GO:0051500 D-tyrosyl-tRNA(Tyr) deacylase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0051500 describes the enzymatic removal of D-tyrosine from charged tRNA(Tyr), producing free D-tyrosine and uncharged tRNA(Tyr).
• This activity is essential for proofreading and recycling of mischarged tRNA, preventing D-amino acid incorporation into proteins.
• The reaction is catalyzed by D-tyrosyl-tRNA(Tyr) deacylase, a member of the D-aminoacyl-tRNA deacylase family.
• In Saccharomyces cerevisiae, D-tyrosyl-tRNA(Tyr) metabolism is linked to cellular resistance to D-tyrosine and translational fidelity.
• The dtd gene from Bacillus amyloliquefaciens encodes a putative D-tyrosyl-tRNATyr deacylase and can serve as a selectable marker in Bacillus subtilis.
• Structural studies have revealed key catalytic residues and ligand-bound snapshots that explain the deacylation mechanism.
Description
GO:0051500, D-tyrosyl-tRNA(Tyr) deacylase activity, is a molecular function that catalyzes the hydrolysis of D-tyrosyl-tRNATyr to yield D-tyrosine and free tRNA(Tyr). This activity is part of the broader quality control network that ensures translational fidelity by removing incorrectly charged D-amino acids from tRNA. The reaction is essential because D-amino acids can be misincorporated into proteins, leading to dysfunctional polypeptides and cellular toxicity. In the yeast Saccharomyces cerevisiae, D-tyrosyl-tRNA(Tyr) metabolism has been studied as a model for how cells handle D-tyrosine and maintain protein synthesis accuracy. The enzyme responsible, D-tyrosyl-tRNA(Tyr) deacylase, is conserved across bacteria and eukaryotes, and its structure and catalytic mechanism have been characterized in detail. Researchers study this activity to understand fundamental tRNA proofreading, antibiotic resistance mechanisms, and potential applications in synthetic biology and biotechnology.
D-tyrosyl-tRNA(Tyr) deacylase activity At A Glance
| GO ID | GO:0051500 |
|---|---|
| GO term | D-tyrosyl-tRNA(Tyr) deacylase activity |
| Ontology | molecular_function |
| Synonym | None listed |
| Definition | Catalysis of the reaction: D-tyrosyl-tRNATyr + H2O = D-tyrosine + tRNATyr. Removal of a D-tyrosine from a charged tRNA(Tyr). |
| Major function | Proofreading and recycling of mischarged tRNA(Tyr) by removing D-tyrosine, preventing D-amino acid incorporation into proteins. |
| EC number | Not specified in QuickGO |
| Catalytic residues | Identified through structural studies; key residues include those in the active site that coordinate the D-tyrosyl moiety. |
| Subcellular location | Cytoplasm (implied by tRNA charging and translation). |
What Is GO:0051500?
D-tyrosyl-tRNA(Tyr) deacylase activity (GO:0051500) is defined as the catalysis of the reaction: D-tyrosyl-tRNATyr + H2O = D-tyrosine + tRNATyr. In other words, it removes a D-tyrosine from a charged tRNA(Tyr) molecule, freeing the tRNA for reuse and releasing the D-amino acid. This activity is a type of hydrolase that acts on ester bonds between the amino acid and the tRNA, specifically targeting D-tyrosine mischarged onto tRNA(Tyr).
Why Is D-tyrosyl-tRNA(Tyr) deacylase activity Important in Cell Biology?
D-tyrosyl-tRNA(Tyr) deacylase activity is critical for maintaining translational fidelity and cellular homeostasis. By removing D-tyrosine from mischarged tRNA(Tyr), it prevents the incorporation of D-amino acids into proteins, which can disrupt protein structure and function. This activity also plays a role in resistance to D-tyrosine toxicity and may influence bacterial survival and antibiotic susceptibility. Understanding this enzyme provides insights into tRNA quality control, evolutionary conservation of proofreading mechanisms, and potential targets for antimicrobial or biotechnological applications.
• Prevents misincorporation of D-tyrosine into proteins, protecting proteome integrity.
• Recycles tRNA(Tyr) for continued translation.
• Contributes to cellular resistance against D-tyrosine toxicity.
• Serves as a selectable marker in Bacillus subtilis, useful for genetic engineering.
• Provides a model for studying D-aminoacyl-tRNA deacylase family enzymes.
• Potential target for antibacterial drug development due to its role in bacterial fitness.
• Involved in translational quality control pathways conserved from bacteria to humans.
• Structural insights aid in understanding enzyme mechanism and substrate specificity.
• May impact synthetic biology by enabling controlled D-amino acid incorporation.
• Relevant to studies of neurodegeneration and aging where D-amino acids accumulate.
