Recombinant Nocardia farcinica Undecaprenyl-diphosphatase (uppP)
Description
Enzymatic Function and Role in Bacterial Physiology
uppP is a membrane-bound hydrolase (EC 3.6.1.27) that facilitates the recycling of UPP in bacterial cell wall synthesis. It operates in a cycle where UP is phosphorylated to UPP during the transport of peptidoglycan precursors across the cytoplasmic membrane. Subsequent dephosphorylation by uppP regenerates UP for reuse .
Key Functions:
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Peptidoglycan Synthesis: Converts UPP to UP, enabling the lipid carrier to transport N-acetylmuramic acid-N-acetylglucosamine precursors .
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Lipopolysaccharide Assembly: Supports the translocation of O-antigen subunits in Gram-negative bacteria .
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Antibiotic Resistance: Overexpression of uppP can confer bacitracin resistance by counteracting bacitracin’s inhibition of UPP dephosphorylation .
Recombinant Production and Applications
Recombinant uppP is synthesized in heterologous systems (e.g., yeast) for biochemical and structural studies.
Production Details:
| Parameter | Specification | Source |
|---|---|---|
| Host Organism | Yeast | |
| Purity | >85% (SDS-PAGE) | |
| Storage Buffer | Tris-based, 50% glycerol | |
| Stability | 6 months at -20°C/-80°C (liquid) |
Key Applications:
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Antibiotic Target Validation: Testing inhibitors of peptidoglycan synthesis.
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Structural Studies: X-ray crystallography to map active-site interactions .
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Mechanistic Insights: Mutagenesis to identify residues critical for catalysis .
Functional Redundancy and Antibiotic Resistance
In Bacillus subtilis, uppP and BcrC are functionally redundant UPP phosphatases. Depletion of both enzymes leads to cell lysis and activation of the σᴹ stress response, highlighting their essential role in cell wall integrity . Similarly, N. farcinica may employ uppP to counteract bacitracin by regenerating UP despite antibiotic-mediated UPP sequestration .
Comparative Analysis of UPP Phosphatases
The following table contrasts N. farcinica uppP with homologs in other pathogens:
| Organism | Enzyme | Key Features |
|---|---|---|
| N. farcinica | uppP (Q5YVL4) | Bacitracin resistance, periplasmic activity |
| E. coli | UppP (P60933) | Membrane-bound, His30/Arg174 critical residues |
| B. subtilis | UppP/BcrC | Redundant, σᴹ-dependent stress response |
Challenges and Future Directions
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Structural Elucidation: High-resolution crystal structures of N. farcinica uppP remain lacking.
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In Vivo Validation: Testing recombinant uppP’s role in N. farcinica pathogenicity models.
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Antibiotic Synergy: Exploring combinations targeting uppP and other cell wall enzymes.
Product Specs
Note: While we will prioritize shipping the format currently in stock, we understand your unique needs. Please specify any format preference when placing your order, and we will fulfill it to the best of our ability.
Note: All proteins are shipped with standard blue ice packs. Should you require dry ice shipping, please inform us in advance, as additional fees may apply.
Generally, the shelf life of liquid forms is 6 months at -20°C/-80°C, while lyophilized forms have a shelf life of 12 months at -20°C/-80°C.
Target Background
KEGG: nfa:NFA_29300
STRING: 247156.nfa29300
Q&A
What is the basic function of Nocardia farcinica Undecaprenyl-diphosphatase (uppP)?
Nocardia farcinica Undecaprenyl-diphosphatase (uppP) catalyzes the dephosphorylation of undecaprenyl diphosphate (UPP) . This enzymatic activity is crucial for the lipid II cycle of cell wall biosynthesis, as it enables the recycling of the carrier molecule undecaprenyl phosphate (UP). During cell wall synthesis, building blocks are assembled inside the cytoplasm and transported to the outside for incorporation into the cell wall. After this process, the carrier molecule must be recycled through dephosphorylation from UPP to UP . This recycling step is indispensable for maintaining cell wall integrity and continued bacterial growth.
What is the subcellular localization and structure of uppP in Nocardia farcinica?
