Recombinant Rhodopirellula baltica D-tyrosyl-tRNA (Tyr) deacylase (dtd)

What is the biological function of D-tyrosyl-tRNA (Tyr) deacylase in Rhodopirellula baltica?

D-tyrosyl-tRNA (Tyr) deacylase (DTD) in R. baltica likely functions as a quality control enzyme that prevents the incorporation of D-amino acids into proteins during translation. Similar to the human DTD1 enzyme, it hydrolyzes D-tyrosyl-tRNA(Tyr) into D-tyrosine and free tRNA(Tyr), serving as a defense mechanism against the potentially harmful effects of D-amino acids in protein synthesis . This function is particularly important for organisms like R. baltica that may encounter diverse environmental conditions in marine habitats, where D-amino acids could be present in sediments or produced by other microorganisms.

The enzyme belongs to a highly conserved family of proteins that play crucial roles in maintaining translational fidelity. While not explicitly characterized in the provided proteome studies of R. baltica, inferences can be made based on the conservation of this enzyme across different domains of life. The enzyme's importance in maintaining proper protein folding and function makes it an essential component of cellular protein quality control mechanisms.

How is the DTD gene organized in the R. baltica genome?

Based on the genomic analysis of R. baltica, the DTD gene would be one of the 6,644 coding sequences identified in the genome annotation . The R. baltica genome has a size of approximately 7.4 Mb with a G+C content of 55.3% . While specific information about the DTD gene organization is not explicitly provided in the search results, it is likely located within the main chromosome, given that most housekeeping genes in bacteria tend to be chromosomally encoded.

The gene may be part of an operon related to translation or protein quality control functions, though this would require specific experimental verification through transcriptomic analysis. The genome annotation performed with PATRIC version 3.3.15 would provide the specific location and context of the DTD gene within the genome .

What expression systems are suitable for producing recombinant R. baltica DTD?

For expressing recombinant R. baltica DTD, researchers should consider several expression systems based on the properties of the protein and experimental needs:

• E. coli expression system: Most commonly used for recombinant protein production due to its simplicity, rapid growth, and high yields. This system would be particularly suitable for R. baltica DTD given that bacterial proteins often express well in E. coli. Typical vectors would include pET series with IPTG-inducible promoters.

• Native expression in R. baltica: For studying the protein in its natural context, expression in R. baltica itself could be considered. R. baltica can be grown in mineral medium with various carbon sources such as ribose, glucose, or N-acetylglucosamine . The cells would typically be harvested in exponential growth phase by centrifugation (10,000×g, 15 min, 4°C) .

• Fusion tag selection: A C-terminal 6×His tag (similar to what's used for human DTD1 ) would facilitate purification through affinity chromatography. This approach would enable single-step purification while minimizing interference with enzyme activity.

The expression conditions should be optimized considering R. baltica's natural growth environment, which includes marine conditions. Protein extraction could follow protocols similar to those used in proteome studies of R. baltica, involving cell lysis in buffer containing urea, thiourea, DTT, CHAPS, and carrier ampholytes .

How does the structure-function relationship of R. baltica DTD compare to DTD enzymes from other species?

The structure-function relationship of R. baltica DTD likely shares conserved features with DTD enzymes from other species while possessing unique adaptations reflective of its marine environment:

Conserved features:

• The catalytic mechanism likely involves key residues for recognizing the D-configuration of amino acids

• The binding pocket probably maintains specificity for tyrosyl-tRNA

• Structural elements for ATP binding may be present, as seen in human DTD1 which exhibits ATPase activity

Potential unique features:

• Adaptations to marine conditions, possibly including salt tolerance mechanisms

• Structural modifications that might enhance stability under the specific cellular compartmentalization in R. baltica

• Possible adaptations related to the unusual cell morphology of R. baltica, which includes different compartments (pirellulosome containing the riboplasm, and paryphoplasm)

Comparative structural analysis would require protein crystallography studies of the recombinant R. baltica DTD. This could be achieved by purifying the protein with affinity chromatography using a His-tag system, followed by crystallization trials and X-ray diffraction analysis. The resulting structure could be compared with known DTD structures from other organisms to identify conserved motifs and species-specific adaptations.

