • Size exclusion chromatography to remove aggregates

  • Ion exchange chromatography for higher purity

• [4Fe-4S] cluster reconstitution: If the cluster is damaged during purification, in vitro reconstitution can be performed using:

The reconstitution mixture should be incubated anaerobically at 4°C for 2-4 hours, followed by desalting to remove excess reagents .

What electron transfer systems support ispG catalytic activity in P. syringae and how can they be reconstituted in vitro?

The catalytic activity of ispG requires an electron transfer system to supply the electrons needed for the reduction reaction. For bacterial ispG enzymes, including those from P. syringae, the following electron transfer systems can be used:

• Physiological electron donors:

  • NADPH/flavodoxin/flavodoxin reductase system (bacterial)

  • Ferredoxin/ferredoxin reductase system (some bacteria and plants)

• Artificial electron donors for in vitro assays:

  • 5-deazaflavin semiquinone radical (photoactivated deazaflavin)

  • Sodium dithionite

  • Methyl viologen radical

To reconstitute these systems in vitro, researchers should include:

The reaction mixture should be incubated anaerobically at 30°C, and product formation can be monitored by HPLC or LC-MS analysis .

What are the proposed catalytic mechanisms for ispG and how can they be experimentally distinguished?

Multiple catalytic mechanisms have been proposed for ispG, with three predominant theories:

• Radical-based mechanism: Involves formation of an allylic radical intermediate through homolytic cleavage of the C-O bond.

• Bioorganometallic mechanism: Involves direct coordination of the substrate to the [4Fe-4S] cluster, forming metallacycle intermediates.

• Allyl cation mechanism: Proposes that the [4Fe-4S] cluster acts as a Lewis acid to facilitate C-O bond cleavage and formation of an allyl cation intermediate.

To experimentally distinguish between these mechanisms, the following approaches can be employed:

• EPR spectroscopy: To detect and characterize radical intermediates

• Mössbauer spectroscopy: To analyze changes in the [4Fe-4S] cluster during catalysis

• ENDOR spectroscopy: To study the interaction between the substrate and the [4Fe-4S] cluster

• Substrate analogs: Synthesize analogs with specific isotopic labels or chemical modifications to trap intermediates

• Site-directed mutagenesis: Modify key active site residues to probe their roles in the proposed mechanisms

• Computational studies: Perform density functional theory calculations to evaluate energetic barriers for different mechanistic pathways

These complementary approaches can provide evidence for or against specific mechanistic proposals .

How can recombineering techniques be applied to study ispG function in Pseudomonas syringae?

Recombineering offers powerful approaches for genetic manipulation of ispG in P. syringae. Specifically, the RecTE system from P. syringae can be utilized for precise genomic modifications:

• Gene disruption methodology:

  • Express the RecTEPsy proteins in P. syringae pv. tomato DC3000

  • Design PCR products with 80-83 bp homology flanking regions targeting the ispG gene

  • Transform the PCR product into cells expressing RecTEPsy

  • Select recombinants using appropriate antibiotic markers

  • Verify gene disruption by PCR and sequencing

• Point mutation introduction:

  • Design ssDNA oligonucleotides (70-100 nucleotides) containing the desired mutation flanked by homologous sequences

  • Transform the oligonucleotide into cells expressing RecTPsy

  • Implement a selection strategy if possible

  • Screen colonies by PCR and sequence verification

The recombineering efficiency for dsDNA with the RecTEPsy system is approximately 11-45 recombinants per 10⁸ viable cells, significantly higher than controls without RecTEPsy expression .

What phenotypic effects result from ispG mutations or deletions in P. syringae, and how can they be measured?

Mutations or deletions in the ispG gene in P. syringae can have profound effects on cellular physiology due to the essential nature of the MEP pathway. These effects can be observed and measured through:

• Growth phenotypes:

  • Complete deletion is likely lethal unless complemented or in mevalonate-supplemented media

  • Partial activity mutations may show temperature-sensitive growth

  • Quantitative growth curve analysis to measure growth rates

• Metabolite profiling:

  • LC-MS or GC-MS analysis of MEP pathway intermediates, particularly accumulation of MEcPP

  • Quantification of downstream isoprenoids using targeted metabolomics

  • Measurement of IPP and DMAPP levels in conditional mutants

• Virulence assessment:

  • Plant infection assays to evaluate bacterial fitness and virulence

  • Measurement of symptom development in host plants

  • Bacterial population dynamics in planta

• Volatile organic compound (VOC) analysis:

  • GC-MS analysis of terpene emissions

  • Particularly relevant as monoterpenes like α-pinene and β-pinene are implicated in plant-pathogen interactions and are derived from MEP pathway products

These multifaceted approaches can provide comprehensive insights into the functional consequences of ispG perturbation in P. syringae.

How does P. syringae ispG differ structurally and functionally from homologs in other organisms?

