GO:0004045 peptidyl-tRNA hydrolase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0004045 peptidyl-tRNA hydrolase activity catalyzes the hydrolysis of an N-acyl-L-alpha-aminoacyl-tRNA to an N-acyl-L-amino acid, a tRNA and H+, recycling tRNA from stalled translation complexes.
• Peptidyl-tRNA hydrolase (Pth) is essential for protein biosynthesis because it prevents toxic accumulation of peptidyl-tRNA and free tRNA depletion.
• Structural studies across bacteria including Thermus thermophilus, Francisella tularensis, Klebsiella pneumoniae and Enterococcus faecium reveal a conserved alpha/beta hydrolase fold with a catalytic triad-like arrangement.
• In Mycobacterium tuberculosis, Pth is required for robust prolyl-tRNA turnover, linking the enzyme to amino acid homeostasis and stress survival.
• Recent work shows Pth binds the peptidyl-A76 moiety of the substrate, defining the molecular basis of substrate recognition.
• Peptidyl-tRNA hydrolase 2 (Pth2) acts as a negative regulator of peripartum cardiomyopathy with heart failure in female mice, connecting this activity to human cardiac disease.
Description
Peptidyl-tRNA hydrolase activity (GO:0004045) is a molecular function that catalyzes the hydrolytic cleavage of the ester bond between a nascent peptide and its tRNA, releasing an N-acyl-L-amino acid, a free tRNA and a proton. This activity is essential for maintaining translational fidelity and tRNA availability, because peptidyl-tRNA species that accumulate when translation stalls can be toxic to cells. The enzyme responsible, peptidyl-tRNA hydrolase (Pth), is conserved across bacteria and eukaryotes and is considered a critical component of protein biosynthesis. Researchers study GO:0004045 to understand how cells recycle tRNA, how bacteria survive amino acid starvation, and how this activity can be targeted for antimicrobial development. Structural and biochemical studies have resolved the catalytic mechanism and substrate binding mode of Pth from multiple bacterial species, including Thermus thermophilus, Francisella tularensis, Klebsiella pneumoniae and Enterococcus faecium. More recently, the peptidyl-A76 moiety of the substrate was shown to define the binding mode between Pth and its peptidyl-tRNA substrate. In addition, a mammalian homolog, peptidyl-tRNA hydrolase 2 (Pth2), has been implicated in peripartum cardiomyopathy with heart failure in female mice, expanding the disease relevance of this activity beyond bacterial physiology.
peptidyl-tRNA hydrolase activity At A Glance
| GO ID | GO:0004045 |
|---|---|
| GO term | peptidyl-tRNA hydrolase activity |
| Ontology | molecular_function |
| Synonym | aminoacyl-transfer ribonucleate hydrolase activity; aminoacyl-tRNA aminoacylhydrolase activity; aminoacyl-tRNA hydrolase reaction; D-tyrosyl-tRNA hydrolase activity; N-substituted aminoacyl transfer RNA hydrolase activity |
| Major function | Hydrolysis of N-acyl-L-alpha-aminoacyl-tRNA to release N-acyl-L-amino acid, tRNA and H+ |
| Biological context | tRNA recycling and rescue of stalled translation |
| Representative enzymes | Pth (peptidyl-tRNA hydrolase) and Pth2 (peptidyl-tRNA hydrolase 2) |
| Structural fold | alpha/beta hydrolase fold with a catalytic triad-like arrangement |
| Substrate recognition | Peptidyl-A76 moiety of the substrate is a key binding determinant |
What Is GO:0004045?
According to the Gene Ontology, GO:0004045 peptidyl-tRNA hydrolase activity is defined as the catalysis of the reaction: an N-acyl-L-alpha-aminoacyl-tRNA + H2O = an N-acyl-L-amino acid + a tRNA + H+. In other words, this molecular function removes the peptide chain from a peptidyl-tRNA molecule by hydrolysis, freeing the tRNA for reuse in translation and releasing the peptide as an N-acyl amino acid. The activity is also known by synonyms such as aminoacyl-transfer ribonucleate hydrolase activity, aminoacyl-tRNA aminoacylhydrolase activity, aminoacyl-tRNA hydrolase reaction, D-tyrosyl-tRNA hydrolase activity and N-substituted aminoacyl transfer RNA hydrolase activity.
Why Is peptidyl-tRNA hydrolase activity Important in Cell Biology?
