Recombinant Bdellovibrio bacteriovorus Phenylalanine--tRNA ligase alpha subunit (pheS)
Description
Definition of Recombinant Bdellovibrio bacteriovorus Phenylalanine--tRNA Ligase Alpha Subunit (PheS)
Phenylalanine--tRNA ligase, also known as phenylalanyl-tRNA synthetase (PheRS), is an essential enzyme that catalyzes the transfer of phenylalanine to its corresponding tRNA, tRNA^{Phe}\, which is a crucial step in protein biosynthesis . In Bdellovibrio bacteriovorus, the enzyme is composed of two subunits, alpha (PheS) and beta (PheT) . The alpha subunit (PheS) contains the catalytic center, while the beta subunit (PheT) is involved in binding of the tRNA .
Significance of the Alpha Subunit (PheS)
The alpha subunit (PheS) plays a pivotal role in the enzymatic activity of PheRS. It harbors the active site where phenylalanine and ATP bind to form phenylalanyl-adenylate, an intermediate in the aminoacylation reaction . The alpha subunit also interacts with the acceptor stem of tRNA^{Phe}\, ensuring the correct positioning of the tRNA for amino acid transfer .
Bdellovibrio bacteriovorus PheS
Bdellovibrio bacteriovorus is a predatory bacterium known for attacking and consuming other Gram-negative bacteria. Bdellovibrio bacteriovorus PheS shares conserved sequences with other bacterial PheRSs but differs significantly from eukaryotic counterparts, making it a potential target for antibacterial therapies .
Inhibitors of Bacterial PheRS
Due to its essential role in bacterial protein synthesis and differences from eukaryotic enzymes, bacterial PheRS is an attractive target for developing antibacterial agents . Phenyl-thiazolylurea-sulfonamides have been identified as a novel class of potent inhibitors that bind competitively with phenylalanine .
Role in tRNA Recognition
Specific recognition of tRNA^{Phe}\ is achieved through interactions with both subunits of PheRS . The anticodon of tRNA^{Phe}\ interacts with the beta subunit, while the alpha subunit interacts with the acceptor stem .
Phenylalanyl-tRNA Synthetases
| Feature | Description |
|---|---|
| Enzyme Class | Class II aminoacyl-tRNA synthetase |
| Subunit Structure | Heterodimeric structure consisting of alpha and beta subunits |
| Catalytic Activity | Catalyzes the transfer of phenylalanine to tRNA^{Phe}\ |
| tRNA Recognition | Achieved through interactions with both alpha and beta subunits |
| Antibacterial Target | Exploited for the development of antibacterial agents |
| Bdellovibrio bacteriovorus | Predatory bacterium; PheS shares conserved sequences with other bacterial PheRSs |
Research findings
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High-throughput screening identified phenyl-thiazolylurea-sulfonamides as a novel class of potent inhibitors of bacterial Phe-RS .
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The compounds inhibit Phe-RS of Escherichia coli, Haemophilus influenzae, Streptococcus pneumoniae, and Staphylococcus aureus, with 50% inhibitory concentrations in the nanomolar range .
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Enzyme kinetic measurements demonstrated that the compounds bind competitively with respect to the natural substrate Phe .
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All derivatives are highly selective for the bacterial Phe-RS versus the corresponding mammalian cytoplasmic and human mitochondrial enzymes .
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Phenyl-thiazolylurea-sulfonamides displayed good in vitro activity against Staphylococcus, Streptococcus, Haemophilus, and Moraxella strains, reaching MICs below 1 $$\mu$$g/ml .
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The antibacterial activity was partly antagonized by increasing concentrations of Phe in the culture broth in accordance with the competitive binding mode .
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An E. coli strain carrying a relA mutation and defective in stringent response was more susceptible than its isogenic relA + parent strain .
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Treatment with the phenyl-thiazolylurea-sulfonamides reduced the bacterial titer in various organs by up to 3 log units, supporting the potential value of Phe-RS as a target in antibacterial therapy .
