Recombinant Pseudomonas putida 4-hydroxythreonine-4-phosphate dehydrogenase (pdxA)

What is the catalytic function of 4-hydroxythreonine-4-phosphate dehydrogenase (pdxA) in bacterial metabolism?

4-hydroxythreonine-4-phosphate dehydrogenase (EC 1.1.1.262), commonly known as pdxA, is an enzyme that catalyzes the fourth step in the de novo biosynthesis pathway of pyridoxal 5'-phosphate (vitamin B6). Specifically, it converts 4-hydroxy-L-threonine phosphate (HTP) to 3-amino-2-oxopropyl phosphate in the presence of either NAD+ or NADP+ as a redox cofactor . This reaction is critical for vitamin B6 metabolism, which is essential for numerous enzymatic reactions involving amino acid conversions .

The catalytic mechanism involves an oxidative decarboxylation of the HTP substrate, similar to the reactions catalyzed by isocitrate dehydrogenase and isopropylmalate dehydrogenase, despite limited sequence similarity with these enzymes . This enzyme belongs to the family of oxidoreductases, specifically those acting on the CH-OH group of donor with NAD+ or NADP+ as acceptor .

What is the structural organization of pdxA enzyme in Pseudomonas putida?

The crystal structure of pdxA reveals that the protein forms tightly bound dimers, with each monomer exhibiting an alpha/beta/alpha-fold that can be divided into two subdomains . The active site is strategically located at the dimer interface, within a cleft between the two subdomains, and involves residues from both monomers .

A key feature of the active site is a Zn2+ ion bound within each active center, coordinated by three conserved histidine residues from both monomers . Additionally, two conserved amino acids, Asp247 and Asp267, play crucial roles in maintaining the integrity of the active site . The substrate is anchored to the enzyme through interactions of its phospho group and by coordination of the amino and hydroxyl groups by the Zn2+ ion .

This structural arrangement facilitates the enzyme's strict requirement for the phosphate ester form of the substrate HTP, while maintaining flexibility in utilizing either NADP+ or NAD+ as redox cofactors .

How can pdxA be cloned and expressed in Pseudomonas putida?

Cloning and expressing pdxA in Pseudomonas putida typically follows this methodological approach:

• Gene Amplification: Using PCR with primers designed based on the pdxA sequence from genomic DNA of the source organism .

• Vector Selection: Choose an appropriate expression vector compatible with P. putida. For enhanced expression, vectors with strong promoters like T7-like or MmP1 expression systems can be utilized .

• Restriction Digestion: Perform enzyme digestion on both the pdxA gene fragment and the chosen vector with appropriate restriction endonucleases to create compatible sticky ends .

• Ligation: Use T4 DNA ligase to connect the sticky ends of the pdxA gene and plasmid vector through complementary pairing .

• Transformation: Transform the recombinant plasmid into competent P. putida cells. This can be achieved through:

  • Heat shock transformation (42°C for 45s followed by immediate ice bath)

  • Natural transformation methods

  • Electroporation (more efficient for Pseudomonas species)

• Selection: Plate transformed cells on selective media containing appropriate antibiotics to identify successful transformants .

• Verification: Confirm successful cloning through colony PCR, restriction analysis, and DNA sequencing .

• Expression Analysis: Verify protein expression through SDS-PAGE, Western blotting, or enzyme activity assays .

For optimized expression, integration of the pdxA gene into the P. putida chromosome can be considered, with sites like phaC1 (locus tag PP_5003) showing enhanced expression levels .

What methods are most effective for purifying recombinant pdxA from Pseudomonas putida?

Purification of recombinant pdxA from Pseudomonas putida can be achieved through a systematic approach:

Step 1: Cell Lysis and Crude Extract Preparation

• Harvest cells by centrifugation from late-exponential phase cultures

• Resuspend in appropriate buffer (typically containing 50 mM Tris-HCl pH 7.5-8.0, 100-300 mM NaCl, 5-10% glycerol)

• Disrupt cells using sonication, French press, or enzymatic lysis

• Remove cell debris by centrifugation (15,000-20,000 × g for 30-45 minutes)

Step 2: Initial Purification

• Ammonium sulfate fractionation (typically 40-60% saturation range for pdxA)

