Recombinant Nitrosomonas europaea Nucleoside diphosphate kinase (ndk)

What is the genomic context of the ndk gene in Nitrosomonas europaea?

The complete genome sequence of Nitrosomonas europaea (ATCC 19718) consists of a single circular chromosome of 2,812,094 bp with 2,460 protein-encoding genes . The ndk gene is part of the nucleotide metabolism pathway. Within the genome, genes are distributed relatively evenly, with approximately 47% transcribed from one strand and 53% from the complementary strand . Unlike the ammonia monooxygenase (amo) and hydroxylamine oxidoreductase (hao) genes that exist in multiple copies, the ndk gene is present as a single copy. This reflects the general pattern observed in N. europaea where genes involved in core metabolic functions typically exist as single copies, while those directly involved in ammonia oxidation often appear as duplicates.

How is recombinant N. europaea NDK typically expressed and purified for research studies?

Recombinant N. europaea NDK is typically expressed in E. coli expression systems using vectors that introduce a polyhistidine (His) tag for simplified purification. The methodology involves:

• PCR amplification of the ndk gene from N. europaea genomic DNA

• Cloning into an expression vector (commonly pET-based systems)

• Transformation into an E. coli expression strain (BL21(DE3) or derivatives)

• Induction of protein expression using IPTG at reduced temperatures (16-25°C) to enhance solubility

• Cell lysis via sonication or high-pressure homogenization

• Purification via immobilized metal affinity chromatography (IMAC)

• Secondary purification using size exclusion chromatography

This approach yields highly pure recombinant NDK suitable for biochemical and structural studies. When preserving enzymatic activity is crucial, care must be taken to include appropriate metal cofactors (typically Mg²⁺) in all buffers throughout the purification process.

What are the basic biochemical properties of N. europaea NDK?

N. europaea NDK, like other bacterial NDKs, is a small protein (approximately 15-17 kDa per monomer) that typically forms homohexameric structures. The enzyme catalyzes the transfer of the γ-phosphate from nucleoside triphosphates to nucleoside diphosphates via a ping-pong mechanism involving a phosphorylated histidine intermediate:

N₁TP + N₂DP ⟷ N₁DP + N₂TP

The enzyme demonstrates broad substrate specificity across different nucleotides but may show preferences similar to those observed in other bacterial NDKs. The activity requires divalent metal ions, particularly Mg²⁺, which coordinates with the phosphate groups during catalysis. The enzyme has a phosphatase activity that generates a net loss of organic phosphate during extended reaction periods, while simultaneously incorporating inorganic phosphate into organic fractions including ATP and ADP .

How does oxygen limitation affect NDK expression and activity in N. europaea?

N. europaea is an obligate aerobe that can adapt to oxygen-limited conditions by modifying its metabolic pathways. Under oxygen-limited conditions, N. europaea undergoes significant transcriptional changes affecting various metabolic processes . While specific data on NDK regulation under oxygen limitation is limited, we can infer that:

• The transition to oxygen-limited growth likely affects nucleotide metabolism, potentially altering NDK expression patterns

• Under oxygen limitation, N. europaea shifts resources toward maintaining essential metabolic functions

• NDK activity may be indirectly affected by changes in the availability of nucleoside triphosphates, which are dependent on ATP production via oxidative phosphorylation

The phosphorylation mechanisms in N. europaea extracts demonstrate that even under varying oxygen conditions, the bacterium maintains the ability to generate high-energy phosphate units (ATP and ADP) that serve as substrates for NDK . This suggests a potential regulatory role for NDK in balancing nucleotide pools during metabolic adjustments to oxygen limitation.

What methodologies are recommended for assessing NDK activity in N. europaea extracts?

