Recombinant Nitrosomonas europaea ATP phosphoribosyltransferase regulatory subunit (hisZ)
Description
Introduction to Recombinant Nitrosomonas europaea ATP Phosphoribosyltransferase Regulatory Subunit (hisZ)
The Recombinant Nitrosomonas europaea ATP phosphoribosyltransferase regulatory subunit, denoted as hisZ, is a protein derived from the bacterium Nitrosomonas europaea. This bacterium is well-known for its role in the nitrogen cycle, specifically in the process of nitrification, where it oxidizes ammonia to nitrite . The hisZ protein is part of the histidine biosynthesis pathway, which is crucial for the synthesis of the amino acid histidine. In this pathway, ATP phosphoribosyltransferase plays a key role as the first committed step in histidine biosynthesis.
Function and Role of hisZ
The hisZ protein acts as a regulatory subunit of ATP phosphoribosyltransferase, which catalyzes the transfer of a phosphoribosyl group from phosphoribosyl pyrophosphate (PRPP) to ATP, forming N-(5'-phosphoribosyl)-ATP (PR-ATP). This reaction is a critical step in the biosynthesis of histidine, as it commits the pathway towards the production of this essential amino acid. The regulatory subunit hisZ is important for modulating the activity of the enzyme in response to cellular needs.
Recombinant Production and Characteristics
Recombinant production of the hisZ protein involves expressing the gene encoding this protein in a suitable host organism, such as Escherichia coli. This method allows for large-scale production of the protein for research and potential therapeutic applications. The recombinant hisZ protein from Nitrosomonas europaea has been characterized with a purity of >85% as determined by SDS-PAGE .
Characteristics of Recombinant hisZ:
| Characteristic | Description |
|---|---|
| Purity | >85% (SDS-PAGE) |
| Immunogen Species | Nitrosomonas europaea |
| Sequence | MRNWLLPEYI EDVLPRDAYR IEKIRRLIMD MLFAHGYQFV MPPLLEYVES LLAGSGSGMN LRMFKVVDQL SGRMMGLRAD MTPQAARIDA HLLNISGVTR LCYASSVVHT VPDEITRTRE PFQVGAELYG HSGIESDLEI QCLLLECLSV |
| Storage Conditions | Liquid form: 6 months at -20°C/-80°C; Lyophilized form: 12 months at -20°C/-80°C |
Research Findings and Applications
While specific research on the recombinant hisZ protein from Nitrosomonas europaea is limited, studies on similar proteins in other organisms highlight the importance of ATP phosphoribosyltransferase in histidine biosynthesis. The regulatory subunit hisZ plays a crucial role in modulating enzyme activity based on cellular conditions. This could have implications for understanding metabolic regulation in bacteria and potentially for biotechnological applications, such as improving histidine production in industrial settings.
References:
- Cusabio. Recombinant Nitrosomonas europaea ATP phosphoribosyltransferase regulatory subunit (hisZ).
- Chain, P. et al. Complete Genome Sequence of the Ammonia-Oxidizing Bacterium Nitrosomonas europaea.
- UniProt. Ammonia monooxygenase alpha subunit.
Product Specs
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Target Background
ATP phosphoribosyltransferase regulatory subunit (HisZ) is essential for the initial step of histidine biosynthesis. It may also mediate feedback regulation of ATP phosphoribosyltransferase activity by histidine.
KEGG: neu:NE1282
STRING: 228410.NE1282
Q&A
What is the role of ATP phosphoribosyltransferase regulatory subunit (hisZ) in N. europaea metabolism?
ATP phosphoribosyltransferase regulatory subunit (hisZ) functions as a regulatory component of the ATP phosphoribosyltransferase complex, which catalyzes the first committed step in histidine biosynthesis. In N. europaea, which has a specialized chemolithoautotrophic lifestyle, this regulatory protein helps control the flux through the histidine biosynthesis pathway in response to cellular needs. The enzyme catalyzes the condensation of ATP and phosphoribosyl pyrophosphate (PRPP) to form phosphoribosyl-ATP, a rate-limiting step subject to feedback inhibition by histidine.
As a specialized ammonia oxidizer, N. europaea must carefully regulate amino acid biosynthesis in coordination with its unique energy metabolism, making hisZ an important regulatory node in its metabolic network. Considering that N. europaea has limited carbon source options, efficient regulation of energetically costly biosynthetic pathways like histidine production is particularly critical for optimizing growth and energy utilization.
