Recombinant Nitrosomonas europaea Leucyl/phenylalanyl-tRNA--protein transferase (aat)
Description
Introduction to Recombinant Nitrosomonas europaea Leucyl/phenylalanyl-tRNA--protein transferase (aat)
Recombinant Nitrosomonas europaea Leucyl/phenylalanyl-tRNA--protein transferase (aat) is a recombinant enzyme produced in yeast, which belongs to the family of leucyl/phenylalanyl-tRNA--protein transferases. These enzymes are crucial in the process of conjugating leucine or phenylalanine to the N-terminal arginine or lysine residues of proteins, utilizing aminoacyl-tRNAs as substrates. This process plays a significant role in the N-end rule protein degradation pathway, which affects protein stability and turnover in bacteria.
Function and Mechanism
The primary function of Leucyl/phenylalanyl-tRNA--protein transferase (aat) is to modify proteins by adding leucine or phenylalanine to their N-terminus, specifically targeting proteins with N-terminal arginine or lysine residues. This modification acts as a degradation signal, marking the proteins for degradation via the ClpS-ClpAP pathway in bacteria. The enzyme's activity is highly specific, preferring hydrophobic amino acids like leucine and phenylalanine over others due to its hydrophobic pocket structure.
Production and Purification
Recombinant Nitrosomonas europaea Leucyl/phenylalanyl-tRNA--protein transferase (aat) is produced in yeast, ensuring high purity and efficiency. For storage and handling, it is recommended to reconstitute the protein in deionized sterile water at a concentration of 0.1-1.0 mg/mL, with the addition of 5-50% glycerol to enhance stability .
Research Findings and Applications
Research on Leucyl/phenylalanyl-tRNA--protein transferase (aat) has provided insights into its role in protein degradation pathways and its potential applications in biotechnology. The enzyme's specificity for hydrophobic amino acids makes it a valuable tool for studying protein modification and degradation mechanisms.
Data Table: Characteristics of Recombinant Nitrosomonas europaea Leucyl/phenylalanyl-tRNA--protein transferase (aat)
| Characteristic | Description |
|---|---|
| Species | Nitrosomonas europaea (strain ATCC 19718 / CIP 103999 / KCTC 2705 / NBRC 14298) |
| Length | 236 amino acids |
| Production Host | Yeast |
| Purity | High purity |
| Function | Conjugates leucine or phenylalanine to N-terminal Arg or Lys residues of proteins |
| Substrate | Aminoacyl-tRNAs (e.g., Leu-tRNA Leu, Phe-tRNA Phe) |
Product Specs
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Target Background
KEGG: neu:NE2219
STRING: 228410.NE2219
Q&A
What is the genomic context of the Leucyl/phenylalanyl-tRNA--protein transferase (aat) gene in Nitrosomonas europaea?
The aat gene in Nitrosomonas europaea is part of the organism's single circular chromosome (2,812,094 bp). N. europaea has a relatively compact genome with genes distributed evenly, with approximately 47% transcribed from one strand and 53% from the complementary strand . The genomic architecture of N. europaea is organized into two unequal replichores as indicated by GC skew analysis . When examining the genetic context, the aat gene should be analyzed in relation to other protein synthesis machinery genes, particularly those involved in tRNA processing, as N. europaea contains multiple tRNA genes including those encoding threonyl tRNA synthetase (thrS) and phenylalanyl tRNA synthetase α and β subunits (pheS and pheT) .
What expression systems are most effective for producing recombinant Nitrosomonas europaea aat protein?
Recombinant expression of N. europaea aat protein presents unique challenges due to the specialized metabolic nature of this chemolithoautotroph. When designing expression systems, consider the following methodological approaches:
Table 1: Comparative Efficiency of Expression Systems for N. europaea aat Protein
| Expression System | Advantages | Limitations | Optimal Conditions |
|---|---|---|---|
| E. coli BL21(DE3) | High yield, established protocols | Potential misfolding | IPTG induction at 16°C, 18h |
| Mammalian cell lines | Proper folding and processing | Lower yield, higher cost | Viral delivery vectors at MOI <10 |
| Cell-free systems | Rapid results, no toxicity issues | Scalability challenges | Supplementation with tRNA components |
For bacterial expression, a strategy similar to that employed for other N. europaea proteins should be used, incorporating tags that facilitate purification while minimizing interference with protein function . For mammalian expression, optimized tRNA systems similar to those described for leucyl-tRNA in the literature can significantly improve expression efficiency . The viral delivery vectors developed for improved genetic incorporation of non-canonical amino acids could be particularly beneficial when working with this challenging enzyme .
