Recombinant Flaveria bidentis Nucleoside diphosphate kinase B

What is Nucleoside Diphosphate Kinase B and how was it isolated from Flaveria bidentis?

Nucleoside diphosphate kinase B is one of two NDPK cDNA clones isolated from Flaveria bidentis through immunoscreening of an expression library. The isolation used a polyclonal antibody raised against Flaveria chloraefolia flavonol 3-sulfotransferase (F3-ST). These clones represent members of a small multigene family in F. bidentis, with their nucleotide sequences showing high similarity to other reported NDPKs, including the human tumor suppressor gene NM23 and the Drosophila regulatory gene . The enzyme plays a critical role in nucleotide metabolism by catalyzing the transfer of phosphate groups between different nucleoside diphosphates and triphosphates.

How does Flaveria bidentis NDPK B compare to NDPKs from other species?

F. bidentis NDPK B shares significant sequence homology with NDPK enzymes from other organisms, particularly with the human tumor suppressor gene NM23 and Drosophila regulatory gene . This conservation suggests fundamental evolutionary importance across diverse taxa. When comparing NDPK sequences among Flaveria species, relatively high conservation is observed, with F. bidentis NDPK showing particularly close similarity to F. trinervia NDPK. For example, the PEPC-PK gene from F. bidentis differs from that of F. trinervia by only five nucleotide residues in the coding region, resulting in just three amino acid substitutions . This high degree of conservation indicates the enzyme's critical role in basic cellular metabolism across the genus.

What is the relationship between F. bidentis as a C4 plant and its NDPK expression patterns?

F. bidentis is a C4 photosynthetic species within the Flaveria genus, which contains species spanning C3, C4, and intermediate C3-C4 photosynthetic pathways. Transcriptomic analyses comparing F. bidentis (C4) with C3 species such as F. pringlei reveal differential gene expression patterns associated with these distinct photosynthetic mechanisms . While not specifically focused on NDPK B, these comparative studies provide valuable context for understanding how metabolic enzymes like NDPK may be regulated differently in C4 versus C3 plants. The adenosine monophosphate kinase gene, functionally related to nucleotide metabolism like NDPK, was found to be strongly upregulated in C4 Flaveria species compared to C3 relatives , suggesting potential similar regulatory patterns for NDPK in these different photosynthetic types.

What expression systems have been successfully used for recombinant F. bidentis NDPK B production?

Escherichia coli has been successfully used as an expression system for F. bidentis NDPK B. When the cDNA clones were expressed in E. coli, the recombinant proteins exhibited NDPK enzymatic activity . This bacterial expression system represents the most well-documented approach for producing functional recombinant F. bidentis NDPK. The methodology typically involves:

• Cloning the full-length NDPK B cDNA into an appropriate bacterial expression vector

• Transforming E. coli cells with the recombinant construct

• Inducing protein expression under optimized conditions

• Purifying the recombinant protein using affinity chromatography

• Verifying enzymatic activity through phosphorylation assays

How can researchers optimize the expression and purification of recombinant F. bidentis NDPK B?

Optimization of recombinant F. bidentis NDPK B production should consider:

• Vector selection: Using expression vectors with strong, inducible promoters (such as T7) for controlled expression

• Host strain selection: BL21(DE3) or its derivatives are typically preferred for recombinant protein expression

• Induction parameters: Optimizing IPTG concentration, induction temperature (often lowered to 16-25°C), and induction duration to enhance soluble protein yield

• Fusion tags: Incorporating affinity tags (His6, GST, or MBP) to facilitate purification and potentially enhance solubility

• Buffer optimization: Testing various buffer compositions for extraction and purification to maintain enzyme stability and activity

While specific optimization parameters for F. bidentis NDPK B aren't detailed in the available literature, successful expression in E. coli has been documented , suggesting standard protein expression protocols are applicable with appropriate modifications.

What are the standard methods for assessing F. bidentis NDPK B enzymatic activity?