What is the optimal protocol for purifying recombinant R. baltica DTD while maintaining enzyme activity?

A comprehensive purification protocol for recombinant R. baltica DTD would involve:

• Expression optimization:

  • Clone the R. baltica DTD gene into an expression vector with a C-terminal 6×His tag

  • Transform into E. coli BL21(DE3) or similar expression strain

  • Optimize induction conditions (IPTG concentration, temperature, induction time)

  • Culture cells in LB or 2×YT medium at 25-30°C to enhance protein solubility

• Cell lysis:

  • Harvest cells by centrifugation (10,000×g, 15 min, 4°C)

  • Resuspend in lysis buffer containing 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 10 mM imidazole, 1 mM DTT, and protease inhibitors

  • Lyse cells via sonication or French press

  • Clarify lysate by centrifugation (20,000×g, 30 min, 4°C)

• Affinity purification:

  • Apply clarified lysate to Ni-NTA column equilibrated with lysis buffer

  • Wash extensively with wash buffer (lysis buffer with 20 mM imidazole)

  • Elute with elution buffer (lysis buffer with 250 mM imidazole)

• Secondary purification:

  • Apply eluted protein to size exclusion chromatography column (Superdex 75 or 200)

  • Collect fractions and analyze by SDS-PAGE

  • Pool pure fractions and concentrate using ultrafiltration

• Activity preservation:

  • Add glycerol to final concentration of 10-20%

  • Store at -80°C in small aliquots to avoid freeze-thaw cycles

  • Include DTT or β-mercaptoethanol in storage buffer to maintain reduced state of cysteine residues

Enzyme activity should be monitored throughout purification using a deacylation assay that measures the release of D-tyrosine from D-tyrosyl-tRNA(Tyr) substrates, using either radioactive substrates or HPLC-based methods for D-tyrosine detection.

How does growth phase affect R. baltica DTD expression and activity?

Growth phase significantly impacts protein expression profiles in R. baltica, which likely extends to DTD expression and activity:

Growth phase-dependent protein regulation patterns:

• The number of regulated proteins (fold changes > |2|) increases from early stationary phase (10 proteins) to late stationary phase (179 proteins)

• Fold changes in protein abundance can reach values up to 40

• Growth phase transitions show opposing regulation of key metabolic pathways, including tricarboxylic acid cycle and oxidative pentose phosphate cycle

Potential DTD regulation patterns:

• As a quality control enzyme, DTD might show differential expression patterns depending on growth phase

• If DTD functions similarly to other protein quality control mechanisms, it might be upregulated during stationary phase when stress responses are activated

• DTD activity might correlate with the alternative sigma factor sigmaH, which is upregulated in stationary phase and often associated with stress responses

Experimental approach to study growth phase dependence:

• Grow R. baltica cultures in mineral medium with ribose as carbon source

• Harvest cells at different growth phases (early exponential, late exponential, early stationary, late stationary)

• Analyze DTD protein levels using western blot with anti-DTD antibodies

• Measure DTD enzyme activity in cell extracts from different growth phases

• Compare with 2D-DIGE profiles to correlate DTD expression with other regulated proteins

What is the subcellular localization of DTD in R. baltica and how does it relate to the unique cell compartmentalization?