How do the catalytic properties of recombinant N. europaea aat differ from homologous enzymes in other bacteria?
The catalytic properties of recombinant N. europaea Leucyl/phenylalanyl-tRNA--protein transferase reflect its adaptation to the unique metabolic lifestyle of this specialized ammonia-oxidizing bacterium. Unlike most bacteria that can utilize organic carbon sources, N. europaea is an obligate chemolithoautotroph that derives energy almost exclusively from ammonia oxidation .
Methodological approaches to characterizing the kinetic parameters should include:
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Comparative substrate specificity analysis using purified recombinant aat with various tRNA substrates
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Determination of optimal reaction conditions (pH, temperature, salt concentration)
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Assessment of metal ion requirements and inhibition patterns
The specialized metabolism of N. europaea may have driven evolutionary adaptations in its aat enzyme, potentially resulting in unique structural features or catalytic mechanisms compared to homologs from heterotrophic bacteria. The limited genetic capacity for catabolism of organic compounds in N. europaea suggests its protein synthesis machinery, including aat, may have evolved distinctive properties to function efficiently within its specialized metabolic context.
What is the role of N. europaea aat in protein N-terminal modification and how does this affect protein stability?
N. europaea Leucyl/phenylalanyl-tRNA--protein transferase likely plays a crucial role in post-translational modification of protein N-termini, a process important for protein stability and turnover. In many bacteria, this enzyme transfers specific amino acids (leucine or phenylalanine) from aminoacyl-tRNAs to the N-terminus of target proteins, marking them for recognition by cellular degradation machinery.
The methodological approach to studying this function should include:
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Identification of native protein substrates in N. europaea using proteomics approaches
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Site-directed mutagenesis of key catalytic residues to assess functional mechanisms
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In vitro reconstitution of the N-terminal modification pathway using purified components
Given N. europaea's specialized metabolism and adaptation to ammonia oxidation , its protein turnover mechanisms may have unique features compared to heterotrophic bacteria. Exploring whether aat activity is regulated in response to ammonia availability or oxidative stress could provide insights into how this specialized bacterium maintains proteostasis under its unique metabolic constraints.
What structural features of N. europaea aat contribute to its substrate specificity?
The structural features of N. europaea aat that determine its substrate specificity can be investigated through comparative structural biology approaches. While the specific structure of N. europaea aat has not been directly reported in the provided search results, methodological approaches should include:
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Homology modeling based on related bacterial aat structures
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Identification of conserved domains and catalytic residues through sequence alignment
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Molecular docking simulations with tRNA and amino acid substrates
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Site-directed mutagenesis of predicted substrate-binding residues
The unique metabolic constraints of N. europaea as an obligate chemolithoautotroph may have driven evolutionary adaptations in its aat enzyme's structure. The genomic analysis of N. europaea reveals it has genes necessary for specialized biosynthetic pathways , which suggests its protein modification machinery may contain structural adaptations to function within its unique metabolic network.
How can directed evolution approaches be applied to enhance the catalytic efficiency of N. europaea aat?
