Recombinant Photobacterium profundum Ribose-5-phosphate isomerase A (rpiA)
Description
Introduction to Recombinant Photobacterium profundum Ribose-5-Phosphate Isomerase A (RpiA)
Ribose-5-phosphate isomerase (Rpi), specifically RpiA, is an enzyme that facilitates the interconversion of ribose-5-phosphate (R5P) and ribulose-5-phosphate (Ru5P) . RpiA is a member of the broader class of isomerases, which catalyze the interconversion of chemical isomers; in this instance, structural isomers of pentose . This enzyme plays a crucial role in biochemical metabolism, particularly in the pentose phosphate pathway and the Calvin cycle . The systematic name for this class of enzymes is D-ribose-5-phosphate aldose-ketose-isomerase . The Photobacterium profundum variant refers to the RpiA enzyme derived from the deep-sea bacterium Photobacterium profundum.
Function and Mechanism
RpiA catalyzes the reversible conversion of ribose-5-phosphate to ribulose 5-phosphate . This enzymatic reaction is essential for the synthesis of purine and pyrimidine nucleotides, NAD, and amino acids like histidine and tryptophan, achieved through the production of R5P, and in the synthesis of riboflavins .
Role in Metabolic Pathways
RpiA is integral to both the pentose phosphate pathway and the Calvin cycle .
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Pentose Phosphate Pathway (PPP): In the non-oxidative phase of the PPP, RPIA converts Ru5P to R5P, which is then converted by ribulose-phosphate 3-epimerase to xylulose-5-phosphate . This process effectively converts pentose phosphates into intermediates used in the glycolytic pathway. In the oxidative part, RpiA converts Ru5P to R5P through isomerization . The oxidative branch is a primary source of NADPH, crucial for biosynthetic reactions and protection against reactive oxygen species .
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Calvin Cycle: In the Calvin cycle, energy from electron carriers is used in carbon fixation, converting carbon dioxide and water into carbohydrates. RPIA is vital because Ru5P, generated from R5P, is converted to ribulose-1,5-bisphosphate (RuBP), the acceptor of carbon dioxide in the first dark reaction of photosynthesis . The product of the RuBP carboxylase reaction is glyceraldehyde-3-phosphate, which is then used to create larger carbohydrates. Glyceraldehyde-3-phosphate is converted to glucose, which the plant later converts to storage forms like starch or cellulose or uses for energy .
RpiA Isoforms and Distribution
Ribose 5-phosphate isomerase exists in two isoforms, RpiA and RpiB . Although both catalyze the same reaction, they are evolutionarily distinct, sharing little sequence or structural homology . RpiA is widespread throughout all kingdoms of life, whereas RpiB is found in bacterial sources and some pathogenic eukaryotes . Most organisms, such as Escherichia coli, contain both RpiA and RpiB, while others have only one class of Rpi .
Clinical Significance
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Ribose-5-phosphate Isomerase Deficiency: Mutations in the RPIA gene can lead to ribose-5-phosphate isomerase deficiency, a rare disorder .
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Hepatocarcinogenesis: Human RpiA plays a role in human hepatocellular carcinoma (HCC). Increased RpiA expression has been detected in tumor biopsies of HCC patients and in liver cancer tissue arrays. RpiA mRNA levels are correlated with clinical stage, grade, tumor size, types, invasion, and alpha-fetoprotein levels in HCC patients. RpiA regulates cell proliferation and colony formation in liver cancer cell lines via ERK signaling and modulation of PP2A activity, suggesting that RpiA overexpression can induce oncogenesis in HCC .
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Malaria Parasite: RpiA is essential in the pathogenesis of Plasmodium falciparum, the malaria parasite. Plasmodium cells require a large supply of NADPH via PPP for rapid growth and to detoxify heme, a product of hemoglobin degradation. R5P produced via increased pentose phosphate pathway activity is used to generate 5-phospho-D-ribose α-1-pyrophosphate (PRPP), needed for nucleic acid synthesis. PRPP concentrations are significantly increased in infected erythrocytes. Thus, targeting RpiA in Plasmodium falciparum could have therapeutic potential for malaria patients .
