Recombinant Nitrosomonas europaea Non-canonical purine NTP pyrophosphatase (NE0277)

What is Non-canonical purine NTP pyrophosphatase and how is it classified?

Non-canonical purine NTP pyrophosphatase (NE0277) from Nitrosomonas europaea belongs to the CYTH superfamily of proteins, which includes the CyaB adenylyl cyclase from Aeromonas hydrophila and human 25-kDa thiamine triphosphatase. This enzyme family is also known as triphosphate tunnel metalloenzymes (TTM) due to their characteristic β-barrel structure . The enzyme is functionally characterized by its ability to bind triphosphorylated substrates and divalent metal ions, with specific catalytic activity toward non-canonical purine nucleotides . Unlike many other TTM proteins that form closed β-barrel structures, crystallographic data indicates that NeuTTM (the common designation for this enzyme from N. europaea) forms an open β-barrel structure .

How does the structure of NeuTTM differ from other CYTH superfamily proteins?

NeuTTM has several distinctive structural features compared to other members of the CYTH superfamily:

What genomic context does NE0277 have in the Nitrosomonas europaea genome?

Nitrosomonas europaea has a single circular chromosome of 2,812,094 bp containing approximately 2,460 protein-encoding genes . While the specific genomic context of NE0277 is not directly mentioned in the search results, the genome of N. europaea is known to contain complex repetitive elements constituting approximately 5% of the genome, including 85 predicted insertion sequence elements in eight different families . The genome is relatively compact, with genes averaging 1,011 bp in length and intergenic regions averaging 117 bp . Understanding the genomic context of NE0277 would require further investigation into the gene neighborhood and potential operon structures that might indicate functional relationships with other genes.

What expression systems are most effective for recombinant production of NeuTTM?

Multiple expression systems have been successfully used for recombinant production of Non-canonical purine NTP pyrophosphatase, with varying advantages:

For laboratory-scale research, E. coli expression systems offer the best balance of yield and convenience . Experimental data shows that transformation of E. coli with a pET15b plasmid containing the sequence of NeuTTM with an N-terminal His6-tag allows for efficient expression when induced with isopropyl β-d-1-thiogalactopyranoside (IPTM) .

What is the optimal purification protocol for obtaining active NeuTTM?

Based on published methodologies, the following purification protocol has been successfully employed to obtain active NeuTTM:

• Culture transformed E. coli in 2× YT medium containing ampicillin until A600 reaches 0.6–0.8

• Induce protein expression with 1 mM isopropyl β-d-1-thiogalactopyranoside

• Incubate for 3 hours at 37°C

• Harvest cells by centrifugation (15 min at 8000 × g, 4°C)

• Resuspend in modified binding buffer (20 mM HEPES, 0.5 M NaCl, 30 mM imidazole, pH 7.5)

• Lyse cells using French press disruptor (two cycles)

• Clear lysate by centrifugation (30 min at 50,000 × g)

• Purify tagged protein using FPLC system with HisTrap FF column

• Elute protein with elution buffer (20 mM HEPES, 0.5 M NaCl, 0.5 M imidazole, pH 7.5)

• If needed, remove His-tag by incubating with AcTEV protease

• Perform second HisTrap purification to remove cleaved tag and protease

Importantly, removal of the His-tag has been shown to considerably increase the triphosphatase activity of the enzyme, suggesting that the tag may partially interfere with catalytic function .

How can selenomethionine-substituted NeuTTM be produced for crystallographic studies?

For crystallization studies, selenomethionine-substituted NeuTTM can be expressed to facilitate phase determination through single-wavelength anomalous dispersion (SAD) or multiple-wavelength anomalous dispersion (MAD) methods. The general procedure involves:

• Grow E. coli transformants in minimal medium containing all amino acids except methionine

• Deplete methionine reserves by allowing limited growth

• Add selenomethionine to the culture medium before induction

• Induce protein expression and purify as with the native protein, taking care to include reducing agents in all buffers to prevent selenomethionine oxidation

This approach has been successfully used to express selenomethionine-substituted NeuTTM for crystallographic studies, resulting in high-quality diffraction data that revealed the distinctive open β-barrel structure of the enzyme .