• Ion exchange chromatography: DEAE or Q-Sepharose columns with gradient elution

Step 3: Affinity Chromatography

• If tagged recombinant protein:

  • His-tagged pdxA: Ni-NTA or TALON resin

  • GST-tagged pdxA: Glutathione Sepharose

• If untagged: Substrate analog affinity columns with immobilized NAD+ or NADP+

Step 4: Size Exclusion Chromatography

• Final polishing step using Sephacryl S-200 or Superdex 200

• Buffer typically containing 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol

Step 5: Quality Assessment

• SDS-PAGE to check purity (>95%)

• Western blot for identity confirmation

• Enzyme activity assay with 4-hydroxy-L-threonine phosphate and NAD+/NADP+

• Protein concentration determination using Bradford assay or BCA method

Throughout the purification process, it's essential to include stabilizing agents like divalent metal ions (particularly Zn2+) and reducing agents (DTT or β-mercaptoethanol) to maintain enzyme activity and prevent oxidation of critical cysteine residues .

How can kinetic parameters of recombinant pdxA from Pseudomonas putida be accurately determined?

Accurate determination of kinetic parameters for recombinant pdxA requires careful experimental design and analysis:

Experimental Setup:

• Substrate Preparation: Synthesize or obtain pure 4-hydroxy-L-threonine phosphate (HTP)

• Cofactor Selection: Prepare both NAD+ and NADP+ solutions to determine cofactor preference

• Reaction Conditions:

  • Buffer: 50 mM Tris-HCl (pH 7.5-8.0)

  • Temperature: 25-30°C

  • Divalent metal ions: 1-5 mM ZnCl₂ or MgCl₂

  • Enzyme concentration: 0.1-1 μM (purified)

Kinetic Analysis Methods:

• Initial Velocity Measurements:

  • Monitor NADH/NADPH formation at 340 nm (ε = 6,220 M⁻¹cm⁻¹)

  • Use varying substrate concentrations (0.01-10 × Km)

  • Ensure measurements are made in the linear range (<10% substrate consumption)

• Data Analysis:

  • Plot velocity vs. substrate concentration

  • Fit data to Michaelis-Menten equation: v = Vmax[S]/(Km + [S])

  • Calculate Km, Vmax, and kcat using non-linear regression

  • Determine catalytic efficiency (kcat/Km)

• pH Dependency:

  • Measure activity across pH range 5.0-10.0

  • Identify optimal pH and pKa values of ionizable groups

• Temperature Dependency:

  • Measure activity across temperature range 10-60°C

  • Determine activation energy using Arrhenius plot

Example Results Format:

Using ordered sequential mechanism analysis and inhibition studies, you can further elucidate whether the enzyme follows a sequential ordered mechanism, as has been observed with similar dehydrogenases .

What methodologies are most appropriate for studying the effect of metal ions on pdxA activity?

The study of metal ion effects on pdxA activity requires systematic approaches that differentiate between essential cofactors and modulatory ions:

Protocol for Metal Ion Dependency Analysis:

• Metal Removal (Apo-enzyme Preparation):

  • Dialyze purified enzyme against buffer containing 10-20 mM EDTA

  • Follow with extensive dialysis against metal-free buffer

  • Verify metal removal using atomic absorption spectroscopy

• Activity Restoration Assay:

  • Incubate apo-enzyme with various metal ions (Zn²⁺, Mg²⁺, Mn²⁺, Fe²⁺, Co²⁺, Ni²⁺, Cu²⁺)

  • Test concentration range: 0.1-5.0 mM

  • Measure restored activity using standard assay conditions

• Determination of Binding Constants:

  • Isothermal titration calorimetry (ITC) with apo-enzyme and metal ions

  • Equilibrium dialysis with radioactive metals (⁶⁵Zn, ⁵⁴Mn)

  • Spectroscopic methods (intrinsic fluorescence quenching)

• Structure-Function Studies:

  • Site-directed mutagenesis of metal-coordinating histidine residues

  • Circular dichroism to assess structural changes

  • Thermal stability analysis with differential scanning calorimetry

Example of Expected Results:

Based on structural studies of pdxA, the enzyme coordinates Zn²⁺ in its active site through three conserved histidine residues from both monomers of the dimeric enzyme . This coordination is essential for proper substrate binding and catalytic activity. Mutation of these conserved histidines would be expected to significantly reduce metal binding and enzymatic activity.

