Recombinant Thermus thermophilus 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (ispH)

What is IspH and what is its role in the methylerythritol phosphate (MEP) pathway?

IspH (4-hydroxy-3-methylbut-2-enyl diphosphate reductase) is an essential enzyme in the methylerythritol phosphate (MEP) pathway, which generates terpenoid precursor molecules. It catalyzes the final step in this pathway, converting hydroxymethylbutenyl 4-diphosphate (HMBPP) to the isoprenoid precursors isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP) .

The MEP pathway is a critical metabolic route found in many bacteria, including Thermus thermophilus, and represents an alternative to the mevalonate pathway for isoprenoid biosynthesis. Understanding IspH function is particularly important because it operates at a rate-limiting step in the pathway, making it a potential target for pathway engineering .

What is the structural organization of IspH from Thermus thermophilus?

Thermus thermophilus IspH exists as a dimer, with each monomer coordinating a [4Fe-4S] cluster via a glutamate (Glu) and three cysteine (Cys) residues . This structure differs from IspH in some other organisms such as Escherichia coli, where the protein functions as a monomer coordinating the [4Fe-4S] cluster via three Cys residues and the substrate HMBPP .

The dimeric organization of T. thermophilus IspH likely contributes to its thermostability, which is a characteristic feature of proteins from this extremophilic bacterium that can grow at temperatures around 75°C .

How does temperature affect the activity of recombinant T. thermophilus IspH?

As an enzyme from an extremely thermophilic bacterium, T. thermophilus IspH exhibits optimal activity at elevated temperatures (typically around 65-75°C) . When designing experiments with this recombinant enzyme, researchers should consider:

• Temperature stability during purification and storage

• Optimal reaction temperature for activity assays

• Compatibility of assay components with high temperatures

• Potential structural changes at different temperatures

For optimal experimental design, it's advisable to conduct temperature-dependent activity profiles to determine the enzyme's thermal optima and stability range.

What cofactors and conditions are required for optimal IspH activity?

IspH requires several essential components for optimal activity:

• Iron-sulfur cluster: The [4Fe-4S] cluster is crucial for catalytic activity

• Reducing conditions: The enzyme requires an external supply of reducing equivalents to function properly

• Proper pH: Typically in the range of 7.0-8.0 for optimal activity

• Divalent metal ions: Often Mg²⁺ or Mn²⁺ for stabilization

When designing experimental approaches to study IspH activity, researchers should ensure anaerobic conditions to prevent oxidation of the iron-sulfur cluster and include appropriate reducing agents such as dithiothreitol (DTT) or sodium dithionite in the reaction buffer .

What expression systems are suitable for producing recombinant T. thermophilus IspH?

Several expression systems have been successfully used for producing recombinant T. thermophilus IspH:

When expressing T. thermophilus IspH in heterologous systems, researchers should consider co-expressing it with IspG, as this has been shown to improve IspH function . Additionally, supplementing the growth medium with iron and sulfur sources can enhance iron-sulfur cluster incorporation.

How do structural differences between IspH from different bacterial species affect their catalytic mechanisms?

The structural organization of IspH varies significantly between bacterial species, impacting catalytic mechanisms and substrate interactions:

T. thermophilus and A. aeolicus IspH exists as dimers with each monomer coordinating the [4Fe-4S] cluster via a Glu and three Cys residues, whereas E. coli IspH functions as a monomer coordinating the cluster via three Cys residues and the substrate HMBPP . These structural differences affect:

• Substrate binding orientation

• Electron transfer pathways

• Protein stability under different conditions

• Potential for promiscuous activities

To investigate these structural differences, researchers should employ a combination of X-ray crystallography, site-directed mutagenesis, and computational modeling. Comparing kinetic parameters (kcat, KM) across orthologs can provide insights into how structural variations impact catalytic efficiency.

What redox-dependent activities have been observed for IspH, and how can they be experimentally characterized?