Comparative analysis reveals important differences between ispG from P. syringae and homologs from other organisms:

• Structural conservation and differences:

  • Core catalytic domain with [4Fe-4S] cluster binding motif is highly conserved across bacteria and plants

  • Three cysteine residues that coordinate the [4Fe-4S] cluster are strictly conserved

  • Species-specific variations exist in substrate binding pocket residues

• Functional differences:

  • Bacterial ispG enzymes (including P. syringae) utilize NADPH/flavodoxin/flavodoxin reductase as electron donors

  • Plant ispG (such as from Arabidopsis thaliana) cannot use bacterial electron transfer systems and instead require plant-specific ferredoxin systems

  • The plant enzyme shows activity only in the presence of 5-deazaflavin semiquinone radical under experimental conditions

• Evolutionary implications:

  • Despite the MEP pathway being present in both bacteria and plant plastids, the electron transfer requirements have diverged

  • This divergence suggests evolutionary adaptation to different cellular environments and redox systems

These differences have important implications for developing selective inhibitors and understanding the evolution of the MEP pathway across different kingdoms of life.

How can structural modeling be used to predict substrate specificity and inhibitor binding in P. syringae ispG?

Structural modeling of P. syringae ispG can provide valuable insights into substrate specificity and potential inhibitor binding sites:

• Homology modeling approach:

  • Generate models based on crystal structures of ispG homologs (e.g., from E. coli or T. thermophilus)

  • Refine models through molecular dynamics simulations

  • Validate models using experimental data on known mutants

• Active site analysis:

  • Identify residues involved in substrate binding and catalysis

  • Analyze the [4Fe-4S] cluster environment

  • Compare with known structures to identify unique features of P. syringae ispG

• Virtual screening methodology:

  • Perform docking simulations with potential inhibitors

  • Use scoring functions to rank ligand binding affinities

  • Verify predictions through experimental enzyme inhibition assays

• Substrate specificity prediction:

  • Dock natural substrate (MEcPP) and substrate analogs

  • Analyze binding energy and catalytic site interactions

  • Identify residues that contribute to substrate recognition

• Experimental validation:

  • Site-directed mutagenesis of predicted key residues

  • Enzyme kinetics with substrate analogs

  • Inhibition studies with compounds identified through virtual screening

This integrated computational and experimental approach can guide the development of selective inhibitors targeting P. syringae ispG while minimizing effects on plant homologs .

How does ispG activity in P. syringae contribute to plant-pathogen interactions?

The activity of ispG in P. syringae plays multiple roles in plant-pathogen interactions through its contribution to isoprenoid biosynthesis:

• Bacterial survival and fitness:

  • The MEP pathway provides essential isoprenoids for bacterial membrane and cellular functions

  • IspG activity is required for bacterial growth and multiplication in planta

• Virulence factor production:

  • Isoprenoid-derived molecules may contribute to bacterial virulence

  • The MEP pathway products serve as precursors for secondary metabolites involved in pathogenesis

• Modulation of plant defense responses:

  • MEP pathway products can potentially interfere with plant signaling

  • Bacterial isoprenoids may mimic or antagonize plant defense molecules

• Impact on plant volatile signaling:

  • P. syringae infection induces plant monoterpene emission, particularly α-pinene, β-pinene, and camphene

  • These monoterpenoids are associated with systemic acquired resistance (SAR) and can induce resistance against virulent P. syringae

Understanding these interactions can provide insights into both bacterial pathogenesis mechanisms and potential targets for disease control strategies.

What techniques can be used to study the impact of ispG inhibition on P. syringae pathogenicity?

Several methodological approaches can be employed to investigate how ispG inhibition affects P. syringae pathogenicity:

• Chemical biology approaches:

  • Use fosmidomycin or other MEP pathway inhibitors at sub-lethal concentrations

  • Apply controlled gene expression systems (e.g., inducible promoters) to modulate ispG expression levels

  • Utilize conditional mutants (temperature-sensitive or nutrient-dependent)

• Plant infection assays:

  • Measure bacterial growth curves in planta under ispG inhibition

  • Assess disease symptom development (chlorosis, necrosis)

  • Quantify bacterial population dynamics using fluorescent or luminescent reporter strains

• Defense response monitoring:

  • Analyze plant defense gene expression (e.g., PR1) upon infection with ispG-inhibited bacteria

  • Measure salicylic acid (SA) and pipecolic acid (Pip) accumulation in infected tissues

  • Monitor systemic acquired resistance (SAR) development

• Metabolite profiling:

  • Analyze changes in bacterial and plant isoprenoid profiles

  • Measure volatile organic compound (VOC) emissions, particularly monoterpenes

  • Quantify MEP pathway intermediates and end products

• Imaging techniques:

  • Use confocal microscopy with fluorescently labeled bacteria to track infection progress

  • Apply MALDI-imaging to map metabolite distributions during infection

These techniques provide complementary information about the role of ispG and the MEP pathway in P. syringae pathogenicity and plant-pathogen interactions .

How can the ispG enzyme be engineered to enhance isoprenoid production in bacterial systems?