How does hisZ differ from other regulatory proteins in N. europaea?
Unlike many regulatory proteins that act as transcription factors, hisZ functions through direct protein-protein interactions with the catalytic HisG subunit. This makes it part of a relatively small group of metabolic enzymes regulated through subunit interactions rather than transcriptional control.
N. europaea's proteome includes numerous regulatory proteins that respond to environmental conditions. For instance, transcriptomic studies have shown that regulatory proteins like MraZ correlate strongly with generation time (Spearman's correlation = 0.85) . Similarly, ATP-dependent zinc metalloprotease FtsH shows a negative correlation with generation time (Spearman's correlation = -0.85) . The hisZ protein likely functions within this broader regulatory network, helping coordinate histidine biosynthesis with growth requirements and environmental conditions.
What experimental evidence confirms the function of hisZ in N. europaea?
While direct experimental characterization of N. europaea hisZ is limited in the literature, comparative proteomics studies have identified and quantified numerous proteins in N. europaea, including enzymes involved in central metabolism and amino acid biosynthesis . These studies demonstrate that N. europaea expresses a complete complement of proteins necessary for histidine biosynthesis, and that many metabolic enzymes show differential expression under varying environmental conditions.
Proteomic analysis has shown that approximately 32% of N. europaea's predicted proteome is detectable in laboratory cultures, with 107 proteins (13% of expressed proteins) listed as uncharacterized . This suggests that while core metabolic functions are relatively well-characterized, many regulatory proteins like hisZ may benefit from further functional studies to fully elucidate their roles.
What are the optimal expression systems for recombinant N. europaea hisZ?
For recombinant expression of N. europaea hisZ, several expression systems can be optimized based on research requirements:
| Expression Parameter | Recommended Condition | Notes |
|---|---|---|
| Expression strain | BL21(DE3) or Rosetta | Rosetta provides rare codons often needed for N. europaea proteins |
| Induction temperature | 16-20°C | Lower temperatures improve solubility for N. europaea proteins |
| IPTG concentration | 0.1-0.5 mM | Lower concentrations favor proper folding |
| Media composition | TB or 2YT | Rich media improve protein yield |
| Affinity tag | N-terminal His6 or MBP | MBP tag may improve solubility |
Codon optimization of the hisZ gene sequence for E. coli expression is recommended due to potential codon usage differences between N. europaea and E. coli. Proteomics studies have successfully identified numerous N. europaea proteins, indicating that its proteins can be expressed using standard molecular biology techniques with appropriate optimization .
What purification strategies yield the highest purity of functional recombinant hisZ?
A multi-step purification approach is recommended for obtaining high-purity recombinant N. europaea hisZ:
| Purification Stage | Recommended Method | Purpose |
|---|---|---|
| Initial capture | IMAC (for His-tagged protein) | Separation from bulk cellular proteins |
| Intermediate purification | Ion exchange chromatography | Removal of impurities with different charge properties |
| Polishing | Size exclusion chromatography | Separation of oligomeric states and removal of aggregates |
Buffer composition significantly impacts purification success:
-
50 mM Tris-HCl or HEPES (pH 7.5-8.0)
-
150-300 mM NaCl
-
5-10% glycerol (for stability)
-
1-5 mM reducing agent (DTT or β-mercaptoethanol)
Throughout purification, it's essential to monitor protein activity, as N. europaea proteins may have specific requirements for maintaining functionality during purification. Comparative proteomics studies have used similar approaches to successfully purify and analyze numerous N. europaea proteins .
What are effective methods for assessing the functional activity of purified hisZ?
As a regulatory subunit, hisZ activity should be evaluated in the context of its effect on the catalytic HisG subunit:
| Assay Type | Methodology | Measurement |
|---|---|---|
| Regulatory function | ATP phosphoribosyltransferase activity with/without hisZ | Change in kinetic parameters |
| Binding assays | Isothermal titration calorimetry (ITC) | Direct measurement of binding affinity |
| Response to histidine | Activity across histidine concentration range | Changes in inhibition profile |
Optimal assay conditions generally include:
-
50 mM HEPES buffer (pH 8.0)
-
10 mM MgCl₂ (essential for ATP binding)
-
1-5 mM DTT (maintains reduced state)
-
0.1-0.5 mM ATP and PRPP as substrates
Controls should include assays with known inhibitors of ATP phosphoribosyltransferase and comparisons with well-characterized homologs from other bacterial species. It's important to note that hisZ function is primarily regulatory rather than catalytic, so assays should focus on how it modifies HisG activity rather than direct enzymatic activity.