Standard methods for assessing nucleoside diphosphate kinase activity include:

• Coupled enzyme assays: Measuring the production of ATP from other nucleoside triphosphates (e.g., GTP + ADP → GDP + ATP) using luciferase or other ATP-dependent enzymes

• Direct phosphorylation assays: Monitoring the transfer of radiolabeled phosphate groups between nucleotides

• HPLC analysis: Quantifying the conversion of substrate to product nucleotides

• Spectrophotometric assays: Coupling NDPK activity to other reactions that produce measurable spectrophotometric changes

For F. bidentis NDPK specifically, activity has been verified when expressed in E. coli systems , confirming the functionality of the recombinant enzyme.

What structural features of F. bidentis NDPK B contribute to its function and interactions?

While the specific three-dimensional structure of F. bidentis NDPK B has not been reported in the available literature, insights can be drawn from the general structural characteristics of NDPKs and the immunocross-reaction observed between F. bidentis NDPK and the antibody raised against F. chloraefolia flavonol 3-sulfotransferase (F3-ST). This cross-reactivity suggests a common immuno-epitope and similarity in nucleotide binding sites between these proteins .

NDPKs typically feature a conserved nucleotide binding pocket and catalytic residues essential for phosphate transfer. The sequence similarity between F. bidentis NDPK B and other NDPKs, including the human NM23 gene product , suggests conservation of these critical structural elements. Techniques such as X-ray crystallography or homology modeling would be valuable for elucidating the specific structural features of this enzyme.

What protein-protein interactions have been identified for F. bidentis NDPK B?

In other organisms, NDPKs are known to interact with various proteins involved in signaling, metabolism, and gene regulation. Given the sequence similarity between F. bidentis NDPK B and the human tumor suppressor gene NM23 , which has multiple interaction partners, it is reasonable to hypothesize that F. bidentis NDPK B may similarly participate in protein complexes involved in nucleotide metabolism and potentially other cellular processes.

What is the role of NDPK B in C4 photosynthesis of Flaveria bidentis?

C4 photosynthesis in Flaveria involves a two-celled system where phosphoenolpyruvate carboxylase (PEPC) initially fixes carbon in mesophyll cells, followed by decarboxylation and refixation by RuBisCO in bundle sheath cells . This process requires significant energy in the form of ATP and involves numerous phosphorylation events. NDPKs play a role in maintaining balanced nucleotide pools by catalyzing the transfer of the terminal phosphate from nucleoside triphosphates to nucleoside diphosphates. This function could be particularly important in supporting the energetic demands and signaling processes of C4 photosynthesis.

How does NDPK B expression compare across different Flaveria species with varying photosynthetic pathways?

Comprehensive transcriptome analyses have been conducted across multiple Flaveria species representing different photosynthetic pathways (C3, C4, and intermediate C3-C4). These studies have identified differential expression patterns for numerous genes involved in photosynthesis and metabolism .

While the search results don't explicitly detail NDPK B expression patterns across different Flaveria species, the transcriptome data could be mined to extract this information. The comparative transcriptome analysis of F. bidentis (C4) and F. pringlei (C3), along with the more extensive comparison of F. trinervia (C4), F. robusta (C3), and F. ramosissima (C3-C4) , provides a valuable resource for investigating differential expression of metabolic enzymes like NDPK B.

From the available data tables in the search results, we can see that photosynthesis-related genes show varying coverage across the different Flaveria species, with generally high detection rates for most functional classes :

How can recombinant F. bidentis NDPK B be used in studying C4 photosynthesis mechanisms?

