Recombinant Kosmotoga olearia Nucleoside diphosphate kinase (ndk)

What is Kosmotoga olearia and why is its nucleoside diphosphate kinase of interest to researchers?

Kosmotoga olearia is a thermophilic, anaerobic heterotrophic bacterium first isolated from oil production fluid at the Troll B oil platform in the North Sea. It belongs to the order Thermotogales and has the remarkable ability to grow over an exceptionally wide temperature range (20-79°C), with optimal growth at 65°C . This extreme temperature adaptability makes K. olearia's enzymes, including nucleoside diphosphate kinase (ndk), particularly interesting for studying thermal adaptation mechanisms. Nucleoside diphosphate kinase catalyzes the transfer of terminal phosphate groups between nucleoside diphosphates and triphosphates, playing crucial roles in nucleotide metabolism and potentially in other cellular functions, as observed in other bacterial species .

How does K. olearia's broad temperature growth range potentially influence its ndk properties?

K. olearia can grow at temperatures ranging from 20°C to 79°C, with optimal growth at 65°C . This remarkable thermal flexibility suggests its enzymes, including ndk, may possess unique structural features that maintain functionality across diverse thermal conditions. Transcriptomic studies show that K. olearia undergoes significant gene expression changes at different temperatures, with metabolic reprogramming occurring between low and high temperatures . While specific data for ndk expression across temperatures isn't available in the search results, the organism's ability to adjust its molecular machinery suggests the ndk might have evolved specialized features that maintain catalytic efficiency across varying thermal conditions. Research protocols should account for this by examining enzyme activity across the organism's growth temperature range to fully characterize the recombinant protein's properties.

What are the expected biochemical characteristics of K. olearia ndk based on its phylogenetic classification?

Based on phylogenetic analysis, K. olearia belongs to the Thermotogae phylum, specifically within the family Thermotogaceae . Proteins from this phylum often display characteristic conserved sequence indels (CSIs) that differentiate them from other bacterial groups . Though the search results don't specifically describe K. olearia ndk, we can infer that as a thermophilic enzyme, it likely exhibits:

• Enhanced thermostability compared to mesophilic homologs

• Potential structural adaptations such as increased hydrophobic interactions, ionic bonds, or disulfide bridges

• Possible activity across a broader temperature range than typical ndks

• Conservation of the catalytic core structure common to ndks while potentially having unique sequence features characteristic of Thermotogae proteins

Researchers should perform comparative sequence analysis with other bacterial ndks, particularly focusing on conserved regions identified in the Thermotogae phylum, to identify distinctive features of K. olearia ndk.

What expression systems are most suitable for producing recombinant K. olearia ndk?

When selecting an expression system for K. olearia ndk, researchers should consider:

• E. coli-based systems: While the search results don't specifically mention expression of K. olearia ndk, E. coli systems have been successfully used for other recombinant ndks, including those from Leishmania . For thermophilic proteins like K. olearia ndk, E. coli BL21(DE3) with pET vector systems often provide good expression levels.

• Temperature considerations: Since K. olearia is thermophilic with optimal growth at 65°C , expression conditions should be optimized. Consider:

  • Initial expression at lower temperatures (20-30°C) to prevent inclusion body formation

  • Post-induction temperature shifts to improve folding

  • Use of heat-shock promoters or cold-inducible systems for controlled expression

• Codon optimization: The G+C content of K. olearia genomic DNA is 42.5 mol% , which differs from E. coli. Codon optimization of the synthetic gene may improve expression efficiency.

• Solubility enhancement: Consider fusion tags (His6, GST, MBP) that can improve solubility while facilitating purification.

For functional studies, researchers should compare activity of the recombinant enzyme with that of the native form to ensure proper folding and function.

What purification strategy would yield the highest activity for recombinant K. olearia ndk?

A multi-step purification strategy for recombinant K. olearia ndk should consider the thermophilic nature of the source organism:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using a His-tag is recommended for initial purification. Consider using heat treatment (65°C for 10-20 minutes) before chromatography, as K. olearia proteins should remain soluble while many E. coli proteins denature.

• Secondary purification: Size exclusion chromatography or ion exchange chromatography to remove impurities.

