GO:0004379 glycylpeptide N-tetradecanoyltransferase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0004379 describes the enzymatic activity that transfers a myristoyl group from tetradecanoyl-CoA to the N-terminal glycine of a peptide or protein, producing N-tetradecanoylglycyl-peptide and CoA.
• This activity is essential for protein N-myristoylation, a co-translational modification that anchors proteins to membranes and regulates their localization and function.
• The enzymes responsible are N-myristoyltransferases (NMT1 and NMT2 in humans), which recognize an N-terminal glycine after removal of the initiator methionine.
• N-myristoylation is critical in signal transduction, apoptosis, and cancer; dysregulation of NMT activity is linked to prostate cancer and other malignancies.
• Studying GO:0004379 requires biochemical assays, CRISPR knockout models, and proteomic methods to identify substrates and inhibitors.
• EDITGENE provides CRISPR services to generate knockout, point-mutation, knock-in, and overexpression cell models for genes encoding N-myristoyltransferases and their substrates.
Description
Glycylpeptide N-tetradecanoyltransferase activity (GO:0004379) is a molecular function that catalyzes the covalent attachment of a myristoyl group (a 14-carbon saturated fatty acid) to the N-terminal glycine residue of a peptide or protein. This reaction, known as protein N-myristoylation, is a co-translational modification that occurs on many eukaryotic proteins and is essential for their membrane targeting and function. The enzyme responsible, N-myristoyltransferase (NMT), was first characterized in rat brain and tissues, and its activity is conserved from yeast to humans. Researchers study GO:0004379 to understand how protein lipidation controls cellular signaling, and to develop inhibitors for cancer and infectious diseases. The reaction requires tetradecanoyl-CoA as the acyl donor and produces CoA as a byproduct, and it is highly specific for N-terminal glycine residues exposed after methionine cleavage.
glycylpeptide N-tetradecanoyltransferase activity At A Glance
| GO ID | GO:0004379 |
|---|---|
| GO term | glycylpeptide N-tetradecanoyltransferase activity |
| Ontology | molecular_function |
| Synonym | N-myristoyltransferase activity; myristoyl-CoA:protein N-myristoyltransferase activity; peptide N-tetradecanoyltransferase activity; protein N-myristoyltransferase activity |
| Major function | Catalyzes the transfer of a myristoyl group from tetradecanoyl-CoA to the N-terminal glycine of a peptide, producing N-tetradecanoylglycyl-peptide and CoA. |
| Reaction | tetradecanoyl-CoA + glycyl-peptide = CoA + N-tetradecanoylglycyl-peptide |
| Substrates | Tetradecanoyl-CoA (myristoyl-CoA) and a glycyl-peptide with a free alpha-amino group. |
| Localization | Cytosol and membrane fractions; associated with ribosomes for co-translational modification. |
| Enzymes | NMT1 and NMT2 in humans; orthologs in yeast, plants, and parasites. |
What Is GO:0004379?
GO:0004379, glycylpeptide N-tetradecanoyltransferase activity, is defined by the Gene Ontology as the catalysis of the reaction: tetradecanoyl-CoA + glycyl-peptide = CoA + N-tetradecanoylglycyl-peptide. In other words, it is the enzyme activity that transfers a myristoyl group from myristoyl-CoA to the alpha-amino group of an N-terminal glycine residue on a peptide or protein, forming an amide bond and releasing coenzyme A. This activity is synonymous with N-myristoyltransferase (NMT) activity and is a key step in protein N-myristoylation.
Why Is glycylpeptide N-tetradecanoyltransferase activity Important in Cell Biology?
GO:0004379 is important because protein N-myristoylation is a fundamental co-translational modification that affects thousands of proteins involved in cell signaling, apoptosis, and oncogenesis. The activity is essential for the function of proteins such as Src family kinases, ADP-ribosylation factors, and caspases, and its dysregulation has been implicated in cancer, neurodegenerative diseases, and infections. Understanding this activity provides a basis for developing NMT inhibitors as therapeutic agents, and for using CRISPR models to dissect its role in disease.
• Protein N-myristoylation is required for membrane targeting of many signaling proteins, including Src and Ras family GTPases.
• NMT activity is essential for apoptosis; myristoylation of PAK2 potentiates late apoptotic events.
• Inhibition of NMT activity promotes androgen receptor degradation in prostate cancer, highlighting its therapeutic potential.
• NMT is a validated drug target in parasitic infections and cancer, with several inhibitors in preclinical development.
• The activity is conserved across eukaryotes, making model organisms useful for functional studies.
• CRISPR knockout of NMT genes can reveal essential roles in cell viability and signaling.
• Point mutations in the N-terminal glycine of substrate proteins can abolish myristoylation and alter localization.
• NMT activity can be measured biochemically using radiolabeled myristoyl-CoA or fluorescent peptides.
• Dysregulation of NMT expression is observed in various cancers, including prostate and colorectal cancer.
• Studying GO:0004379 helps link genotype to phenotype in lipid modification disorders.
What Happens During glycylpeptide N-tetradecanoyltransferase activity?
Substrate recognition and binding
In simple terms: The enzyme grabs a myristoyl group and a target protein that starts with glycine.
N-myristoyltransferase (NMT) binds tetradecanoyl-CoA and a peptide substrate that has an N-terminal glycine residue. The enzyme recognizes the glycine after the initiator methionine is removed by methionine aminopeptidases, and it forms a ternary complex with both substrates. The binding is highly specific for myristoyl-CoA, although other acyl-CoAs can compete.
Catalytic transfer of myristate
In simple terms: The enzyme snaps the myristoyl group onto the glycine, releasing CoA.
The catalytic mechanism involves nucleophilic attack of the glycine alpha-amino group on the thioester carbonyl of tetradecanoyl-CoA, forming an amide bond and releasing coenzyme A. This reaction is ordered and requires a conserved catalytic base, typically a glutamate or histidine residue in the active site. The product is N-tetradecanoylglycyl-peptide, which is now anchored to membranes.
Co-translational modification
In simple terms: The modification happens while the protein is still being made on the ribosome.
In human cells, N-myristoylation occurs co-translationally, when the nascent polypeptide chain exposes its N-terminal glycine as it emerges from the ribosome. NMT associates with ribosomes and modifies the protein before folding is complete. This timing ensures efficient membrane targeting and prevents aggregation of hydrophobic proteins.
Post-translational myristoylation
In simple terms: Sometimes the modification happens after the protein is made, especially during cell death.
In addition to co-translational modification, NMT can myristoylate proteins post-translationally after caspase cleavage exposes a new N-terminal glycine. For example, caspase-cleaved PAK2 is myristoylated by NMT, which potentiates late apoptotic events. This alternative mode expands the repertoire of NMT substrates and links the activity to apoptosis.
Key Genes Involved in GO:0004379 glycylpeptide N-tetradecanoyltransferase activity
The following genes encode enzymes, substrates, and regulators directly related to GO:0004379, based on published literature.
| Gene | Major Role | Research Relevance |
|---|---|---|
| NMT1 | Major human N-myristoyltransferase; catalyzes myristoyl transfer to many proteins | Knockout reduces myristoylation of Src and other substrates; target for cancer therapy |
| NMT2 | Second human N-myristoyltransferase with overlapping and distinct substrates | Knockout models show redundancy with NMT1; involved in cell survival |
| SRC | Proto-oncogene tyrosine kinase; myristoylated by NMT for membrane localization | Myristoylation is required for Src transforming activity; point mutation of Gly2 blocks it |
| PAK2 | Serine/threonine kinase; post-translationally myristoylated after caspase cleavage | Myristoylation potentiates apoptosis; knockout or point mutation affects cell death |
| ARF1 | ADP-ribosylation factor; myristoylated for Golgi membrane binding | Myristoylation is essential for vesicular transport; knockout is lethal in model organisms |
| ARF6 | ADP-ribosylation factor; myristoylated for endosomal recycling | Regulates membrane trafficking; myristoylation mutants show mislocalization |
| GNAI1 | G protein alpha subunit; myristoylated for membrane anchoring | Myristoylation is required for signaling; point mutation alters function |
| GNAO1 | G protein alpha subunit; myristoylated in neurons | Mutations in myristoylation site cause neurological disorders |
| CASP3 | Caspase-3; cleaves substrates exposing myristoylation sites | Involved in apoptosis; regulates post-translational myristoylation |
| CASP8 | Caspase-8; similar role in apoptosis | Links apoptotic signaling to NMT activity |
| LYN | Src family kinase; myristoylated for membrane targeting | Myristoylation is required for B-cell signaling; knockout affects immune function |
| YES1 | Src family kinase; myristoylated | Involved in cell growth; myristoylation inhibitors affect its localization |
| FYN | Src family kinase; myristoylated | Regulates neuronal signaling; myristoylation is essential for function |
| NMT1 (yeast) | Yeast ortholog; essential for viability | Model for studying NMT function and inhibitors |
| NMT2 (yeast) | Yeast ortholog; redundant with NMT1 | Double knockout is lethal; useful for genetic studies |
| NMT (parasite) | NMT from Trypanosoma brucei and Leishmania; drug target | Inhibitors show antiparasitic activity; CRISPR models validate target |
| NMT (plant) | Plant NMT; involved in development and stress responses | Knockout affects growth and pathogen resistance |
| NMT (Drosophila) | Fly NMT; essential for development | Genetic models reveal roles in signaling and morphogenesis |
How Is glycylpeptide N-tetradecanoyltransferase activity Regulated?
N-myristoyltransferase activity is regulated at multiple levels. Expression of NMT1 and NMT2 is controlled by transcription factors and can be induced by growth factors and hormones. The activity is also regulated by substrate availability, as the N-terminal glycine must be exposed after methionine removal. Post-translational modifications of NMT, such as phosphorylation, can modulate its activity and localization. In cancer cells, NMT1 is often overexpressed and its inhibition leads to degradation of oncoproteins like the androgen receptor. Additionally, competitive inhibitors of NMT, such as myristic acid analogs, can block the activity and are being developed as therapeutics.
glycylpeptide N-tetradecanoyltransferase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| NMT1 | Prostate cancer; androgen receptor stability | CRISPR knockout of NMT1 in LNCaP cells; overexpression for inhibitor testing |
| NMT2 | Cancer; redundant with NMT1 | Double knockout in cancer cell lines to assess synthetic lethality |
| SRC | Cancer; membrane targeting and transformation | Point mutation of Gly2 to Ala to block myristoylation; knock-in in cancer models |
| PAK2 | Apoptosis; post-translational myristoylation | Knockout or point mutation of the myristoylation site; apoptosis assays |
| GNAO1 | Neurological disorders; myristoylation site mutations | Knock-in of patient mutations in neurons; imaging of localization |
Cancer
N-myristoyltransferase activity is frequently upregulated in human cancers, including prostate, colorectal, and brain tumors. In prostate cancer, inhibition of NMT activity promotes androgen receptor degradation, suggesting that NMT inhibitors could be used to treat castration-resistant prostate cancer. Myristoylation of Src family kinases is required for their oncogenic signaling, and blocking this modification reduces tumor growth in preclinical models.
Neurodegeneration
Defects in protein N-myristoylation have been linked to neurodegenerative disorders. For example, mutations in the myristoylation site of GNAO1 cause neurological phenotypes, and impaired myristoylation of neuronal proteins may contribute to synaptic dysfunction. NMT activity is also important for the function of proteins involved in neuronal survival and apoptosis.
Infectious diseases
N-myristoyltransferases from parasites such as Trypanosoma brucei, Leishmania, and Plasmodium are essential for their survival and are validated drug targets. Inhibitors of NMT activity have shown efficacy in animal models of trypanosomiasis and leishmaniasis, and CRISPR knockout of parasite NMT genes confirms their essentiality.
Apoptosis and cell death
Post-translational myristoylation of caspase-cleaved PAK2 by NMT potentiates late apoptotic events, linking GO:0004379 to programmed cell death. Dysregulation of this process can contribute to cancer cell survival or excessive tissue damage.
From glycylpeptide N-tetradecanoyltransferase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Is NMT1 essential for cell viability? | CRISPR knockout of NMT1 in human cell lines; viability assays |
| Does myristoylation of Src regulate its oncogenic activity? | Point mutation of Src Gly2 to Ala; knock-in in cancer cells |
| What are the substrates of NMT in a specific cell type? | Knock-in of tagged NMT1; proteomics and click chemistry |
| Can NMT inhibitors reduce tumor growth? | Overexpression of NMT1 in xenograft models; treatment with inhibitors |
| Does post-translational myristoylation of PAK2 affect apoptosis? | Knockout of PAK2 myristoylation site; apoptosis induction |
| Is NMT2 redundant with NMT1? | Double knockout of NMT1 and NMT2; synthetic lethality screens |
How to Study the glycylpeptide N-tetradecanoyltransferase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Radiolabeled NMT assay | Enzymatic transfer of [3H]myristate to peptide | Kinetic studies and inhibitor screening |
| Click chemistry proteomics | Identification of myristoylated proteins | Substrate profiling in cells |
| CRISPR knockout screen | Gene essentiality and synthetic lethality | Identifying NMT dependencies in cancer |
| Fluorescence microscopy | Subcellular localization of myristoylated proteins | Assessing membrane targeting |
| Western blot | Expression and stability of NMT and substrates | Validating knockout or overexpression |
| Mass spectrometry | Precise mass of myristoylated peptides | Confirming modification sites |
| Apoptosis assays | Caspase activity and cell death | Studying PAK2 myristoylation |
| Xenograft models | Tumor growth in vivo | Testing NMT inhibitors |
Biochemical NMT activity assays
NMT activity can be measured using radiolabeled [3H]myristoyl-CoA and a peptide substrate, followed by scintillation counting or thin-layer chromatography. Fluorescent or click-chemistry-based assays are also used for high-throughput screening of inhibitors.
Proteomic identification of myristoylated proteins
Click chemistry with azide-modified myristic acid analogs allows selective labeling and enrichment of myristoylated proteins, which can be identified by mass spectrometry. This approach reveals the substrate landscape of GO:0004379 in different cell types.
CRISPR-based genetic screens
Genome-wide CRISPR knockout screens can identify genes required for NMT activity or for the function of myristoylated proteins. For example, knockout of NMT1 in cancer cell lines followed by drug treatment can reveal synthetic lethal interactions.
Imaging and localization studies
Fluorescence microscopy of GFP-tagged myristoylated proteins can assess membrane localization and the effect of NMT inhibitors or mutations. Live-cell imaging can track dynamic changes in myristoylation-dependent trafficking.
How CRISPR Can Be Used to Study GO:0004379 glycylpeptide N-tetradecanoyltransferase activity
Knockout
CRISPR knockout of NMT1 or NMT2 in human cell lines abolishes specific myristoylation events and can reduce cell viability, depending on the cell type. Knockout models are used to identify essential substrates and to validate NMT as a drug target.
Point Mutation
Point mutations of the N-terminal glycine in substrate proteins (e.g., Src Gly2 to Ala) prevent myristoylation and cause mislocalization. CRISPR-mediated point mutation can recreate these effects in endogenous genes to study function.
Knock-in
Knock-in of tagged NMT1 or substrate proteins allows affinity purification and proteomic identification of myristoylated proteins. Knock-in of disease-associated mutations in myristoylation sites can model neurological disorders.
Overexpression
Overexpression of NMT1 or NMT2 in cancer cell lines increases myristoylation of substrates and can promote oncogenic signaling. Overexpression models are used to test NMT inhibitors and to study resistance mechanisms.
How EDITGENE Supports glycylpeptide N-tetradecanoyltransferase activity Research
Researchers studying glycylpeptide N-tetradecanoyltransferase activity-related genes often need to determine whether a candidate gene is causally involved in myristoylation-dependent processes, and CRISPR-based models provide a direct way to test this. EDITGENE offers a comprehensive suite of services to generate and characterize such models.
Contact EDITGENE today to design your custom CRISPR model for glycylpeptide N-tetradecanoyltransferase activity research.
Frequently Asked Questions About glycylpeptide N-tetradecanoyltransferase activity
What is glycylpeptide N-tetradecanoyltransferase activity?
It is the enzyme activity that transfers a myristoyl group from tetradecanoyl-CoA to the N-terminal glycine of a peptide, as defined by GO:0004379.
What genes are involved in glycylpeptide N-tetradecanoyltransferase activity?
The main genes are NMT1 and NMT2, which encode N-myristoyltransferases, along with substrate genes like SRC, PAK2, and ARF1.
What is the reaction catalyzed by GO:0004379?
The reaction is: tetradecanoyl-CoA + glycyl-peptide = CoA + N-tetradecanoylglycyl-peptide.
How is N-myristoylation related to cancer?
NMT activity is often elevated in cancer, and its inhibition promotes degradation of oncoproteins like the androgen receptor in prostate cancer.
What are the substrates of N-myristoyltransferase?
Substrates include Src family kinases, ADP-ribosylation factors, G protein alpha subunits, and caspase-cleaved PAK2.
How can I study glycylpeptide N-tetradecanoyltransferase activity in the lab?
Common methods include radiolabeled enzyme assays, click chemistry proteomics, and CRISPR knockout models.
What is the difference between NMT1 and NMT2?
NMT1 and NMT2 are two human N-myristoyltransferases with overlapping but distinct substrate specificities and tissue distributions.
Can CRISPR be used to study N-myristoylation?
Yes, CRISPR knockout, point mutation, and knock-in models are widely used to dissect the function of NMT and its substrates.
What diseases are associated with defects in N-myristoylation?
Diseases include cancer, neurodegenerative disorders, and parasitic infections.
What inhibitors target glycylpeptide N-tetradecanoyltransferase activity?
Several NMT inhibitors, such as myristic acid analogs and small molecules, are in preclinical development for cancer and parasitic diseases.
Conclusion
GO:0004379, glycylpeptide N-tetradecanoyltransferase activity, is a central enzymatic function in protein N-myristoylation, impacting signal transduction, apoptosis, and disease. Its study offers insights into fundamental cell biology and provides therapeutic opportunities, particularly in cancer and infectious diseases. CRISPR-based models are invaluable for dissecting the roles of NMT and its substrates, and EDITGENE provides the tools to accelerate this research.
References
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- 3. Boutin JA. 1997. Myristoylation.. Cell Signal 9(1):15-35 PMID: 9067626
- 4. Gamerdinger M et al.. 2025. Mechanism of cotranslational protein N-myristoylation in human cells.. Mol Cell 85(14):2749-2758.e8 PMID: 40639378
- 5. Glover CJ et al.. 1988. N-myristoylation of p60src. Identification of a myristoyl-CoA:glycylpeptide N-myristoyltransferase in rat tissues.. Biochem J 250(2):485-91 PMID: 3128285
- 6. Alsaidan OA et al.. 2024. Inhibition of N-myristoyltransferase activity promotes androgen receptor degradation in prostate cancer.. Prostate 84(3):254-268 PMID: 37905842
- 7. Vilas GL et al.. 2006. Posttranslational myristoylation of caspase-activated p21-activated protein kinase 2 (PAK2) potentiates late apoptotic events.. Proc Natl Acad Sci U S A 103(17):6542-7 PMID: 16617111
- 8. Yao S et al.. 2026. N-myristoyltransferases: structure, function, disease, and inhibitors.. Drug Discov Today 31(3):104657 PMID: 41932429