Several complementary approaches can be used to assess NDK activity in N. europaea extracts:

• Coupled Spectrophotometric Assay:

  • Measures the formation of ATP from ADP using pyruvate kinase and lactate dehydrogenase coupled reactions

  • Monitors NADH oxidation at 340 nm

  • Reaction mixture includes ADP, GTP (or other NTP), MgCl₂, phosphoenolpyruvate, NADH, pyruvate kinase, and lactate dehydrogenase

• Radioactive Assay:

  • Uses [γ-³²P]GTP or other labeled NTPs

  • Measures the transfer of labeled phosphate to ADP

  • Quantifies labeled ATP by thin-layer chromatography or HPLC

• Bioluminescence Assay:

  • Measures ATP production through luciferase reaction

  • Highly sensitive for detecting low levels of NDK activity

• Phosphohistidine Detection:

  • Western blotting with anti-phosphohistidine antibodies

  • Identifies the phosphorylated enzyme intermediate

For cell-free preparations of N. europaea, maintaining appropriate conditions is critical since these extracts exhibit strong phosphatase activity that can result in a net loss of organic phosphate during experimental periods .

How does the structure of N. europaea NDK compare to NDKs from other bacterial species?

While specific structural data for N. europaea NDK is limited, comparative analysis with other bacterial NDKs suggests:

• Conserved Catalytic Core: The active site likely contains the highly conserved histidine residue that forms a phosphoenzyme intermediate during catalysis

• Quaternary Structure: Probably forms a homohexamer composed of identical subunits arranged with dihedral symmetry, similar to E. coli and other prokaryotic NDKs

• Metal Binding Sites: Contains conserved residues for coordination of divalent cations (typically Mg²⁺)

• Surface Properties: May have unique surface electrostatic properties reflecting adaptation to the specific physiological environment of N. europaea

• Substrate Binding Pocket: Likely accommodates various nucleotides with subtle differences in affinity compared to other bacterial NDKs

A homology model based on other bacterial NDKs would predict a structure with alternating α-helices and β-sheets forming a compact globular domain with the catalytic histidine positioned within a conserved loop region.

What is the relationship between NDK activity and energy metabolism in N. europaea?

N. europaea is a chemolithoautotroph that derives all its energy from the oxidation of ammonia to nitrite and must fix carbon dioxide to meet its carbon requirements . The relationship between NDK and energy metabolism in this organism involves:

• Nucleotide Balance: NDK maintains equilibrium between different nucleoside triphosphates, ensuring availability of GTP, UTP, and CTP using ATP as the primary energy currency

• Energy Conservation: In N. europaea, ATP is primarily generated through electron transport phosphorylation during ammonia oxidation. NDK plays a crucial role in distributing this energy to various cellular processes by maintaining balanced nucleotide pools

• Metabolic Integration: NDK activity bridges energy metabolism and biosynthetic pathways, particularly:

  • DNA and RNA synthesis

  • Polysaccharide synthesis

  • Phospholipid metabolism

  • Protein synthesis (GTP requirements)

• Response to Metabolic Changes: During transitions between different growth conditions, NDK likely helps maintain metabolic homeostasis by adjusting nucleotide ratios

The unique metabolic capabilities of N. europaea, including its ability to convert energy released in the oxidation of ammonia into high-energy phosphate units , create a distinctive context for NDK function compared to heterotrophic bacteria.

What genetic approaches are most effective for studying ndk function in N. europaea?

Studying ndk function in N. europaea presents unique challenges due to this organism's specialized metabolism. The most effective genetic approaches include:

• Gene Knockout/Knockdown Strategies:

  • Allelic exchange methods using suicide vectors

  • CRISPR-Cas9 based approaches adapted for N. europaea

  • Conditional expression systems if ndk proves essential

• Complementation Studies:

  • Expression of wildtype ndk in knockout strains

  • Heterologous expression of ndk from other organisms

  • Site-directed mutagenesis to study specific residues

• Reporter Gene Fusions:

  • Transcriptional fusions to study promoter activity

  • Translational fusions to study protein localization

  • Dual-reporter systems to study regulation

• Overexpression Analysis:

  • Controlled overexpression to assess metabolic impacts

  • Expression of tagged variants for protein interaction studies

When designing genetic studies, researchers should consider N. europaea's slow growth rate and sensitivity to environmental conditions. Success with genetic manipulation of N. europaea genes has been demonstrated with genes involved in ammonia oxidation, such as the creation of nirK knockout mutants , suggesting similar approaches would be feasible for ndk.