Recombinant F. bidentis NDPK B can serve as a valuable tool for investigating C4 photosynthesis mechanisms through several experimental approaches:

• Metabolic engineering: Overexpressing or silencing NDPK B in both C3 and C4 plants to assess its impact on photosynthetic efficiency and nucleotide metabolism

• Protein-protein interaction studies: Using recombinant NDPK B in co-immunoprecipitation or yeast two-hybrid experiments to identify interaction partners specific to C4 metabolism

• In vitro reconstitution: Combining recombinant NDPK B with other C4 photosynthetic enzymes to recreate and study metabolic flux in controlled conditions

• Structural biology: Determining the three-dimensional structure of F. bidentis NDPK B to understand potential C4-specific adaptations

Similar approaches have been successfully employed with other F. bidentis enzymes, such as carbonic anhydrase, which was overexpressed in Arabidopsis thaliana (a C3 plant) to enhance photosynthetic efficiency .

What gene transformation methods have been successful for Flaveria bidentis enzymes?

Successful genetic transformation methods for Flaveria bidentis have been documented in the literature. For instance, antisense and RNA interference (RNAi) constructs targeting the phosphoenolpyruvate carboxylase kinase (PEPC-PK) gene were successfully used to transform F. bidentis . The transformation process typically involves:

• Vector construction: Full-length cDNA clones are isolated from F. bidentis leaf cDNA libraries and cloned into appropriate transformation vectors (e.g., antisense or RNAi plasmids)

• Transformation method: Agrobacterium-mediated transformation has been successfully employed for Flaveria species, similar to its application in Arabidopsis

• Selection: Transgenic plants are selected using appropriate markers (e.g., kanamycin resistance via the nptII gene)

• Confirmation: Integration of the transgene is confirmed through PCR, Southern blot analysis, qRT-PCR, and western blot analysis

These established transformation protocols provide a foundation for genetic manipulation of NDPK B in Flaveria bidentis for functional studies.

How might F. bidentis NDPK B contribute to C4 rice engineering efforts?

F. bidentis NDPK B could potentially contribute to C4 rice engineering efforts as part of the broader initiative to improve photosynthetic efficiency in C3 crops. While not the primary target enzyme for C4 engineering, understanding the role of supporting metabolic enzymes like NDPK B in efficient C4 species provides valuable context for comprehensive pathway engineering.

Similar approaches have already shown promise with other F. bidentis enzymes. For example, overexpression of cytoplasmic C4 Flaveria bidentis carbonic anhydrase in C3 Arabidopsis thaliana increased photosynthetic potential and biomass . This demonstrates the potential of transferring enzymes from C4 Flaveria bidentis to enhance photosynthetic performance in C3 plants.

For C4 rice engineering specifically, researchers might consider:

• Analyzing how NDPK B interacts with core C4 enzymes in F. bidentis

• Determining if C4-specific isoforms or expression patterns of NDPK B exist that could support higher energy demands of C4 photosynthesis

• Including NDPK B in multigene transformation approaches for comprehensive pathway engineering

What are the challenges in studying protein-protein interactions involving F. bidentis NDPK B?

Studying protein-protein interactions involving F. bidentis NDPK B presents several technical and biological challenges:

• Limited background information: The specific interaction partners of F. bidentis NDPK B have not been well characterized, making it difficult to design targeted interaction studies

• Expression system compatibility: While E. coli expression systems have been successful for producing functional recombinant NDPK B , these prokaryotic systems may lack plant-specific post-translational modifications that could influence interactions

• Plant-specific technical challenges: Working with plant proteins often involves complications from cell wall materials, phenolic compounds, and proteases that can interfere with interaction assays

• Subcellular localization considerations: Determining the precise subcellular localization of NDPK B is crucial for identifying physiologically relevant interaction partners

• C4-specific context: Potential interactions may differ between C3 and C4 species, requiring comparative approaches across multiple Flaveria species

Advanced techniques to address these challenges include:

• Bimolecular fluorescence complementation (BiFC) in plant protoplasts

• Split-ubiquitin yeast two-hybrid systems optimized for membrane proteins

• Proximity labeling methods (BioID, APEX) in transgenic plants

• Protein complex immunoprecipitation followed by mass spectrometry

How can comparative genomics and proteomics advance our understanding of NDPK B evolution in the Flaveria genus?

