Recombinant Gloeobacter violaceus Ribose-phosphate pyrophosphokinase (prs)

What is the biological significance of Ribose-phosphate pyrophosphokinase in Gloeobacter violaceus?

Ribose-phosphate pyrophosphokinase (EC 2.7.6.1) in Gloeobacter violaceus plays a fundamental role in nucleotide and amino acid biosynthesis by catalyzing the formation of PRPP, an activated form of ribose 5-phosphate and a major macromolecular building block of RNA . This enzyme is particularly significant in Gloeobacter violaceus because this organism represents the most primitive living cyanobacterium, characterized by a unique ancestral cell organization with a complete absence of inner membranes . Understanding the structure and function of prs in this organism provides insights into the evolution of nucleotide metabolism in photosynthetic organisms and primitive cellular systems.

What expression systems are available for producing Recombinant Gloeobacter violaceus Ribose-phosphate pyrophosphokinase?

Based on current research methodologies, Recombinant Gloeobacter violaceus Ribose-phosphate pyrophosphokinase can be expressed in multiple heterologous systems:

The E. coli expression system typically offers a good balance of yield and functionality for basic research applications. For specialized studies involving protein-protein interactions, the Avi-tag biotinylated version allows specific immobilization or detection protocols.

How can researchers effectively purify Recombinant Gloeobacter violaceus Ribose-phosphate pyrophosphokinase for structural studies?

For structural studies requiring high-purity preparations of Recombinant Gloeobacter violaceus Ribose-phosphate pyrophosphokinase, the following methodological approach is recommended:

• Initial purification: Following expression in E. coli, perform cell lysis using sonication or French press in a buffer containing 50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 10% glycerol, and protease inhibitors.

• Affinity chromatography: If the recombinant protein contains an affinity tag, use the appropriate resin (Ni-NTA for His-tagged proteins or streptavidin for Avi-tagged versions) .

• Ion exchange chromatography: Apply the protein to a Q-Sepharose column equilibrated with 20 mM Tris-HCl (pH 7.5), and elute with a 0-500 mM NaCl gradient.

• Size exclusion chromatography: Further purify using a Superdex 200 column in a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 5% glycerol.

• Concentration and storage: Concentrate the protein using ultrafiltration devices with appropriate molecular weight cutoffs and store in buffer containing 10-20% glycerol at -80°C.

This multi-step purification strategy typically yields protein with >95% purity suitable for crystallization trials and other structural studies.

What methods are recommended for assessing the enzymatic activity of Recombinant Gloeobacter violaceus Ribose-phosphate pyrophosphokinase?

The enzymatic activity of Recombinant Gloeobacter violaceus Ribose-phosphate pyrophosphokinase can be assessed using several complementary approaches:

• Spectrophotometric coupled assay: This method measures the formation of AMP from ATP during the PRPP synthesis reaction, coupled to NADH oxidation through auxiliary enzymes (myokinase, pyruvate kinase, and lactate dehydrogenase). The decrease in absorbance at 340 nm corresponds to enzyme activity.

• Radiometric assay: Using radiolabeled substrates such as [β-33P]-ATP to monitor the formation of [β-33P]-PRPP. This approach provides high sensitivity and specificity for kinetic studies .

• Equilibrium dialysis: This technique can be used to determine the dissociation constant (Kd) of the enzyme-substrate complex, similarly to methods described for PRPP-aptamer complexes .

• Mass spectrometry: LC-MS/MS can be employed to directly quantify the formation of PRPP, providing an accurate measurement of enzyme activity without the need for coupled reactions.

Standard reaction conditions typically include: 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 1 mM ATP, 1 mM ribose-5-phosphate, and 0.5-5 μg of purified enzyme, incubated at 37°C.

How does Gloeobacter violaceus Ribose-phosphate pyrophosphokinase function within the context of this primitive cyanobacterium's unique metabolism?

Gloeobacter violaceus Ribose-phosphate pyrophosphokinase operates within a unique metabolic context because G. violaceus is the most primitive extant cyanobacterium, lacking thylakoid membranes typically present in other photosynthetic organisms . This distinctive cellular organization necessitates alternative strategies for energy production and metabolite synthesis.

The prs enzyme in G. violaceus plays critical roles in:

• Nucleotide biosynthesis pathway: PRPP produced by the enzyme serves as a precursor for both purine and pyrimidine nucleotides, essential for DNA and RNA synthesis.