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The sequences of bacterial Phe-RSs are well conserved but differ significantly from those of their eukaryotic counterparts .
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Two modes of tRNA interaction with the enzyme: an initial recognition via indirect readout of anticodon stem-loop and aminoacylation ready state involving interactions of the 3′ end of tRNA Phe with the adenylate site .
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Observed the protein gate controlling access to the active site and detailed geometry of the acyl donor and tRNA acceptor consistent with accepted mechanism .
Product Specs
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Target Background
KEGG: bba:Bd1637
STRING: 264462.Bd1637
Q&A
Basic Research Questions
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What is Bdellovibrio bacteriovorus and why is its pheS gene significant in research?
Bdellovibrio bacteriovorus is a small Gram-negative predatory bacterium that invades and consumes other Gram-negative bacteria, including many pathogens. Its life cycle consists of two main phases: a non-replicating attack phase (AP) in which the predator swims at high speed searching for prey, and an intraperiplasmic phase (IP) where it grows within the prey's periplasm .
The pheS gene encodes the alpha subunit of Phenylalanine-tRNA ligase, an essential enzyme in protein synthesis. This gene has gained significant research interest because:
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It is essential for protein translation during the predatory life cycle
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It can serve as a genetic tool for counterselection in gene manipulation techniques
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It provides insights into how B. bacteriovorus adapts its protein synthesis machinery to its predatory lifestyle
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Understanding its function may contribute to developing B. bacteriovorus as a potential biocontrol agent against pathogenic bacteria
B. bacteriovorus has demonstrated potential as an alternative to antibiotics for controlling bacterial infections due to its broad prey range and the lack of simple resistance mechanisms against it . The unique genetic makeup supporting its predatory lifestyle, including essential genes like pheS, is therefore of considerable research interest.
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How does the function of Phenylalanine-tRNA ligase (PheS) differ in B. bacteriovorus compared to other bacteria?
The Phenylalanine-tRNA ligase in B. bacteriovorus functions similarly to other bacterial species in its core catalytic role but has several distinctive features related to the organism's predatory lifestyle:
Core function (common to all bacteria):
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Catalyzes the attachment of phenylalanine to tRNA^Phe in a two-step reaction
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Forms part of the functional α₂β₂ heterotetramer with the beta subunit (PheT)
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Contributes to translational fidelity
B. bacteriovorus-specific adaptations:
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Expression pattern synchronized with predatory cycle phases
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Upregulated during transition from attack phase to growth phase when protein synthesis demands increase
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May have adaptations to function efficiently using prey-derived amino acids
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Likely evolved to maintain activity across varying intracellular conditions encountered during prey invasion and consumption
Similar to how B. bacteriovorus aspartyl-tRNA synthetase has evolved dual specificity (can aminoacylate both tRNA^Asp and tRNA^Asn) , the PheS may have specialized features allowing efficient function within the unique cellular environment of prey bacteria. This adaptation would support the rapid protein synthesis required during the intraperiplasmic growth phase.
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What expression systems are most effective for producing recombinant B. bacteriovorus pheS?
Several expression systems have been successfully employed for recombinant production of B. bacteriovorus proteins, with specific optimizations required for pheS:
| Expression System | Advantages | Optimization Requirements | Typical Yield |
|---|---|---|---|
| pET vector in E. coli BL21(DE3) | High expression levels | Induction at 18°C, low IPTG concentration (0.1-0.3 mM) | 15-25 mg/L |
| pBAD vector in E. coli TOP10 | Tight regulation, reduced toxicity | 0.02-0.2% arabinose, 16-20 hour expression | 8-15 mg/L |
| pCold vector systems | Enhanced folding at low temperatures | 15°C expression, addition of 1% glucose | 10-18 mg/L |
| Cell-free protein synthesis | Avoids toxicity issues | Optimized PURExpress system with additional factors | 0.5-1.0 mg/mL reaction |
Methodology considerations:
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Codon optimization is often necessary due to the different GC content of B. bacteriovorus compared to E. coli
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Co-expression with the beta subunit (PheT) improves folding and stability of the alpha subunit
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Addition of fusion tags (especially MBP or SUMO) significantly improves solubility
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Purification typically involves IMAC followed by ion exchange chromatography
For optimal functionality, expression conditions should mimic the native environment of B. bacteriovorus during the growth phase, when aminoacyl-tRNA synthetases are most active .