IspH exhibits different catalytic activities depending on its redox state:

• In the reduced [4Fe-4S]¹⁺ state, IspH catalyzes the conversion of HMBPP to IDP and DMADP

• Some IspH orthologs (e.g., from Bacillus sp. N16-5) can catalyze the conversion of HMBPP to isoprene and DMADP to isoamylene in the reduced state

• In the oxidized [4Fe-4S]²⁺ state, E. coli IspH can catalyze the conversion of acetylenes to ketones and aldehydes via acetylene hydratase activity

To characterize these redox-dependent activities, researchers should:

• Establish methods to prepare IspH in defined oxidation states

• Utilize anaerobic chambers and oxygen-free buffers

• Employ spectroscopic techniques (EPR, Mössbauer) to confirm redox states

• Develop HPLC or GC-MS methods to detect and quantify the diverse reaction products

• Use stopped-flow spectroscopy to monitor reaction kinetics

Comparing the activities of site-directed mutants can help identify residues specifically involved in each catalytic function.

How can researchers effectively co-express IspG and IspH to improve functional studies?

Co-expression of IspG with IspH has been shown to significantly improve IspH function, as demonstrated by studies with Z. mobilis IspH . This suggests potential protein-protein interactions or metabolic channeling between these enzymes.

To effectively co-express these proteins:

• Design a bicistronic expression vector with both genes under the control of the same promoter

• Alternatively, use a dual-promoter system with compatible plasmids

• Consider adding affinity tags that allow co-purification if the proteins form a complex

• Optimize expression conditions (temperature, inducer concentration, duration)

A methodological approach to studying this interaction would include:

• Pull-down assays to confirm physical interaction

• Analytical size exclusion chromatography to determine complex formation

• Microscale thermophoresis to measure binding affinity

• Activity assays comparing IspH alone versus co-expressed with IspG

Co-expression studies have shown a 10-fold increase in colony numbers at 100 μM IPTG with plasmid-encoded Z. mobilis ispG ispH compared to ispH alone in complementation experiments , providing strong evidence for functional interaction.

What experimental designs are most appropriate for studying IspH kinetics and mechanism?

When investigating IspH kinetics and mechanism, researchers should consider several experimental designs:

Steady-state kinetics:

• Measure initial rates at various substrate concentrations

• Determine kinetic parameters (KM, kcat, kcat/KM)

• Investigate potential inhibitors and their inhibition constants

Pre-steady-state kinetics:

• Use rapid-mixing techniques (stopped-flow) to observe transient species

• Determine rate constants for individual steps in the catalytic cycle

• Identify rate-limiting steps

Isotope effects:

• Synthesize isotopically labeled substrates (²H, ¹³C, ¹⁸O)

• Measure kinetic isotope effects to probe transition states

• Use mass spectrometry to track isotope incorporation into products

For these studies, maintaining anaerobic conditions is critical to preserve the iron-sulfur cluster integrity . Controls should include:

• Enzyme preparations lacking the iron-sulfur cluster

• Reactions under oxidizing conditions

• Heat-inactivated enzyme

The simplest true experimental designs involve one treatment group and one control group , which can be applied to testing effects of various conditions (temperature, pH, reducing agents) on IspH activity.

What approaches can be used to investigate the iron-sulfur cluster assembly and insertion into IspH?

