Recombinant Helicobacter pylori Ribulose-phosphate 3-epimerase (rpe)

What is the functional role of RPE in H. pylori metabolism?

RPE catalyzes the reversible interconversion of D-ribulose 5-phosphate and D-xylulose 5-phosphate in the non-oxidative phase of the pentose phosphate pathway (PPP). This step is critical for generating precursors for nucleotide synthesis and maintaining redox balance via NADPH production . In H. pylori, RPE activity supports survival under oxidative stress by replenishing NADPH pools required for antioxidant defense systems.

• Methodological Insight: To confirm its metabolic role, researchers employ gene knockout strains and measure growth defects under oxidative conditions (e.g., H₂O₂ exposure). Isotopic tracing with ¹³C-labeled glucose can track carbon flux through the PPP .

How is recombinant H. pylori RPE produced and purified?

Recombinant RPE is typically expressed in Escherichia coli using plasmid vectors (e.g., pET or pQE systems) with affinity tags (His₆ or GST) for purification.

What structural determinants govern substrate specificity in RPE?

RPE adopts a triosephosphate isomerase (TIM)-barrel fold with a conserved catalytic site featuring two aspartate residues (Asp72 and Asp143 in H. pylori) for proton transfer. Substrate specificity is mediated by a flexible loop (residues 190–210) that undergoes conformational changes to accommodate ribulose 5-phosphate .

• Structural Analysis Workflow:

  • X-ray crystallography: Resolve ligand-bound structures (e.g., RPE with ribulose 5-phosphate) to identify binding pocket residues .

  • Mutagenesis: Replace key residues (e.g., Ser10 or Arg118) to assess impacts on catalytic efficiency using steady-state kinetics .

Table 1: Kinetic Parameters of Wild-Type vs. Mutant RPE

Data derived from .

Why do kinetic studies report conflicting metal ion dependencies?

Discrepancies arise from variations in assay conditions and isoform-specific requirements:

• Plasmid-encoded RPE (e.g., Bacillus methanolicus Rpe1) shows 2-fold higher activity with Mn²⁺ than Mg²⁺ .

• Chromosomal isoforms (e.g., Trypanosoma cruzi TcRPE2) exhibit strict Mg²⁺ dependence .

• Resolution Strategy:

  • Pre-treat enzymes with EDTA to remove bound metals.

  • Perform activity assays with 1–5 mM Mg²⁺, Mn²⁺, or Fe²⁺ under standardized buffer conditions (pH 7.5–9.0) .

How to reconcile divergent oligomeric state observations?

H. pylori RPE primarily forms hexamers, whereas T. cruzi RPE exists as dimers and tetramers. These differences stem from:

• Sequence variations: Charged residues at subunit interfaces (e.g., Glu250 in H. pylori vs. Lys220 in T. cruzi).

• Methodological factors: SEC buffer composition (e.g., high salt stabilizes hexamers) .

• Experimental Approach:

  • Use native PAGE and analytical ultracentrifugation to assess oligomer distribution.

  • Crosslink subunits with glutaraldehyde to trap transient states .

What explains the biphasic kinetics observed in some RPE isoforms?

Biphasic kinetics (e.g., T. cruzi TcRPE1) suggest coexisting high- and low-affinity enzyme populations. This phenomenon is attributed to:

• Allosteric regulation: Substrate-induced conformational changes.

• Post-translational modifications: Phosphorylation or acetylation altering active site accessibility .

• Validation Method: Pre-incubate enzyme with 5 mM ribulose 5-phosphate to saturate allosteric sites before assay .

How to resolve low recombinant RPE yields in E. coli?

Common issues include codon bias and protein aggregation.

• Optimization Strategies:

  • Use E. coli strains with rare tRNA supplements (e.g., Rosetta2).

  • Reduce induction temperature to 18°C and prolong induction to 16–20 hours .

What functional assays best quantify RPE activity?

• Coupled spectrophotometric assay: Link RPE activity to NADH oxidation via transketolase and triosephosphate isomerase .

• HPLC-based quantification: Resolve ribulose 5-phosphate and xylulose 5-phosphate using anion-exchange chromatography .

Table 2: Comparison of Activity Assays

Can RPE be engineered for altered substrate specificity?

Rational design targeting the substrate-binding loop (residues 190–210) could enable catalysis of non-physiological substrates like D-psicose.

• Approach:

  • Perform saturation mutagenesis at positions 196–198.

  • Screen mutants using high-throughput fluorogenic assays .

How does RPE contribute to H. pylori pathogenicity?

Preliminary evidence links RPE to biofilm formation and antibiotic resistance.

• Hypothesis Testing:

  • Compare virulence of wild-type and Δrpe strains in murine models.

  • Profile NADPH/NADP⁺ ratios in infected gastric epithelial cells .