GO:0098621 O-phosphoseryl-tRNA(Sec) selenium transferase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0098621 defines the molecular function that converts O-phospho-L-seryl-tRNA(Sec) into L-selenocysteinyl-tRNA(Sec) using selenophosphate and water, releasing two phosphate molecules.
• This activity is the committed step in selenocysteine (Sec) biosynthesis, the 21st amino acid that is co-translationally inserted into selenoproteins.
• The enzyme is known as SepSecS (O-phosphoseryl-tRNA(Sec) selenium transferase) and is conserved from archaea to humans.
• Structural studies of the archaeal homolog revealed a pyridoxal phosphate (PLP)-dependent fold and a unique catalytic mechanism.
• Loss of SepSecS function impairs selenoprotein synthesis, which is linked to neurodevelopmental disorders, cancer, and oxidative stress-related pathologies.
• CRISPR knockout, point-mutation, and knock-in models are essential to dissect the role of GO:0098621 in disease and to validate therapeutic targets.
Description
GO:0098621, O-phosphoseryl-tRNA(Sec) selenium transferase activity, is a molecular function that catalyzes the final step of selenocysteine (Sec) biosynthesis. This reaction converts O-phospho-L-seryl-tRNA(Sec) into L-selenocysteinyl-tRNA(Sec) in the presence of selenophosphate and water, producing two phosphate molecules. The enzyme responsible, SepSecS, is a pyridoxal phosphate (PLP)-dependent transferase that ensures the correct aminoacylation of tRNA(Sec) with Sec, a process essential for the synthesis of selenoproteins. Because selenoproteins are critical for redox homeostasis, thyroid hormone metabolism, and immune function, understanding this activity is fundamental to molecular biology and medicine. Researchers study GO:0098621 to uncover how selenoprotein synthesis is regulated and how its dysfunction contributes to human disease. The availability of structural and functional data from archaeal and eukaryotic systems has provided a framework for investigating the catalytic mechanism and for developing CRISPR-based models to probe its physiological roles.
O-phosphoseryl-tRNA(Sec) selenium transferase activity At A Glance
| GO ID | GO:0098621 |
|---|---|
| GO term | O-phosphoseryl-tRNA(Sec) selenium transferase activity |
| Ontology | molecular_function |
| Synonym | SepSecS; O-phospho-L-seryl-tRNA(Sec):L-selenocysteinyl-tRNA synthase; O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase; phosphoseryl-selenocysteinyl-tRNA selenium transferase activity |
| Definition | Catalysis of the reaction: O-phospho-L-seryl-tRNA(Sec) + selenophosphate + H2O = L-selenocysteinyl-tRNA(Sec) + 2 phosphate. |
| Major function | Final step of selenocysteine biosynthesis; converts O-phosphoseryl-tRNA(Sec) to selenocysteinyl-tRNA(Sec) |
| Cofactor | Pyridoxal phosphate (PLP) |
| Reaction direction | Irreversible under physiological conditions |
| Subcellular location | Cytoplasm (in eukaryotes) |
What Is GO:0098621?
In our own words, GO:0098621 describes the catalytic activity of an enzyme that takes a tRNA molecule charged with O-phosphoserine (O-phospho-L-seryl-tRNA(Sec)) and, using selenophosphate as the selenium donor and water, replaces the phosphate group with selenium to form L-selenocysteinyl-tRNA(Sec). The reaction also releases two phosphate ions. This activity is synonymous with SepSecS, O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase, and phosphoseryl-selenocysteinyl-tRNA selenium transferase. It is a molecular_function term in the Gene Ontology and is essential for the biosynthesis of selenocysteine, which is then used in translation.
Why Is O-phosphoseryl-tRNA(Sec) selenium transferase activity Important in Cell Biology?
GO:0098621 is important because it governs the production of selenocysteine, the 21st amino acid that is essential for the function of selenoproteins such as glutathione peroxidases and thioredoxin reductases. These enzymes protect cells from oxidative damage and regulate redox signaling. Without this activity, selenoprotein synthesis stalls, leading to impaired antioxidant defense and contributing to pathologies including neurodevelopmental disorders, cancer, and cardiovascular disease. Thus, understanding the molecular details of GO:0098621 provides insights into fundamental translation mechanisms and offers potential targets for therapeutic intervention.
• Enables the synthesis of selenocysteine, a key component of selenoproteins.
• Supports cellular antioxidant defense via glutathione peroxidases and thioredoxin reductases.
• Plays a role in thyroid hormone metabolism through iodothyronine deiodinases.
• Dysfunction is linked to neurodevelopmental disorders and neurodegeneration.
• Altered expression is observed in various cancers, affecting tumor growth and survival.
• Serves as a model for studying tRNA-dependent amino acid modification.
• Provides a target for CRISPR-based functional genomics.
• Highlights the importance of selenium metabolism in human health.
• Enables structural and mechanistic studies of PLP-dependent enzymes.
• Contributes to the understanding of genetic code expansion.
What Happens During O-phosphoseryl-tRNA(Sec) selenium transferase activity?
Substrate recognition and binding
In simple terms: The enzyme grabs the tRNA that already carries a phosphoserine molecule.
SepSecS specifically recognizes O-phospho-L-seryl-tRNA(Sec), the product of the previous step in selenocysteine biosynthesis. Structural studies of the archaeal enzyme show that it forms a dimer and binds the tRNA through its N-terminal domain, positioning the phosphoseryl moiety near the active site.
Selenophosphate delivery and activation
In simple terms: A selenium donor molecule enters the active site to provide the selenium atom.
Selenophosphate (SeP) is the selenium donor. It is synthesized by selenophosphate synthetase (SPS) and delivered to SepSecS. The enzyme uses a pyridoxal phosphate (PLP) cofactor to form a Schiff base with the phosphoserine, facilitating the elimination of phosphate and the attack by selenium.
Catalytic conversion to selenocysteinyl-tRNA
In simple terms: The enzyme swaps the phosphate group for selenium, creating selenocysteine on the tRNA.
The catalytic mechanism involves the formation of an aminoacrylate intermediate from the phosphoserine moiety, followed by nucleophilic attack by selenophosphate. This results in the formation of L-selenocysteinyl-tRNA(Sec) and the release of two phosphate ions. The reaction is PLP-dependent and requires water.
Product release and tRNA recycling
In simple terms: The finished tRNA with selenocysteine is released to be used in protein synthesis.
After catalysis, the selenocysteinyl-tRNA(Sec) is released from the enzyme. This charged tRNA is then delivered to the ribosome by the elongation factor SelB (in bacteria) or its eukaryotic counterpart, where it decodes UGA codons in selenoprotein mRNAs. The enzyme is ready for another round of catalysis.
Key Genes Involved in GO:0098621 O-phosphoseryl-tRNA(Sec) selenium transferase activity
The following genes and proteins are directly or indirectly involved in O-phosphoseryl-tRNA(Sec) selenium transferase activity and selenocysteine biosynthesis.
| Gene | Major Role | Research Relevance |
|---|---|---|
| SEPSECS (SepSecS) | Catalyzes the final step of selenocysteine biosynthesis | Primary enzyme for GO:0098621; mutations cause neurodevelopmental disorders |
| PSTK | Phosphorylates seryl-tRNA(Sec) to form O-phosphoseryl-tRNA(Sec) | Provides the substrate for SepSecS |
| SPS1 (SEPHS1) | Synthesizes selenophosphate, the selenium donor | Supplies selenophosphate for the reaction |
| SPS2 (SEPHS2) | Alternative selenophosphate synthetase | May compensate for SPS1 in some tissues |
| SARS | Seryl-tRNA synthetase charges tRNA(Sec) with serine | Initiates the pathway by forming Ser-tRNA(Sec) |
| EEFSEC (SelB) | Delivers selenocysteinyl-tRNA(Sec) to the ribosome | Essential for selenoprotein translation |
| SECISBP2 | Binds SECIS elements in selenoprotein mRNAs | Required for recoding UGA as selenocysteine |
| TRU-TCA1-1 | tRNA(Sec) gene | Provides the tRNA backbone for the pathway |
| GPX1 | Glutathione peroxidase 1 | Selenoprotein whose synthesis depends on SepSecS |
| GPX4 | Glutathione peroxidase 4 | Critical for ferroptosis regulation; requires selenocysteine |
| TXNRD1 | Thioredoxin reductase 1 | Selenoprotein involved in redox homeostasis |
| TXNRD2 | Thioredoxin reductase 2 | Mitochondrial selenoprotein |
| DIO1 | Iodothyronine deiodinase 1 | Selenoprotein for thyroid hormone metabolism |
| DIO2 | Iodothyronine deiodinase 2 | Selenoprotein for thyroid hormone activation |
| SELENOP | Selenoprotein P | Major selenium transport protein |
| SELENOK | Selenoprotein K | Involved in ER stress and calcium signaling |
| SELENOS | Selenoprotein S | Involved in ER-associated degradation |
| SELENOT | Selenoprotein T | Involved in redox regulation and neuroprotection |
How Is O-phosphoseryl-tRNA(Sec) selenium transferase activity Regulated?
The activity of SepSecS (GO:0098621) is regulated at multiple levels. Expression of the SEPSECS gene can be influenced by selenium availability, as selenium deficiency reduces selenoprotein synthesis and may feedback on the pathway. Additionally, the enzyme's activity depends on the availability of its substrates, O-phosphoseryl-tRNA(Sec) and selenophosphate, which are produced by PSTK and SPS enzymes, respectively. Post-translational modifications and interactions with other components of the selenocysteine biosynthesis machinery may also modulate its function, though specific regulatory mechanisms remain an active area of research.
O-phosphoseryl-tRNA(Sec) selenium transferase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| SEPSECS | Pontocerebellar hypoplasia type 2D | Knockout mouse, patient-derived iPSCs |
| GPX4 | Ferroptosis-related diseases, cancer | Point mutation knock-in in cancer cell lines |
| TXNRD1 | Cancer, oxidative stress | Overexpression and knockout in tumor models |
| SELENOP | Selenium transport deficiency, male infertility | Knockout mouse |
| DIO2 | Thyroid hormone resistance | Knock-in of patient mutations |
Neurodevelopmental disorders
Mutations in the SEPSECS gene, which encodes the enzyme responsible for GO:0098621, cause pontocerebellar hypoplasia type 2D (PCH2D), a severe neurodevelopmental disorder characterized by microcephaly, intellectual disability, and progressive cerebellar atrophy. This highlights the critical role of selenocysteine biosynthesis in brain development.
Cancer
Altered expression of selenoproteins and selenocysteine biosynthesis enzymes has been observed in various cancers. For example, reduced SepSecS activity may impair antioxidant defense, leading to increased oxidative stress and genomic instability that promotes tumorigenesis. Conversely, some cancers upregulate selenoproteins to survive oxidative stress.
Cardiovascular and metabolic diseases
Selenoproteins such as glutathione peroxidases and thioredoxin reductases are crucial for cardiovascular health. Dysfunction in GO:0098621 can lead to reduced selenoprotein levels, contributing to oxidative damage in cardiomyocytes and endothelial cells, and may exacerbate conditions like atherosclerosis and heart failure.
From O-phosphoseryl-tRNA(Sec) selenium transferase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| What is the effect of SepSecS loss on selenoprotein synthesis? | CRISPR knockout of SEPSECS in HEK293 or HeLa cells |
| How do disease-associated mutations affect catalytic activity? | Point mutation knock-in of SEPSECS variants |
| Can we rescue the phenotype by restoring SepSecS expression? | Knock-in of wild-type SEPSECS under a doxycycline-inducible promoter |
| What is the subcellular localization of SepSecS? | Tagged knock-in with GFP or FLAG |
| How does SepSecS overexpression affect cancer cell growth? | Overexpression of SEPSECS in cancer cell lines |
| What are the interactors of SepSecS? | Knock-in of BirA tag for proximity labeling |
How to Study the O-phosphoseryl-tRNA(Sec) selenium transferase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Ribo-seq | Translation efficiency of selenoprotein mRNAs | Assessing impact of SepSecS loss on selenoprotein synthesis |
| RNA-seq | Global gene expression changes | Identifying pathways affected by SEPSECS knockout |
| Western blot | Protein levels of selenoproteins | Validating changes in GPX4, TXNRD1, etc. |
| Enzymatic assay | SepSecS catalytic activity | Testing mutant enzymes or inhibitors |
| CRISPR knockout | Loss-of-function phenotype | Studying the role of SEPSECS in cell models |
| CRISPR knock-in | Expression of tagged or mutant SepSecS | Localization and interaction studies |
| Proteomics | Selenoproteome profiling | Quantifying global selenoprotein levels |
| Imaging | Subcellular localization | Visualizing SepSecS in live cells |
Ribo-seq and RNA-seq
Ribosome profiling (Ribo-seq) can measure translation efficiency of selenoprotein mRNAs, which depends on functional SepSecS. RNA-seq reveals changes in gene expression upon SEPSECS knockout or overexpression, providing insights into downstream pathways.
Proteomics and selenoprotein detection
Mass spectrometry-based proteomics can quantify selenoprotein levels, which are directly affected by GO:0098621 activity. Labeling with radioactive selenium or using selenium-specific antibodies can also monitor selenocysteine incorporation.
Enzymatic assays
In vitro assays using purified SepSecS and radiolabeled substrates can directly measure the transferase activity, monitoring the conversion of O-phospho-L-seryl-tRNA(Sec) to L-selenocysteinyl-tRNA(Sec).
Structural biology
X-ray crystallography and cryo-EM can elucidate the structure of SepSecS in complex with tRNA and substrates, revealing the catalytic mechanism and guiding drug design.
How CRISPR Can Be Used to Study GO:0098621 O-phosphoseryl-tRNA(Sec) selenium transferase activity
Knockout
CRISPR knockout of SEPSECS eliminates GO:0098621 activity, leading to impaired selenoprotein synthesis. This model is useful to study the cellular consequences of selenocysteine deficiency, such as increased oxidative stress and altered cell growth.
Point Mutation
Introducing patient-derived point mutations into SEPSECS via CRISPR allows researchers to dissect the molecular basis of diseases like PCH2D. These models can reveal how specific amino acid changes affect enzyme stability, substrate binding, or catalysis.
Knock-in
Knock-in of a tagged version of SEPSECS (e.g., GFP or FLAG) enables real-time tracking of the enzyme's localization and interaction partners. Conditional knock-in using Cre-lox or Tet-On systems provides spatial and temporal control of expression.
Overexpression
CRISPR activation (CRISPRa) or lentiviral overexpression of SEPSECS can boost GO:0098621 activity, which may protect cells from oxidative stress or enhance selenoprotein production. This approach is valuable for studying the effects of increased selenocysteine biosynthesis in cancer and neurodegeneration models.
How EDITGENE Supports O-phosphoseryl-tRNA(Sec) selenium transferase activity Research
Researchers studying O-phosphoseryl-tRNA(Sec) selenium transferase activity-related genes often need to determine whether a candidate gene is causally involved in selenoprotein synthesis, stress responses, or disease phenotypes. EDITGENE provides a comprehensive suite of CRISPR services to generate precisely engineered cell models, enabling rigorous functional validation of GO:0098621 and its associated pathways.
Contact EDITGENE today to design your custom CRISPR model for O-phosphoseryl-tRNA(Sec) selenium transferase activity research.
Frequently Asked Questions About O-phosphoseryl-tRNA(Sec) selenium transferase activity
What is O-phosphoseryl-tRNA(Sec) selenium transferase activity?
It is the enzymatic activity (GO:0098621) that converts O-phospho-L-seryl-tRNA(Sec) to L-selenocysteinyl-tRNA(Sec), the final step in selenocysteine biosynthesis.
What genes are involved in O-phosphoseryl-tRNA(Sec) selenium transferase activity?
The primary gene is SEPSECS, which encodes the enzyme SepSecS. Other genes in the pathway include PSTK, SPS1, and SARS.
What is the function of SepSecS?
SepSecS catalyzes the selenium transfer reaction that produces selenocysteinyl-tRNA(Sec), essential for selenoprotein synthesis.
How is O-phosphoseryl-tRNA(Sec) selenium transferase activity regulated?
It is regulated by selenium availability, substrate supply, and potentially post-translational modifications.
What diseases are associated with defects in this activity?
Mutations in SEPSECS cause pontocerebellar hypoplasia type 2D, and altered activity is linked to cancer and cardiovascular diseases.
What is the reaction catalyzed by GO:0098621?
O-phospho-L-seryl-tRNA(Sec) + selenophosphate + H2O = L-selenocysteinyl-tRNA(Sec) + 2 phosphate.
What cofactor does SepSecS require?
SepSecS is a pyridoxal phosphate (PLP)-dependent enzyme.
How can I study GO:0098621 in the lab?
You can use CRISPR knockout, point mutation knock-in, overexpression, and enzymatic assays to study its function.
Is SepSecS conserved across species?
Yes, SepSecS is conserved from archaea to humans, with structural homologs in all domains of life.
What are the synonyms for GO:0098621?
Synonyms include SepSecS, O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase, and phosphoseryl-selenocysteinyl-tRNA selenium transferase activity.
Conclusion
GO:0098621, O-phosphoseryl-tRNA(Sec) selenium transferase activity, is a cornerstone of selenocysteine biosynthesis and thus of selenoprotein function. Its catalytic mechanism, mediated by SepSecS, ensures the proper decoding of UGA codons as selenocysteine, which is vital for antioxidant defense and cellular homeostasis. Dysregulation of this activity has been implicated in neurodevelopmental disorders, cancer, and cardiovascular diseases, making it a compelling target for further research. By leveraging CRISPR-based models and advanced omics technologies, scientists can continue to unravel the precise roles of this activity in health and disease, paving the way for novel therapeutic strategies.
References
- 1. Kaiser JT et al.. 2005. Structural and functional investigation of a putative archaeal selenocysteine synthase.. Biochemistry 44(40):13315-27 PMID: 16201757