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Colorectal Cancer: RPIA is significantly elevated in colorectal cancer (CRC). It modulates cell proliferation and oncogenicity via activation of β-catenin in colon cancer cell lines. Unlike its role in PPP, where RPIA functions in the cytosol, in CRC, RPIA enters the nucleus to form a complex with adenomatous polyposis coli (APC) and β-catenin .
RpiB Enzymes as Drug Targets
Given the important cellular role of ribose-5-phosphate isomerases and that mammals only have RpiA, RpiB enzymes have garnered interest as potential drug targets, particularly in pathogenic organisms, many of which only have RpiB . E. coli RpiB inhibitors have been described, and inhibitor-bound crystal structures have been determined for RpiB from Mycobacterium tuberculosis and Trypanosma cruzi .
Product Specs
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Target Background
KEGG: ppr:PBPRA3122
STRING: 298386.PBPRA3122
Q&A
What is Ribose-5-phosphate isomerase A and its role in Photobacterium profundum?
Ribose-5-phosphate isomerase A (rpiA) is an enzyme widespread in microorganisms that plays a pivotal role in the pentose phosphate pathway, catalyzing the isomerization between D-ribulose 5-phosphate and D-ribose 5-phosphate . In Photobacterium profundum, a deep-sea bacterium that thrives under high-pressure conditions (piezophilic), this enzyme is particularly interesting due to its potential adaptations to function optimally in extreme environments. P. profundum is a gram-negative bacterium that can grow at pressures ranging from 0.1 MPa to 70 MPa and temperatures between 0°C and 25°C, with strain-dependent optimal conditions . The rpiA enzyme in this organism may possess structural and functional adaptations that enable its activity under high-pressure, low-temperature environments characteristic of deep-sea habitats.
What expression systems are optimal for producing recombinant P. profundum rpiA?
For recombinant expression of P. profundum rpiA, E. coli-based expression systems are commonly employed with specific modifications to accommodate the unique properties of deep-sea bacterial proteins. Based on similar studies with piezophilic proteins, the following methodological approaches are recommended:
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Expression vector selection: pET-series vectors with T7 promoter systems offer strong, inducible expression
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Host strain considerations: E. coli BL21(DE3) derivatives with additional cold-shock protein co-expression can improve folding
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Expression conditions: Lowered induction temperatures (15-20°C) and extended expression periods (18-24 hours) improve proper folding
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Pressure adaptation: For highest fidelity expression, consider using specialized high-pressure bioreactors that mimic native conditions
When expressing piezophilic proteins, researchers should be aware that atmospheric pressure conditions may impact proper folding, as P. profundum SS9 upregulates several stress response genes, including molecular chaperones (htpG, dnaK, dnaJ, and groEL), when exposed to atmospheric pressure . Co-expressing these chaperones might enhance proper folding of the recombinant enzyme.
What purification strategies yield highest activity for recombinant P. profundum rpiA?
Purification of recombinant P. profundum rpiA requires careful consideration of pressure effects on protein stability. Recommended methodological approaches include:
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Initial capture: Immobilized metal affinity chromatography (IMAC) using His-tagged constructs
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Buffer composition: Include osmolytes like TMAO or glycine betaine that stabilize proteins under pressure
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Temperature control: Maintain 4-15°C throughout purification to preserve native-like structure
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Pressure considerations: When possible, conduct purification steps under elevated pressure (custom high-pressure chromatography systems)
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Final polishing: Size exclusion chromatography under conditions mimicking native environment
Post-purification assessment should include activity measurements at various pressures to confirm preservation of pressure-adapted characteristics. Enzymatic activity should be measured under both atmospheric and high-pressure conditions to establish the impact of pressure on catalytic efficiency.
How can structural studies of P. profundum rpiA inform protein engineering for high-pressure biocatalysis?
Structural analysis of P. profundum rpiA can provide valuable insights for engineering pressure-stable enzymes for industrial applications. Cold-adapted and pressure-adapted enzymes from organisms like P. profundum often exhibit increased flexibility in specific regions while maintaining rigidity in others, creating a balanced dynamic stability appropriate for their environmental niche .
