Recombinant Photobacterium profundum Isopentenyl-diphosphate Delta-isomerase (idi)

What is Isopentenyl-diphosphate Delta-isomerase (IDI) and what role does it play in Photobacterium profundum?

Isopentenyl-diphosphate delta isomerase (IDI) catalyzes the conversion of the relatively unreactive isopentenyl pyrophosphate (IPP) to the more-reactive electrophile dimethylallyl pyrophosphate (DMAPP). This isomerization represents a critical step in isoprenoid biosynthesis through both the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways . In P. profundum, this enzyme likely plays a significant role in adaptation to deep-sea conditions by facilitating the synthesis of isoprenoid compounds that maintain membrane fluidity at high pressures.

The reaction catalyzed proceeds by an antarafacial transposition of hydrogen through a protonation/deprotonation mechanism. This involves the addition of a proton to the re-face of the C3-C4 double bond, creating a transient carbocation intermediate, followed by removal of the pro-R proton from C2 to form the C2-C3 double bond of DMAPP . Studies with Thermus thermophilus IDI-2 have revealed that this isomerization reaction is not concerted .

What are the two types of IDI enzymes and which type is found in Photobacterium profundum?

There are two structurally unrelated forms of IDI that catalyze the same reaction:

How does high pressure affect the activity of P. profundum IDI and what methodologies are used to study this?

P. profundum SS9 is a piezophilic bacterium with optimal growth at 28 MPa and 15°C . Studying the pressure effects on its IDI enzyme requires specialized methodologies:

Methodology for high-pressure enzyme kinetics:

• High-pressure reaction vessels: Custom-built pressure chambers capable of maintaining pressures up to 100 MPa while allowing spectrophotometric measurements.

• Coupled enzyme assays: IDI activity can be measured by coupling the formation of DMAPP to subsequent enzymatic reactions that produce detectable products.

• Stopped-flow apparatus with pressure cells: For measuring rapid kinetics under pressure.

• High-pressure microscopic chambers: Similar to those used for studying P. profundum motility under pressure , modified for enzyme activity measurements.

Research indicates that proteins from P. profundum often show pressure-optimized activity profiles, with maximal activity at pressures corresponding to the organism's natural habitat. A proteomic analysis of P. profundum grown at high pressure versus atmospheric pressure revealed differential expression of proteins involved in key metabolic pathways , suggesting that IDI expression and activity may also be pressure-regulated to maintain isoprenoid biosynthesis under varying pressure conditions.

What expression systems are recommended for producing recombinant P. profundum IDI?

Based on successful expression of other P. profundum proteins, the following expression systems are recommended:

For optimal expression in E. coli:

• Use a strong promoter (T7) with a 6×His-tag for purification.

• Culture at reduced temperatures (15-20°C) after induction to improve folding.

• Include osmolytes or pressure-mimicking compounds in the buffer system.

• Consider co-expression with chaperones to enhance proper folding.

Similar approaches have been successful for expressing the α-carbonic anhydrase from P. profundum . The recombinant protein showed both monomeric and dimeric forms with activity confirmed by protonography .

How does the isoprenoid biosynthesis pathway in P. profundum compare to other bacteria?

The isoprenoid biosynthesis in bacteria can occur through two distinct pathways:

• Mevalonate (MVA) pathway: Starting with acetyl-CoA and proceeding through mevalonate.

• Methylerythritol phosphate (MEP) pathway: Starting with pyruvate and glyceraldehyde-3-phosphate.

Genomic analysis of P. profundum suggests it primarily utilizes the MEP pathway for isoprenoid biosynthesis. This is evidenced by:

• The absence of HMGR homologs in the complete sequence of P. profundum, while this enzyme is present in related Vibrio species .

• The presence of genes encoding MEP pathway enzymes such as DXS (1-deoxy-D-xylulose-5-phosphate synthase) and DXR (1-deoxy-D-xylulose-5-phosphate reductoisomerase) .

This reliance on the MEP pathway differs from some related Vibrio species that appear to have acquired the HMGR gene through lateral gene transfer, enabling them to use the MVA pathway .