• Integration with photosynthetic machinery: Unlike other cyanobacteria, G. violaceus has unique molecular structures for Photosystems I and II with missing or poorly conserved components (PsaI, PsaJ, PsaK, PsaX for Photosystem I and PsbY, PsbZ, Psb27 for Photosystem II) . This necessitates efficient nucleotide metabolism to support these modified photosystems.

• Amino acid biosynthesis: PRPP is required for the synthesis of histidine and tryptophan, connecting nucleotide metabolism with amino acid production.

• Evolutionary significance: The primitive nature of G. violaceus suggests that its prs enzyme may represent an ancestral form, providing insights into the evolution of PRPP synthesis and utilization.

Research methodologies studying these relationships often employ comparative genomics, metabolic flux analysis, and in vitro reconstitution of metabolic pathways to understand how prs functions within this unique cellular context.

How can researchers address issues with low activity of purified Recombinant Gloeobacter violaceus Ribose-phosphate pyrophosphokinase?

When encountering low enzymatic activity with purified Recombinant Gloeobacter violaceus Ribose-phosphate pyrophosphokinase, consider implementing the following methodological solutions:

• Buffer optimization: Test various buffer compositions, focusing on:

  • pH range (7.0-8.0)

  • Divalent cation concentration (Mg2+ typically at 5-10 mM)

  • Salt concentration (50-300 mM NaCl or KCl)

  • Addition of stabilizing agents (glycerol 5-20%, reducing agents like DTT or β-mercaptoethanol)

• Protein refolding strategies: If inclusion bodies form during expression:

  • Solubilize in 6M guanidine hydrochloride or 8M urea

  • Perform stepwise dialysis with decreasing denaturant concentration

  • Add molecular chaperones during refolding

• Co-factor addition: Ensure sufficient Mg2+ is present (5-10 mM), as this is essential for catalytic activity.

• Substrate quality assessment: Verify the purity and integrity of ribose-5-phosphate and ATP substrates using analytical methods.

• Protein stability analysis: Use differential scanning fluorimetry to identify optimal stabilizing conditions for the enzyme.

Implementing these approaches systematically should help identify and address the specific cause of low enzymatic activity.

What are common pitfalls when characterizing the kinetic properties of Gloeobacter violaceus Ribose-phosphate pyrophosphokinase?

Researchers often encounter several methodological challenges when characterizing the kinetic properties of Gloeobacter violaceus Ribose-phosphate pyrophosphokinase:

• Substrate inhibition phenomena: At high concentrations, ATP can inhibit enzyme activity. Methodological solution: Perform initial velocity studies across a wide range of substrate concentrations (0.05-5 mM) to identify potential inhibitory effects.

• Incorrect interpretation of bi-substrate kinetics: As a bi-substrate enzyme (requiring both ribose-5-phosphate and ATP), simplified Michaelis-Menten treatments may be inadequate. Methodological solution: Use appropriate bi-substrate kinetic models (ping-pong or sequential) and design experiments where one substrate is varied while others are kept constant at saturating levels.

• Neglecting metal ion effects: Activity is highly dependent on Mg2+ concentration and may be affected by other divalent cations. Methodological solution: Characterize the effect of various concentrations of Mg2+, Mn2+, and Ca2+ on enzyme activity.

• Temperature and pH sensitivity: Activity and stability can vary significantly with temperature and pH. Methodological solution: Determine pH and temperature optima before performing detailed kinetic analyses.

• Product inhibition: PRPP can inhibit the enzyme at high concentrations. Methodological solution: Include product inhibition studies in kinetic characterization.

By anticipating these challenges and designing experiments accordingly, researchers can obtain more reliable kinetic parameters for this enzyme.

How does Gloeobacter violaceus Ribose-phosphate pyrophosphokinase compare to homologs from other photosynthetic organisms?

Comparative analysis of Gloeobacter violaceus Ribose-phosphate pyrophosphokinase with homologs from other photosynthetic organisms reveals important evolutionary and functional differences:

Gloeobacter violaceus prs likely represents a more primitive form of the enzyme, lacking some of the sophisticated regulatory mechanisms present in more evolved photosynthetic organisms. This aligns with G. violaceus's status as the most primitive extant cyanobacterium, lacking thylakoid membranes and containing unique photosystems .

Methodologically, these comparative studies typically employ structural modeling, sequence alignment analysis, and heterologous expression of different homologs followed by standardized enzymatic assays to directly compare kinetic parameters.

What insights does Gloeobacter violaceus Ribose-phosphate pyrophosphokinase provide into the evolution of nucleotide metabolism?