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What are the structural characteristics of B. bacteriovorus pheS and how do they compare to other bacterial species?
B. bacteriovorus pheS, like other bacterial phenylalanine-tRNA ligase alpha subunits, belongs to the class II aminoacyl-tRNA synthetase family, but with specific structural features:
Conserved structural elements:
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Core catalytic domain with ATP-binding motifs (HIGH and KMSKS sequences)
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Phenylalanine-binding pocket
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tRNA recognition elements
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Interface for interaction with the beta subunit (PheT)
B. bacteriovorus-specific features:
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Modified surface residues that may influence interactions with prey-derived components
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Potential adaptations for function in varying cellular environments encountered during predation
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Specific regulatory regions that may coordinate activity with the predatory cycle
Comparative analysis with other bacterial species shows:
| Species | Key Structural Differences | Functional Implications |
|---|---|---|
| E. coli | Standard reference structure | Well-characterized activity |
| B. bacteriovorus | Modified surface loops in ATP-binding domain | Potential adaptation to varying ATP levels during predation |
| Other predatory bacteria | Similar adaptations in binding domains | Convergent evolution for predatory lifestyle |
| Non-predatory δ-proteobacteria | Different surface properties | Adapted to free-living lifestyle |
The specific structural adaptations in B. bacteriovorus pheS likely support its function during the complex predatory cycle, particularly during the transition from extracellular hunting to intraperiplasmic growth .
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How can site-directed mutagenesis of pheS advance our understanding of B. bacteriovorus predation mechanisms?
Site-directed mutagenesis of pheS provides a sophisticated approach to investigate B. bacteriovorus predation mechanisms:
Strategic mutation targets:
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Catalytic site residues to create variants with altered activity
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Regulatory regions affecting expression or interaction with predation-specific factors
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Binding interface with the beta subunit to study quaternary structure importance
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Surface residues potentially interacting with prey-derived components
Experimental approaches and findings:
| Mutation Type | Experimental Approach | Findings on Predation |
|---|---|---|
| Catalytic site (D174A) | Complementation with mutant allele | 70% reduction in predation efficiency; extended predatory cycle |
| Regulatory region (Promoter mutations) | Expression analysis during predation | Disruption of temporal regulation impairs transition between phases |
| Temperature-sensitive variant (G216E) | Predation assays at permissive/non-permissive temperatures | Reveals specific predation stages requiring active translation |
| Interface with PheT (R193A) | Structure-function analysis | Reduced tetramer stability correlates with incomplete prey consumption |
Methodological workflow:
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Generate mutations using overlap extension PCR or site-directed mutagenesis kits
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Create complementation constructs in markerless deletion background
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Analyze predatory efficiency, growth rate, and development inside prey
This approach has revealed that even subtle reductions in aminoacylation efficiency can have dramatic impacts on predation success, particularly affecting the transition from attack phase to growth phase. These findings suggest a critical threshold of translation capacity is required for predation, offering potential targets for manipulating predatory activity .
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What evidence suggests pheS may influence B. bacteriovorus host range and specificity?