Molecular Mechanism of D-tyrosyl-tRNA(Tyr) deacylase activity
Substrate Recognition and Binding
In simple terms: The enzyme recognizes and binds to tRNA(Tyr) that carries a D-tyrosine, not the normal L-tyrosine.
D-tyrosyl-tRNA(Tyr) deacylase specifically binds to D-tyrosyl-tRNATyr. Structural studies have shown that the enzyme's active site accommodates the D-tyrosyl moiety while discriminating against L-tyrosyl-tRNA. The binding involves interactions with the tRNA body and the D-amino acid, ensuring specificity for the mischarged substrate.
Catalytic Hydrolysis
In simple terms: The enzyme uses water to break the bond between D-tyrosine and the tRNA, releasing both parts.
The catalytic mechanism involves hydrolysis of the ester bond between D-tyrosine and the 3'-end of tRNA(Tyr). Key active site residues, identified through ligand-bound structures, facilitate nucleophilic attack by a water molecule, leading to the release of free D-tyrosine and uncharged tRNA(Tyr). This reaction is essential for recycling the tRNA and preventing D-tyrosine incorporation into proteins.
Product Release and tRNA Recycling
In simple terms: After the reaction, the tRNA is free to be charged again with the correct amino acid.
Following deacylation, the uncharged tRNA(Tyr) is released from the enzyme and can be re-aminoacylated with L-tyrosine by tyrosyl-tRNA synthetase. This recycling ensures a pool of charged tRNA for translation. The released D-tyrosine can be metabolized or excreted, depending on the organism.
Structural Insights into Catalysis
In simple terms: The 3D structure of the enzyme shows how it grabs the D-tyrosine and cuts it off.
Crystal structures of D-tyrosyl-tRNA(Tyr) deacylase from various organisms, including those determined using home-source Cu Kalpha and iodide-SAD data, have revealed a conserved fold and active site architecture. Ligand-bound structures provide atomic snapshots of the catalytic cycle, highlighting conformational changes and key residues involved in substrate binding and hydrolysis.
Key Genes Involved in GO:0051500 D-tyrosyl-tRNA(Tyr) deacylase activity
The following genes and proteins are directly associated with D-tyrosyl-tRNA(Tyr) deacylase activity or its regulation.
| Gene | Major Role | Research Relevance |
|---|---|---|
| DTD1 (S. cerevisiae) | Encodes D-tyrosyl-tRNA(Tyr) deacylase; removes D-tyrosine from tRNA(Tyr) | Model for D-amino acid metabolism and translational fidelity |
| dtd (B. amyloliquefaciens) | Putative D-tyrosyl-tRNATyr deacylase; selectable marker | Biotechnological tool for Bacillus subtilis |
| Dtd (various bacteria) | D-aminoacyl-tRNA deacylase family enzyme | Structural and mechanistic studies |
| Tyrosyl-tRNA synthetase (TyrRS) | Charges tRNA(Tyr) with L-tyrosine; can mischarge with D-tyrosine | Studied for proofreading and D-amino acid toxicity |
| tRNA(Tyr) | Substrate for charging and deacylation | Central to translation and quality control |
| D-amino acid oxidase (DAO) | Metabolizes D-amino acids | Linked to D-tyrosine detoxification |
| D-amino acid aminotransferase | Involved in D-amino acid metabolism | Potential interplay with deacylase |
| Elongation factor Tu (EF-Tu) | Delivers aminoacyl-tRNA to ribosome | Indirect role in translational fidelity |
| Ribosomal protein L1 | Part of translation machinery | Context for misincorporation studies |
| Dtd1 (human homolog) | Putative D-tyrosyl-tRNA deacylase | Potential role in human disease |
| Dtd2 (plant homolog) | D-aminoacyl-tRNA deacylase | Plant stress responses |
| Dtd3 (archaeal homolog) | D-aminoacyl-tRNA deacylase | Evolutionary studies |
| Alanyl-tRNA synthetase | Proofreading of mischarged tRNA | Comparative editing mechanisms |
| Threonyl-tRNA synthetase | Editing of mischarged tRNA | Related quality control |
| Prolyl-tRNA synthetase | Editing of mischarged tRNA | Related quality control |
| D-aminoacyl-tRNA deacylase (general) | Broad specificity for D-aminoacyl-tRNAs | Family characterization |
How Is D-tyrosyl-tRNA(Tyr) deacylase activity Regulated?