How can isotopic labeling be used to study NDK-mediated phosphate transfer in N. europaea?

Isotopic labeling provides powerful insights into NDK-mediated phosphate transfer pathways in N. europaea:

• ³²P-Labeling Experiments:

  • Incubating cell extracts with [γ-³²P]ATP or other labeled nucleotides

  • Tracking phosphate transfer to different nucleoside diphosphates

  • Monitoring the formation of the phosphohistidine intermediate

• ¹⁸O-Labeled Phosphate Studies:

  • Using ¹⁸O-labeled phosphate to track oxygen atoms during phosphate transfer

  • Mass spectrometry analysis to determine reaction mechanisms

  • Distinguishing between direct transfer versus hydrolysis and resynthesis

• ¹⁵N-Labeling for Protein Structural Studies:

  • Expression of recombinant NDK in media containing ¹⁵N-labeled ammonium sources

  • NMR studies of protein structure and dynamics

  • Analysis of conformational changes during catalysis

Previous studies with N. europaea extracts have demonstrated P³²-labeled inorganic phosphate incorporation into organic fractions, including ATP and ADP , providing precedent for isotopic approaches. When designing isotopic labeling experiments, researchers should consider the background activities in N. europaea extracts, particularly the strong phosphatase activity that may complicate interpretation of results.

What are the recommended protocols for assessing NDK substrate specificity in N. europaea?

Assessing NDK substrate specificity requires systematic evaluation of various nucleotide combinations:

Table 1: Experimental Design for NDK Substrate Specificity Assessment

The recommended protocol includes:

• Enzyme Preparation:

  • Purify recombinant NDK to >95% homogeneity

  • Verify enzyme activity with standard ATP/ADP pair

  • Determine protein concentration accurately

• Reaction Conditions:

  • Buffer: 50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 75 mM KCl, 1 mM DTT

  • Temperature: 30°C (optimal for N. europaea enzymes)

  • Fixed concentration of one substrate, varying concentration of the other

• Data Collection and Analysis:

  • Initial velocity measurements for each substrate pair

  • Construction of Lineweaver-Burk plots

  • Determination of kinetic parameters (Km, Vmax, kcat)

  • Calculation of specificity constants (kcat/Km)

This systematic approach enables comprehensive characterization of NDK substrate preferences, providing insights into its metabolic roles in N. europaea.

How can structural biology approaches enhance our understanding of N. europaea NDK?

Structural biology offers critical insights into N. europaea NDK function:

• X-ray Crystallography:

  • Determination of high-resolution structure

  • Co-crystallization with substrates, products, and analogs

  • Analysis of metal-binding sites and coordination geometry

  • Structural comparison with NDKs from other organisms

• Cryo-Electron Microscopy:

  • Analysis of quaternary structure and oligomeric assembly

  • Visualization of conformational states during catalysis

  • Study of potential protein-protein interactions

• NMR Spectroscopy:

  • Analysis of protein dynamics in solution

  • Characterization of substrate binding events

  • Identification of flexible regions and their roles in catalysis

• Small-Angle X-ray Scattering (SAXS):

  • Low-resolution envelope of the protein in solution

  • Analysis of conformational changes upon substrate binding

  • Validation of oligomeric state in physiological conditions

• Computational Approaches:

  • Homology modeling based on related NDK structures

  • Molecular dynamics simulations to study conformational flexibility

  • Docking studies to predict substrate binding modes

These approaches, combined with functional studies, provide a comprehensive understanding of how the structure of N. europaea NDK relates to its function in nucleotide metabolism and potential adaptation to the specialized metabolism of this ammonia-oxidizing bacterium.

How does NDK activity relate to the chemolithoautotrophic lifestyle of N. europaea?

The relationship between NDK activity and N. europaea's chemolithoautotrophic lifestyle is multifaceted:

• Energy Distribution: N. europaea derives all its energy from the oxidation of ammonia to nitrite . NDK plays a crucial role in distributing this energy currency (primarily generated as ATP) to other nucleotide-dependent metabolic pathways.