Undecaprenyl-diphosphatase is a multi-pass membrane protein localized in the cell membrane of Nocardia farcinica . The full-length protein consists of 313 amino acids. Its transmembrane nature is consistent with its function in processing membrane-associated undecaprenyl diphosphate. The protein belongs to the UppP family, a group of enzymes conserved across various bacterial species that participate in cell envelope biosynthesis . The multi-pass transmembrane structure allows the enzyme to interact with its substrate in the membrane environment where the lipid II cycle components are processed.
What expression systems are optimal for producing recombinant Nocardia farcinica uppP?
The most effective system reported for expression of recombinant Nocardia farcinica uppP is an in vitro E. coli expression system . When designing an expression protocol, researchers should consider the following methodological approaches:
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Using an N-terminal 10xHis-tag for purification purposes
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Expressing the full-length protein (amino acids 1-313)
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Utilizing Tris/PBS-based buffer systems with pH 8.0
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Including 6% Trehalose as a stabilizing agent
This approach yields functional recombinant protein suitable for enzymatic studies. When selecting between liquid and lyophilized forms, consider the specific experimental requirements. Lyophilized protein shows better stability with a shelf life of approximately 12 months at -20°C/-80°C, compared to 6 months for the liquid form .
What are the optimal storage conditions for maintaining recombinant uppP activity?
To maintain optimal activity of recombinant Nocardia farcinica uppP, store the protein at -20°C/-80°C and avoid repeated freeze-thaw cycles by preparing aliquots for multiple use . The shelf life varies depending on the preparation format:
| Preparation Format | Storage Temperature | Approximate Shelf Life |
|---|---|---|
| Liquid form | -20°C/-80°C | 6 months |
| Lyophilized form | -20°C/-80°C | 12 months |
Several factors affect enzyme stability, including buffer composition, storage temperature, and the inherent stability of the protein itself. For long-term studies, the lyophilized preparation with 6% Trehalose provides better stability. When reconstituting the lyophilized protein, use the original buffer system (Tris/PBS-based buffer, pH 8.0) to maintain native conformation and activity .
What PCR-based methods can be used to identify and study Nocardia farcinica uppP?
A highly specific PCR assay has been developed for the rapid identification of Nocardia farcinica, which can be adapted to study the uppP gene . The methodological approach involves:
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Design of specific primer pairs complementary to N. farcinica-specific sequences
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PCR amplification of genomic DNA to generate a specific fragment
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Verification of specificity through restriction enzyme digestion and direct sequencing
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Differentiation from heterologous nocardial species and other related bacterial genera
The Nf1 (5′-CCGCAGACCACGCAAC) and Nf2 (5′-ACGAGGTGACGGCTGC) primer pair has been validated for specific identification of N. farcinica . This PCR assay allows for rapid identification (within 1 day of obtaining DNA) of this medically important pathogen, which is significantly faster than traditional phenotypic characterization methods that can take up to 3 weeks. The approach shows high sensitivity and specificity with no cross-reactivity observed with other Nocardia species or related bacterial genera .
How can researchers effectively measure uppP enzymatic activity in vitro?
To effectively measure Nocardia farcinica uppP enzymatic activity in vitro, researchers should implement a phosphatase assay that quantifies the conversion of undecaprenyl diphosphate (UPP) to undecaprenyl phosphate (UP). A methodological approach would include:
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Preparation of purified recombinant uppP protein (using N-terminal 10xHis-tagged protein)
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Isolation or synthesis of the UPP substrate
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Establishment of an assay buffer system that mimics physiological conditions
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Quantification of released inorganic phosphate using colorimetric methods
Reaction conditions should include appropriate divalent cations (likely Mg²⁺) and optimal pH conditions (generally around pH 7.5-8.0 for phosphatases). Activity can be measured by monitoring the release of inorganic phosphate using malachite green or similar colorimetric assays.
For kinetic analysis, researchers should:
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Determine Km and Vmax values by varying UPP concentrations
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Assess the effects of potential inhibitors on enzyme activity
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Examine the impact of pH and temperature on catalytic efficiency