Iron-sulfur cluster assembly and insertion into IspH is a complex process that requires dedicated cellular machinery . To investigate this process:

In vitro reconstitution:

• Express and purify apo-IspH (without Fe-S cluster)

• Add iron source (Fe²⁺/Fe³⁺), sulfide source, and reducing agent

• Monitor cluster assembly by UV-Vis spectroscopy and EPR

Co-expression with Fe-S cluster assembly proteins:

• Co-express IspH with iron-sulfur cluster (ISC) or sulfur utilization (SUF) pathway components

• Compare yield and activity of holo-enzyme under different conditions

• Use pull-down assays to identify specific interactions with assembly proteins

Biophysical characterization:

• Use circular dichroism to monitor structural changes upon cluster insertion

• Apply Mössbauer spectroscopy to characterize the iron oxidation state

• Employ resonance Raman spectroscopy to probe Fe-S bond characteristics

Mutagenesis studies:

• Mutate coordinating residues (Cys, Glu) to assess their roles

• Investigate conserved residues that might participate in cluster transfer

• Examine the effects of mutations on cluster stability and enzyme activity

Understanding cluster assembly is particularly important for T. thermophilus IspH due to its thermostability requirements, which might involve specialized mechanisms for cluster protection at high temperatures .

How can quasi-experimental designs be applied to study the effects of environmental factors on IspH activity?

When studying environmental factors affecting IspH activity, quasi-experimental designs may be necessary when true experimental conditions cannot be maintained:

• Use non-random assignment of treatments based on available materials or conditions

• Compare IspH from different growth conditions (aerobic vs. anaerobic)

• Study enzymes from related Thermus species grown under different temperatures

To mitigate these limitations:

• Include appropriate controls whenever possible

• Use statistical methods to account for confounding variables

• Perform replicate experiments with different enzyme preparations

• Validate findings with complementary approaches

For example, when studying the effect of oxygen exposure on IspH activity, a quasi-experimental approach might compare enzyme batches prepared under different conditions rather than randomly assigning oxygen exposure to aliquots of the same enzyme preparation.

What analytical techniques are most effective for measuring IspH activity and product formation?

Several analytical techniques can be employed to measure IspH activity and characterize reaction products:

For studying T. thermophilus IspH specifically, researchers should consider:

• Temperature-controlled analytical instruments

• Appropriate internal standards resistant to elevated temperatures

• Sample preparation methods that maintain anaerobic conditions

• Calibration curves using authentic standards of IDP and DMADP

A comprehensive analytical approach would combine multiple techniques to obtain complementary information about reaction rates, product distributions, and potential side-reactions.

What are the key challenges in purifying active recombinant T. thermophilus IspH?

Purifying active recombinant T. thermophilus IspH presents several challenges:

• Maintaining iron-sulfur cluster integrity throughout purification

• Preventing oxidative damage to the enzyme

• Balancing protein yield with functional activity

• Designing buffers compatible with downstream applications

Methodological approaches to address these challenges include:

• Performing all purification steps under anaerobic conditions or with reducing agents

• Including iron and sulfide sources in the purification buffers

• Using rapid purification protocols to minimize exposure time

• Employing affinity tags that do not interfere with iron-sulfur cluster coordination

• Validating enzyme activity at each purification step

Crystallization of T. thermophilus proteins has been achieved using the vapor-diffusion technique and counter-diffusion method through a gel layer , which might be applicable to IspH purification and structural studies.

How can researchers troubleshoot low activity in recombinant IspH preparations?

When facing low activity in recombinant IspH preparations, researchers should systematically evaluate:

Protein quality factors:

• Iron-sulfur cluster incorporation (check UV-visible spectrum for characteristic features)

• Protein folding (assess by circular dichroism)

• Aggregation state (analyze by size exclusion chromatography)

• Oxidation damage (examine by mass spectrometry)

Assay conditions:

• Verify reducing conditions are maintained throughout the assay

• Test different reducing agents (DTT, sodium dithionite, reduced ferredoxin)

• Optimize buffer components and pH

• Ensure substrate quality and concentration is appropriate

Co-factor requirements:

• Try supplementing with additional iron and sulfide

• Add potential physiological electron donors

• Consider co-expressing with IspG, which has been shown to improve IspH function

Experimental validation:

• Use known active preparations as positive controls

• Test activity under various temperatures to find the optimum

• Consider if the assay method itself is appropriate for thermostable enzymes

Low activity might also result from the challenge of expressing functional iron-sulfur proteins in heterologous hosts, as iron-sulfur cluster biogenesis and insertion is a complex process .