Crystallographic studies complemented with molecular dynamics simulations under various pressure conditions can reveal:
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Pressure-sensitive domains that undergo conformational changes
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Amino acid residues that contribute to pressure adaptation
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Hydrogen bonding networks that maintain stability under pressure
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Surface charge distributions that may counteract pressure effects on protein-solvent interactions
These structural insights can guide rational design of enzymes for high-pressure biocatalysis applications, such as rare sugar production. Ribose-5-phosphate isomerases have demonstrated utility in producing rare sugars, including D-allose, L-rhamnulose, L-lyxose, and L-tagatose . P. profundum rpiA may offer enhanced performance for these applications under high-pressure reaction conditions.
What are the kinetic parameters of P. profundum rpiA under varying pressure conditions?
While specific kinetic data for P. profundum rpiA is not provided in the search results, a methodological approach for determining these parameters can be outlined based on similar piezophilic enzyme studies:
| Pressure (MPa) | Expected Kinetic Changes | Methodological Considerations |
|---|---|---|
| 0.1 (atmospheric) | Baseline activity; potentially suboptimal | Standard spectrophotometric assays; coupled enzyme systems |
| 10 | Increased activity for some strains (DSJ4) | Specialized pressure vessels with optical windows |
| 28 | Optimal activity for SS9 strain | High-pressure stopped-flow devices; real-time monitoring |
| 50+ | Potential activity for extreme piezophiles | Ultra-high-pressure diamond anvil cells with spectroscopic coupling |
To accurately measure kinetic parameters under pressure, researchers should:
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Utilize specialized high-pressure equipment with real-time measurement capabilities
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Employ rapid mixing techniques to initiate reactions at pressure
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Consider temperature effects, as pressure and temperature adaptations are often linked
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Compare multiple substrate concentrations to determine Km and Vmax under each pressure condition
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Analyze pH-dependence under pressure, as ionization states can shift with pressure
These experiments would establish the pressure-activity profile of P. profundum rpiA and enable optimization of reaction conditions for potential biotechnological applications.
How do you maintain enzyme stability during experimental manipulations of recombinant P. profundum rpiA?
Maintaining the stability of piezophilic enzymes during experimental manipulations at atmospheric pressure presents significant challenges. P. profundum proteins have evolved to function optimally at high pressures, and decompression can lead to structural perturbations or denaturation. Researchers should consider the following methodological approaches:
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Buffer optimization: Include stabilizing agents like glycerol (10-20%), reducing agents, and osmolytes
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Temperature control: Maintain samples at 4-10°C throughout all manipulations
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Pressure cycling: When possible, apply periodic pressurization to prevent irreversible denaturation
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Chemical stabilization: Consider chemical crosslinking or immobilization techniques to preserve structure
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Rapid analysis: Minimize time at atmospheric pressure by preparing all experimental setups before decompression
Additionally, researchers should monitor stability using techniques like dynamic light scattering, circular dichroism, or fluorescence spectroscopy to detect early signs of aggregation or unfolding.
What are the optimal conditions for assaying recombinant P. profundum rpiA activity?
The optimal assay conditions for recombinant P. profundum rpiA should reflect the native environment of the enzyme while accounting for practical experimental constraints:
How does genetic manipulation of P. profundum rpiA contribute to understanding piezophilic adaptation?
Genetic studies of P. profundum rpiA can provide valuable insights into the molecular basis of piezophilic adaptation. P. profundum SS9 has become a model organism for studying pressure adaptation because it can grow over a wide range of pressures and temperatures, including at atmospheric pressure, which enables the application of genetic tools . Researchers can employ several approaches:
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Site-directed mutagenesis: Introducing specific mutations in the rpiA gene can help identify residues critical for high-pressure activity
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Domain swapping: Exchanging domains between piezophilic and non-piezophilic rpiA variants can identify pressure-adaptive regions
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Expression analysis: Quantifying rpiA expression under various pressure conditions can reveal regulatory mechanisms
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Complementation studies: Expressing P. profundum rpiA in mesophilic bacteria can test if pressure tolerance can be conferred
These genetic approaches should be complemented with biochemical characterization to establish structure-function relationships. As demonstrated with other genes in P. profundum SS9, processes affecting DNA and the bacterial cell envelope play important roles in piezophilic growth , suggesting that studying rpiA in context with these processes may provide a more comprehensive understanding.
