Recombinant Synechococcus sp. Nucleoside diphosphate kinase (ndk)

What is the fundamental function of nucleoside diphosphate kinase in Synechococcus sp.?

Nucleoside diphosphate kinase (ndk) in Synechococcus sp. plays a major role in the synthesis of nucleoside triphosphates other than ATP. The enzyme catalyzes the transfer of the gamma phosphate from ATP to the beta phosphate of nucleoside diphosphates via a ping-pong mechanism, using a phosphorylated active-site intermediate . This reaction is critical for maintaining balanced pools of various nucleoside triphosphates required for DNA replication, RNA synthesis, and protein translation. In Synechococcus, as in other organisms, ndk helps coordinate metabolic processes by ensuring appropriate nucleotide availability for different cellular functions. The enzyme belongs to the NDK family, which is highly conserved across prokaryotes and eukaryotes, indicating its fundamental importance in cellular metabolism .

How does ndk structure relate to its catalytic function in Synechococcus?

In Synechococcus sp., ndk typically exists as a hexameric protein composed of identical subunits of approximately 149 amino acids each . The active site contains a conserved histidine residue that becomes phosphorylated during the catalytic cycle, forming a high-energy phosphohistidine intermediate. This phosphate group is subsequently transferred to the acceptor nucleoside diphosphate substrate. The enzyme's quaternary structure is essential for its function, with the hexameric arrangement providing stability and creating an optimal environment for catalysis. Key structural elements include:

• The nucleotide-binding pocket that recognizes both purine and pyrimidine nucleotides

• A conserved active site with the catalytic histidine residue

• Interfaces between subunits that stabilize the hexameric structure

• Surface features that may mediate interactions with other cellular components

These structural characteristics enable ndk to efficiently maintain balanced nucleotide pools in Synechococcus cells.

What is known about the regulation of ndk expression in Synechococcus?

The regulation of ndk expression in Synechococcus likely involves multiple mechanisms responding to cellular energy status, nutrient availability, and environmental conditions. In thermophilic Synechococcus strains from hot spring microbial mats, many metabolic enzymes show diel (day-night) expression patterns . While ndk-specific expression patterns are not directly reported in the search results, similar temporal regulation may occur to coordinate nucleotide synthesis with photosynthetic activity and other metabolic processes that fluctuate over the diel cycle.

Phosphorus availability may also influence ndk expression, as Synechococcus OS-B′ induces a suite of genes involved in phosphorus acquisition and metabolism under phosphate-limited conditions . Given the central role of phosphate in nucleotide metabolism, ndk expression and activity may be coordinated with the broader phosphorus management network in these organisms.

Additionally, stress conditions such as high light and high temperature may trigger adjustments in ndk expression as part of the broader metabolic adaptation response in Synechococcus .

How does ndk interact with other metabolic pathways in Synechococcus?

Nucleoside diphosphate kinase occupies a central position in Synechococcus metabolism, connecting multiple pathways through its role in nucleotide interconversion. STRING database analysis reveals strong predicted functional interactions between ndk and various ribosomal proteins in Synechococcus sp. JA33Ab, including rpsJ, rpsO, rpsB, rpsP, rpsL, and rpsG, with interaction scores above 0.99 . These associations suggest a potential role for ndk in coordinating nucleotide availability with protein synthesis machinery.

Additional metabolic connections likely include:

• Photosynthetic electron transport, which generates ATP used by ndk

• Nucleotide salvage pathways that provide substrate nucleoside diphosphates

• Polyphosphate metabolism, which serves as a phosphate storage system in Synechococcus

• Cell division processes requiring GTP for FtsZ polymerization

• RNA synthesis pathways utilizing various nucleoside triphosphates

These interconnections position ndk as a key integrator of energy metabolism, nucleotide homeostasis, and macromolecular synthesis in Synechococcus.

What are the optimal expression systems for producing recombinant Synechococcus sp. ndk?

The selection of an appropriate expression system for recombinant Synechococcus sp. ndk depends on the specific research objectives and the properties of the particular ndk variant. Based on approaches used for similar cyanobacterial proteins, several systems warrant consideration:

E. coli-based expression systems:

• pET series vectors with T7 promoter control for high-level expression

• Cold-shock expression vectors (pCold) for improved folding

• Co-expression with molecular chaperones to enhance solubility

• Codon optimization for E. coli if rare codons are present in the Synechococcus gene

Cyanobacterial expression systems:

• Homologous expression in Synechococcus using native or strong synthetic promoters

• Heterologous expression in model cyanobacteria such as Synechocystis sp. PCC 6803

For thermophilic Synechococcus ndk variants, expression conditions should be optimized to accommodate their unique folding requirements. The development of transformation protocols for thermophilic Synechococcus strains, as demonstrated for other genes , provides additional options for homologous expression of ndk in its native context. Expression in E. coli typically yields higher protein amounts but may require additional optimization for proper folding and activity.