• Carbon Fixation Support: As an autotroph, N. europaea fixes CO₂ via the Calvin-Benson-Bassham cycle, which requires ATP and reducing power. NDK ensures the provision of appropriate nucleotides for these energy-intensive carbon fixation processes.

• Metabolic Homeostasis During Stress: Under conditions where energy generation is limited (such as oxygen limitation), NDK likely helps maintain essential nucleotide pools to support critical cellular functions.

• Integration with Nitrogen Metabolism: The primary energy-generating pathway in N. europaea involves ammonia oxidation through ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO) . NDK activity must be coordinated with the energy output from these nitrogen metabolism pathways.

• Adaptation to Nutrient Limitation: N. europaea has evolved to function in environments with limited organic nutrients, reflected in its genome structure with abundant transporters for inorganic ions but limited transporters for organic molecules . NDK plays a role in this adaptation by efficiently recycling nucleotides.

The chemolithoautotrophic lifestyle places unique demands on nucleotide metabolism, making NDK an important enzyme for metabolic integration in this specialized bacterium.

What experimental approaches can determine if NDK has moonlighting functions in N. europaea?

NDK enzymes from various organisms have been shown to perform secondary "moonlighting" functions beyond their canonical nucleotide phosphorylation role. To investigate potential moonlighting functions in N. europaea NDK:

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation with anti-NDK antibodies

  • Bacterial two-hybrid screening

  • Pull-down assays with recombinant tagged NDK

  • Cross-linking followed by mass spectrometry identification

• DNA/RNA Binding Assays:

  • Electrophoretic mobility shift assays

  • Filter binding assays

  • Chromatin immunoprecipitation (if DNA binding is suspected)

  • RNA immunoprecipitation (if RNA binding is suspected)

• Phenotypic Analysis of Mutants:

  • Creation of ndk knockout or knockdown strains

  • Complementation with NDK variants (e.g., catalytically inactive)

  • Phenotypic characterization beyond nucleotide metabolism

  • Transcriptomic and proteomic analysis of mutants

• Subcellular Localization Studies:

  • Immunofluorescence microscopy

  • Fractionation studies with Western blot analysis

  • Fusion with fluorescent proteins for live-cell imaging

• Enzymatic Activity Screening:

  • Testing for phosphatase, kinase, or nuclease activities

  • DNase/RNase protection assays

  • Protein phosphorylation assays using proteome extracts

These approaches would help identify potential roles of NDK in processes such as gene regulation, stress response, or cell signaling that extend beyond its primary metabolic function.

How does NDK activity in N. europaea compare with other nitrifying bacteria?

Comparative analysis of NDK activity across nitrifying bacteria reveals both conserved features and potential adaptations:

Table 2: Comparative Analysis of NDK Properties Across Nitrifying Bacteria

• Ammonia-oxidizing bacteria (like N. europaea): NDK activity closely linked to energy generation from ammonia oxidation

• Nitrite-oxidizing bacteria (like Nitrobacter): NDK activity potentially linked to energy generation from nitrite oxidation

• Complete ammonia oxidizers (Comammox Nitrospira): NDK activity potentially integrated with the complete ammonia-to-nitrate oxidation pathway

While core enzymatic properties remain conserved, regulatory mechanisms and metabolic integration of NDK likely vary to support the distinct ecological niches and metabolic strategies of different nitrifying bacteria.

What are the potential biotechnological applications of recombinant N. europaea NDK?