Directed evolution represents a powerful approach for enhancing the catalytic properties of N. europaea aat. Drawing from methods successfully applied to tRNA-related enzymes, the following methodological framework is recommended:
Table 2: Directed Evolution Strategies for N. europaea aat Engineering
| Evolutionary Approach | Methodology | Selection System | Expected Outcome |
|---|---|---|---|
| Error-prone PCR | Random mutagenesis of entire aat gene | Activity-based selection in E. coli | Variants with enhanced catalytic rate |
| DNA shuffling | Recombination with homologous aat genes | Survival under selective pressure | Chimeric enzymes with novel properties |
| VADER technique | Virus-assisted evolution in mammalian cells | AAV-based selection | Variants with improved in vivo activity |
The VADER (virus-assisted directed evolution for engineering tRNAs) approach, which has been successfully applied to enhance the activities of suppressor tRNAs in mammalian cells , could be particularly effective. This method links enzyme activity to viral proliferation, allowing for the identification of highly active aat variants. By applying selection pressure that mimics the metabolic constraints N. europaea faces as an ammonia oxidizer , evolved variants may exhibit improved catalytic efficiency under physiologically relevant conditions.
What are the optimal conditions for assaying N. europaea aat activity in vitro?
Establishing robust assay conditions is critical for accurately measuring N. europaea aat activity. Based on the biochemical characteristics of N. europaea and related aat enzymes, the following methodological approach is recommended:
Table 3: Optimized Assay Conditions for N. europaea aat Activity
| Parameter | Optimal Range | Measurement Method | Critical Considerations |
|---|---|---|---|
| pH | 7.5-8.0 | Spectrophotometric coupled assay | Buffer compatibility with aminoacyl-tRNA stability |
| Temperature | 25-30°C | Thermal stability analysis | N. europaea optimal growth temperature |
| Ionic strength | 50-100 mM KCl | Activity measurements at varying salt concentrations | Effect on protein-tRNA interactions |
| Metal requirements | 5-10 mM Mg²⁺ | Chelation and reconstitution experiments | Essential for tRNA structural integrity |
When developing activity assays, the unique biochemical environment of N. europaea should be considered. As an ammonia-oxidizing bacterium that generates energy through nitrification , its intracellular environment may have distinctive characteristics that affect enzyme function. The assay should incorporate relevant aminoacyl-tRNAs as substrates, potentially utilizing the leucyl-tRNA engineering approaches described in the literature to generate defined substrate molecules.
How can isotope labeling be used to track N. europaea aat-mediated protein modifications in vivo?
Isotope labeling offers powerful approaches for tracking aat-mediated protein modifications in the N. europaea cellular context. A comprehensive methodological strategy would include:
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Metabolic labeling with stable isotopes (¹⁵N, ¹³C) to track amino acid transfer
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Pulse-chase experiments to determine modification kinetics
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Combined mass spectrometry approaches to identify modified protein substrates
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Integration with proteomics data to correlate modification with protein turnover
Given N. europaea's ability to fix carbon dioxide , experiments must control for potential dilution of isotope labels through autotrophic metabolism. The unique metabolism of N. europaea as an obligate chemolithoautotroph presents both challenges and opportunities for isotope labeling studies, as its limited capacity for organic carbon assimilation may result in higher incorporation efficiency of labeled amino acids compared to heterotrophic bacteria.
How should researchers account for the impact of N. europaea's unusual metabolism when interpreting aat activity data?
The interpretation of experimental data for N. europaea aat must consider the unique metabolic context of this organism. As an obligate chemolithoautotroph, N. europaea derives energy from ammonia oxidation and fixes carbon dioxide for biosynthesis , which creates a distinctive cellular environment that may influence aat function.
Methodological considerations for data interpretation should include:
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Normalizing enzyme activity to account for differences in cellular energy status
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Considering the potential impact of oxidative stress from ammonia metabolism on protein stability
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Evaluating tRNA aminoacylation efficiency in the context of autotrophic vs. heterotrophic growth
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Comparing results against appropriate controls, such as aat enzymes from metabolically diverse bacteria
The specialized genome of N. europaea, with its abundance of genes for inorganic ion transport but limited capacity for organic molecule transport , suggests that its protein synthesis machinery operates in a unique ionic environment. Researchers should consider how these factors might affect aat activity both in vitro and in vivo when analyzing experimental results.
What statistical approaches are most appropriate for analyzing large-scale datasets from N. europaea aat structure-function studies?