How can researchers distinguish between different ndk isoforms or variants in Synechococcus samples?

Distinguishing between different ndk isoforms or variants in Synechococcus samples requires a multi-faceted approach:

Genomic and proteomic techniques:

• PCR amplification with isoform-specific primers targeting unique sequence regions

• Mass spectrometry to identify peptide sequences unique to specific variants

• Antibodies raised against isoform-specific epitopes for immunoblotting

• 2D gel electrophoresis to separate variants based on both pI and molecular weight

Functional approaches:

• Kinetic characterization with various substrates to identify catalytic differences

• Thermal stability profiles to distinguish thermophilic from mesophilic variants

• Activity staining following native PAGE to identify functional variants

Structural biology methods:

• Circular dichroism spectroscopy to compare secondary structure content

• Limited proteolysis to identify structural differences affecting protease accessibility

• Differential scanning calorimetry to determine thermal transition profiles

These approaches can be particularly valuable when studying mixed communities of Synechococcus strains or when investigating natural variants with potentially different functional properties. For research involving hot spring microbial mats containing multiple Synechococcus strains , these techniques could help attribute specific ndk activities to different community members.

What role does ndk play in nucleotide homeostasis during diel cycles in Synechococcus?

In thermophilic Synechococcus from hot spring microbial mats, metabolic activities show pronounced diel regulation, with transcripts and proteins for various pathways fluctuating over the 24-hour cycle . While ndk-specific diel patterns are not directly reported in the search results, its role in nucleotide homeostasis likely varies throughout the day-night cycle to support changing metabolic priorities:

Daytime role:

• Utilizing ATP from photosynthesis to generate diverse nucleoside triphosphates

• Supporting DNA replication and RNA synthesis during active growth

• Coordinating with increased phosphate transport activity

Nighttime role:

• Maintaining essential nucleotide pools during non-photosynthetic periods

• Supporting transcription of genes expressed preferentially at night

• Potential coordination with polyphosphate metabolism, as polyphosphate kinase (PPK) levels peak at night in thermophilic Synechococcus

Dawn/dusk transitions:

• Rapid adjustment of nucleotide pools to changing metabolic demands

• Possible coordination with polyphosphatase (PPX) activity, which peaks during early morning hours

This temporal orchestration would allow Synechococcus to optimize resource allocation throughout the diel cycle, ensuring that appropriate nucleotide pools are available to match the changing metabolic priorities from day to night.

How does ndk contribute to stress responses in Synechococcus under extreme conditions?

Nucleoside diphosphate kinase likely plays important roles in stress adaptation in Synechococcus, particularly for thermophilic strains living in extreme environments:

Thermal stress response:

• Maintenance of nucleotide pool balance at high temperatures

• Support of heat shock protein synthesis through GTP provision

• Coordination with other thermotolerance mechanisms

Oxidative stress management:

• Maintenance of GTP pools for antioxidant systems

• Support of redox balance mechanisms through nucleotide homeostasis

• Possible direct roles in ROS scavenging reported in some organisms

Nutrient limitation response:

• Adjustment of activity under phosphate limitation conditions

• Coordination with polyphosphate metabolism, which serves as phosphorus storage in Synechococcus OS-B′

• Optimization of nucleotide synthesis when resources are scarce

High light stress adaptation:

• Support for photosystem repair processes requiring nucleotides

• Coordination with photoprotective mechanisms

• Assistance in metabolic adjustments during combined high light and high temperature stress

The ability of thermophilic Synechococcus strains to thrive in hot spring environments suggests their ndk has evolved to function effectively under these extreme conditions, potentially making it a valuable model for studying enzyme adaptation to thermal stress.

How can site-directed mutagenesis be used to enhance the catalytic properties of Synechococcus ndk?