Recombinant N. europaea NDK offers several potential biotechnological applications:

• Nucleotide Biosynthesis:

  • Enzymatic production of modified or rare nucleotides

  • Regeneration of ATP in coupled enzymatic systems

  • Synthesis of labeled nucleotides for research applications

• Bioremediation Enhancement:

  • Improving nitrogen removal efficiency in wastewater treatment

  • Enhancing metabolic capabilities of engineered N. europaea strains

  • Supporting bioremediation of sites contaminated with chlorinated aliphatic hydrocarbons

• Biosensor Development:

  • NDK-based ATP detection systems

  • Environmental monitoring of ammonia levels

  • Integration into multi-enzyme biosensing platforms

• Structural Biology Tools:

  • Model system for studying phosphoryl transfer mechanisms

  • Platform for protein engineering and directed evolution

  • Template for designing novel catalysts with expanded substrate ranges

• Diagnostic Applications:

  • Development of high-sensitivity nucleotide detection methods

  • Coupling with other diagnostic enzymes in multi-step assays

  • Creation of stable enzyme preparations for field-deployable tests

Understanding the unique properties of N. europaea NDK, particularly its activity under the specialized metabolic conditions of this chemolithoautotroph, may reveal advantageous characteristics for specific biotechnological applications.

What are the current methodological challenges in studying N. europaea NDK and potential solutions?

Research on N. europaea NDK faces several methodological challenges:

• Protein Expression Challenges:

  • Challenge: Low expression levels in heterologous systems

  • Solution: Optimization of codon usage, expression as fusion proteins, use of specialized expression strains

• Enzyme Stability Issues:

  • Challenge: Maintaining stability during purification and storage

  • Solution: Addition of stabilizing agents (glycerol, reducing agents), immobilization techniques, nanodisk incorporation

• Activity Assay Limitations:

  • Challenge: Background phosphatase activity interfering with measurements

  • Solution: Development of highly specific assays, use of phosphatase inhibitors, careful design of control experiments

• Genetic Manipulation Difficulties:

  • Challenge: Slow growth and limited genetic tools for N. europaea

  • Solution: Adaptation of CRISPR-Cas systems, development of specialized vectors, optimization of transformation protocols

• Physiological Relevance Assessment:

  • Challenge: Connecting in vitro findings to in vivo function

  • Solution: Development of cell-based assays, in situ activity measurements, metabolomic approaches

• Structural Analysis Barriers:

  • Challenge: Obtaining sufficient quantities of protein for structural studies

  • Solution: High-density cultivation approaches, fusion with crystallization chaperones, advanced cryo-EM techniques for smaller samples

• Moonlighting Function Detection:

  • Challenge: Identifying non-canonical functions

  • Solution: Unbiased screening approaches, high-throughput interaction studies, advanced proteomics techniques

Addressing these challenges requires interdisciplinary approaches combining molecular biology, biochemistry, structural biology, and systems biology to fully characterize this enzyme and its roles in N. europaea.

How might emerging technologies enhance our understanding of NDK function in N. europaea?

Emerging technologies offer new avenues for investigating NDK function in N. europaea:

• Single-Cell Approaches:

  • Single-cell RNA-seq to capture cell-to-cell variation in ndk expression

  • Microfluidic cultivation systems for studying NDK function in individual cells

  • Live-cell imaging with new-generation fluorescent reporters

• Advanced Structural Techniques:

  • Time-resolved crystallography to capture catalytic intermediates

  • Cryo-electron tomography for in situ structural analysis

  • Integrative structural biology combining multiple data sources

• Systems Biology Tools:

  • Multi-omics integration to place NDK in the context of cellular networks

  • Constraint-based metabolic modeling to predict NDK flux control

  • Kinetic modeling of nucleotide metabolism with NDK as a central component

• Genome Editing Technologies:

  • CRISPR-Cas9 adaptations for precise engineering of ndk variants

  • Base editing for introducing specific mutations without double-strand breaks

  • Prime editing for more complex genetic modifications

• Microbiome-Based Approaches:

  • Study of NDK function in natural microbial communities

  • Metaproteomics to analyze NDK expression in environmental samples

  • Synthetic microbial communities to study inter-species effects on NDK function

• Computational Advances:

  • Machine learning for predicting NDK interaction networks

  • Molecular dynamics simulations with enhanced sampling techniques

  • Quantum mechanical/molecular mechanical (QM/MM) calculations for reaction mechanism studies

These emerging technologies promise to provide unprecedented insights into the function of NDK within the unique metabolic context of N. europaea, potentially revealing new roles and applications for this enzyme.