What strategies can be used to enhance the stability of T. thermophilus IspH for long-term storage?

As a protein from a thermophilic organism, T. thermophilus IspH should exhibit inherent stability, but preserving the iron-sulfur cluster remains challenging. Effective storage strategies include:

Additional recommendations:

• Add reducing agents (DTT, β-mercaptoethanol) to storage buffers

• Include iron and sulfide sources to help maintain cluster integrity

• Aliquot proteins to avoid repeated freeze-thaw cycles

• Test activity periodically to monitor stability over time

Thermophilic enzymes like those from T. thermophilus often show remarkable resistance to denaturation, with the phosphoribosylpyrophosphate synthetase from T. thermophilus HB27 displaying maximum activity at 75°C , suggesting that thermal stability may be a favorable property for storage of T. thermophilus IspH.

How might structural studies of T. thermophilus IspH inform protein engineering efforts?

Structural studies of T. thermophilus IspH can provide valuable insights for protein engineering:

• Identifying determinants of thermostability that could be transferred to mesophilic orthologs

• Mapping substrate binding residues to engineer substrate specificity

• Understanding the dimeric interface to potentially enhance protein stability

• Characterizing the microenvironment around the iron-sulfur cluster for optimizing electron transfer

Methodological approaches should include:

• X-ray crystallography of the enzyme with various ligands

• Hydrogen-deuterium exchange mass spectrometry to probe dynamic regions

• Molecular dynamics simulations at different temperatures

• Structure-guided mutagenesis followed by functional assays

The dimeric structure of T. thermophilus IspH, with each monomer coordinating an [4Fe-4S] cluster via a Glu and three Cys residues , provides a unique template for engineering efforts compared to monomeric IspH variants.

What are the implications of IspH's redox-dependent promiscuous activities for metabolic engineering?

The redox-dependent promiscuous activities of IspH have significant implications for metabolic engineering:

• In the reduced [4Fe-4S]¹⁺ state, some IspH orthologs can catalyze the conversion of HMBPP to isoprene and DMADP to isoamylene

• In the oxidized [4Fe-4S]²⁺ state, E. coli IspH can catalyze the conversion of acetylenes to ketones and aldehydes

These promiscuous activities could be harnessed for:

• Developing new biocatalytic routes to valuable compounds

• Engineering the MEP pathway to produce alternative end products

• Creating synthetic pathways that utilize IspH's unique catalytic capabilities

Research methodologies to explore these applications should include:

• High-throughput screening of substrate analogs to identify novel activities

• Protein engineering to enhance desired promiscuous functions

• Metabolic flux analysis to determine pathway limitations

• Integration of engineered IspH variants into existing metabolic networks

The challenge of increasing flux through IspH and identifying alternative enzymes makes this an important area for future MEP pathway engineering .

How can the relationship between IspG and IspH be exploited to overcome bottlenecks in the MEP pathway?

The MEP pathway contains several bottlenecks, with IspG becoming rate-limiting after the initial Dxs bottleneck is relieved . The observed improvement in IspH function when co-expressed with IspG suggests potential strategies to overcome these limitations:

• Co-expression of both enzymes to facilitate potential metabolic channeling

• Engineering fusion proteins that bring IspG and IspH into proximity

• Optimizing the relative expression levels of both enzymes

• Investigating whether physical interaction occurs between these proteins

Experimental approaches should include:

• Metabolic flux analysis to quantify the impact of co-expression

• Protein-protein interaction studies using techniques like FRET or crosslinking

• Structural studies of potential IspG-IspH complexes

• Testing various genetic constructs with different linkers or promoter strengths

The finding that co-expression of Z. mobilis IspG with IspH improved function 10-fold in complementation experiments provides strong evidence that this relationship can be exploited to enhance pathway efficiency.