Site-directed mutagenesis offers powerful approaches for enhancing various properties of Synechococcus ndk:

Enhancing thermal stability:

• Introduction of additional salt bridges through charge optimization

• Substitution of glycine residues with alanine to reduce conformational flexibility

• Introduction of proline residues in loop regions to reduce entropy

• Creation of disulfide bonds to constrain flexible regions

Improving catalytic efficiency:

• Modification of active site residues to optimize substrate binding

• Engineering of the phosphohistidine microenvironment to enhance phosphate transfer

• Alteration of substrate specificity through binding pocket modifications

• Introduction of allosteric regulation sites

Experimental design approach:

• Identify conserved vs. variable residues through sequence alignment of multiple ndk proteins

• Use structural modeling to predict impacts of specific mutations

• Create site-directed mutants using established PCR-based methods

• Express and purify mutant proteins for comparative analysis

• Evaluate thermal stability, catalytic parameters, and substrate preference

Mutagenesis approaches could be particularly valuable for adapting ndk from thermophilic Synechococcus strains for biotechnological applications requiring thermal stability combined with specific catalytic properties. The hypermutation systems developed for Synechococcus could potentially be adapted to accelerate this engineering process through directed evolution approaches.

What are the most effective protocols for purifying active recombinant Synechococcus sp. ndk?

An optimized protocol for purifying active recombinant Synechococcus sp. ndk typically includes:

Expression conditions:

• E. coli BL21(DE3) or Rosetta(DE3) strains for efficient expression

• Induction at OD₆₀₀ = 0.6-0.8 with 0.2-0.5 mM IPTG

• Post-induction growth at 25-30°C for 4-6 hours (mesophilic variants) or 30-37°C for thermophilic variants

• Addition of 5 mM MgCl₂ to the culture medium to stabilize the enzyme

Extraction and initial purification:

• Lysis buffer: 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM MgCl₂, 1 mM DTT, 5% glycerol

• Addition of protease inhibitor cocktail

• Cell disruption by sonication (10 cycles of 15s on/45s off) or pressure homogenization

• Clarification by centrifugation at 15,000 × g for, 30 min at 4°C

Chromatography sequence:

• Immobilized metal affinity chromatography (if His-tagged):

  • Ni-NTA resin with imidazole gradient elution (20-250 mM)

  • Buffer containing 5 mM MgCl₂ throughout

• Size exclusion chromatography:

  • Superdex 200 column to isolate properly formed hexamers

  • Running buffer: 25 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM MgCl₂, 1 mM DTT

• Optional polishing step:

  • Anion exchange chromatography (Q Sepharose)

  • Salt gradient: 50-500 mM NaCl

Quality control:

• SDS-PAGE for purity assessment

• Native PAGE to confirm oligomeric state

• Activity assay at each purification stage

• Dynamic light scattering to confirm homogeneity

• Circular dichroism to verify proper folding

For thermophilic Synechococcus variants, maintaining higher temperatures (30-40°C) during purification steps may improve stability and activity retention. Storage should include 5 mM MgCl₂, with addition of glycerol (20-25%) for long-term preservation at -80°C.

What approaches are most effective for studying ndk protein-protein interactions in Synechococcus?

Investigating ndk protein-protein interactions in Synechococcus requires approaches tailored to cyanobacterial systems:

Affinity-based methods:

• Pull-down assays using recombinant tagged ndk (His, GST, or MBP tags)

• Co-immunoprecipitation with specific anti-ndk antibodies

• Crosslinking combined with mass spectrometry (XL-MS) to capture transient interactions

Protein complementation assays:

• Split-GFP systems adapted for cyanobacterial expression

• Bacterial two-hybrid systems using adenylate cyclase or phage repressor domains

• FRET-based approaches using fluorescent protein fusions

In situ approaches:

• Proximity labeling methods (BioID, APEX) to identify proteins in close proximity to ndk

• Fluorescence microscopy to visualize co-localization of ndk with potential partners

• Immunogold electron microscopy for high-resolution localization studies

Biophysical techniques:

• Surface plasmon resonance (SPR) to quantify binding kinetics

• Isothermal titration calorimetry (ITC) for thermodynamic parameters

• Microscale thermophoresis for interaction studies in complex solutions

Validation strategies:

• Mutational analysis of predicted interaction interfaces

• Competition assays with peptides corresponding to interaction domains

• Functional assays to determine effects of disrupting specific interactions

The STRING database analysis showing strong predicted interactions between ndk and various ribosomal proteins in Synechococcus sp. JA33Ab provides a starting point for targeted investigation of these associations using the techniques outlined above.

How should researchers design experiments to study the impact of temperature on Synechococcus ndk activity?