Gloeobacter violaceus Ribose-phosphate pyrophosphokinase offers several key insights into the evolution of nucleotide metabolism:

• Ancestral enzyme characteristics: As G. violaceus represents one of the earliest branches of cyanobacterial evolution (lacking thylakoid membranes) , its prs enzyme likely retains ancestral features. Structural and functional studies suggest a simpler regulatory architecture compared to homologs from more derived organisms.

• Metabolic integration: Analysis of the G. violaceus genome (a single circular chromosome of 4,659,019 bp with 62% GC content) reveals how prs functions within the context of primitive photosynthetic machinery. The absence or poor conservation of certain photosystem components (PsaI, PsaJ, PsaK, PsaX, PsbY, PsbZ, Psb27) suggests alternative nucleotide utilization patterns.

• Substrate specificity evolution: Comparative enzymatic studies indicate that G. violaceus prs may exhibit broader substrate specificity than more specialized homologs, potentially accommodating alternative pentose phosphates as substrates.

• Connection to RNA world hypothesis: The central role of PRPP in both nucleotide and amino acid biosynthesis makes G. violaceus prs an interesting model for studying the transition from RNA-based to protein-based metabolic systems in early life.

Research methodologies that have proven valuable in extracting these evolutionary insights include ancestral sequence reconstruction, comparative genomics, and metabolic network analysis incorporating prs function.

What emerging techniques could advance our understanding of Gloeobacter violaceus Ribose-phosphate pyrophosphokinase function and regulation?

Several cutting-edge methodological approaches show promise for deepening our understanding of Gloeobacter violaceus Ribose-phosphate pyrophosphokinase:

• Cryo-EM structural analysis: High-resolution cryo-electron microscopy could resolve the enzyme's structure in various conformational states, providing insights into the catalytic mechanism beyond what crystallography has revealed.

• Single-molecule enzymology: Techniques such as FRET-based assays could track the conformational changes of individual enzyme molecules during catalysis, revealing heterogeneity in enzyme behavior.

• Systems biology integration: Multi-omics approaches combining transcriptomics, proteomics, and metabolomics could elucidate how prs activity is coordinated with other metabolic pathways in G. violaceus, particularly in response to changing light conditions.

• Optogenetic control of enzyme activity: Engineering light-responsive domains into the enzyme could enable precise spatiotemporal control of PRPP synthesis, facilitating studies on its metabolic impact.

• CRISPR-Cas9 genome editing in G. violaceus: Developing genetic manipulation tools for this primitive cyanobacterium would allow direct investigation of prs function in its native context.

• Riboswitch-based sensors: Building on knowledge of PRPP-binding riboswitches , researchers could develop biosensors to monitor PRPP levels in vivo, providing real-time data on enzyme activity.

These methodological innovations would collectively provide a more comprehensive understanding of how this enzyme functions within the unique cellular and evolutionary context of Gloeobacter violaceus.

How might the study of Gloeobacter violaceus Ribose-phosphate pyrophosphokinase contribute to synthetic biology applications?

The study of Gloeobacter violaceus Ribose-phosphate pyrophosphokinase offers several promising avenues for synthetic biology applications:

• Engineering nucleotide biosynthesis pathways: The enzyme's role in PRPP formation makes it a key target for optimizing nucleotide production in synthetic systems. By understanding its catalytic mechanism and regulatory properties, researchers could engineer strains with enhanced nucleotide biosynthetic capacity.

• Developing minimal photosynthetic systems: G. violaceus's status as a primitive cyanobacterium with unique photosynthetic machinery suggests that its prs enzyme could be incorporated into minimal artificial photosynthetic systems, particularly those designed without thylakoid membranes.

• Creating PRPP-responsive genetic circuits: Knowledge of PRPP-binding riboswitches coupled with engineered variants of G. violaceus prs could enable the development of genetic circuits responsive to PRPP levels, allowing metabolic feedback control in synthetic organisms.

• Temperature-adaptive enzymatic systems: As G. violaceus inhabits diverse environments including hot springs, its prs enzyme may possess thermostability properties that could be valuable for high-temperature bioprocesses.

• Evolutionary-inspired enzyme design: Comparative analysis between G. violaceus prs and homologs from more derived organisms could inform the design of novel enzymes with tailored regulatory properties for synthetic applications.

Methodologically, these applications would require protein engineering approaches, directed evolution, and in vitro reconstitution of metabolic pathways incorporating the engineered enzymes.