R. baltica possesses a unique cellular compartmentalization that divides the cell into distinct regions:

R. baltica cell compartments:

• The intracytoplasmic membrane encloses the pirellulosome, which contains the riboplasm with ribosome-like particles and the condensed nucleoid

• The region between the intracytoplasmic and cytoplasmic membranes contains the paryphoplasm, which harbors some RNA but no ribosome-like particles

• Protein biosynthesis likely only takes place in the riboplasm compartment

Predicted DTD localization:

• Given its function in translation quality control, DTD is most likely localized to the riboplasm compartment where protein synthesis occurs

• The enzyme would need to be in proximity to ribosomes to efficiently hydrolyze misaminoacylated tRNAs

• Similar to human DTD1, which is localized to both nucleus and cytoplasm , R. baltica DTD might also show dual localization patterns

Experimental approach to determine localization:

• Generate antibodies against recombinant R. baltica DTD or create a fluorescently tagged version

• Perform immunogold electron microscopy to visualize DTD localization within the compartmentalized cell structure

• Alternatively, perform subcellular fractionation to separate the paryphoplasm and pirellulosome compartments

• Analyze fractions by western blotting to detect DTD

• Correlate findings with the presence/absence of signal peptides, as 146 of the identified proteins in R. baltica contained predicted signal peptides suggesting translocation

Understanding DTD localization would provide insights into the spatial organization of translation quality control mechanisms in this uniquely compartmentalized bacterium.

What approaches can be used to study the kinetics of R. baltica DTD?

Studying the kinetics of R. baltica DTD requires specialized techniques to measure its deacylation activity:

Substrate preparation:

• Synthesize D-tyrosyl-tRNA(Tyr) substrate by:

  • In vitro transcription of tRNA(Tyr) gene

  • Aminoacylation using D-tyrosyl-tRNA synthetase or chemical aminoacylation methods

  • Purification of aminoacylated tRNA by chromatography

Kinetic measurement techniques:

• Radiometric assay:

  • Use 14C or 3H-labeled D-tyrosine to prepare labeled D-tyrosyl-tRNA(Tyr)

  • Measure release of labeled D-tyrosine after incubation with DTD

  • Determine initial velocities at different substrate concentrations

  • Calculate Km and kcat values using Michaelis-Menten kinetics

• HPLC-based assay:

  • Incubate DTD with D-tyrosyl-tRNA(Tyr)

  • Analyze reaction mixture by reverse-phase HPLC

  • Quantify released D-tyrosine using UV detection or fluorescence after derivatization

  • Plot reaction rates versus substrate concentrations

• Coupled enzyme assay:

  • Design a coupled assay where D-tyrosine release is linked to a detectable signal

  • For example, couple with D-amino acid oxidase and horseradish peroxidase

  • Measure H2O2 production spectrophotometrically using a chromogenic substrate

Kinetic parameters to determine:

• Km for D-tyrosyl-tRNA(Tyr) substrate

• kcat for deacylation reaction

• Substrate specificity by testing other D-aminoacyl-tRNAs

• Effects of pH, temperature, and ionic strength on enzyme activity, particularly considering R. baltica's marine environment

These approaches would provide comprehensive insights into the catalytic efficiency and substrate preferences of R. baltica DTD.

How can differential proteomics be used to study the role of DTD in R. baltica under various stress conditions?

Differential proteomics offers powerful approaches to investigate DTD's role in R. baltica stress responses:

Experimental design for stress studies:

• Culture R. baltica under various stress conditions:

  • D-amino acid stress (addition of D-tyrosine or D-amino acid mixture)

  • Oxidative stress (H2O2 exposure)

  • Temperature stress (heat shock or cold shock)

  • Nutrient limitation

  • pH stress (relevant to marine environments)

• Apply two-dimensional difference gel electrophoresis (2D-DIGE) methodology:

  • Label control and stressed samples with different fluorescent dyes (Cy2, Cy3, Cy5)

  • Combine samples and separate by 2D electrophoresis

  • Analyze differential protein expression using specialized software

  • This methodology has been successfully applied to study growth phase-dependent protein changes in R. baltica

• Identify regulated proteins by mass spectrometry:

  • Excise differentially expressed protein spots

  • Perform peptide mass fingerprinting using MALDI-TOF-MS

  • Identify proteins by database searching against the R. baltica genome (BX119912)

  • Analyze data using probability-based MOWSE scoring (score >51 considered significant, p<0.05)

Data analysis and interpretation:

• Create protein interaction networks centered on DTD

• Correlate DTD expression with other stress response proteins

• Compare expression changes across different stress conditions

• Identify potential functional partners of DTD under stress

This approach would reveal whether DTD is part of specific stress response pathways and how its expression correlates with other proteins involved in translation quality control or stress response mechanisms.