The MEP pathway begins with the condensation of pyruvate and glyceraldehyde-3-phosphate to form 1-deoxy-D-xylulose-5-phosphate (enzyme DXS), followed by reduction and rearrangement (enzyme DXR) to yield 2-C-methyl-D-erythritol-4-phosphate (MEP) . Further steps involve cyclophosphorylation and reduction, ultimately producing IPP, which is then converted to DMAPP by IDI.

What purification strategies yield the highest activity for recombinant P. profundum IDI?

Based on successful purification of other P. profundum proteins and typical protocols for IDI enzymes, the following purification strategy is recommended:

Recommended purification protocol:

• Cell lysis: Use gentle methods such as enzymatic lysis or mild sonication in a buffer containing 50 mM HEPES (pH 7.5), 300 mM NaCl, 10% glycerol, and 5 mM β-mercaptoethanol.

• Initial purification: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin if a His-tag is incorporated.

• Secondary purification: Size exclusion chromatography to separate monomeric and dimeric forms, as P. profundum proteins often exist in multiple oligomeric states .

• Buffer optimization: Include osmolytes like trimethylamine N-oxide (TMAO) or glycine betaine that can stabilize proteins from piezophiles.

• Storage conditions: Add 50% glycerol and store at -80°C to maintain activity.

A similar approach was successful for purifying P. profundum α-carbonic anhydrase, which yielded both monomeric and dimeric forms with distinct activity bands on protonography gels .

How can site-directed mutagenesis be used to investigate pressure-adapted features of P. profundum IDI?

Site-directed mutagenesis represents a powerful approach to explore pressure adaptation mechanisms in P. profundum IDI:

Experimental design for mutagenesis studies:

• Target selection: Identify amino acid residues unique to P. profundum IDI compared to homologs from non-piezophilic bacteria using multiple sequence alignment.

• Volume-change mutants: Replace bulky amino acids with smaller ones or vice versa to alter the volume change of enzyme-substrate interactions under pressure.

• Flexibility mutants: Target glycine or proline residues that might confer specific flexibility patterns important for pressure adaptation.

• Cavity-filling mutants: Identify and modify internal cavities that might contribute to pressure sensitivity.

• Hydrogen-bonding network modifications: Alter residues involved in hydrogen bonding to test their contribution to pressure stability.

Each mutant should be characterized for:

• Enzyme kinetics (Km, kcat) at different pressures (0.1 MPa to 50 MPa)

• Thermal stability at different pressures

• Structural changes using circular dichroism or fluorescence spectroscopy

This approach has successfully identified pressure-adaptation mechanisms in other P. profundum proteins, such as the RecD function required for high-pressure growth .

What analytical techniques are most effective for studying P. profundum IDI activity under pressure?

Several specialized techniques are particularly valuable for characterizing P. profundum IDI under pressure:

• High-pressure stopped-flow spectroscopy: Allows measurement of rapid kinetics under pressure, critical for determining if pressure affects the rate-limiting step of the IDI reaction.

• NMR spectroscopy with high-pressure cells: Can provide insights into structural changes and dynamics of the enzyme under pressure.

• FTIR spectroscopy under pressure: Useful for monitoring conformational changes in protein secondary structure elements at different pressures.

• Mass spectrometry with hydrogen-deuterium exchange: Can identify regions of the protein with altered flexibility or solvent accessibility under pressure.

• High-pressure X-ray crystallography: Although technically challenging, this can provide direct structural evidence of pressure-induced conformational changes.

• Computational approaches: Molecular dynamics simulations under pressure conditions can predict structural adaptations and help direct experimental efforts.

For the IDI reaction specifically, a coupled spectrophotometric assay using a UV-vis spectrophotometer equipped with a high-pressure cell would allow continuous monitoring of enzyme activity under varying pressure conditions.

How does temperature affect the catalytic efficiency of recombinant P. profundum IDI?

P. profundum SS9 is both a piezophile and a psychrophile, with optimal growth at 15°C and 28 MPa . The temperature dependence of its IDI enzyme likely reflects these adaptations:

Expected temperature effects:

• Optimal temperature range: Likely between 10-20°C, significantly lower than mesophilic enzymes (which typically show optimal activity around 37°C).

• Cold adaptation features: Higher catalytic efficiency (kcat/Km) at low temperatures compared to mesophilic homologs.