When analyzing comprehensive datasets from structure-function studies of N. europaea aat, researchers should implement robust statistical frameworks tailored to the specific experimental design:
Table 4: Statistical Approaches for N. europaea aat Structure-Function Datasets
| Data Type | Recommended Statistical Method | Visualization Approach | Validation Strategy |
|---|---|---|---|
| Mutational scanning | Multiple hypothesis testing with FDR correction | Heatmap clustering of functional effects | Cross-validation with structural data |
| Evolutionary conservation | Phylogenetic comparative methods | Sequence-structure mappings | Ancestral sequence reconstruction |
| Kinetic parameters | Nonlinear regression with global fitting | Michaelis-Menten and Lineweaver-Burk plots | Bootstrap resampling for confidence intervals |
| Structural dynamics | Principal component analysis of molecular dynamics | Energy landscape visualizations | Comparison with experimental B-factors |
How can N. europaea aat be leveraged for incorporating non-canonical amino acids in protein engineering?
The potential application of N. europaea aat for non-canonical amino acid incorporation represents an exciting frontier in protein engineering. Drawing from advances in tRNA engineering for genetic code expansion, researchers could develop the following methodological approach:
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Engineer the aat enzyme to recognize tRNAs charged with non-canonical amino acids
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Develop specialized expression systems incorporating evolved tRNAs similar to the LeuIGIs (leucyl tRNAs for improved genetic incorporation)
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Establish selection systems to identify optimized aat variants with enhanced specificity for non-canonical substrates
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Design synthetase-tRNA-aat systems that function coordinately to facilitate N-terminal protein modification with novel chemical groups
The optimization of tRNA EcLeu through directed evolution in mammalian cells provides a valuable template for similar engineering of the N. europaea aat system. By adapting the virus-assisted directed evolution strategy for engineering tRNAs (VADER) , researchers could develop specialized aat variants with novel specificities. The established structural diversity of non-canonical amino acids successfully incorporated using engineered leucyl-tRNA synthetase systems suggests that aat engineering could similarly expand the chemical toolkit for protein modification.
What are the challenges in establishing a CRISPR-Cas9 gene editing system for manipulating the aat gene in N. europaea?
Establishing effective genetic manipulation systems for N. europaea presents significant challenges due to its specialized metabolism and relatively limited genetic tools compared to model organisms. For CRISPR-Cas9 editing of the aat gene, researchers should consider the following methodological approach:
Table 5: CRISPR-Cas9 Implementation Challenges for N. europaea aat Editing
| Challenge | Technical Solution | Validation Method | Expected Outcomes |
|---|---|---|---|
| Delivery of CRISPR components | Electroporation optimization | Transformation efficiency measurement | Protocol for reliable transformation |
| Guide RNA design | Target unique regions avoiding repetitive elements | In silico off-target prediction | Specific targeting of aat gene |
| Homology-directed repair | Design HDR templates accounting for GC content | PCR and sequencing verification | Precise genome editing |
| Selection of edited cells | Develop ammonia-dependent selection markers | Growth assays under selective conditions | Enrichment of edited populations |
The genome of N. europaea contains complex repetitive elements that constitute approximately 5% of its genome, including 85 predicted insertion sequence elements in eight different families . This repetitive nature could complicate CRISPR targeting, requiring careful guide RNA design to ensure specificity. Additionally, the unique physiology of N. europaea as an obligate chemolithoautotroph necessitates specialized cultivation conditions for screening and maintaining edited strains.
What are the most common pitfalls in purification of recombinant N. europaea aat and how can they be avoided?
Purification of recombinant N. europaea aat can present several challenges that require careful methodological considerations:
Table 6: Troubleshooting Guide for N. europaea aat Purification
| Challenge | Probable Cause | Solution Strategy | Quality Control Method |
|---|---|---|---|
| Low solubility | Improper folding in expression host | Expression at lower temperature (16-20°C) | Solubility screening with different detergents |
| Co-purifying contaminants | Non-specific binding to affinity resin | Addition of low imidazole in binding buffer | SDS-PAGE and mass spectrometry verification |
| Loss of activity during purification | Metal ion depletion | Include stabilizing agents (glycerol, reducing agents) | Activity assays at each purification step |
| Aggregation during concentration | Hydrophobic interactions | Addition of stabilizing osmolytes | Dynamic light scattering analysis |
The specialized metabolism of N. europaea may result in unique post-translational modifications or cofactor requirements for its aat enzyme . Researchers should consider the potential impacts of N. europaea's obligate chemolithoautotrophic lifestyle on protein structure and function when designing purification strategies. Comparative analysis with aat enzymes from heterotrophic bacteria may reveal specific considerations needed for the N. europaea enzyme.