What genomic and proteomic approaches can be combined to identify DTD-interacting partners in R. baltica?

A multi-omics approach can be employed to comprehensively identify DTD-interacting partners:

Genomic approaches:

• Comparative genomics: Analyze DTD gene neighborhood across related species to identify conserved gene clusters that might function together

• Transcriptomic analysis: Perform RNA-Seq to identify genes co-expressed with DTD under various conditions

• Genome-wide CRISPR interference: Systematically knock down genes and screen for effects on DTD function or expression

Proteomic approaches:

• Affinity purification-mass spectrometry (AP-MS):

  • Express His-tagged or FLAG-tagged DTD in R. baltica

  • Perform gentle cell lysis to preserve protein-protein interactions

  • Capture DTD and associated proteins using affinity chromatography

  • Identify interacting proteins by LC-MS/MS

  • Distinguish true interactors from background using controls

• Proximity-dependent biotin identification (BioID):

  • Fuse DTD to a biotin ligase (BirA*)

  • Express in R. baltica

  • Biotin-label proximal proteins in vivo

  • Purify biotinylated proteins using streptavidin beads

  • Identify by mass spectrometry

• Cross-linking mass spectrometry (XL-MS):

  • Treat R. baltica cells with chemical cross-linkers

  • Enrich for DTD-containing complexes

  • Digest and analyze by specialized mass spectrometry

  • Identify cross-linked peptides that reveal direct interactions

Data integration:

• Create interaction networks combining evidence from all approaches

• Assign confidence scores based on detection across multiple methods

• Validate key interactions using targeted approaches like co-immunoprecipitation or yeast two-hybrid

This integrated approach would provide a comprehensive view of the DTD interactome in R. baltica, revealing both stable and transient interactions that contribute to its function in translation quality control.

How does R. baltica DTD differ from DTD enzymes in other bacterial species and eukaryotes?

R. baltica DTD likely exhibits both conserved features and unique characteristics compared to DTD enzymes from other organisms:

Comparative analysis with other bacterial DTDs:

Comparison with eukaryotic DTDs:

• Human DTD1 is both nuclear and cytoplasmic , while R. baltica DTD is likely confined to the riboplasm compartment

• Human DTD1 shows ATPase activity and involvement in DNA replication , functions not typically associated with bacterial DTDs

• Eukaryotic DTDs often have additional domains or regulatory regions absent in bacterial counterparts

• Human DTD1 is preferentially phosphorylated in cells arrested in S phase , representing a regulatory mechanism likely absent in R. baltica

Evolutionary considerations:

• DTD is a highly conserved enzyme across all domains of life, suggesting its fundamental importance

• R. baltica belongs to the phylum Planctomycetes, which has a unique evolutionary position

• Comparative genomic analysis could reveal whether R. baltica DTD shows unique adaptations related to its unusual cellular compartmentalization

These differences would have implications for experimental design when studying R. baltica DTD, particularly regarding assay conditions, substrate selection, and interpretation of functional data.

What role might DTD play in R. baltica's adaptation to its marine environment?