Designing experiments to study temperature effects on Synechococcus ndk activity should focus on both functional and structural aspects:

Temperature-dependent activity profiles:

• Reaction setup:

  • Standard ndk activity assay buffer with temperature control

  • Temperature range: 20-80°C for thermophilic variants

  • Pre-equilibration of all reagents at each temperature point

  • Inclusion of thermostable coupled enzymes for continuous assays

• Key measurements:

  • Optimal temperature determination (Topt)

  • Temperature coefficient (Q10) calculation

  • Arrhenius plot analysis to determine activation energy

  • Comparative analysis between different Synechococcus ndk variants

Thermal stability assessment:

• Differential scanning calorimetry (DSC):

  • Determine melting temperature (Tm)

  • Assess cooperativity of unfolding

  • Measure enthalpy of denaturation

• Thermal inactivation kinetics:

  • Incubate enzyme at various temperatures

  • Sample at regular intervals for residual activity

  • Calculate inactivation rate constants

  • Determine half-life at different temperatures

Structural studies:

• Temperature-dependent circular dichroism:

  • Monitor secondary structure changes with temperature

  • Detect structural transitions before complete unfolding

• Limited proteolysis at different temperatures:

  • Assess conformational flexibility changes

  • Identify thermally sensitive regions

Comparative analysis:

• Compare thermophilic Synechococcus ndk (e.g., from Yellowstone hot spring strains ) with mesophilic variants

• Correlate structural features with thermal properties

• Identify potential determinants of thermostability

This comprehensive approach provides insights into how temperature affects both the structural integrity and catalytic function of ndk, particularly relevant for thermophilic Synechococcus strains adapted to high-temperature environments.

What genomic tools are available for manipulating ndk in different Synechococcus strains?

Genetic manipulation of ndk in Synechococcus strains can be accomplished using various tools and approaches, with consideration for strain-specific characteristics:

Transformation systems:

• Natural transformation protocols for amenable strains

• Electroporation methods optimized for various Synechococcus strains

• Conjugation-based approaches using helper E. coli strains

• For thermophilic strains, specialized transformation protocols similar to those developed for Synechococcus OS-B′

Vectors and selectable markers:

• Shuttle vectors with appropriate origins of replication

• Thermostable kanamycin resistance cassette (Km Te) for thermophilic strains

• Neutral site integration vectors for stable transformants

• Inducible promoter systems for controlled expression

Gene editing approaches:

• Homologous recombination for gene knockout or replacement

• CRISPR-Cas9 systems adapted for cyanobacterial genomes

• Recombineering methods for precise genomic modifications

• Transposon mutagenesis for random insertional inactivation

Expression control elements:

• Strong constitutive promoters like PcpcB560

• Inducible promoters responsive to metals, nutrients, or light

• Native ndk promoter for physiologically relevant expression

• Synthetic ribosome binding sites optimized for Synechococcus

Mutant screening and analysis:

• Activity-based screening methods for ndk variants

• Phenotypic assays for growth under various conditions

• PCR-based verification of genomic modifications

• Whole-genome sequencing to confirm mutations and detect off-target effects

The development of hypermutation systems in Synechococcus elongatus PCC 7942 through manipulation of DNA repair pathways offers additional tools for generating and screening ndk variants with altered properties. For thermophilic Synechococcus strains from hot springs, consideration must be given to the higher operating temperatures when designing genetic tools and selectable markers .

How can recombinant Synechococcus ndk be applied in nucleotide synthesis biotechnology?

Recombinant Synechococcus ndk offers several advantages for nucleotide synthesis applications:

Enzymatic synthesis of modified nucleotides:

• Production of isotopically labeled nucleotides for NMR studies

• Synthesis of fluorescently tagged nucleotides for imaging applications

• Generation of nucleotide analogs for pharmaceutical research

• Preparation of non-standard nucleotides for aptamer selection

Biocatalysis applications:

• Coupled enzyme systems for regeneration of nucleoside triphosphates

• Continuous-flow bioreactors for nucleotide interconversion

• Scale-up production of specialty nucleotides

Technical advantages of Synechococcus ndk:

• Thermostable variants from hot spring strains enable operation at elevated temperatures

• Tolerance to various reaction conditions compared to mammalian enzymes

• Potential for engineered substrate specificity through directed evolution

• Compatibility with immobilization technologies for reusable systems

Process development considerations:

• Immobilization strategies (covalent attachment, entrapment, cross-linked enzyme aggregates)

• Reactor design for continuous nucleotide production

• Coupling with nucleoside kinases for complete phosphorylation pathways

• Downstream processing for nucleotide purification

The development of efficient expression systems for Synechococcus ndk and protein engineering approaches to enhance stability and activity could make this enzyme particularly valuable for industrial nucleotide synthesis applications.