How does rpiA interact with other components of the pentose phosphate pathway in P. profundum under high pressure?
The pentose phosphate pathway (PPP) in piezophilic organisms likely involves coordinated adaptation of multiple enzymes to function under high pressure. While specific information about PPP protein-protein interactions in P. profundum is limited in the provided research, metabolic pathway analysis suggests several important considerations:
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Enzyme complex formation: Under high pressure, protein-protein interactions may be strengthened or weakened, potentially affecting metabolic channeling
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Regulatory interactions: Pressure may influence allosteric regulation of rpiA by metabolic intermediates
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Metabolite stability: The chemical equilibria of PPP intermediates shift under pressure, potentially affecting substrate availability
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Competitive pathways: Alternative metabolic routes may be differentially affected by pressure
To study these interactions methodologically, researchers could employ:
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Protein-protein interaction studies under pressure (e.g., FRET-based approaches in high-pressure cells)
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Metabolomic analysis under various pressure conditions to map flux through the PPP
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In vitro reconstitution of partial or complete PPP enzyme systems to measure coordinated activity
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Comparative analysis with mesophilic systems to identify pressure-specific adaptations
Understanding these pathway interactions is crucial for both fundamental knowledge of pressure adaptation and potential biotechnological applications of P. profundum enzymes.
How can recombinant P. profundum rpiA be utilized for rare sugar production?
Ribose-5-phosphate isomerase has received considerable attention as a multipurpose biocatalyst for the production of rare sugars, including D-allose, L-rhamnulose, L-lyxose, and L-tagatose . P. profundum rpiA, with its adaptation to high-pressure environments, offers unique advantages for rare sugar production under non-conventional reaction conditions:
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Pressure-enhanced catalysis: High hydrostatic pressure can shift reaction equilibria and accelerate certain isomerization reactions
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Low-temperature operation: The psychrophilic nature of P. profundum enzymes allows energy-efficient processes at reduced temperatures
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Novel substrate specificity: Piezophilic enzymes may exhibit altered substrate preferences compared to mesophilic counterparts
A methodological approach for utilizing P. profundum rpiA in rare sugar production would involve:
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Substrate screening under pressure to identify optimal rare sugar precursors
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Process optimization including pressure cycling to maximize conversion while maintaining enzyme stability
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Immobilization strategies to enable enzyme reuse and continuous processing
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Integration with other piezophilic enzymes for multi-step conversions
By leveraging the unique properties of P. profundum rpiA, researchers may develop novel bioprocesses that operate under conditions inaccessible to conventional biocatalysts.
What analytical methods are most effective for monitoring rpiA-catalyzed reactions under high pressure?
Monitoring enzymatic reactions under high pressure presents significant technical challenges. For effective analysis of rpiA-catalyzed reactions, researchers should consider:
| Analytical Method | Advantages | Limitations | Technical Implementation |
|---|---|---|---|
| High-pressure NMR | Real-time monitoring; structural information | Expensive equipment; limited sensitivity | Specialized high-pressure NMR tubes and probes |
| Optical spectroscopy | Continuous monitoring; relatively simple | Requires transparent pressure vessels | Sapphire/diamond window high-pressure cells |
| Quenched-flow sampling | Compatible with standard analytics | Discontinuous data; potential decompression artifacts | Custom high-pressure sampling systems with rapid quenching |
| In situ Raman spectroscopy | Molecular vibration information; high specificity | Complex data interpretation | Fiber optic probes designed for high-pressure vessels |
For most academic laboratories, a practical approach combines:
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Custom-built high-pressure reactors with sampling capability
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Rapid quenching of reactions upon decompression
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Analysis of samples by conventional HPLC or LC-MS methods
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Time-course studies with multiple pressure cycles to establish reproducibility
These methodological approaches enable quantitative monitoring of rpiA-catalyzed reactions under conditions that mimic the native deep-sea environment of P. profundum.