How can researchers verify the specificity of N. europaea aat for different tRNA substrates?
Verifying the substrate specificity of N. europaea aat requires systematic methodological approaches:
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In vitro aminoacylation assays using purified tRNAs and recombinant aminoacyl-tRNA synthetases
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Competition experiments with different aminoacyl-tRNAs to determine relative affinity
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Structural analysis of aat-tRNA complexes through crystallography or cryo-electron microscopy
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Mutagenesis of tRNA determinants to map recognition elements
Researchers can adapt approaches similar to those used in the directed evolution of tRNA EcLeu , which identified key acceptor stem variants with improved activity. The virus-assisted selection scheme developed for tRNA evolution could potentially be modified to assess aat-tRNA interactions by linking successful aminoacyl transfer to viral replication. When analyzing results, researchers should consider how N. europaea's unique genome structure and specialized metabolism might influence tRNA diversity and abundance in its native context.
How might the study of N. europaea aat contribute to understanding protein quality control in specialized bacteria?
The study of N. europaea aat opens avenues for understanding protein quality control mechanisms in specialized metabolic niches. As an obligate chemolithoautotroph that derives energy from ammonia oxidation , N. europaea likely has adapted protein turnover mechanisms to support its unique lifestyle. Research approaches should include:
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Comparative genomics analysis of protein quality control machinery across bacteria with diverse metabolic strategies
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Identification of aat-dependent degradation pathways specific to ammonia-oxidizing bacteria
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Investigation of regulatory mechanisms linking ammonia oxidation to protein turnover
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Development of systems biology models integrating metabolic and proteostatic networks
N. europaea's genome encodes a variety of stress response systems that may interact with aat-mediated protein modification pathways. Understanding these interactions could reveal how specialized bacteria maintain proteome integrity under the unique constraints of chemolithoautotrophic growth. This knowledge could potentially inform strategies for engineering robust biocatalysts for environmental applications, such as wastewater treatment where ammonia-oxidizing bacteria like N. europaea play crucial roles .
What implications does the study of N. europaea aat have for understanding the evolution of aminoacyl-tRNA transferases across different bacterial phyla?
The evolutionary analysis of N. europaea aat can provide valuable insights into the adaptation of aminoacyl-tRNA transferases across diverse bacterial lineages. Methodological approaches should include:
Table 7: Evolutionary Analysis Framework for N. europaea aat
| Analytical Approach | Methodological Tools | Key Evolutionary Questions | Expected Insights |
|---|---|---|---|
| Phylogenetic reconstruction | Maximum likelihood tree building | Vertical vs. horizontal gene transfer patterns | Evolutionary history of aat in proteobacteria |
| Selection pressure analysis | dN/dS ratio calculation across lineages | Sites under positive/purifying selection | Functional constraints on aat evolution |
| Ancestral sequence reconstruction | Bayesian phylogenetic methods | Functional shifts during aat evolution | Correlation with metabolic transitions |
| Structural evolution mapping | Homology modeling of evolutionary intermediates | Conservation of catalytic vs. substrate-binding regions | Structural basis of functional divergence |
The genome of N. europaea shows evidence of both vertical inheritance and horizontal gene transfer , making it an interesting model for studying the evolution of essential cellular machinery. By comparing aat sequence and structure across bacteria with diverse metabolic strategies, researchers can gain insights into how protein modification systems have adapted to different ecological niches. The comparative genomics between N. europaea and related Nitrosomonas species reveals both conserved gene arrangements and dramatic rearrangements , providing a framework for understanding the evolutionary forces shaping aminoacyl-tRNA transferase function across bacterial diversity.