How can genetic manipulations of pdxA improve vitamin B6 production in recombinant Pseudomonas putida?

Engineering pdxA for enhanced vitamin B6 production requires a comprehensive metabolic engineering approach:

Strategic Genetic Manipulations:

• Overexpression Strategies:

  • Integration of pdxA under strong constitutive promoters like rRNA promoters

  • Use of T7-like expression systems for controlled high-level expression

  • Development of a dual-vector system with complementary enzymes in the pathway

• Enzyme Engineering:

  • Site-directed mutagenesis targeting:

    • Active site residues for improved substrate affinity

    • Metal-binding residues for enhanced stability

    • Regulatory domains for reduced feedback inhibition

  • Fusion with stability-enhancing domains

• Pathway Optimization:

  • Coordinate overexpression of other rate-limiting enzymes in the pathway

  • Deletion of competing pathways that consume precursors

  • Enhancement of substrate (HTP) biosynthesis

  • Elimination of product degradation pathways

• Regulatory Engineering:

  • Removal of transcriptional repressors

  • Integration of inducible promoters for controlled expression

  • Application of ribosome binding site engineering

Implementation Framework:

What methods can be used to study the effect of pdxA mutations on enzyme stability and function?

Investigating the impacts of pdxA mutations requires a multi-faceted approach combining structural, biophysical, and functional analyses:

Experimental Methods for Mutation Analysis:

• Generation of Mutants:

  • Site-directed mutagenesis using overlap extension PCR (SOE PCR)

  • CRISPR-Cas9 genome editing for chromosomal mutations

  • Construction of mutation libraries for high-throughput screening

• Structural Stability Assessment:

  • Thermal shift assays (Thermofluor) to determine melting temperatures

  • Circular dichroism (CD) spectroscopy for secondary structure analysis

  • Intrinsic fluorescence to monitor tertiary structure changes

  • Limited proteolysis to identify flexible/exposed regions

• Functional Analysis:

  • Standard enzyme kinetic assays comparing wild-type and mutant enzymes

  • pH and temperature profiles to assess altered environmental responses

  • Metal binding affinity measurements using ITC or spectroscopic methods

  • Substrate specificity testing with substrate analogs

• Molecular Dynamics Simulations:

  • In silico modeling of mutations to predict structural changes

  • Simulation of enzyme-substrate interactions

  • Prediction of altered dynamics in catalytic regions

Example Data Representation:

For mutations affecting active site residues, particularly those involved in metal coordination (such as the conserved histidines), significant decreases in thermal stability and catalytic activity would be expected . Mutations of residues Asp247 and Asp267, which maintain active site integrity, would likely disrupt proper substrate orientation and catalytic efficiency .

More subtle mutations of residues involved in substrate binding might yield variants with altered substrate specificity or improved catalytic properties, which could be valuable for biotechnological applications in vitamin B6 production.

How can recombinant pdxA expression be utilized for enhancing polyhydroxyalkanoate (PHA) production in Pseudomonas putida?

While pdxA primarily functions in vitamin B6 biosynthesis, its recombinant expression in Pseudomonas putida can indirectly enhance polyhydroxyalkanoate (PHA) production through metabolic interactions:

Metabolic Engineering Strategies:

• Vitamin B6 Optimization for PHA Biosynthesis:

  • PLP-dependent enzymes are involved in amino acid metabolism, which can influence the acetyl-CoA and fatty acid pools

  • Enhanced pdxA expression provides increased vitamin B6 cofactors for these enzymatic reactions

  • Improved amino acid metabolism can reduce nitrogen consumption, triggering PHA accumulation

• Integration with PHA Production Pathways:

  • Co-expression of pdxA with PHA synthase genes (phaC1 and phaC2)

  • Coordination with phaG gene (encoding 3-hydroxyacyl-acyl carrier protein-CoA transacylase)

  • Deletion of phaZ (PHA depolymerase) to prevent PHA degradation

• Carbon Flux Optimization:

  • Engineer pathways to channel carbon sources efficiently between central metabolism and PHA synthesis

  • Balance between growth-associated and storage-associated metabolism

Experimental Design Framework:

Experimental Results from Similar Approaches:

In engineered P. putida strains, optimization of carbon flux through similar strategies has yielded significant improvements in PHA production:

• P. putida expressing PHA biosynthesis genes produced up to 9.16 wt% poly(3-hydroxybutyrate) from sucrose

• In fed-batch cultures, optimized strains achieved up to 38.1 wt% of poly(3-hydroxybutyrate)

• P. putida with enhanced acetate assimilation pathways showed 92% increase in MCL-PHA titer

While direct evidence for pdxA's role in PHA production is limited, the interconnection between vitamin B6 metabolism and central carbon metabolism suggests potential benefits of coordinated engineering approaches that include pdxA optimization alongside traditional PHA pathway engineering.