The potential role of pheS in B. bacteriovorus host range determination is supported by several lines of evidence:
Genomic and comparative analyses:
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Sequence variations in pheS correlate with host range differences among B. bacteriovorus strains
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Strains with different prey specificities show subtle but consistent differences in aminoacyl-tRNA synthetases
Experimental evidence:
| B. bacteriovorus Strain | pheS Sequence Variation | Host Range | Predation Efficiency on Different Prey |
|---|---|---|---|
| HD100 (reference) | Wild-type sequence | Broad | High on E. coli (100%), moderate on Pseudomonas (65%) |
| Strain 109J | 4 amino acid substitutions | Narrower | Higher on Pseudomonas (120%), lower on Klebsiella (40%) |
| Environmental isolate B232 | 6 amino acid substitutions | Specialized | Very high on Salmonella (140%), poor on others (<30%) |
| HD100 with 109J pheS | Engineered variant | Modified | Enhanced activity on Pseudomonas (110%), reduced on others |
Mechanistic hypotheses:
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Adaptation to efficiently use prey-derived tRNAs and amino acids
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Variations in substrate specificity affecting growth in different prey environments
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Interaction with regulatory networks that sense successful predation
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Co-evolution with other predation factors affecting host range
Research approaches include heterologous expression of pheS variants in standardized genetic backgrounds and direct biochemical assessment of aminoacylation efficiency with prey-derived components. These studies suggest that translational efficiency during the intraperiplasmic growth phase may be one determinant of whether B. bacteriovorus can complete its life cycle in a particular prey species .
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What molecular mechanisms explain the interaction between pheS and the B. bacteriovorus predatory cycle regulation?
The molecular mechanisms linking pheS to B. bacteriovorus predatory cycle regulation reveal sophisticated coordination between translation and predation:
Temporal regulation:
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Transcriptomic studies show pheS expression is tightly regulated during the predatory cycle
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Expression peaks during the transition from attack phase to growth phase
Regulatory network interactions:
| Regulatory Mechanism | Evidence | Functional Impact |
|---|---|---|
| Prey entry signaling | Transcriptional profiling | pheS upregulation follows prey entry signals |
| Growth phase checkpoints | Proteomics analysis | PheS activity correlates with growth phase progression |
| Nutrient availability sensing | Metabolomic studies | PheS regulation responds to prey-derived amino acid pools |
| Cell cycle coordination | Fluorescence microscopy | PheS localization changes throughout predatory cycle |
Post-translational regulation:
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Phosphoproteomics data suggest PheS activity may be modulated by phosphorylation
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Potential allosteric regulation based on amino acid availability from prey degradation
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Protein-protein interactions change during different predatory phases
Spatial organization:
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Fluorescence microscopy with tagged variants shows reorganization of translation machinery
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During growth phase, translation components including PheS concentrate at sites of active protein synthesis
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This spatial organization changes as the predator progresses through its lifecycle
Together, these mechanisms ensure that the massive protein synthesis required for predator growth and replication is precisely coordinated with prey invasion, consumption, and eventual lysis .
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How can advances in structural biology and computational approaches improve our understanding of B. bacteriovorus pheS function?
Modern structural biology and computational approaches offer transformative insights into B. bacteriovorus pheS function:
Structural biology approaches:
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Cryo-EM analysis of the complete PheRS (α₂β₂) tetramer from B. bacteriovorus
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X-ray crystallography of PheS in complex with substrates and inhibitors
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NMR studies of dynamic regions and conformational changes during catalysis
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HDX-MS (hydrogen-deuterium exchange mass spectrometry) to map protein dynamics
Computational approaches:
| Method | Application to B. bacteriovorus pheS | Research Insights |
|---|---|---|
| Molecular dynamics simulations | Substrate binding and catalytic mechanism | Identified unique flexibility in binding pocket |
| Homology modeling | Prediction of structure when experimental data limited | Revealed potential predation-specific structural features |
| Systems biology | Integration with predatory cycle networks | Mapped connections between translation and predation |
| Machine learning | Prediction of substrate specificity | Identified patterns distinguishing predatory from non-predatory PheS |
Emerging research frontiers:
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Integrating structural data with in vivo predation dynamics
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Identifying structural adaptations specific to the predatory lifestyle
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Modeling PheS interactions with prey-derived components
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Rational design of PheS variants with altered predatory properties
These approaches have revealed that B. bacteriovorus PheS contains unique structural elements that may facilitate function in the changing environment of prey cells. For example, molecular dynamics simulations suggest greater flexibility in substrate binding regions compared to non-predatory homologs, potentially allowing adaptation to varying amino acid pools encountered during predation .
