Recombinant Lactobacillus plantarum Non-canonical purine NTP pyrophosphatase (lp_2267)

What is Lactobacillus plantarum Non-canonical purine NTP pyrophosphatase (lp_2267)?

Lactobacillus plantarum Non-canonical purine NTP pyrophosphatase (lp_2267) is an enzyme classified under EC 3.6.1.66 that catalyzes the hydrolysis of non-canonical purine nucleoside triphosphates. The enzyme is also known as dITP/XTP pyrophosphatase and functions primarily to cleave non-standard purine nucleotides such as XTP, dITP, and ITP into their corresponding monophosphates and diphosphate molecules . This enzyme plays a critical role in nucleotide pool sanitization by eliminating potentially mutagenic non-canonical nucleotides from the cellular environment, thereby maintaining genomic integrity. The full-length protein consists of 202 amino acids and is found in Lactobacillus plantarum strain ATCC BAA-793/NCIMB 8826/WCFS1 .

What are the alternative names and classifications for this enzyme?

The enzyme is known by several alternative designations in scientific literature and databases:

• dITP/XTP pyrophosphatase

• EC 3.6.1.66

• Non-canonical purine NTP pyrophosphatase

• Non-standard purine NTP pyrophosphatase

• Nucleoside-triphosphate diphosphatase

• Nucleoside-triphosphate pyrophosphatase

• NTPase

This diverse nomenclature reflects the enzyme's various functional aspects and its classification within different enzyme systems. The EC number 3.6.1.66 places it within the hydrolase class, specifically acting on acid anhydrides in phosphorus-containing anhydrides .

What reactions does lp_2267 catalyze?

Lactobacillus plantarum Non-canonical purine NTP pyrophosphatase (lp_2267) catalyzes the following reactions:

• XTP + H₂O → XMP + diphosphate

• dITP + H₂O → dIMP + diphosphate

• ITP + H₂O → IMP + diphosphate

In these reactions, the enzyme hydrolyzes the bond between the α and β phosphates of non-canonical purine nucleotides, releasing a monophosphate nucleotide and a diphosphate molecule. This activity is crucial for removing potentially mutagenic nucleotides from the cellular nucleotide pool, as incorporation of these non-canonical nucleotides into DNA could lead to errors during replication and transcription.

What cofactors are required for optimal lp_2267 activity?

The activity of Non-canonical purine NTP pyrophosphatase is dependent on divalent cations, with magnesium (Mg²⁺) being the preferred cofactor . The magnesium ion likely coordinates with the phosphate groups of the substrate, facilitating proper substrate binding and catalysis. While other divalent cations such as manganese (Mn²⁺) or calcium (Ca²⁺) may support activity to some extent, they typically result in lower enzymatic efficiency compared to magnesium. Researchers working with this enzyme should ensure buffer conditions contain appropriate concentrations of Mg²⁺ (typically 1-10 mM) to achieve optimal enzymatic activity in experimental settings.

How does the substrate specificity of lp_2267 compare to similar enzymes from other organisms?

The substrate specificity of lp_2267 from Lactobacillus plantarum appears to be consistent with the general characteristics of this enzyme family across different species. Enzymes with similar function from Escherichia coli and the archaea Methanococcus jannaschii and Archaeoglobus fulgidus have been reported to be highly specific for XTP, dITP, and ITP . This conservation of substrate specificity across evolutionarily diverse organisms suggests the fundamental importance of this enzymatic function for cellular health.

What are the key structural domains and functional motifs in lp_2267?

While detailed structural information specific to lp_2267 is limited in the provided search results, this enzyme likely shares structural features with other members of the NTP pyrophosphatase family. Based on related enzymes, several key structural elements can be inferred:

• Nucleotide-binding pocket: A conserved region that accommodates the purine base of non-canonical nucleotides

• Catalytic site: Contains amino acid residues essential for hydrolysis of the α-β phosphodiester bond

• Divalent cation-binding site: Coordinates Mg²⁺ or other divalent cations necessary for catalysis

• Substrate specificity determinants: Structural elements that confer selectivity for XTP, dITP, and ITP over canonical nucleotides

Detailed structural studies, including X-ray crystallography or cryo-electron microscopy of lp_2267, would be necessary to fully elucidate these features and understand their roles in enzyme function.

What expression systems are available for producing recombinant lp_2267?