Engineering ispG for enhanced isoprenoid production requires addressing several key aspects of enzyme function:

• Protein engineering strategies:

  • Directed evolution to improve catalytic efficiency

  • Site-directed mutagenesis of residues involved in:

    • Substrate binding (to improve affinity)

    • Product release (often rate-limiting)

    • [4Fe-4S] cluster stability (to enhance oxygen tolerance)

  • Domain swapping with homologs from thermophilic organisms to improve stability

• Redox partner engineering:

  • Co-expression of optimal electron transfer proteins (flavodoxin and flavodoxin reductase)

  • Engineering of electron transfer interface between ispG and redox partners

  • Balancing NADPH supply through cofactor regeneration systems

• Expression optimization:

  • Codon optimization for target host

  • Implementation of appropriate promoters and ribosome binding sites

  • Balancing expression with other MEP pathway enzymes to prevent bottlenecks

  • Subcellular compartmentalization or membrane association to improve pathway flux

• Oxygen sensitivity mitigation:

  • Development of oxygen-tolerant variants through protein engineering

  • Implementation of oxygen scavenging systems

  • Creation of microaerobic cultivation strategies

These approaches can be combined in a systematic metabolic engineering framework to enhance the MEP pathway flux and increase isoprenoid production .

What analytical methods are most appropriate for assessing ispG activity and product formation in vitro and in vivo?

A comprehensive analytical toolkit is essential for accurate assessment of ispG activity and product formation:

In vitro analytical methods:

• Spectrophotometric assays:

  • Monitoring NADPH oxidation at 340 nm in coupled assays

  • UV-Vis spectroscopy to track [4Fe-4S] cluster state changes

• Chromatographic techniques:

  • HPLC with UV detection for HMBPP quantification

  • LC-MS/MS for sensitive and specific quantification of MEcPP and HMBPP

  • Ion-pairing reversed-phase chromatography for separation of phosphorylated intermediates

• Enzymatic coupled assays:

  • Using IspH to convert HMBPP to IPP/DMAPP

  • Coupling with prenyltransferases that utilize IPP/DMAPP

In vivo analytical methods:

• Metabolite extraction protocols:

  • Quenching with cold methanol (-40°C)

  • Extraction with acidified acetonitrile/water mixtures

  • Solid-phase extraction for sample cleanup

• Advanced MS techniques:

  • High-resolution LC-MS for untargeted metabolomics

  • Multiple reaction monitoring (MRM) for targeted quantification

  • Isotope dilution methods using labeled standards

• Real-time monitoring:

  • Implementation of biosensors responsive to MEP pathway intermediates

  • Transcriptional reporters linked to pathway activity

  • GC-MS analysis of headspace samples for volatile isoprenoid products

These analytical approaches enable rigorous characterization of ispG function and its impact on isoprenoid biosynthesis .

What are the most promising strategies for studying the structure-function relationship of P. syringae ispG?

The most promising strategies for elucidating structure-function relationships in P. syringae ispG include:

• Advanced structural biology approaches:

  • Cryo-electron microscopy to capture different catalytic states

  • Neutron diffraction to identify hydrogen positions in the active site

  • Time-resolved X-ray crystallography to capture reaction intermediates

• Integrated spectroscopic techniques:

  • Combined EPR, ENDOR, and Mössbauer spectroscopy to characterize [4Fe-4S] cluster states

  • Resonance Raman spectroscopy to analyze substrate-cluster interactions

  • NMR studies with isotopically labeled substrates to track reaction progress

• Synthetic biology platforms:

  • Development of minimal synthetic systems reconstituting ispG and its redox partners

  • Cell-free expression systems for rapid mutant screening

  • Reconstitution in nanodiscs or liposomes to mimic membrane environments

• Computational approaches:

  • Quantum mechanics/molecular mechanics (QM/MM) simulations of catalytic mechanisms

  • Machine learning to identify patterns in sequence-function relationships

  • Network analysis to understand interactions with other cellular components

These multidisciplinary approaches will provide deeper insights into the fundamental properties and biological roles of P. syringae ispG, potentially leading to new applications in biocatalysis and antimicrobial development .

How might research on P. syringae ispG contribute to the development of novel antimicrobial strategies?

Research on P. syringae ispG has significant potential to inform the development of novel antimicrobial strategies through several avenues:

• Targeted inhibitor development:

  • Design of transition state analogs based on mechanistic understanding

  • Identification of allosteric inhibitors through structure-based drug design

  • Development of prodrugs activated by P. syringae-specific enzymes

• Selective targeting advantages:

  • Bacterial ispG differs from mammalian isoprenoid biosynthesis (which uses the mevalonate pathway)

  • Structural differences between bacterial and plant ispG can be exploited for specificity

  • The essential nature of the MEP pathway makes it an attractive antibiotic target

• Combination therapy strategies:

  • Synergistic effects when combining ispG inhibitors with other MEP pathway inhibitors

  • Potential for enhancing efficacy of existing antibiotics through metabolic weakening

  • Development of multi-target approaches addressing both virulence and bacterial survival

• Innovative delivery mechanisms:

  • Nanoparticle-based delivery of ispG inhibitors

  • Phage-based delivery systems for targeted antimicrobial action

  • Plant-expressed RNAi constructs targeting bacterial ispG expression

The unique position of ispG in bacterial metabolism and its divergence from host pathways make it a promising target for next-generation antimicrobial development, particularly for agricultural applications against P. syringae and related plant pathogens .