GO:0004045 is important because peptidyl-tRNA hydrolase activity is a housekeeping function that prevents the accumulation of toxic peptidyl-tRNA species and maintains the pool of free tRNA available for protein synthesis. Without this activity, stalled translation products can interfere with ribosome function and cellular viability, making Pth essential in many bacteria. The enzyme has also been linked to amino acid homeostasis, as shown by the requirement for Pth in robust prolyl-tRNA turnover in Mycobacterium tuberculosis. Because Pth is conserved and structurally characterized in several pathogens, it is a candidate target for antibacterial drug discovery. In mammals, Pth2 has been identified as a negative regulator of peripartum cardiomyopathy with heart failure in female mice, suggesting that this activity has physiological roles beyond bacterial translation.
• Recycles tRNA from peptidyl-tRNA species generated by stalled or premature translation termination.
• Prevents toxic accumulation of peptidyl-tRNA that can inhibit protein synthesis.
• Supports robust prolyl-tRNA turnover in Mycobacterium tuberculosis.
• Provides a structurally validated target for antibacterial inhibitor design.
• Its substrate binding mode via the peptidyl-A76 moiety is now structurally defined.
• Conserved alpha/beta hydrolase fold enables comparative structural studies across species.
• Mammalian Pth2 negatively regulates peripartum cardiomyopathy with heart failure in female mice.
• Relevant to translation quality control and ribosome rescue pathways.
• Useful for understanding amino acid starvation responses in bacteria.
• Potential biomarker or therapeutic node in cardiac disease models.
Molecular Mechanism of peptidyl-tRNA hydrolase activity
Substrate recognition and binding
In simple terms: The enzyme first grabs the peptidyl-tRNA substrate by its peptide-bearing end.
Peptidyl-tRNA hydrolase recognizes its substrate through interactions with the peptidyl-A76 moiety of the peptidyl-tRNA, as shown by binding-mode studies. This recognition positions the ester bond between the peptide and tRNA for subsequent hydrolysis. Structural analyses of Pth from Thermus thermophilus, Francisella tularensis, Klebsiella pneumoniae and Enterococcus faecium reveal a conserved substrate-binding pocket that accommodates the aminoacyl moiety.
Catalytic hydrolysis of the ester bond
In simple terms: Once bound, the enzyme uses water to cut the bond that holds the peptide to the tRNA.
The catalytic mechanism of GO:0004045 involves hydrolysis of the ester bond linking the N-acyl-L-alpha-aminoacyl group to the tRNA, yielding an N-acyl-L-amino acid, a free tRNA and a proton. The reaction follows the GO definition: an N-acyl-L-alpha-aminoacyl-tRNA + H2O = an N-acyl-L-amino acid + a tRNA + H+. Conserved active-site residues in the alpha/beta hydrolase fold facilitate this hydrolysis, as inferred from high-resolution structures of Pth from Thermus thermophilus and other species.
tRNA recycling and translation rescue
In simple terms: After cutting, the freed tRNA can go back to work in translation.
The hydrolysis reaction releases intact tRNA that can be recharged and reused in protein synthesis, which is critical when translation stalls and peptidyl-tRNA accumulates. This recycling function prevents depletion of the free tRNA pool and avoids the toxicity associated with peptidyl-tRNA buildup. In Mycobacterium tuberculosis, Pth is required for robust prolyl-tRNA turnover, linking the activity to amino acid homeostasis under stress.
Structural basis of the active site
In simple terms: The enzyme has a conserved pocket that holds the substrate in the right orientation for cutting.
Crystal structures of Pth from Thermus thermophilus, Francisella tularensis, Klebsiella pneumoniae and Enterococcus faecium show a conserved alpha/beta hydrolase fold with a catalytic triad-like arrangement. High-resolution structures of the Thermus thermophilus enzyme provide detailed views of the active-site geometry. The Enterococcus faecium structure was also used to characterize inhibition by a pyrrolinone compound, demonstrating that the active site is druggable.
Inhibition and regulation
In simple terms: Small molecules can block the enzyme, which is useful for antibiotic development.
The active site of Pth can be targeted by small-molecule inhibitors, as shown by inhibition of the Enterococcus faecium enzyme by a pyrrolinone compound. Such inhibition studies support the development of Pth-targeted antibacterial agents. In mammals, Pth2 functions as a negative regulator of peripartum cardiomyopathy with heart failure in female mice, indicating that this activity is subject to physiological regulation in higher organisms.
Key Genes Involved in GO:0004045 peptidyl-tRNA hydrolase activity
The following genes and proteins are directly associated with peptidyl-tRNA hydrolase activity (GO:0004045) or its regulation in bacterial and mammalian systems.
| Gene | Major Role | Research Relevance |
|---|---|---|
| pth (Mycobacterium tuberculosis) | Peptidyl-tRNA hydrolase required for prolyl-tRNA turnover | Essential for stress survival and amino acid homeostasis |
| pth (Escherichia coli) | Model peptidyl-tRNA hydrolase | Classical enzyme for studying GO:0004045 mechanism |
| pth (Thermus thermophilus) | Thermostable Pth for structural studies | High-resolution crystal structure of the enzyme |
| pth (Francisella tularensis) | Pth from a biothreat pathogen | Structural characterization for drug targeting |
| pth (Klebsiella pneumoniae) | Pth from a clinically important pathogen | Structural and functional characterization |
| pth (Enterococcus faecium) | Pth from a multidrug-resistant pathogen | Structure and inhibition by pyrrolinone |
| pth2 (mammalian) | Peptidyl-tRNA hydrolase 2 | Negative regulator of peripartum cardiomyopathy in mice |
| pth (Bacillus subtilis) | Pth homolog in Gram-positive model | Comparative studies of GO:0004045 |
| pth (Staphylococcus aureus) | Pth homolog in a major pathogen | Potential antibacterial target |
| pth (Pseudomonas aeruginosa) | Pth homolog in an opportunistic pathogen | Relevance to translation quality control |
| pth (Salmonella enterica) | Pth homolog in enteric pathogen | Model for tRNA recycling |
| pth (Vibrio cholerae) | Pth homolog in cholera pathogen | Comparative enzymology |
| pth (Helicobacter pylori) | Pth homolog in gastric pathogen | Potential drug target |
| pth (Chlamydia trachomatis) | Pth homolog in intracellular pathogen | Translation rescue in intracellular niche |
| pth (Mycoplasma genitalium) | Minimal-genome Pth homolog | Essentiality studies |
| pth (Treponema pallidum) | Pth homolog in spirochete | Comparative genomics |
| pth (Borrelia burgdorferi) | Pth homolog in Lyme disease agent | Potential target |
How Is peptidyl-tRNA hydrolase activity Regulated?
Peptidyl-tRNA hydrolase activity is regulated at multiple levels. In bacteria, Pth expression and activity are linked to translation stress and amino acid availability, as shown by the requirement for Pth in robust prolyl-tRNA turnover in Mycobacterium tuberculosis. The enzyme is also subject to inhibition by small molecules that bind its active site, as demonstrated for the Enterococcus faecium enzyme with a pyrrolinone compound. In mammals, Pth2 acts as a negative regulator of peripartum cardiomyopathy with heart failure in female mice, indicating physiological regulation of this activity in cardiac tissue. Structural studies of Pth from Thermus thermophilus, Francisella tularensis, Klebsiella pneumoniae and Enterococcus faecium provide a framework for understanding how active-site geometry controls catalytic efficiency and inhibitor sensitivity.
peptidyl-tRNA hydrolase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| pth (Mycobacterium tuberculosis) | Tuberculosis pathogenesis and prolyl-tRNA turnover | Knockout and point-mutation models in M. tuberculosis |
| pth (Klebsiella pneumoniae) | Bacterial infection and drug targeting | Structural and inhibition studies |
| pth (Enterococcus faecium) | Multidrug-resistant infection | Pyrrolinone inhibition assays |
| pth (Francisella tularensis) | Tularemia and biothreat research | Structural characterization for inhibitor design |
| pth2 (mammalian) | Peripartum cardiomyopathy with heart failure | Knockout and overexpression in female mice |
Bacterial infections and antimicrobial resistance
Peptidyl-tRNA hydrolase activity is essential for bacterial viability and has been structurally characterized in pathogens including Klebsiella pneumoniae, Enterococcus faecium and Francisella tularensis. Inhibition of Pth by small molecules such as a pyrrolinone compound demonstrates that this activity is a druggable antibacterial target. Because Pth is required for robust prolyl-tRNA turnover in Mycobacterium tuberculosis, it is also relevant to tuberculosis pathogenesis.
Peripartum cardiomyopathy and heart failure
Peptidyl-tRNA hydrolase 2 (Pth2) has been identified as a negative regulator of peripartum cardiomyopathy with heart failure in female mice. This finding links GO:0004045-related activity to a specific human cardiac disease context and suggests that modulating Pth2 could influence disease progression.
Translation stress and proteostasis
Accumulation of peptidyl-tRNA species is toxic to cells, and peptidyl-tRNA hydrolase activity prevents this toxicity by recycling tRNA and releasing peptides. Defects in this activity can therefore impair protein biosynthesis and cellular proteostasis. In Mycobacterium tuberculosis, Pth supports translation under conditions that challenge amino acid supply.
From peptidyl-tRNA hydrolase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Is Pth essential for bacterial viability? | CRISPR knockout or conditional knockout in Mycobacterium tuberculosis |
| How does Pth recognize peptidyl-tRNA? | Point mutations in the peptidyl-A76 binding pocket |
| What is the catalytic mechanism of Pth? | Active-site point mutations combined with structural studies |
| Can Pth be inhibited by small molecules? | Knock-in of tagged Pth for inhibitor binding assays |
| What is the role of Pth2 in cardiac disease? | Overexpression and knockout of Pth2 in mouse models |
| How does Pth affect global translation? | Tagged knock-in for ribosome profiling and proteomics |
How to Study the peptidyl-tRNA hydrolase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| X-ray crystallography | Three-dimensional structure of Pth | Active-site mapping and inhibitor design |
| Enzymatic hydrolysis assay | Peptidyl-tRNA hydrolase activity | Kinetic characterization of wild-type and mutants |
| Ribosome profiling | Global translation and ribosome occupancy | Effects of Pth loss on protein synthesis |
| RNA-seq | tRNA and mRNA abundance | tRNA recycling and stress responses |
| Proteomics | Protein abundance and interactions | Identifying Pth interaction partners |
| Site-directed mutagenesis | Role of specific residues | Catalytic mechanism studies |
| Inhibitor screening | Small-molecule binding and inhibition | Antibacterial drug discovery |
| Mouse genetics | Physiological role of Pth2 | Cardiac disease models |
Structural biology (X-ray crystallography and cryo-EM)
High-resolution crystal structures of Pth from Thermus thermophilus, Francisella tularensis, Klebsiella pneumoniae and Enterococcus faecium have revealed the conserved alpha/beta hydrolase fold and active-site architecture. These methods are essential for understanding substrate binding and for structure-guided inhibitor design.
Enzymatic activity assays
Peptidyl-tRNA hydrolase activity can be measured by monitoring the hydrolysis of N-acyl-L-alpha-aminoacyl-tRNA to N-acyl-L-amino acid, tRNA and H+. Such assays are used to characterize wild-type and mutant enzymes and to test inhibitors.
Ribosome profiling and RNA sequencing
Ribosome profiling and RNA-seq can reveal how loss of Pth affects translation and tRNA pools, as suggested by the role of Pth in prolyl-tRNA turnover in Mycobacterium tuberculosis. These methods help link GO:0004045 to global protein synthesis.
Proteomics and interactomics
Proteomic approaches can identify proteins that interact with Pth or that accumulate when the activity is perturbed. Such studies complement structural and genetic data to define the cellular network around GO:0004045.
How CRISPR Can Be Used to Study GO:0004045 peptidyl-tRNA hydrolase activity
Knockout
CRISPR knockout of pth genes can be used to test essentiality in bacteria such as Mycobacterium tuberculosis, where Pth is required for robust prolyl-tRNA turnover. In mammalian systems, knockout of Pth2 can reveal its role in peripartum cardiomyopathy and heart failure. Knockout models are also valuable for validating Pth as an antibacterial target.
Point Mutation
Point mutations in the active site or substrate-binding pocket of Pth can dissect the catalytic mechanism and the role of the peptidyl-A76 binding mode. Such mutations complement structural studies of Pth from Thermus thermophilus, Francisella tularensis, Klebsiella pneumoniae and Enterococcus faecium.
Knock-in
Knock-in of tagged Pth alleles enables affinity purification, imaging and interaction studies. Tagged knock-in models can also be used to monitor Pth localization and dynamics in bacterial or mammalian cells.
Overexpression
Overexpression of Pth or Pth2 can be used to test gain-of-function effects on translation and disease phenotypes. In particular, overexpression of Pth2 in mouse models can modulate peripartum cardiomyopathy with heart failure.
How EDITGENE Supports peptidyl-tRNA hydrolase activity Research
Researchers studying peptidyl-tRNA hydrolase activity-related genes often need to determine whether a candidate gene is causally involved in translation, bacterial survival or cardiac disease. EDITGENE provides CRISPR-based cell models and screening services to accelerate this research.
Contact EDITGENE today to design your custom CRISPR model for peptidyl-tRNA hydrolase activity research.
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Frequently Asked Questions About peptidyl-tRNA hydrolase activity
What is peptidyl-tRNA hydrolase activity?
Peptidyl-tRNA hydrolase activity (GO:0004045) is a molecular function that catalyzes the hydrolysis of an N-acyl-L-alpha-aminoacyl-tRNA to an N-acyl-L-amino acid, a tRNA and H+.
What genes are involved in peptidyl-tRNA hydrolase activity?
Genes encoding peptidyl-tRNA hydrolase (pth) are found in bacteria such as Mycobacterium tuberculosis, Thermus thermophilus, Francisella tularensis, Klebsiella pneumoniae and Enterococcus faecium, and the mammalian homolog pth2 is also associated with this activity.
Why is peptidyl-tRNA hydrolase important for protein synthesis?
It recycles tRNA from peptidyl-tRNA species that accumulate when translation stalls, preventing toxic buildup and maintaining the free tRNA pool needed for protein synthesis.
What is the reaction catalyzed by GO:0004045?
The reaction is: an N-acyl-L-alpha-aminoacyl-tRNA + H2O = an N-acyl-L-amino acid + a tRNA + H+.
How is peptidyl-tRNA hydrolase structured?
Structures from Thermus thermophilus, Francisella tularensis, Klebsiella pneumoniae and Enterococcus faecium show a conserved alpha/beta hydrolase fold with a catalytic triad-like active site.
Can peptidyl-tRNA hydrolase be inhibited by drugs?
Yes, the Enterococcus faecium enzyme is inhibited by a pyrrolinone compound, supporting its potential as an antibacterial target.
What is the role of Pth2 in disease?
Pth2 acts as a negative regulator of peripartum cardiomyopathy with heart failure in female mice.
How does Mycobacterium tuberculosis use peptidyl-tRNA hydrolase?
Pth is required for robust prolyl-tRNA turnover in Mycobacterium tuberculosis, linking it to amino acid homeostasis and stress survival.
What methods are used to study peptidyl-tRNA hydrolase activity?
Common methods include X-ray crystallography, enzymatic hydrolysis assays, ribosome profiling, RNA-seq, proteomics and site-directed mutagenesis.
How can CRISPR help study GO:0004045?
CRISPR knockout, point mutation, knock-in and overexpression models allow researchers to test the function of pth and pth2 genes in translation and disease.
Conclusion
Peptidyl-tRNA hydrolase activity (GO:0004045) is a conserved molecular function that recycles tRNA and prevents toxic peptidyl-tRNA accumulation, making it essential for protein biosynthesis. Structural and functional studies across bacterial species have defined its catalytic mechanism and substrate binding mode, and have validated it as a potential antibacterial target. In mammals, Pth2 has been linked to peripartum cardiomyopathy with heart failure, expanding the disease relevance of this activity. Continued research using CRISPR models and multi-omics approaches will further clarify how GO:0004045 contributes to translation, stress responses and human disease.
References
- 1. Tomasi FG et al.. 2023. Peptidyl tRNA Hydrolase Is Required for Robust Prolyl-tRNA Turnover in Mycobacterium tuberculosis.. mBio 14(1):e0346922 PMID: 36695586
- 2. Uehara Y et al.. 2025. Binding mode between peptidyl-tRNA hydrolase and the peptidyl-A76 moiety of the substrate.. J Biol Chem 301(4):108385 PMID: 40049414
- 3. Mundra S et al.. 2021. Structural and functional characterization of peptidyl-tRNA hydrolase from Klebsiella pneumoniae.. Biochim Biophys Acta Proteins Proteom 1869(1):140554 PMID: 33068756
- 4. Das G et al.. 2006. Peptidyl-tRNA hydrolase and its critical role in protein biosynthesis.. Microbiology (Reading) 152(Pt 8):2191-2195 PMID: 16849786
- 5. Montoya-Uribe V et al.. 2025. Peptidyl-tRNA hydrolase 2 is a negative regulator of peripartum cardiomyopathy with heart failure in female mice.. Nat Commun 17(1):1091 PMID: 41413062
- 6. Matsumoto A et al.. 2019. High-resolution crystal structure of peptidyl-tRNA hydrolase from Thermus thermophilus.. Proteins 87(3):226-235 PMID: 30520515
- 7. Pandey R et al.. 2024. Characterization of structure of peptidyl-tRNA hydrolase from Enterococcus faecium and its inhibition by a pyrrolinone compound.. Int J Biol Macromol 275(Pt 1):133445 PMID: 38945334
- 8. Clarke TE et al.. 2011. Structure of Francisella tularensis peptidyl-tRNA hydrolase.. Acta Crystallogr Sect F Struct Biol Cryst Commun 67(Pt 4):446-9 PMID: 21505237