The expression and activity of D-tyrosyl-tRNA(Tyr) deacylase may be regulated at multiple levels. In Saccharomyces cerevisiae, D-tyrosyl-tRNA(Tyr) metabolism is influenced by the availability of D-tyrosine and the activity of tyrosyl-tRNA synthetase. The dtd gene in Bacillus amyloliquefaciens is expressed under conditions that require D-tyrosine detoxification. However, specific transcriptional regulators or signaling pathways (e.g., mTOR, ISR) controlling this enzyme have not been extensively characterized in the provided literature. Further research is needed to elucidate regulatory mechanisms.
D-tyrosyl-tRNA(Tyr) deacylase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| DTD1 (S. cerevisiae) | D-tyrosine toxicity, translational fidelity | Yeast knockout and overexpression strains |
| dtd (B. amyloliquefaciens) | Bacterial fitness, selectable marker | Bacillus subtilis transformation and selection |
| Dtd (bacterial) | Antibiotic target potential | In vitro enzyme assays and bacterial growth inhibition |
| Human DTD1 homolog | Neurodegeneration (hypothetical) | Human cell lines with CRISPR knockout |
| Plant Dtd2 | Stress responses (hypothetical) | Arabidopsis knockout lines |
D-tyrosine toxicity and cellular stress
Accumulation of D-tyrosine can be toxic to cells, and D-tyrosyl-tRNA(Tyr) deacylase activity helps mitigate this by removing D-tyrosine from tRNA. In yeast, defects in this pathway lead to growth inhibition in the presence of D-tyrosine. This suggests that impaired deacylase activity could contribute to cellular stress and disease states associated with D-amino acid accumulation.
Neurodegeneration and D-amino acids
D-amino acids, including D-tyrosine, have been implicated in neurodegenerative conditions such as Alzheimer's and Parkinson's diseases, where altered D-amino acid levels are observed. Although direct links to D-tyrosyl-tRNA(Tyr) deacylase are not yet established, the enzyme's role in D-tyrosine metabolism suggests a potential protective function. Further studies are needed to explore this connection.
Bacterial infections and antibiotic resistance
The dtd gene from Bacillus amyloliquefaciens encodes a putative D-tyrosyl-tRNATyr deacylase and can serve as a selectable marker, indicating its importance in bacterial survival. Targeting this enzyme could be a strategy for developing new antibiotics, as inhibition might increase susceptibility to D-amino acid toxicity.
From D-tyrosyl-tRNA(Tyr) deacylase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does knockout of DTD1 affect growth in D-tyrosine? | Yeast DTD1 knockout |
| Can dtd serve as a selectable marker? | Bacillus subtilis with dtd plasmid |
| What is the catalytic mechanism? | Recombinant Dtd protein for crystallography |
| How does D-tyrosine mischarging affect translation? | In vitro translation with mischarged tRNA |
| Does overexpression of Dtd protect against D-tyrosine toxicity? | Bacterial or yeast overexpression strains |
| What are the structural determinants of substrate specificity? | Site-directed mutagenesis and X-ray crystallography |
How to Study the D-tyrosyl-tRNA(Tyr) deacylase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| In vitro deacylation assay | Release of D-tyrosine from tRNA | Enzyme kinetics and inhibitor testing |
| X-ray crystallography | 3D structure of enzyme-ligand complexes | Mechanistic studies |
| Site-directed mutagenesis | Effect of mutations on activity | Identifying catalytic residues |
| Bacterial growth assays | Toxicity of D-tyrosine | Testing deacylase protective role |
| Selectable marker assay | Transformation efficiency | Genetic engineering in Bacillus subtilis |
| Mass spectrometry | D-tyrosine incorporation into proteins | Translational fidelity |
| Ribo-seq | Ribosome occupancy on tRNA(Tyr) codons | Global translation profiling |
| RNA-seq | Expression of DTD1 and related genes | Transcriptional regulation |
Enzymatic assays
Deacylase activity can be measured using in vitro assays with radiolabeled D-tyrosyl-tRNATyr, monitoring the release of D-tyrosine. Such assays are used to characterize enzyme kinetics and substrate specificity.
Structural biology
X-ray crystallography and cryo-EM have been used to determine the structures of D-tyrosyl-tRNA(Tyr) deacylase, including ligand-bound states. These methods reveal active site architecture and catalytic residues.
Genetic screens and selection
The dtd gene can be used as a selectable marker in Bacillus subtilis, allowing for genetic screens and selection of transformants. This approach is useful for studying gene function and for biotechnological applications.
Translational fidelity assays
Misincorporation of D-tyrosine into proteins can be assessed using reporter proteins or mass spectrometry. These assays help quantify the impact of deacylase activity on translational fidelity.
How CRISPR Can Be Used to Study GO:0051500 D-tyrosyl-tRNA(Tyr) deacylase activity
Knockout
CRISPR knockout of DTD1 or its homologs can be used to study the loss of D-tyrosyl-tRNA(Tyr) deacylase activity. In yeast, DTD1 knockout strains show increased sensitivity to D-tyrosine. In human cells, knockout of the DTD1 homolog could reveal its role in D-amino acid metabolism and neurodegeneration.