How does oxygen limitation affect hisZ expression and activity in N. europaea?
While specific data on hisZ expression under oxygen limitation isn't directly provided in the search results, we can infer from N. europaea's broader metabolic responses:
Under oxygen-limited conditions, N. europaea undergoes significant metabolic shifts, with reduced growth yield and non-stoichiometric ammonia-to-nitrite conversion . These conditions likely affect amino acid biosynthesis pathways, including the histidine pathway. Transcriptomic studies show that under oxygen limitation, N. europaea significantly remodels its proteome, with differential expression of enzymes involved in central metabolism .
What role does hisZ play in N. europaea's adaptation to environmental stressors?
Environmental stressors trigger significant changes in N. europaea's metabolism. For example, under salinity stress, N. europaea shows differential expression of proteins involved in oxidative stress response . Similarly, oxygen limitation leads to upregulation of specific cytochrome oxidases, including B-type heme-copper oxidase .
Regulatory proteins in N. europaea show stress-specific expression patterns. For instance, the heat shock protein ClpB shows dramatically different expression levels between N. europaea (0.06%) and related species like N. ureae (0.82%) . Such differential expression of regulatory proteins suggests complex adaptation mechanisms to environmental conditions.
As a regulator of histidine biosynthesis, hisZ likely participates in these broader stress responses by modulating amino acid production based on cellular needs during stress. This would help balance the energetic cost of histidine synthesis against the requirement for this amino acid in stress-response proteins.
How does growth rate correlate with hisZ expression in N. europaea?
While specific data on hisZ expression correlation with growth rates isn't directly provided, insights can be drawn from related regulatory proteins:
Comparative proteomics studies have identified strong correlations between certain regulatory proteins and growth rates in ammonia-oxidizing bacteria. For example, the MraZ transcriptional regulator shows a strong positive correlation with generation time (Spearman's correlation = 0.85, p < 0.005) . Conversely, the ATP-dependent zinc metalloprotease FtsH shows a strong negative correlation with generation time (Spearman's correlation = -0.85, p < 0.005) .
Given that histidine biosynthesis is essential for protein synthesis and thus growth, hisZ expression likely correlates with growth parameters. Since ATP phosphoribosyltransferase catalyzes an ATP-consuming reaction, its regulation through hisZ may be particularly important during growth rate transitions when cellular energy demands change significantly.
Studies comparing N. europaea, N. multiformis, and N. ureae found distinct protein expression patterns correlating with their different growth rates , suggesting that metabolic enzymes and their regulators, including those involved in amino acid biosynthesis, are differentially expressed based on growth parameters.
What structural features determine substrate specificity in N. europaea hisZ?
Key structural determinants of hisZ function likely include:
The hisZ protein contains domains involved in interaction with the catalytic HisG subunit, allosteric binding of histidine or other regulatory molecules, and potential oligomerization interfaces. While the specific structure of N. europaea hisZ has not been fully characterized in the provided search results, we can infer that it shares conserved features with homologous proteins from other bacteria.
Residues at the HisG-hisZ interface would be crucial for determining the regulatory effect of hisZ. The histidine-binding pocket, if present in hisZ, would contain residues determining sensitivity to feedback inhibition. Given N. europaea's unique ecological niche as an ammonia oxidizer, its hisZ might have evolved specific features to integrate histidine biosynthesis with nitrogen metabolism.
Structural studies combining X-ray crystallography or cryo-EM with site-directed mutagenesis would be necessary to fully characterize these structural determinants. Proteomics studies have shown that N. europaea expresses numerous proteins with regulatory functions , suggesting active regulatory networks that likely include specialized structural adaptations.
How do protein-protein interactions regulate hisZ function in N. europaea?
The activity of hisZ is likely regulated through a network of protein-protein interactions:
The primary interaction partner for hisZ is the catalytic HisG subunit, forming the functional ATP phosphoribosyltransferase complex. This interaction is essential for the regulatory function of hisZ. Beyond this core interaction, hisZ may interact with other histidine biosynthesis pathway enzymes to facilitate metabolic channeling.