What is the substrate specificity profile of NeuTTM?

The substrate specificity of NeuTTM is notably distinct from other CYTH family members, showing strong preference for specific substrates:

Despite structural similarities to mammalian 25-kDa thiamine triphosphatase, NeuTTM shows only slight thiamine triphosphatase (ThTPase) activity . Similarly, nucleoside triphosphatase activity is negligible in the presence of Mg²⁺, though a small activity is observed with Mn²⁺, particularly with GTP . The enzyme demonstrates a strong preference and high affinity for tripolyphosphate (PPPi), making it the first characterized specific tripolyphosphatase (PPPase) .

How do divalent metal ions influence the catalytic activity of NeuTTM?

Divalent metal ions play a crucial role in the catalytic activity of NeuTTM, with different ions showing variable effects:

The enzyme's preference for Mn²⁺ over Mg²⁺, particularly for NTP substrates, suggests that the metal ion influences both substrate binding and catalytic efficiency. This metal ion dependency is a characteristic feature of the CYTH superfamily proteins, which typically require divalent metal ions for their catalytic function .

What residues are critical for catalysis in NeuTTM?

Site-directed mutagenesis studies have identified several residues that are important for the catalytic function of NeuTTM:

These residues are highly conserved in CYTH proteins, suggesting their fundamental importance to the catalytic mechanism . The exact roles of these residues in the catalytic mechanism remain to be fully elucidated, but they likely participate in substrate binding, metal coordination, or direct catalysis of the hydrolytic reaction.

How can site-directed mutagenesis be optimally performed to study structure-function relationships in NeuTTM?

For conducting structure-function studies of NeuTTM through site-directed mutagenesis, the following methodology has proven effective:

• Identify conserved residues through multiple sequence alignment of CYTH family proteins

• Design mutagenic primers (forward and reverse) containing the desired mutations

• Perform QuikChange mutagenesis using the expression plasmid containing the NeuTTM sequence as template

• Amplify with Pfu DNA polymerase to ensure high fidelity

• Remove template DNA by DpnI digestion (2 hours at 37°C)

• Transform E. coli cells with the mutagenized plasmid

• Isolate single clones and verify mutations by DNA sequencing

• Express and purify mutant proteins using the same protocol as for wild-type

• Compare enzymatic activities to assess the impact of mutations

This approach has successfully been used to generate and characterize mutations in key residues such as Lys-52 and Tyr-28, demonstrating their importance for catalytic function .

What challenges might researchers encounter when studying the physiological role of NeuTTM in Nitrosomonas europaea?

Investigating the physiological role of NeuTTM in N. europaea presents several significant challenges:

N. europaea participates in the biogeochemical nitrogen cycle through nitrification , and understanding how NeuTTM contributes to this process would require careful correlation of enzyme activity with nitrogen metabolism under various environmental conditions.

How does NeuTTM compare to similar enzymes in other organisms, and what evolutionary insights can be gained?

Comparative analysis of NeuTTM with related enzymes reveals interesting evolutionary patterns:

While NeuTTM shares structural features with mammalian 25-kDa ThTPase (open cleft rather than closed tunnel), it has evolved a distinct substrate specificity as a tripolyphosphatase rather than a thiamine triphosphatase . This suggests that structural similarities between these enzymes may be due to convergent evolution or adaptation of a common ancestral scaffold for different biochemical functions. Further phylogenetic analysis incorporating additional CYTH family members from diverse organisms could provide deeper insights into the evolutionary history and functional diversification of this enzyme family.

What is the potential role of NeuTTM in the metabolism of Nitrosomonas europaea?

The precise physiological role of NeuTTM in N. europaea metabolism remains to be fully elucidated, but several hypotheses can be proposed based on its biochemical properties:

• Tripolyphosphate metabolism: As a specific tripolyphosphatase, NeuTTM may participate in the metabolism of inorganic polyphosphates, which can serve as energy reserves in bacteria.

• Protection against non-canonical nucleotides: By hydrolyzing non-canonical purine nucleotides, NeuTTM might protect the organism from potentially mutagenic effects of these nucleotides if incorporated into DNA.