What role might ndk play in the adaptation of Synechococcus to changing environments?

Nucleoside diphosphate kinase likely serves as an important component in the adaptive response of Synechococcus to environmental changes:

Temperature adaptation:

• Thermophilic Synechococcus strains from hot springs possess thermostable variants of metabolic enzymes

• ndk activity must be maintained across the temperature range experienced in natural habitats

• Thermal stability of ndk affects cellular resilience during temperature fluctuations

Nutrient limitation responses:

• In phosphate-limited environments, ndk activity may be coordinated with polyphosphate metabolism

• Polyphosphate serves as a phosphorus storage compound in Synechococcus OS-B′, with synthesis and degradation showing diel regulation

• ndk likely participates in the careful allocation of phosphorus resources under limiting conditions

Diel cycle adaptation:

• Thermophilic Synechococcus from hot spring mats show complex diel regulation of metabolic pathways

• ndk activity may be temporally regulated to match nucleotide demand with resource availability

• Coordination with photosynthesis, respiration, and nitrogen metabolism throughout the day-night cycle

Environmental stress responses:

• During combined high light and high temperature stress, metabolic adjustments require coordinated nucleotide provision

• ndk likely supports stress response pathways requiring specific nucleotides

• Adaptation to oxidative stress may involve ndk-mediated maintenance of nucleotide pool integrity

Understanding ndk's role in these adaptive processes could provide insights into how cyanobacteria maintain metabolic homeostasis under fluctuating environmental conditions, with potential implications for predicting responses to climate change and extreme environments.

How can structural biology approaches enhance our understanding of Synechococcus ndk?

Structural biology offers powerful tools for understanding the molecular basis of Synechococcus ndk function:

X-ray crystallography:

• High-resolution structures of different conformational states

• Substrate and product complex structures to elucidate binding determinants

• Comparative analysis of thermophilic vs. mesophilic variants

• Identification of structural features contributing to oligomerization

Cryo-electron microscopy:

• Visualization of hexameric assembly

• Analysis of conformational heterogeneity

• Structures of ndk in complex with interacting partners

• Investigation of potential large macromolecular assemblies

NMR spectroscopy:

• Dynamic analysis of protein motion during catalysis

• Investigation of conformational changes upon substrate binding

• Characterization of protein-protein interaction interfaces

• Study of temperature effects on protein dynamics

Computational approaches:

• Molecular dynamics simulations to understand thermal adaptation

• Protein-protein docking with predicted interaction partners

• Evolutionary analysis to identify co-evolving residues

• Virtual screening for potential inhibitors or activators

Integration with functional data:

• Structure-guided mutagenesis to test mechanistic hypotheses

• Correlation of structural features with kinetic parameters

• Mapping of temperature-sensitive regions in thermophilic variants

• Identification of binding interfaces with interaction partners

These structural insights can guide protein engineering efforts to enhance specific properties of Synechococcus ndk and provide a molecular understanding of how these enzymes have adapted to extreme environments such as hot springs .

What emerging technologies might advance research on Synechococcus ndk?

Several emerging technologies hold promise for advancing Synechococcus ndk research:

Advanced genetic tools:

• CRISPR interference (CRISPRi) for tunable gene repression

• Base editing for precise nucleotide substitutions without double-strand breaks

• Engineered hypermutation systems adapted for targeted evolution of ndk

• Single-cell genomics for studying ndk variation in natural populations

Protein engineering approaches:

• Deep mutational scanning to comprehensively map sequence-function relationships

• Computational design tools for enhancing thermostability

• Non-canonical amino acid incorporation for specialized function

• Directed evolution in continuous culture systems

Imaging technologies:

• Super-resolution microscopy for visualizing ndk localization

• Single-molecule tracking to study dynamics in living cells

• Label-free imaging methods for monitoring enzyme activity

• Correlative light and electron microscopy for contextual localization

Synthetic biology applications:

• Cell-free expression systems for rapid ndk variant screening

• Minimal cell systems for studying ndk in simplified contexts

• Biosensor development using ndk-based detection systems

• Integration of ndk into designed metabolic pathways

Multi-omics integration:

• Combined transcriptomic, proteomic, and metabolomic analysis

• Kinetic modeling of ndk in the context of whole-cell metabolism

• Elucidation of temporal dynamics using time-resolved omics

• Network analysis to identify system-level effects of ndk perturbation

These technologies could significantly accelerate understanding of Synechococcus ndk function and application, particularly for thermophilic variants from extreme environments that may possess unique properties valuable for biotechnological applications.