Proteomics studies have shown that even under controlled laboratory conditions, N. europaea expresses a diverse complement of proteins , creating ample opportunity for regulatory interactions. Investigation approaches like co-immunoprecipitation coupled with mass spectrometry would be necessary to fully map hisZ's interactome.
What experimental approaches can elucidate the molecular mechanism of hisZ regulation?
Several complementary approaches can reveal the regulatory mechanisms of hisZ:
| Experimental Approach | Specific Technique | Information Gained |
|---|---|---|
| Structural analysis | X-ray crystallography or cryo-EM | Atomic-level structure, binding interfaces |
| Binding studies | Surface plasmon resonance (SPR) | Binding kinetics, affinity constants |
| Mutational analysis | Alanine scanning mutagenesis | Identification of critical residues |
| In vitro reconstitution | Purified component enzymes | Direct regulatory effects without cellular complexity |
For comprehensive mechanistic understanding, these approaches should be combined with functional assays measuring ATP phosphoribosyltransferase activity under varying conditions. Proteomic studies of N. europaea have successfully employed mass spectrometry-based approaches to characterize protein expression patterns , suggesting that similar techniques could be applied to study hisZ regulation.
Additionally, transcriptomic approaches similar to those used to study N. europaea under oxygen limitation could reveal how hisZ expression responds to varying environmental conditions, providing insights into its regulatory role in cellular adaptation.
How can solubility and stability be improved for recombinant N. europaea hisZ?
Optimizing solubility and stability requires a multi-faceted approach:
| Strategy | Specific Methods | Expected Outcome |
|---|---|---|
| Expression conditions | Growth at 16-20°C after induction | Slower expression promoting proper folding |
| Fusion partners | MBP or SUMO tags | Enhanced solubility through highly soluble partners |
| Buffer additives | 5-10% glycerol, 0.5-1 mM TCEP | Stabilization of folded state, prevention of oxidation |
| Co-expression | With HisG partner | Formation of natural complex improving stability |
The search results indicate that proteomics studies have successfully analyzed numerous N. europaea proteins , suggesting that with appropriate optimization, recombinant hisZ can be expressed with good solubility. Maintaining reducing conditions throughout purification (inclusion of 1-5 mM DTT or 2-mercaptoethanol) may be particularly important for preventing aggregation.
Co-expression with the HisG catalytic subunit may be especially effective, as hisZ naturally functions in complex with HisG. This approach mimics the native environment of the protein and may promote proper folding and stability.
What are the common pitfalls in experimental studies of N. europaea hisZ?
Several challenges commonly arise when working with hisZ:
| Challenge | Underlying Cause | Mitigation Strategy |
|---|---|---|
| Low protein yield | Toxicity to expression host | Use tightly controlled expression systems |
| Improper folding | Different cellular environment | Try various expression temperatures and chaperone co-expression |
| Loss of activity during purification | Sensitivity to oxidation | Include reducing agents throughout purification |
| Inconsistent activity assays | Complex regulatory function | Ensure proper complex formation with HisG partner |
It's essential to remember that hisZ functions as a regulatory subunit rather than having direct catalytic activity. Therefore, activity assays should focus on how hisZ modifies HisG activity under various conditions rather than expecting independent enzymatic function.
The search results indicate that N. europaea proteins can exhibit stress responses involving oxidative damage protection mechanisms , suggesting that attention to redox conditions may be particularly important when working with proteins from this organism.
How can site-directed mutagenesis be optimized for studying N. europaea hisZ function?
Site-directed mutagenesis provides powerful insights into structure-function relationships:
| Aspect | Recommendation | Rationale |
|---|---|---|
| Target selection | Conserved residues identified by multiple sequence alignment | Focus on functionally important residues |
| Primer design | 25-35 bp matching sequences, minimal secondary structure | Ensure specific amplification |
| PCR conditions | High-fidelity polymerase, DMSO for GC-rich regions | Account for N. europaea's genomic characteristics |
| Functional assessment | Multi-parameter analysis (activity, binding, stability) | Comprehensive characterization of mutant effects |
When designing mutations, focus on residues likely involved in:
-
Interaction with the HisG catalytic subunit
-
Histidine binding for feedback regulation
-
Oligomerization interfaces affecting quaternary structure
The search results indicate that comparative studies of N. europaea proteins have yielded valuable insights , suggesting that comparing wild-type and mutant hisZ properties would be a productive approach to understanding its functional mechanisms.