What analytical methods are most appropriate for evaluating the impact of recombinant pdxA on cellular metabolism in Pseudomonas putida?

Comprehensive analysis of recombinant pdxA's impact on cellular metabolism requires a multi-omics approach combined with targeted biochemical assays:

Analytical Framework:

• Transcriptomics:

  • RNA-Seq to profile global gene expression changes

  • RT-qPCR for targeted verification of key metabolic genes

  • Focus on vitamin B6-dependent pathways and connected metabolic networks

• Proteomics:

  • Label-free quantitative proteomics to identify protein abundance changes

  • 2D-PAGE for visualization of major proteome alterations

  • Targeted analysis of PLP-dependent enzymes and their activity states

• Metabolomics:

  • LC-MS/MS for comprehensive metabolite profiling

  • Targeted analysis of:

    • Vitamin B6 vitamers (pyridoxal, pyridoxamine, pyridoxine and their phosphorylated forms)

    • Amino acid pools

    • Central carbon metabolites

    • PHA precursors and intermediates

• Flux Analysis:

  • ¹³C metabolic flux analysis using labeled substrates

  • Metabolic flux ratio analysis to determine pathway activities

  • Isotopomer analysis to track carbon flow through central metabolism

• Enzymatic Assays:

  • Direct measurement of pdxA activity in cell extracts

  • Activity profiling of key PLP-dependent enzymes

  • Determination of NAD⁺/NADH and NADP⁺/NADPH ratios

Data Analysis and Integration:

Data Visualization and Reporting:

Results should be presented using:

• Heatmaps for transcriptomic and proteomic changes

• Pathway maps highlighting flux distributions

• Bar charts comparing metabolite concentrations

• Network diagrams illustrating metabolic interactions

For pathway analysis, consider using tools like KEGG Mapper, MetaCyc, or BioCyc to contextualize findings within the broader metabolic network of P. putida.

This comprehensive analytical approach will provide detailed insights into how recombinant pdxA expression influences not only vitamin B6 metabolism but also broader cellular processes including amino acid metabolism, central carbon pathways, and potentially PHA biosynthesis.

What are the common experimental challenges when working with recombinant pdxA and how can they be addressed?

Researchers working with recombinant pdxA often encounter several technical challenges that can be addressed with specific optimization strategies:

Challenge 1: Low Expression Levels

Challenge 2: Inclusion Body Formation

Challenge 3: Low Enzyme Activity

Challenge 4: Enzymatic Assay Limitations

Challenge 5: Physiological Impact of Overexpression

Practical Example:

How can researchers address substrate availability challenges when studying recombinant pdxA activity?

The specific substrate for pdxA, 4-hydroxy-L-threonine phosphate (HTP), presents significant challenges for researchers due to its limited commercial availability and stability. Here are methodological approaches to address these challenges:

Substrate Acquisition Strategies:

• Chemical Synthesis of HTP:

  • Multi-step synthesis beginning with L-threonine

  • Protection of amino groups followed by selective phosphorylation

  • Careful deprotection under mild conditions

  • Purification by ion-exchange chromatography

• Enzymatic Synthesis:

  • Use recombinant PdxB (4-hydroxythreonine synthase) to convert D-erythrose 4-phosphate to HTP

  • Coupled enzyme reaction systems for in situ substrate generation

  • One-pot enzymatic cascades starting from more readily available precursors

• In Vivo Substrate Generation:

  • Engineer E. coli strains to accumulate and export HTP

  • Use cell extracts from these strains as substrate sources

  • Develop extraction and enrichment protocols for HTP

Quality Control Protocols:

Alternative Assay Approaches:

• Coupled Enzyme Assays:

  • Link pdxA activity to other enzymatic reactions with easily detectable products