Based on the product information, several expression systems are available for producing recombinant Lactobacillus plantarum Non-canonical purine NTP pyrophosphatase:

Each expression system offers distinct advantages and limitations. E. coli systems typically provide high yields but may face challenges with protein folding or post-translational modifications. Yeast and mammalian expression systems often produce properly folded proteins with appropriate modifications but at lower yields. The biotinylated version utilizes AviTag-BirA technology for in vivo biotinylation, offering advantages for protein detection, immobilization, and protein-protein interaction studies .

What are the recommended protocols for measuring lp_2267 enzymatic activity?

For researchers investigating the enzymatic activity of lp_2267, several methodological approaches can be employed:

• Colorimetric phosphate detection: This method monitors the release of inorganic pyrophosphate (PPi) during the reaction. The pyrophosphate can be further cleaved by inorganic pyrophosphatase to release inorganic phosphate (Pi), which can be quantified using malachite green or other colorimetric reagents.

• HPLC-based assays: High-performance liquid chromatography can be used to separate and quantify substrate depletion (XTP, dITP, or ITP) and product formation (XMP, dIMP, or IMP).

• Coupled enzyme assays: The activity can be coupled to secondary reactions that produce detectable signals, such as changes in absorbance or fluorescence.

Standard reaction conditions should include:

• Buffer: Typically Tris-HCl or HEPES at pH 7.5-8.0

• Divalent cation: 1-10 mM MgCl₂

• Substrate concentration: 50-500 μM XTP, dITP, or ITP

• Temperature: 25-37°C

• Enzyme concentration: Determined empirically to achieve linear reaction kinetics

How does lp_2267 contribute to nucleotide metabolism and genomic integrity?

The primary biological role of Non-canonical purine NTP pyrophosphatase is to maintain the purity of the cellular nucleotide pool by eliminating potentially mutagenic non-canonical nucleotides. In the context of cellular metabolism, this enzyme functions as a safeguard against the incorporation of non-standard nucleotides into DNA and RNA, which could otherwise lead to mutations or inhibition of nucleic acid synthesis.

The enzyme's involvement in purine metabolism is evidenced by its inclusion in the KEGG metabolic pathway database (ec00230 - Purine metabolism) . Within this pathway, lp_2267 specifically targets the non-canonical purine nucleotides XTP, dITP, and ITP, converting them to their respective monophosphates. This activity prevents these non-standard nucleotides from competing with canonical nucleotides (ATP, GTP, etc.) in DNA and RNA synthesis.

Additionally, the enzyme's classification in the "Drug metabolism - other enzymes" pathway (ec00983) suggests potential roles in the metabolism of nucleotide analogs used as therapeutic agents.

What approaches are most effective for optimizing recombinant lp_2267 expression and purification?

Optimizing the expression and purification of recombinant lp_2267 requires consideration of several factors:

• Expression system selection: Based on available information, researchers can choose from E. coli, yeast, baculovirus, or mammalian cell systems . The choice depends on research needs:

  • For high yield: E. coli systems typically provide the highest protein yields

  • For proper folding and activity: Eukaryotic systems (yeast or mammalian) may be preferable

  • For specific applications: Biotinylated versions (CSB-EP801659LMS-B) offer advantages for immobilization and protein interaction studies

• Purification strategy:

  • Initial capture: Affinity chromatography based on the specific tag (information about the tag type would be determined during the manufacturing process)

  • Polishing steps: Ion exchange chromatography and size exclusion chromatography to achieve >85% purity (as specified in the product details)

• Protein stabilization: The lyophilized powder format suggests stability concerns in solution . Consider:

  • Buffer optimization: Testing various pH conditions and buffer components

  • Addition of stabilizing agents: Glycerol, reducing agents, or specific cofactors (Mg²⁺)

  • Storage conditions: Temperature, concentration, and freeze-thaw considerations

How do mutations in key residues affect the catalytic mechanism of lp_2267?

Structure-function relationship studies through site-directed mutagenesis provide valuable insights into lp_2267's catalytic mechanism. Though specific mutagenesis data for lp_2267 is not provided in the search results, research approaches would typically target:

• Catalytic residues: Mutations in amino acids directly involved in bond cleavage would likely abolish or severely reduce enzymatic activity. These often include conserved aspartate, glutamate, histidine, or lysine residues that coordinate water molecules or directly participate in catalysis.

• Substrate binding residues: Mutations in the nucleotide-binding pocket could alter substrate specificity or affinity. For instance, changing residues that interact with the purine base might shift specificity from XTP/ITP toward other nucleotides.