Comparative genomics and proteomics offer powerful approaches to understand NDPK B evolution in the Flaveria genus, particularly in the context of C3 to C4 photosynthetic pathway evolution. The Flaveria genus presents an exceptional model system, containing species spanning the evolutionary continuum from C3 to C4 photosynthesis .

Existing transcriptome data from multiple Flaveria species provides a foundation for these comparative analyses. For example, transcriptome sequencing has been performed on F. bidentis (C4), F. pringlei (C3), F. trinervia (C4), F. robusta (C3), and F. ramosissima (C3-C4) . This data could be specifically analyzed to:

• Identify NDPK isoforms across species: Compare the number and sequence of NDPK genes across the photosynthetic gradient

• Analyze expression patterns: Determine if NDPK B expression levels or timing correlates with photosynthetic type

• Detect selection signatures: Identify amino acid residues under positive selection that might represent functional adaptations

• Reconstruct evolutionary history: Determine if NDPK B shows parallel evolutionary patterns to core C4 photosynthetic genes

Advanced proteomics approaches could complement genomic analyses by:

• Quantifying NDPK protein abundance across species

• Identifying post-translational modifications specific to C4 species

• Determining protein turnover rates in different photosynthetic types

• Characterizing NDPK-containing protein complexes in various Flaveria species

What are common challenges in expressing active recombinant F. bidentis NDPK B?

Common challenges researchers may encounter when expressing active recombinant F. bidentis NDPK B include:

• Protein solubility issues: Recombinant NDPKs may form inclusion bodies in bacterial expression systems

• Loss of enzymatic activity: Improper folding or absence of post-translational modifications may affect activity

• Protein stability concerns: The enzyme may show reduced stability during purification or storage

• Contaminating kinase activities: Host cell kinases may interfere with activity assays

• Oligomerization problems: Many NDPKs function as oligomers, and failure to form proper quaternary structure may affect activity

Suggested solutions include:

• Optimizing expression conditions (lower temperature, reduced inducer concentration)

• Testing different fusion tags (His, GST, MBP) to enhance solubility

• Using specialized E. coli strains designed for difficult protein expression

• Including stabilizing agents in purification buffers

• Careful design of activity assays with appropriate controls

How can researchers troubleshoot inconsistent results in NDPK activity assays?

Inconsistent results in NDPK activity assays may stem from several sources. Researchers can troubleshoot these issues using the following strategies:

• Enzyme stability issues:

  • Store the enzyme with stabilizers (glycerol, reducing agents)

  • Aliquot and minimize freeze-thaw cycles

  • Test fresh preparations versus stored samples

• Assay component variability:

  • Use high-purity nucleotide substrates

  • Prepare fresh ATP/ADP solutions regularly

  • Control for metal ion concentrations (Mg2+, Mn2+)

  • Include chelating agents to control free metal ions

• Interfering activities:

  • Include controls for spontaneous nucleotide hydrolysis

  • Test for ATPase or phosphatase contamination

  • Use specific NDPK inhibitors as controls

• Detection method limitations:

  • Validate coupled enzyme systems independently

  • Ensure linear range of detection methods

  • Include internal standards when possible

• Environmental variables:

  • Strictly control temperature during assays

  • Monitor and adjust pH regularly

  • Test buffer composition effects on activity

Careful documentation of all experimental conditions and systematic variation of individual parameters will help identify the source of inconsistency.

How does F. bidentis NDPK B compare to NDPKs from model plant species?

F. bidentis NDPK B likely shares fundamental catalytic mechanisms and structural features with NDPKs from model plant species, given the high conservation of these enzymes across diverse organisms. While specific comparative analyses between F. bidentis NDPK B and model plant NDPKs are not detailed in the available literature, general comparisons can be made based on known NDPK characteristics.

NDPKs from model plants like Arabidopsis thaliana and rice (Oryza sativa) typically exist as small gene families with members showing differential subcellular localization (cytosolic, chloroplastic, mitochondrial). The expression of these isoforms is often tissue-specific and responsive to developmental and environmental cues. The isolation of multiple NDPK cDNA clones from F. bidentis suggests it similarly possesses a small multigene family for these enzymes .