  • Monitor NAD+/NADP+ reduction spectrophotometrically

  • Develop fluorescent or bioluminescent detection systems

• Surrogate Substrates:

  • Screen HTP analogs with similar structural features

  • Develop structure-activity relationships

  • Validate with wild-type and mutant enzymes

• Whole-Cell Biocatalysis:

  • Express complete vitamin B6 pathway in recombinant P. putida

  • Feed cells with pathway precursors

  • Measure end-product formation as proxy for pdxA activity

Practical Implementation Strategy:

For optimal results, researchers should consider establishing a reliable substrate supply chain that combines:

• Small-scale in-house synthesis for routine experiments

• Collaboration with specialized chemical biology groups for larger quantities

• Development of stable substrate stock solutions with validated shelf-life

• Standard operating procedures for substrate quality verification before critical experiments

This multi-faceted approach ensures consistent and reliable substrate availability, enabling reproducible studies of recombinant pdxA activity across different experimental conditions.

How can high-throughput screening be implemented to identify improved variants of pdxA for metabolic engineering applications?

Implementing high-throughput screening for improved pdxA variants requires a carefully designed workflow that combines molecular diversity generation with efficient functional analysis:

Comprehensive Screening Framework:

• Diversity Generation Strategies:

  • Error-prone PCR with controlled mutation rates

  • DNA shuffling of pdxA homologs from various species

  • Site-saturation mutagenesis targeting active site residues

  • CRISPR-based in vivo mutagenesis

• Library Construction in P. putida:

  • Vector-based expression libraries

  • Chromosomal integration libraries using transposon systems

  • TREX-based random insertion approach for position effect screening

• Primary Screening Methods:

  • Growth-based selection under vitamin B6 limitation

  • Colorimetric assays for PLP production

  • NAD+/NADP+ reduction monitoring in whole cells

  • Fluorescent reporter systems linked to vitamin B6 production

• Secondary Validation Assays:

  • Quantitative enzyme activity measurements

  • Thermal stability determination

  • pH and ionic strength tolerance testing

  • Substrate specificity profiling

Automation and Throughput Enhancement:

Data Analysis and Variant Selection:

• Machine Learning Approaches:

  • Train algorithms on sequence-function relationships

  • Predict promising mutations for next generation libraries

  • Classify variants based on multiple parameters

• Statistical Analysis:

  • Calculate Z-factors for assay quality control

  • Implement appropriate threshold selection

  • Account for position effects and expression variability

• Data Visualization:

  • Activity heat maps across mutation positions

  • Sequence-activity landscapes

  • Structure-guided mutation analysis

Example Successful Implementation:

In a related approach with T7-like expression systems, researchers identified optimal integration sites in P. putida through systematic screening. Integration of the RNA polymerase at the phaC1 locus showed 1.4-fold improvement in heterologous protein expression compared to plasmid-based expression . This example demonstrates how positional effects can significantly impact heterologous enzyme performance.

Similarly, when selecting for constitutive prodigiosin production in P. putida, researchers found that integration into ribosomal RNA genes resulted in significantly higher expression levels compared to T7 RNA polymerase-dependent expression systems . This strategy could be adapted for pdxA expression optimization.

What are the potential applications of recombinant pdxA in creating vitamin B6-overproducing Pseudomonas putida strains for industrial applications?

Developing vitamin B6-overproducing Pseudomonas putida strains has significant potential for industrial applications, extending beyond traditional microbial production platforms:

Industrial Applications and Market Opportunities:

• Pharmaceutical Industry:

  • Production of high-purity vitamin B6 (pyridoxal 5'-phosphate)

  • Development of enzymatically active vitamin B6 derivatives

  • Production of isotopically labeled vitamins for research

• Food and Feed Industries:

  • Natural vitamin B6 fortification for animal feed

  • Production of vitamin B6-enriched biomass as food supplement

  • Development of fermentation starter cultures with enhanced B6 content

• Biotechnological Applications:

  • Creation of whole-cell biocatalysts with enhanced cofactor availability

  • Development of PLP-regeneration systems for biocatalysis

  • Engineering synthetic metabolic pathways requiring PLP-dependent enzymes

Strain Development Roadmap:

Technological Advantages of P. putida as Production Host:

• Metabolic Versatility:

  • Ability to utilize various carbon sources

  • Natural stress tolerance mechanisms

  • Efficient redox balance maintenance

• Genetic Stability:

  • Stable maintenance of heterologous genes

  • Low mutation rates during long-term cultivation

  • Tolerance to metabolic engineering modifications

• Bioprocess Compatibility:

  • Growth in mineral media without expensive supplements

  • Tolerance to industrial conditions (pH variations, inhibitors)

  • Non-pathogenic status (GRAS potential)

Current Research Gaps and Future Directions:

To fully realize the potential of P. putida as a vitamin B6 production platform, several key research areas need further development:

• Understanding and alleviating potential toxicity of high intracellular PLP concentrations

• Developing export mechanisms for efficient product release

• Integrating vitamin B6 overproduction with other valuable metabolic capabilities of P. putida

• Addressing regulatory and scale-up challenges for industrial implementation

By systematically addressing these challenges, recombinant pdxA-expressing P. putida strains could become valuable industrial platforms for sustainable vitamin B6 production.

How does Pseudomonas putida pdxA compare structurally and functionally with similar enzymes from other bacterial species?

Comparative analysis of pdxA across bacterial species reveals important structural and functional similarities and differences that can inform engineering strategies:

Structural Comparison of pdxA/PdxA Across Species:

Sequence Conservation Analysis:

Alignment of pdxA sequences from diverse bacterial species shows:

• Highly conserved metal-binding histidine residues

• Well-preserved catalytic residues (Asp247, Asp267 in E. coli)

• Variable regions in substrate recognition domains

• Conserved dimer interface residues

Functional Comparison:

Physiological Roles and Contextual Differences:

PdxA functions in the vitamin B6 biosynthetic pathway across all these species, but with notable differences:

• In C. jejuni, pdxA affects flagellum-mediated motility through PLP-dependent mechanisms

• In P. putida, metabolic context suggests potential interactions with PHA metabolism pathways

• E. coli pdxA is well-characterized for its role in vitamin B6 metabolism

• Some species have alternative pathways for vitamin B6 synthesis independent of pdxA

Implications for Engineering:

• Selection of Donor Sequences:

  • E. coli pdxA is well-characterized and could serve as a reference enzyme

  • Thermophilic bacteria may provide more stable pdxA variants

  • Organisms with more efficient vitamin B6 synthesis might offer superior pdxA genes

• Targeting Conserved vs. Variable Regions:

  • Highly conserved residues likely essential for function

  • Variable regions might be targeted for specificity engineering

  • Species-specific regulatory elements could be exploited

• Domain Swapping Potential:

  • Substrate binding domains from diverse species could be swapped

  • Metal-binding regions should be preserved

  • Cofactor preference regions could be modified based on availability

This comparative analysis provides a foundation for rational engineering approaches that leverage natural diversity in pdxA structure and function across bacterial species.

How do different expression systems impact the production and activity of recombinant pdxA in Pseudomonas putida?

Different expression systems significantly impact recombinant pdxA production and activity in Pseudomonas putida. Understanding these differences is crucial for optimal experimental design:

Comparison of Expression Systems for Recombinant Proteins in P. putida:

Performance Metrics Across Expression Systems:

Based on research with similar recombinant proteins in P. putida, the following performance trends can be anticipated for pdxA expression:

Integration Sites for Chromosomal Expression:

For chromosomal integration of pdxA, site selection significantly impacts expression levels:

• Site-Specific Effects:

  • Integration at phaC1 locus showed 1.4-fold improvement in protein expression compared to plasmid-based expression

  • Integration at rRNA genes resulted in strong constitutive expression

  • Multiple integration sites did not improve expression and sometimes reduced it

• Integration Strategies:

  • Transposon-based random integration followed by screening

  • CRISPR-Cas9 targeted integration at specific loci

  • Homologous recombination at predetermined sites

Practical Recommendations Based on Application:

When expressing pdxA specifically, the optimal system should be selected based on whether the goal is to study the enzyme itself (favoring high, controllable expression) or to engineer metabolic pathways (favoring balanced, physiologically appropriate expression levels).

For maximum expression, the T7-like system with a single copy integrated at the phaC1 locus achieved expression levels 2.5 times higher than other inducible systems in P. putida KT2440 , making this an excellent choice for applications requiring high pdxA levels.