R. baltica's marine environment presents unique challenges that may influence DTD function and importance:

Environmental factors affecting DTD function:

• Marine environments can contain D-amino acids from bacterial cell walls and algal peptides

• Fluctuating salinity may impact protein folding and stability, potentially increasing translational errors

• Temperature variations in marine habitats could affect tRNA charging fidelity

Potential adaptive roles of DTD:

• Enhanced quality control: DTD may be particularly important for preventing incorporation of environmental D-amino acids into proteins

• Stress response: DTD could play a role in R. baltica's ability to survive in changing marine conditions

• Nutrient recycling: By reclaiming misaminoacylated tRNAs, DTD may contribute to efficient resource utilization in potentially nutrient-limited environments

Genomic context evidence:

• R. baltica possesses genes associated with aromatic compound metabolism , suggesting adaptations to utilizing diverse carbon sources

• The complex cell morphology of R. baltica, including motile swarmer cells and sessile rosettes , may require specialized quality control mechanisms during differentiation

• Growth phase-dependent regulation of 179 proteins indicates complex adaptations to environmental changes

Research approaches to investigate environmental adaptation:

• Compare DTD expression and activity under different salinity, temperature, and nutrient conditions

• Assess DTD importance for survival when R. baltica is exposed to environmental D-amino acids

• Examine whether DTD is differentially expressed in different R. baltica morphotypes (swarmer cells vs. rosettes)

Understanding DTD's role in environmental adaptation could provide insights into the molecular mechanisms underlying R. baltica's ecological success in marine habitats.

What are the best methods for analyzing the substrate specificity of recombinant R. baltica DTD?

Comprehensive analysis of R. baltica DTD substrate specificity requires multiple complementary approaches:

Substrate library preparation:

• Generate a diverse set of D-aminoacyl-tRNAs including:

  • D-tyrosyl-tRNA(Tyr) (primary predicted substrate)

  • Other D-aromatic-aminoacyl-tRNAs (D-Phe, D-Trp)

  • D-aliphatic-aminoacyl-tRNAs (D-Ala, D-Val, D-Leu)

  • D-charged-aminoacyl-tRNAs (D-Lys, D-Glu)

• Prepare substrates using either:

  • Chemical aminoacylation with activated D-amino acids

  • Enzymatic aminoacylation using engineered aminoacyl-tRNA synthetases

Activity measurement techniques:

• Parallel reaction monitoring:

  • Incubate DTD with a mixture of different D-aminoacyl-tRNAs

  • Analyze reaction products by LC-MS/MS

  • Quantify deacylation rates for each substrate simultaneously

  • Calculate relative specificity constants (kcat/Km)

• Thin-layer chromatography (TLC) assay:

  • Use radioactively labeled D-amino acids

  • Separate reaction products by TLC

  • Quantify using phosphorimager

  • Compare relative deacylation efficiencies

• Real-time fluorescence assay:

  • Label tRNAs with fluorescent dyes

  • Monitor conformational changes during deacylation in real-time

  • Calculate reaction rates for different substrates

Data analysis and presentation:

• Create a substrate specificity profile showing relative activity against different D-aminoacyl-tRNAs

• Determine kinetic parameters (Km, kcat, kcat/Km) for each substrate

• Correlate specificity with amino acid properties (size, hydrophobicity, charge)

This comprehensive approach would provide detailed insights into the substrate preferences of R. baltica DTD, which could be related to its ecological niche and the types of D-amino acids it might encounter in its marine environment.

How can high-throughput screening be used to identify inhibitors or enhancers of R. baltica DTD activity?

High-throughput screening (HTS) for DTD modulators can be implemented using the following systematic approach:

Assay development for HTS:

• Fluorescence-based primary assay:

  • Develop a fluorogenic substrate (e.g., D-tyrosyl-tRNA(Tyr) with fluorescence quenched by aminoacylation)

  • Deacylation by DTD results in measurable fluorescence increase

  • Optimize in 384 or 1536-well format for miniaturization

  • Validate with known controls (heat-inactivated enzyme, varying enzyme concentrations)

• Assay quality parameters:

  • Achieve Z' factor >0.5 for statistical reliability

  • Signal-to-background ratio >10

  • Coefficient of variation <10%

  • DMSO tolerance up to 1% for compound solubility

Screening strategy:

• Primary screen:

  • Test compounds at single concentration (10-20 μM)

  • Include positive controls (known inhibitors if available) and negative controls

  • Set thresholds for hit selection (>50% inhibition or >150% enhancement)

• Secondary validation:

  • Counterscreen against related hydrolases to assess selectivity

  • Dose-response curves to determine IC50 or EC50 values

  • Rule out interference mechanisms (fluorescence quenching, aggregation)

• Tertiary characterization:

  • Determine mechanism of inhibition (competitive, non-competitive)

  • Assess reversibility

  • Evaluate effects in cellular context (if cellular system available)

Data analysis and compound prioritization matrix:

This screening approach would identify chemical tools to study DTD function and potentially lead to compounds for investigating DTD's role in R. baltica physiology and adaptation.

What are the most promising research directions for understanding DTD's role in R. baltica cell biology?

Future research on R. baltica DTD should focus on several key areas that would advance our understanding of its biological significance:

Priority research directions:

• Functional genomics approach:

  • Generate DTD knockout or depletion strains in R. baltica

  • Perform phenotypic characterization under various growth conditions

  • Analyze effects on proteome quality and D-amino acid tolerance

  • This would establish the importance of DTD for R. baltica survival and adaptation

• Structural biology investigations:

  • Determine crystal structure of R. baltica DTD

  • Compare with structures from other organisms

  • Identify unique structural features that might relate to marine adaptation

  • Perform structure-guided mutagenesis to identify key catalytic residues

• Systems biology integration:

  • Map the position of DTD in R. baltica's protein quality control network

  • Integrate proteomics, transcriptomics, and metabolomics data

  • Develop computational models of translation quality control

  • This would place DTD in the broader context of cellular homeostasis

• Evolutionary studies:

  • Compare DTD sequences and functions across Planctomycetes

  • Investigate horizontal gene transfer events that might have shaped DTD evolution

  • Reconstruct the evolutionary history of DTD in relation to cellular compartmentalization

• Environmental adaptation mechanisms:

  • Study DTD expression and activity across different R. baltica morphotypes

  • Investigate regulation during transitions between swarmer cells and rosettes

  • Examine DTD's role in biofilm formation and surface attachment

These research directions would provide comprehensive insights into DTD's biological significance in R. baltica and potentially reveal novel aspects of translation quality control in bacteria with complex cellular organizations.

How might recombinant R. baltica DTD be used as a tool in biotechnology applications?

Recombinant R. baltica DTD offers several promising biotechnology applications based on its predicted enzymatic properties:

Potential biotechnology applications:

• Biocatalysis and chiral chemistry:

  • Development of enzymatic methods for D-amino acid purification

  • Creation of stereospecific biocatalysts for pharmaceutical synthesis

  • DTD could be used to selectively remove D-amino acids from mixtures

• Protein production quality control:

  • Addition of recombinant DTD to cell-free protein synthesis systems

  • Reduction of mistranslation events involving D-amino acids

  • Improvement of recombinant protein quality in biotechnology applications

• Biosensor development:

  • Creation of DTD-based biosensors for D-amino acid detection

  • Environmental monitoring of D-amino acids in marine samples

  • Potential applications in food safety and fermentation monitoring

• Synthetic biology tools:

  • Integration into synthetic genetic circuits as a quality control component

  • Development of orthogonal translation systems with enhanced fidelity

  • Creation of minimal synthetic cells with defined quality control mechanisms

• Marine biotechnology applications:

  • Utilization of R. baltica DTD's potential adaptations to marine conditions

  • Development of enzyme technologies optimized for high-salt environments

  • Applications in marine bioremediation or resource utilization

To realize these applications, further characterization of R. baltica DTD's biochemical properties, substrate specificity, and stability under various conditions would be required. The enzyme's potential adaptations to marine environments might provide unique advantages for certain biotechnology applications compared to DTD enzymes from other organisms.