• Stress response: In E. coli, thiamine triphosphate (ThTP) is involved in responses to environmental stress . Although NeuTTM has low ThTPase activity, it might play a role in stress response pathways in N. europaea.

• Nitrogen metabolism: Given that N. europaea is an ammonia-oxidizing bacterium that participates in the biogeochemical nitrogen cycle , NeuTTM might indirectly contribute to nitrogen metabolism through regulation of energy or phosphate homeostasis.

Understanding the exact role would require integrating enzymatic, metabolomic, and transcriptomic approaches to correlate NeuTTM activity with specific metabolic pathways or stress responses in N. europaea.

How might NeuTTM be utilized in biotechnological applications?

The unique properties of NeuTTM suggest several potential biotechnological applications:

The high specificity of NeuTTM for tripolyphosphate makes it particularly valuable as a potential analytical tool for detecting and quantifying this compound in environmental or biological samples. Furthermore, as the first characterized specific tripolyphosphatase , NeuTTM provides a new enzymatic activity that could be exploited for novel biotechnological processes involving phosphate metabolism.

What methodological approaches would be most effective for studying NeuTTM in the context of the nitrogen cycle?

To investigate the role of NeuTTM in the context of the nitrogen cycle and ammonia oxidation by N. europaea, the following methodological approaches would be most effective:

• Gene expression analysis: Quantify NeuTTM expression under different nitrogen availability conditions to identify correlations with ammonia oxidation rates.

• Gene knockout/knockdown: Generate NeuTTM-deficient strains of N. europaea to assess impacts on growth, ammonia oxidation, and response to environmental stressors.

• Metabolomic profiling: Compare the metabolome of wild-type and NeuTTM-deficient strains to identify metabolic pathways affected by the enzyme.

• In situ activity assays: Develop methods to measure NeuTTM activity in intact cells under various environmental conditions.

• Environmental sampling: Assess NeuTTM expression in natural environments where nitrification occurs to correlate enzyme activity with environmental parameters.

These approaches would need to consider the specialized growth requirements of N. europaea as an obligate chemolithoautotroph that derives all its energy and reductant from the oxidation of ammonia to nitrite .

What are the most significant unresolved questions regarding NeuTTM?

Despite significant progress in characterizing NeuTTM, several important questions remain unanswered:

• What is the physiological substrate of NeuTTM in vivo? While in vitro studies show preference for tripolyphosphate, the biological relevance of this activity needs confirmation.

• How is NeuTTM expression regulated in response to environmental conditions, particularly those relevant to nitrification?

• What is the detailed catalytic mechanism of tripolyphosphate hydrolysis by NeuTTM, and how do the identified key residues (Lys-52 and Tyr-28) participate in this mechanism?

• Does NeuTTM interact with other proteins in N. europaea, potentially forming functional complexes involved in phosphate or nitrogen metabolism?

• How widespread are specific tripolyphosphatases in other organisms, and what evolutionary relationships exist between these enzymes?

Addressing these questions will require integrated approaches combining structural biology, enzymology, microbial physiology, and environmental microbiology to place the biochemical activities of NeuTTM in their proper biological context.

What technological advances might facilitate deeper understanding of NeuTTM function?

Several emerging technologies could significantly advance our understanding of NeuTTM:

These technologies, applied in combination, could provide a multidimensional view of NeuTTM function from the molecular to the ecosystem level, ultimately clarifying its role in bacterial physiology and the global nitrogen cycle.

How might research on NeuTTM contribute to broader understanding of bacterial metabolism and enzyme evolution?

Research on NeuTTM has implications extending beyond this specific enzyme:

• As the first characterized specific tripolyphosphatase , NeuTTM provides insights into a previously underappreciated aspect of phosphate metabolism.

• The structural similarity but functional divergence between NeuTTM and mammalian 25-kDa ThTPase illustrates how enzyme scaffolds can be adapted for different functions during evolution.

• Understanding the role of NeuTTM in N. europaea could illuminate specialized metabolic adaptations of chemolithoautotrophic bacteria, which play crucial roles in biogeochemical cycles.

• Investigating the regulation and activity of NeuTTM in response to environmental conditions could provide insights into how bacteria sense and respond to stress, particularly in organisms with specialized metabolic capabilities.