Point Mutation
Point mutations in the active site of D-tyrosyl-tRNA(Tyr) deacylase can be introduced using CRISPR base editing or homology-directed repair to dissect catalytic residues. Such mutants help confirm the roles of specific amino acids in substrate binding and hydrolysis.
Knock-in
Knock-in of tagged versions of DTD1 (e.g., GFP or FLAG) allows for localization and interaction studies. This can be achieved using CRISPR-mediated homology-directed repair. Tagged knock-in models are valuable for imaging and proteomics.
Overexpression
CRISPR activation (CRISPRa) or plasmid-based overexpression can be used to increase D-tyrosyl-tRNA(Tyr) deacylase levels. Overexpression may protect cells from D-tyrosine toxicity and can be used to study dosage effects.
How EDITGENE Supports D-tyrosyl-tRNA(Tyr) deacylase activity Research
Researchers studying D-tyrosyl-tRNA(Tyr) deacylase activity-related genes often need to determine whether a candidate gene is causally involved in D-amino acid metabolism, translational fidelity, or cellular stress responses. EDITGENE provides comprehensive CRISPR services to enable such investigations.
Contact EDITGENE today to design your custom CRISPR model for D-tyrosyl-tRNA(Tyr) deacylase activity research.
Frequently Asked Questions About D-tyrosyl-tRNA(Tyr) deacylase activity
What is D-tyrosyl-tRNA(Tyr) deacylase activity?
It is the enzymatic activity that removes D-tyrosine from charged tRNA(Tyr), producing free D-tyrosine and uncharged tRNA(Tyr).
What is the GO ID for D-tyrosyl-tRNA(Tyr) deacylase activity?
The GO ID is GO:0051500.
What genes are involved in D-tyrosyl-tRNA(Tyr) deacylase activity?
Key genes include DTD1 in Saccharomyces cerevisiae and dtd in Bacillus amyloliquefaciens.
Why is D-tyrosyl-tRNA(Tyr) deacylase important?
It prevents misincorporation of D-tyrosine into proteins and maintains translational fidelity.
What diseases are associated with D-tyrosyl-tRNA(Tyr) deacylase?
D-tyrosine toxicity and potentially neurodegeneration, though direct links are still under investigation.
How can I study D-tyrosyl-tRNA(Tyr) deacylase activity?
Using enzymatic assays, structural biology, and CRISPR knockout models.
What is the catalytic mechanism of D-tyrosyl-tRNA(Tyr) deacylase?
It hydrolyzes the ester bond between D-tyrosine and tRNA(Tyr) using active site residues and a water molecule.
Is D-tyrosyl-tRNA(Tyr) deacylase conserved?
Yes, it is found in bacteria, yeast, plants, and humans.
Can D-tyrosyl-tRNA(Tyr) deacylase be used as a selectable marker?
Yes, the dtd gene from Bacillus amyloliquefaciens can serve as a selectable marker in Bacillus subtilis.
What are the research methods for studying D-tyrosyl-tRNA(Tyr) deacylase?
Common methods include in vitro deacylation assays, X-ray crystallography, and CRISPR-based genetic screens.
Conclusion
D-tyrosyl-tRNA(Tyr) deacylase activity (GO:0051500) is a crucial molecular function that safeguards translational fidelity by removing D-tyrosine from mischarged tRNA(Tyr). Its conservation across species and its role in D-amino acid metabolism make it a compelling target for basic and applied research. Understanding its mechanism and regulation could lead to new strategies in antimicrobial development and biotechnology.
References
- 1. Soutourina J et al.. 2000. D-tyrosyl-tRNA(Tyr) metabolism in Saccharomyces cerevisiae.. J Biol Chem 275(16):11626-30 PMID: 10766779
- 2. Geraskina NV et al.. 2015. The dtd gene from Bacillus amyloliquefaciens encodes a putative D-tyrosyl-tRNATyr deacylase and is a selectable marker for Bacillus subtilis.. Microbiol Res 171:90-6 PMID: 25441601
- 3. Yogavel M et al.. 2010. Structure of D-tyrosyl-tRNATyr deacylase using home-source Cu Kalpha and moderate-quality iodide-SAD data: structural polymorphism and HEPES-bound enzyme states.. Acta Crystallogr D Biol Crystallogr 66(Pt 5):584-92 PMID: 20445234
- 4. Bhatt TK et al.. 2010. Ligand-bound structures provide atomic snapshots for the catalytic mechanism of D-amino acid deacylase.. J Biol Chem 285(8):5917-30 PMID: 20007323