• Activity preservation: Throughout purification, maintain:

  • Appropriate pH (around 6.8, based on K. olearia's optimal growth pH)

  • Include stabilizing agents (glycerol, reducing agents)

  • Consider the presence of NaCl (25-30 g/L) to mimic the organism's optimal growth conditions

• Activity assays: Monitor ndk activity throughout purification using standard assays measuring phosphoryl transfer between nucleoside diphosphates and triphosphates. A simple assay system similar to that used for Leishmania ndk could be employed, measuring ATP utilization to produce nucleoside triphosphates in the presence of excess nucleoside diphosphates .

How can temperature affect the functional stability of purified K. olearia ndk?

The temperature stability profile of K. olearia ndk should reflect the organism's broad growth temperature range (20-79°C) . When handling purified enzyme:

• Storage recommendations:

  • Short-term: 4°C in appropriate buffer with stabilizing agents

  • Long-term: -80°C with cryoprotectants (glycerol, trehalose)

  • Avoid repeated freeze-thaw cycles

• Temperature-dependent activity:

  • Expect functional activity across a broad temperature range (20-80°C)

  • Optimal activity likely around 65°C (matching organism's optimal growth)

  • Activity may persist at lower temperatures, reflecting K. olearia's ability to grow at temperatures as low as 20°C

• Thermal inactivation profiling:

  • Determine half-life at various temperatures

  • Compare with mesophilic ndks to quantify thermostability advantage

• Structure-stability relationship:

  • Consider thermal unfolding studies (DSC, CD spectroscopy)

  • Investigate potential temperature-dependent conformational changes

This temperature flexibility might make K. olearia ndk particularly valuable for applications requiring enzymatic activity across varying thermal conditions.

What structural features might contribute to the thermal adaptability of K. olearia ndk?

While specific structural data for K. olearia ndk is not provided in the search results, we can infer potential structural adaptations based on our knowledge of thermophilic proteins:

• Primary structure features:

  • Increased proportion of charged amino acids forming salt bridges

  • Higher number of hydrophobic residues in the protein core

  • Reduced number of thermolabile amino acids (Asn, Gln, Cys, Met)

  • Potential unique conserved sequence indels (CSIs) characteristic of Thermotogae proteins

• Secondary and tertiary structure stabilization:

  • Enhanced hydrogen bonding networks

  • Increased number of ionic interactions

  • More compact protein folding with reduced surface loops

  • Potentially unique structural elements that enable function across the broad temperature range (20-79°C)

• Quaternary structure considerations:

  • Most ndks function as hexamers or tetramers

  • Stronger subunit interactions could contribute to thermostability

Researchers should perform comparative structural analysis with mesophilic ndks to identify specific thermal adaptation mechanisms, potentially through X-ray crystallography, cryo-EM, or computational modeling approaches.

What enzymatic assays are most appropriate for characterizing K. olearia ndk activity?

For comprehensive characterization of K. olearia ndk activity, researchers should employ:

• Standard phosphoryl transfer assays:

  • Based on techniques similar to those used for Leishmania ndk

  • Measure conversion of nucleoside diphosphates to triphosphates

  • Monitor ATP utilization or GTP formation using spectrophotometric methods

• Temperature-dependent kinetics:

  • Determine enzyme kinetics (Km, kcat, kcat/Km) across temperature range (20-80°C)

  • Calculate activation energy using Arrhenius plots

  • Compare thermal activity profile with organism's growth temperature range

• Substrate specificity profiling:

  • Test various nucleoside diphosphates as phosphoryl acceptors

  • Evaluate different nucleoside triphosphates as phosphoryl donors

  • Create a specificity matrix of relative activities

• Effect of pH and salt concentration:

  • Determine optimal pH (likely near 6.8 based on organism's growth)

  • Assess activity at varying NaCl concentrations (optimal likely 25-30 g/L)

How might K. olearia ndk function differ from mesophilic bacterial ndks?