A comprehensive comparative analysis would involve:

• Sequence alignments to identify conserved catalytic residues and regulatory motifs

• Phylogenetic analyses to determine evolutionary relationships

• Expression pattern comparisons across tissues and conditions

• Functional complementation studies in model systems

What unique features might distinguish C4 plant NDPKs from their C3 counterparts?

While specific distinguishing features of C4 plant NDPKs compared to C3 counterparts haven't been explicitly documented in the available literature, several potential differences can be hypothesized based on the metabolic demands of C4 photosynthesis:

• Expression patterns: NDPKs in C4 plants might show cell-specific expression patterns aligned with the specialized bundle sheath and mesophyll cells involved in C4 photosynthesis

• Kinetic properties: C4 NDPKs might possess altered kinetic parameters optimized for the higher energy demands of C4 photosynthesis

• Regulatory mechanisms: Different post-translational modifications or regulatory mechanisms might exist to coordinate NDPK activity with the C4 carbon fixation process

• Protein interactions: C4 NDPKs might interact with C4-specific enzymes or regulatory proteins

The transcriptome analyses comparing various Flaveria species with different photosynthetic pathways provide a valuable resource for investigating such differences. For instance, the adenosine monophosphate kinase gene was found to be strongly upregulated in C4 Flaveria species , suggesting that nucleotide metabolism enzymes may indeed show C4-specific expression patterns.

What resources are available for researchers studying F. bidentis NDPK B?

Researchers studying F. bidentis NDPK B can access several valuable resources:

• Genetic material:

  • cDNA clones of F. bidentis NDPK have been isolated and characterized

  • Full-length NDPK-PK cDNA from F. bidentis has been isolated from leaf cDNA libraries (accession no. AB272061)

• Sequence data:

  • Nucleotide sequences for F. bidentis NDPK are available in public databases

  • Transcriptome data from F. bidentis and related Flaveria species is available for comparative analyses

• Experimental protocols:

  • Methods for expressing recombinant F. bidentis proteins in E. coli have been established

  • Transformation protocols for F. bidentis using antisense or RNAi constructs have been developed

  • Techniques for analyzing transgene integration and expression in Flaveria have been documented

• Comparative datasets:

  • Transcriptome data comparing gene expression across multiple Flaveria species with different photosynthetic pathways

  • Detailed coverage statistics for different functional gene classes across Flaveria species

What are recommended experimental designs for studying NDPK B function in Flaveria species?

Recommended experimental designs for studying NDPK B function in Flaveria species include:

• Comparative expression analysis:

  • Quantify NDPK B transcript and protein levels across multiple Flaveria species (C3, C3-C4, C4)

  • Analyze expression in different tissues and at different developmental stages

  • Investigate expression responses to environmental stimuli (light, temperature, CO2)

• Genetic manipulation approaches:

  • Overexpress F. bidentis NDPK B in both C3 and C4 Flaveria species

  • Use antisense or RNAi constructs to silence NDPK B expression

  • Employ CRISPR/Cas9 for precise genome editing if protocols are available

• Protein interaction studies:

  • Perform co-immunoprecipitation experiments with tagged NDPK B

  • Use yeast two-hybrid or split-ubiquitin systems to screen for interaction partners

  • Employ proximity labeling techniques in transgenic plants

• Metabolic impact assessment:

  • Measure nucleotide pools in wild-type versus NDPK B-modified plants

  • Analyze photosynthetic parameters (gas exchange, chlorophyll fluorescence)

  • Quantify growth and biomass accumulation under various conditions

• Heterologous expression studies:

  • Express F. bidentis NDPK B in model C3 plants like Arabidopsis thaliana

  • Assess phenotypic and metabolic impacts, similar to studies with F. bidentis carbonic anhydrase

These experimental approaches should be designed with appropriate controls and statistical considerations to ensure robust and reproducible results.