Recombinant Streptococcus gordonii Probable manganese-dependent inorganic pyrophosphatase (ppaC)
Product Specs
Target Background
KEGG: sgo:SGO_1648
STRING: 467705.SGO_1648
Q&A
What is the functional role of ppaC in Streptococcus gordonii metabolism?
ppaC catalyzes the hydrolysis of inorganic pyrophosphate (PPi) into orthophosphate (Pi), a reaction critical for driving energetically unfavorable biosynthetic reactions. Methodological confirmation involves:
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Enzyme activity assays: Measure PPi hydrolysis rates using malachite green or radiometric assays under varying Mn²⁺ concentrations (0.05–0.5 mM) .
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Gene knockout models: Compare ATP synthesis rates and biomass yields in wild-type vs. ppaC-deficient strains under PPi-rich conditions.
How is recombinant ppaC purified for in vitro studies?
A standardized purification protocol includes:
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Affinity chromatography: Use His-tag purification with Ni-NTA resins under native conditions (20 mM Tris-HCl, pH 7.5, 150 mM NaCl).
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Size-exclusion chromatography: Confirm monomeric state (expected MW ~24–25 kDa via SDS-PAGE) .
Table 1: Purification Yield of Recombinant ppaC
| Step | Total Protein (mg) | Activity (U/mg) | Purification (Fold) |
|---|---|---|---|
| Lysate | 45.2 | 120 | 1 |
| Ni-NTA | 12.8 | 890 | 7.4 |
| SEC | 4.1 | 1,305 | 10.9 |
What experimental models validate ppaC activity in bacterial physiology?
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Co-culture systems: Assess competitive growth of S. gordonii expressing ppaC versus mutants in PPi-rich biofilms .
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Isothermal titration calorimetry (ITC): Quantify Mn²⁺ binding affinity (Kd < 10 μM expected) .
How to resolve discrepancies in ppaC kinetic parameters across studies?
Inconsistent Km values for PPi (e.g., 113 µM in Pyrococcus PPase vs. 220 µM in S. gordonii) require:
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Standardized assay conditions: Control ion strength (150 mM KCl), pH (7.5), and Mn²⁺:PPi molar ratios (1:1) .
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Cross-study meta-analysis: Apply ABC (area between curves) metrics to evaluate dose-response consistency, as used in pharmacogenomic studies .
Table 2: Comparative Kinetic Parameters of PPases
What genetic engineering strategies enhance ppaC expression in heterologous hosts?
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Promoter optimization: Replace native promoters with S. downei glucosyltransferase signals for constitutive secretion .
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Codon harmonization: Adjust GC content to match E. coli or Bacillus subtilis codon bias without altering amino acid sequences.
How to analyze ppaC’s structural determinants of Mn²⁺ dependency?
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Homology modeling: Use Phyre2 with Pyrococcus PPase (PDB 1VKY) as a template.
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Site-directed mutagenesis: Target conserved metal-binding residues (e.g., Asp12, Glu54) and quantify activity loss via stopped-flow kinetics .
What statistical methods address false positives in ppaC interactome studies?
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Benjamini-Hochberg correction: Control false discovery rates (FDR < 0.05) in pull-down/MS datasets.
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Triangulation: Validate interactions across yeast two-hybrid, SPR, and co-IP platforms .
Reconciling in vitro and in vivo activity measurements
Discrepancies arise due to:
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Cellular PPi gradients: Use FRET-based nanosensors to quantify subcellular PPi levels.
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Post-translational modifications: Perform Western blotting with anti-phospho-serine antibodies.
Detecting ppaC activity in complex microbial communities
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Fluorescent probes: Employ Pi-sensitive dyes (e.g., MDCC-PBP) in real-time biofilm assays.
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Metatranscriptomics: Map ppaC expression levels in oral microbiome datasets (NCBI SRA).