• Temperature-pressure interrelationship: The optimal temperature may shift under different pressure conditions, with higher optimal temperatures at higher pressures.

• Thermostability: Likely lower thermal stability than mesophilic counterparts, with denaturation occurring at relatively low temperatures (30-40°C).

To characterize these effects, activity assays should be conducted across a temperature range (0-40°C) at different pressures. This approach has been used successfully with other P. profundum enzymes, revealing temperature-pressure interdependence in their activity profiles .

What regulatory mechanisms control IDI expression in P. profundum under different pressure conditions?

Understanding the regulation of IDI expression in P. profundum requires consideration of several mechanisms that have been observed in this organism's response to pressure:

• Pressure-responsive promoters: Proteomics studies have shown that P. profundum differentially expresses proteins involved in key metabolic pathways at high versus atmospheric pressure . The IDI gene may be controlled by similar pressure-responsive elements.

• Stress response systems: In P. profundum SS9, several stress response genes (htpG, dnaK, dnaJ, and groEL) are upregulated in response to atmospheric pressure . These systems may indirectly affect IDI expression.

• Membrane composition feedback: As IDI is involved in isoprenoid biosynthesis, which contributes to membrane components, its expression may be regulated by feedback mechanisms tied to membrane composition, which changes with pressure .

• Transcriptional regulators: P. profundum contains various transcriptional regulators, including the RNA polymerase sigma factor (ECF subfamily) , which might control IDI expression under different pressure conditions.

To investigate these mechanisms, researchers can employ:

• Promoter-reporter fusion constructs to monitor IDI promoter activity under different pressures

• RT-qPCR to quantify IDI transcript levels

• Chromatin immunoprecipitation to identify transcription factors binding to the IDI promoter

• Metabolomic analysis to correlate isoprenoid intermediate levels with IDI expression

How does the reaction mechanism of P. profundum IDI compare to IDIs from non-piezophilic organisms?

The basic reaction mechanism of IDI involves the conversion of IPP to DMAPP through a protonation/deprotonation process. Studies with type-2 IDI from Thermus thermophilus (non-piezophilic) suggest a stepwise rather than concerted mechanism . For P. profundum IDI:

Mechanistic considerations:

• Pressure effects on transition state: High pressure typically favors reactions with negative activation volumes. The P. profundum IDI may have evolved structural features that create a more compact transition state.

• Solvent interactions: Pressure affects water structure and hydration of proteins. The P. profundum IDI active site may have evolved to maintain optimal hydration patterns under pressure.

• Proton transfer steps: The rate-determining step in IDI catalysis often involves proton transfer. P. profundum IDI may have adapted its active site architecture to facilitate efficient proton transfer under pressure.

• Carbocation stabilization: The reaction proceeds through a carbocation intermediate . P. profundum IDI may have evolved specific mechanisms to stabilize this intermediate under pressure.

To investigate these differences, researchers could:

• Compare kinetic isotope effects (using deuterated substrates) at different pressures

• Conduct pH-rate profiles at varying pressures to identify changes in rate-limiting steps

• Use transition state analogs to probe binding differences between P. profundum IDI and mesophilic homologs

What are the challenges in crystallizing P. profundum IDI for structural studies?

Obtaining high-resolution crystal structures of P. profundum IDI presents several challenges, similar to those encountered with other proteins from this organism:

Crystallization challenges and solutions:

• Protein stability: Psychrophilic proteins often have increased flexibility and reduced stability, making crystallization difficult. Solution: Include stabilizing additives (osmolytes, specific ions) in crystallization buffers.

• Oligomeric heterogeneity: P. profundum proteins often exist in multiple oligomeric states, as observed with its α-carbonic anhydrase . Solution: Rigorous size exclusion chromatography to isolate specific oligomeric forms.

• Pressure effects on structure: The native conformation under high pressure may differ from that at atmospheric pressure. Solution: Crystallize under pressure or use pressure-mimicking conditions.

• Cold-adaptation features: The protein's cold adaptation may cause increased surface hydrophilicity and reduced core packing. Solution: Screen crystallization conditions at lower temperatures (4-15°C).

• Post-translational modifications: If present, these may create heterogeneity. Solution: Mass spectrometric analysis to identify and account for modifications.