K. olearia ndk likely exhibits several distinctive functional characteristics compared to mesophilic counterparts:

• Temperature-activity relationship:

  • Broader temperature activity range (20-80°C)

  • Thermal stability at higher temperatures

  • Potential retained activity at lower temperatures, reflecting K. olearia's growth capability at temperatures as low as 20°C

• Kinetic parameters:

  • Potentially different Km values across temperatures

  • Altered catalytic efficiency at different temperatures

  • Possible trade-off between stability and activity at various temperatures

• Structural dynamics:

  • Modified conformational flexibility that balances rigidity needed for thermostability with flexibility required for catalysis

  • Potentially unique adaptation mechanisms that allow function across the wide temperature range

• Additional functions:

  • Based on findings from other bacterial ndks, including Leishmania , investigate potential moonlighting functions beyond nucleotide metabolism

  • Consider possible roles in stress response, given K. olearia's adaptation to varying temperatures

Comparative studies with mesophilic ndks will help elucidate the molecular basis for these functional differences and provide insights into thermal adaptation mechanisms.

How might K. olearia ndk contribute to understanding bacterial temperature adaptation mechanisms?

K. olearia's extraordinary growth temperature range (20-79°C) makes its ndk an excellent model for studying enzymatic thermal adaptation:

• Thermal flexibility mechanisms:

  • K. olearia exhibits remarkable transcriptional reprogramming across temperatures, with differential regulation of 573 out of 2224 genes

  • Studying ndk expression and function across temperatures could reveal whether this enzyme employs specialized adaptations for thermal flexibility

  • Comparing with ndks from strict thermophiles and mesophiles could identify unique features enabling function across the wider temperature range

• Structure-function studies:

  • Mutational analysis to identify residues critical for temperature adaptability

  • Mapping temperature-sensitive regions through hydrogen-deuterium exchange or thermal unfolding studies

  • Computational simulations of protein dynamics at different temperatures

• Evolutionary perspectives:

  • Comparative genomic analysis with other Thermotogae members

  • Investigation of potential horizontal gene transfer events that might have contributed to K. olearia's unique thermal range

  • Analysis of conserved sequence indels (CSIs) specific to Thermotogae that might contribute to thermostability

This research could provide fundamental insights into how enzymes maintain functionality across broad temperature ranges, relevant for understanding bacterial adaptation to thermally fluctuating environments.

What experimental approaches should be used to investigate K. olearia ndk's potential role in stress response?

Transcriptomic data shows that K. olearia undergoes significant gene expression changes at different temperatures , suggesting complex stress response mechanisms. To investigate ndk's potential role:

• Expression analysis:

  • Quantify ndk expression levels across temperature ranges using RT-qPCR

  • Determine if ndk is co-expressed with known stress response genes

  • Analyze promoter regions for stress-responsive elements

• Protein-protein interaction studies:

  • Identify potential interactors through pull-down assays coupled with mass spectrometry

  • Yeast two-hybrid or bacterial two-hybrid screening

  • In vitro validation of identified interactions

• Subcellular localization:

  • Develop fluorescent protein fusions to track ndk localization under different stress conditions

  • Examine if localization changes with temperature, pH, or other stressors

  • Fractionate cells to determine if ndk is secreted under certain conditions, similar to Leishmania ndk

• Functional assays:

  • Assess if ndk activity changes under various stress conditions

  • Determine if the enzyme exhibits additional activities beyond phosphoryl transfer in stress situations

  • Investigate potential protective roles, similar to how Leishmania ndk prevents ATP-mediated cytolysis

These approaches could reveal whether K. olearia ndk has moonlighting functions related to thermal adaptation or other stress responses.

What are the methodological considerations for using K. olearia ndk in nucleotide metabolic engineering applications?

K. olearia ndk's potential thermostability and broad temperature activity range make it a candidate for various biotechnological applications:

• Enzyme immobilization strategies:

  • Covalent attachment to various supports (resins, magnetic particles)

  • Entrapment in polymeric matrices

  • Cross-linked enzyme aggregates (CLEAs)

  • Evaluate stability and activity retention after immobilization across temperature ranges

• Biocatalytic process design:

  • Determine optimal reaction conditions (temperature, pH, substrate concentrations)

  • Investigate compatibility with organic solvents or ionic liquids

  • Develop continuous flow systems leveraging the enzyme's thermostability

• Multienzyme cascade systems:

  • Integration with other thermostable enzymes for nucleotide modification

  • Compatibility with enzymes from mesophilic sources at intermediate temperatures

  • Optimization of reaction conditions for maximum productivity

• Stability enhancement strategies:

  • Site-directed mutagenesis to further enhance thermostability

  • Computational design for optimizing activity at specific temperatures

  • Formulation with stabilizing excipients for long-term storage

What are common challenges in recombinant K. olearia ndk expression and how can they be addressed?