• Metal-coordinating residues: Since activity is dependent on Mg²⁺ , mutations in residues that coordinate this cofactor would likely impact catalytic efficiency.

• Structural elements: Mutations affecting protein folding or domain organization could have indirect effects on activity by altering the positioning of catalytic or substrate-binding residues.

A comprehensive mutagenesis study would involve:

• Identification of conserved residues through sequence alignment with homologous enzymes

• Generation of single and multiple point mutations

• Enzymatic characterization (Km, kcat, substrate specificity)

• Structural analysis of mutant proteins when possible

What bioinformatic approaches can predict substrate binding and specificity determinants in lp_2267?

Advanced computational methods provide powerful tools for investigating lp_2267 function:

• Homology modeling: Using the amino acid sequence provided and structurally characterized homologs as templates, researchers can generate three-dimensional models of lp_2267. This is particularly valuable given the absence of a solved crystal structure in the search results.

• Molecular docking: Virtual screening of various nucleotides (XTP, dITP, ITP, ATP, GTP, etc.) against the homology model can predict binding modes and relative affinities. This approach helps identify key interactions that determine substrate specificity.

• Molecular dynamics simulations: These can provide insights into protein flexibility, water networks, and transient interactions that might not be captured in static models.

• Sequence conservation analysis: Multiple sequence alignment of lp_2267 with homologs across diverse species can identify evolutionarily conserved residues likely critical for function.

• Protein-ligand interaction fingerprinting: Systematic analysis of predicted binding poses can identify common interaction patterns across preferred substrates.

These computational predictions should guide experimental validation through site-directed mutagenesis and enzyme kinetics studies.

How can lp_2267 be utilized in biotechnological applications?

The unique substrate specificity of lp_2267 for non-canonical purine nucleotides presents several potential biotechnological applications:

• Nucleotide pool sanitization: Recombinant lp_2267 could be used in vitro to remove non-canonical nucleotides from nucleotide preparations, ensuring high purity for molecular biology applications.

• Biosensor development: The enzyme could be incorporated into biosensors for detecting specific non-canonical nucleotides in biological samples or environmental monitoring.

• Therapeutic potential: As abnormal accumulation of non-canonical nucleotides has been associated with certain genetic disorders, recombinant lp_2267 could potentially serve as an enzymatic therapy to reduce these harmful metabolites.

• Structural biology tools: The biotinylated version of the enzyme (CSB-EP801659LMS-B) could serve as a useful tool for protein-protein interaction studies or for immobilization on streptavidin-coated surfaces.

• Enzymatic production of nucleoside monophosphates: The specific conversion of XTP, dITP, and ITP to their respective monophosphates could be exploited for the enzymatic synthesis of these compounds.

What are the emerging research directions for studying the physiological role of lp_2267 in Lactobacillus plantarum?

Future research on lp_2267 could explore several promising directions:

What are common challenges in working with recombinant lp_2267 and how can they be addressed?

Researchers working with recombinant lp_2267 may encounter several technical challenges:

• Protein solubility issues: If the recombinant protein forms inclusion bodies or aggregates:

  • Optimize expression conditions (temperature, induction time, inducer concentration)

  • Consider fusion tags that enhance solubility (MBP, SUMO, etc.)

  • Explore different buffer compositions during purification

  • Try alternative expression systems (from the available options: E. coli, yeast, baculovirus, or mammalian cells)

• Low enzymatic activity: If purified protein shows suboptimal activity:

  • Ensure the presence of sufficient Mg²⁺ as the preferred cofactor

  • Verify protein folding using circular dichroism or fluorescence spectroscopy

  • Check for potential inhibitors in the buffer components

  • Consider protein storage conditions (avoid repeated freeze-thaw cycles)

• Reconstitution challenges: Since the product is provided as a lyophilized powder :

  • Follow manufacturer recommendations for reconstitution

  • Use gentle mixing rather than vortexing to avoid denaturation

  • Consider adding stabilizing agents (glycerol, reducing agents)

  • Aliquot reconstituted protein to avoid repeated freeze-thaw cycles

• Assay optimization: For activity measurements:

  • Ensure linear reaction kinetics by optimizing enzyme concentration

  • Use appropriate controls for background phosphate or product levels

  • Consider potential interference from buffer components with detection methods

How can researchers differentiate between the activity of lp_2267 and other cellular phosphatases or pyrophosphatases?

Designing experiments that specifically measure lp_2267 activity in complex biological samples requires careful consideration: