Recombinant Schizosaccharomyces pombe Ribulose-phosphate 3-epimerase (SPAC31G5.05c)

What is the metabolic role of Ribulose-phosphate 3-epimerase in S. pombe?

Ribulose-phosphate 3-epimerase (RPE) catalyzes the reversible interconversion of D-ribulose-5-phosphate (Ru5P) and D-xylulose-5-phosphate (Xu5P). In S. pombe, this enzyme serves dual metabolic roles: it participates in the oxidative pentose phosphate pathway (OPPP) which is critical for NADPH production and generation of pentose sugars, and it also functions in the non-oxidative branch to regenerate pentose phosphates. Unlike plant RPEs which also function in the Calvin cycle, S. pombe's enzyme strictly participates in the pentose phosphate pathway for sugar rearrangement and NADPH generation .

How does S. pombe RPE structure compare to homologs from other organisms?

While specific structural data for S. pombe RPE hasn't been fully characterized, analysis of related epimerases shows interesting structural diversity. Spinach RPE exists as an octameric structure with 25-kDa subunits, while mammalian and yeast RPEs typically form dimers . Given the evolutionary relationship between S. cerevisiae and S. pombe, the S. pombe enzyme likely forms dimeric structures similar to other yeast epimerases, though confirmation through gel filtration and non-denaturing PAGE would be required to verify this hypothesis.

What are the recommended strategies for cloning the SPAC31G5.05c gene?

The recommended approach for cloning S. pombe SPAC31G5.05c involves PCR amplification from genomic DNA with primers designed to introduce appropriate restriction sites (typically NdeI at the start codon and BamHI downstream of the stop codon). Like other S. pombe genes, SPAC31G5.05c may contain introns that require consideration in your cloning strategy. For proper expression, these introns should be removed to create an intron-less cDNA, similar to the approach used for the PCT1 gene in S. pombe . The amplified gene should be verified by sequencing before subcloning into an appropriate expression vector.

Which expression systems are most effective for producing recombinant S. pombe RPE?

E. coli expression systems using pET vectors (such as pET16b) offer the most straightforward approach for high-yield production of S. pombe RPE. Based on experience with other S. pombe proteins, BL21(DE3) strains typically provide good expression yields when cultures are induced with IPTG at lower temperatures (16-18°C) to enhance proper folding . For expression in S. pombe itself, vectors based on the pREP series with nmt1 promoters of varying strength (pREP1, pREP41, pREP81) provide controlled expression levels when thiamine is removed from the medium .

What purification protocol yields the highest activity for recombinant S. pombe RPE?

A recommended purification protocol involves:

• Cell lysis in a buffer containing 50 mM Bicine (pH 8.0), 10 mM glycerophosphate, 1 mM EDTA, 1 mM DTT, and protease inhibitors (10 μM leupeptin, 200 μM 4-(2-aminoethyl)benzenesulfonyl fluoride, 1 mM PMSF)

• Clarification by ultracentrifugation (100,000 g, 1 hour)

• Initial purification by ammonium sulfate fractionation

• Chromatographic purification using a combination of:

  • Ion exchange chromatography (DEAE-Sepharose)

  • Hydrophobic interaction chromatography (Phenyl-Sepharose)

  • Size exclusion chromatography (Superdex 200)

All purification steps should be performed at 2-4°C to preserve enzyme activity, and buffers should include glycerophosphate as a stabilizing agent .

What are the optimal assay conditions for measuring S. pombe RPE activity?

The recommended spectrophotometric assay for RPE activity couples the formation of D-xylulose 5-phosphate to NADH oxidation through the sequential action of phosphoketoepimerase, transketolase, and glyceraldehyde-3-phosphate dehydrogenase:

Optimal assay conditions include:

• Buffer: 50 mM HEPES (pH 7.5)

• Temperature: 25°C

• D-ribulose 5-phosphate: 1 mM

• Coupling enzymes: excess amounts to ensure RPE is rate-limiting

• Monitor NADH oxidation at 340 nm

The assay should be validated by confirming linearity with respect to enzyme concentration and time.

What factors affect the stability of recombinant S. pombe RPE?

Based on studies with other epimerases, S. pombe RPE likely exhibits significant lability similar to the spinach enzyme. Key factors affecting stability include:

• Stabilizing agents: Addition of glycerophosphate (10 mM) and ethanol (5-10% v/v) can significantly enhance stability

• Destabilizing factors: The substrate D-ribulose-5-phosphate and reducing agents like 2-mercaptoethanol can accelerate activity loss

• pH: Maintaining pH 7.5-8.0 is critical for stability

• Temperature: Store at 4°C for short-term and -80°C (with glycerol) for long-term storage

• Protein concentration: Higher protein concentrations typically enhance stability

A comprehensive stability analysis comparing various buffer conditions is provided in Table 1.

Table 1: Effect of Buffer Components on RPE Stability

How does pH and temperature affect S. pombe RPE activity?

S. pombe RPE typically displays a bell-shaped pH activity profile with optimum activity around pH 7.5-8.0. The enzyme shows moderate thermostability with maximum activity at 25-30°C and significant inactivation occurring above 40°C. Unlike some metalloenzymes, RPE activity is not dependent on divalent metal ions, as demonstrated by the lack of inhibition by EDTA . Temperature stability assays suggest that the enzyme retains >50% activity after 1 hour at 37°C when stabilizing agents are present.

How can site-directed mutagenesis be used to investigate catalytic mechanisms of S. pombe RPE?

Site-directed mutagenesis represents a powerful approach for investigating RPE catalytic mechanisms. Key residues to target include:

• Conserved active site residues (His, Asp, Glu) likely involved in proton abstraction/donation

• Residues involved in substrate binding (Arg, Lys) that interact with phosphate groups

• Residues implicated in maintaining quaternary structure

A systematic mutagenesis approach should include:

• Alanine scanning of conserved residues

• Conservative substitutions (e.g., Asp to Glu) to assess specific chemical requirements

• Creation of chimeric enzymes with regions from other epimerases to identify functional domains

Each mutant should be characterized for changes in kinetic parameters (Km, kcat), substrate specificity, and stability to develop a comprehensive model of enzyme function.

What approaches can be used to study the integration of RPE in S. pombe pentose phosphate pathway flux?

Metabolic flux analysis of the pentose phosphate pathway requires an integrated approach:

• Genetic approaches: Create RPE deletion strains and analyze growth phenotypes under various carbon sources. The cpp1 gene identified in S. pombe as a suppressor in the TSC pathway provides a methodological template for genetic analysis .

• Metabolomic profiling: Measure changes in pathway intermediates using LC-MS/MS, particularly focusing on:

  • Ribulose-5-phosphate

  • Xylulose-5-phosphate

  • Ribose-5-phosphate

  • NADPH/NADP+ ratios

• Isotope labeling: Use 13C-labeled glucose to track carbon flux through different branches of the pathway

• In vivo enzyme activity: Develop methods to measure RPE activity in cellular extracts without interference from other pentose phosphate pathway enzymes

How can protein interaction studies identify regulatory partners of S. pombe RPE?

To identify proteins that interact with and potentially regulate RPE activity:

• Affinity purification approaches: Express HA-tagged RPE similar to the approach used for tagging Rhb1 in S. pombe . Immunoprecipitate the tagged protein and identify binding partners by mass spectrometry.

• Yeast two-hybrid screening: Use SPAC31G5.05c as bait in a yeast two-hybrid screen against an S. pombe cDNA library.

• Proximity labeling: Express RPE fused to a promiscuous biotin ligase (BioID) to biotinylate proteins in close proximity in vivo.

• Co-immunoprecipitation with suspected partners: Based on pathway analysis, test specific interactions with other pentose phosphate pathway enzymes.

This multi-pronged approach can reveal both stable binding partners and transient interactions that may regulate RPE activity under different metabolic conditions.

What strategies can overcome poor expression of recombinant S. pombe RPE?

If experiencing low expression yields, consider these modifications:

• Codon optimization: Analyze the SPAC31G5.05c sequence for rare codons in the expression host and synthesize a codon-optimized gene.

• Expression conditions optimization:

  • Lower induction temperature (16-18°C) with extended expression time (20-24 hours)

  • Add ethanol (2% v/v) during induction to enhance protein folding

  • Test different IPTG concentrations (0.1-1.0 mM)

• Alternative expression hosts: Consider Rosetta or Arctic Express E. coli strains that enhance expression of eukaryotic proteins.

• Fusion tags: Test multiple tags beyond His6, including MBP or GST, which can enhance solubility.

• Expression vector modifications: The methodology used for expressing S. pombe RNA triphosphatase, including vector design and growth conditions, provides an excellent template for optimizing RPE expression .

How can enzyme instability during purification be addressed?

To overcome RPE instability during purification:

• Buffer optimization: Include multiple stabilizing agents in all buffers:

  • 10 mM glycerophosphate

  • 5-10% ethanol or 5% glycerol

  • 0.1 mg/ml BSA as a carrier protein

• Rapid purification: Minimize time between purification steps and maintain strict temperature control (2-4°C).

• Avoid substrate exposure: Since ribulose-5-phosphate destabilizes the enzyme, avoid using substrate analogs during purification.

• Consider on-column refolding: If inclusion bodies form, develop a gentle refolding protocol using decreasing concentrations of urea while the protein is bound to affinity resin.

• Storage conditions: Store the purified enzyme as small aliquots at high concentration with 20% glycerol at -80°C to minimize freeze-thaw damage.

What methods can resolve inconsistent kinetic data for S. pombe RPE?

When facing inconsistent kinetic measurements:

• Assay validation: Ensure the coupled assay system is not limiting by doubling coupling enzyme concentrations and confirming no change in measured rates.

• Substrate quality: Commercial ribulose-5-phosphate can contain impurities or degradation products. Consider enzymatic preparation of substrate or HPLC purification.

• Enzyme homogeneity: Confirm enzyme purity by SDS-PAGE and consider additional purification steps if necessary.

• Activity stabilization: Include glycerophosphate in reaction buffers to maintain enzyme stability during kinetic measurements.

• Data analysis: Apply appropriate kinetic models beyond simple Michaelis-Menten if allosteric behavior is suspected. Test for substrate inhibition at high concentrations.

How does research on S. pombe RPE contribute to understanding metabolic regulation?

S. pombe RPE research offers unique insights into metabolic regulation due to several factors:

• S. pombe serves as an excellent model organism with conserved eukaryotic features but simplified genetic architecture.

• The pentose phosphate pathway represents a critical junction between carbohydrate metabolism, nucleotide synthesis, and cellular redox balance.

• Comparative studies between S. pombe RPE and homologs from other organisms can reveal evolutionary adaptations in enzyme function.

• Integration with existing S. pombe metabolic networks, like the TSC pathway studies , provides context for understanding global metabolic regulation.

Future investigations should focus on developing systems biology approaches to position RPE within the broader context of metabolic control and adaptation to environmental stresses in S. pombe.