Recombinant expression of thermophilic proteins like K. olearia ndk presents several challenges:

• Protein misfolding and inclusion body formation:

  • Solution: Lower induction temperature (15-25°C), reduce inducer concentration

  • Consider co-expression with chaperones (GroEL/GroES, DnaK/DnaJ)

  • Use solubility-enhancing fusion partners (MBP, SUMO, TrxA)

• Low expression levels:

  • Strategy: Optimize codon usage for expression host

  • Test different promoter systems and expression strains

  • Optimize media composition and induction conditions

• Protein inactivity despite solubility:

  • Approach: Verify proper folding through circular dichroism or fluorescence spectroscopy

  • Ensure metal cofactors or other essential components are present

  • Test different buffer conditions that mimic K. olearia's natural environment (pH 6.8, 25-30 g/L NaCl)

• Proteolytic degradation:

  • Mitigation: Include protease inhibitors during purification

  • Use protease-deficient expression strains

  • Optimize purification speed and conditions

• Unexpected oligomerization state:

  • Analysis: Verify quaternary structure by size exclusion chromatography or native PAGE

  • Optimize buffer conditions to maintain proper oligomeric state

  • Consider engineering stabilizing interfaces if necessary

These troubleshooting approaches should be systematically applied with careful documentation of conditions and outcomes.

How can researchers optimize activity assays for K. olearia ndk across different temperature ranges?

Given K. olearia's broad temperature growth range (20-79°C) , optimizing activity assays across temperatures requires special considerations:

• Buffer stability across temperatures:

  • Use buffers with minimal temperature-dependent pH changes (e.g., phosphate)

  • Pre-equilibrate all components to target temperature

  • Account for temperature effects on pH (approximately -0.017 pH units/°C for phosphate buffer)

• Assay component stability:

  • Verify stability of substrates and cofactors at elevated temperatures

  • Account for potential increased spontaneous hydrolysis of ATP at higher temperatures

  • Consider thermal stability of coupled enzymes if using coupled assays

• Reaction rate normalization:

  • Adjust enzyme concentrations for different temperatures to achieve measurable rates

  • Use appropriate controls at each temperature

  • Develop temperature correction factors for comparative analysis

• Equipment considerations:

  • For high-temperature assays (65-80°C), prevent evaporation with sealed reaction vessels

  • Ensure temperature control accuracy in instruments

  • Consider rapid-mixing approaches for fast kinetics at elevated temperatures

• Data analysis adaptations:

  • Apply Arrhenius plots to understand temperature dependence

  • Use appropriate models that account for potential temperature-dependent changes in mechanism

  • Compare with temperature dependence of mesophilic ndks as reference

These optimization strategies will enable reliable activity measurements across K. olearia ndk's functional temperature range.

What controls and validation experiments are essential when working with recombinant K. olearia ndk?

Rigorous experimental design for K. olearia ndk research should include:

• Activity validation controls:

  • Positive control: Commercial ndk from well-characterized source

  • Negative control: Heat-inactivated K. olearia ndk

  • Substrate specificity controls with non-substrate nucleotides

  • No-enzyme controls to account for spontaneous phosphoryl transfer

• Protein quality controls:

  • Mass spectrometry verification of protein identity

  • Size exclusion chromatography to confirm proper oligomeric state

  • Circular dichroism to verify secondary structure integrity

  • Thermal shift assays to confirm expected thermal stability profile

• Temperature-specific considerations:

  • Verify buffer component stability at experimental temperatures

  • Include temperature-matched controls for each experiment

  • Perform replicate experiments across the temperature range (20-80°C)

  • Include internal standards for normalizing results across temperatures

• Comparison with native enzyme (if available):

  • Activity profile comparison across temperatures

  • Substrate specificity comparison

  • Thermal stability comparison

  • Structural comparison (if possible)

• Reproducibility validation:

  • Inter-batch comparison of recombinant enzyme preparations

  • Statistical analysis of replicate experiments

  • Blinded experimental design where possible

  • Independent verification of key findings using different methodological approaches