GO:0047444 N-acylneuraminate-9-phosphate synthase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0047444 describes the enzyme activity that converts N-acyl-D-mannosamine 6-phosphate and phosphoenolpyruvate into N-acylneuraminate 9-phosphate and phosphate, a key step in sialic acid biosynthesis.
• The human enzyme is encoded by NANS (also known as N-acetylneuraminic acid phosphate synthase), and its activity is essential for producing N-acetylneuraminic acid (Neu5Ac), the most common sialic acid in humans.
• NANS can also synthesize 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid 9-phosphate (KDN-9-P) in some species, but the mouse enzyme lacks this activity.
• A single mutation in human NANS can eliminate KDN-9-P synthase activity while retaining Neu5Ac-9-P synthase activity, highlighting distinct catalytic determinants.
• Biallelic mutations in NANS cause a severe neurodevelopmental disorder with brain and skeletal abnormalities, demonstrating the critical role of this activity in human development.
• Studying GO:0047444 requires a combination of enzymatic assays, CRISPR knockout/knock-in models, and analytical methods such as mass spectrometry and lectin staining.
Description
N-acylneuraminate-9-phosphate synthase activity (GO:0047444) is a molecular function that catalyzes the formation of N-acylneuraminate 9-phosphate from N-acyl-D-mannosamine 6-phosphate and phosphoenolpyruvate, releasing phosphate. This activity is a critical step in the de novo biosynthesis of sialic acids, a family of nine-carbon acidic sugars that decorate cell surface glycoproteins and glycolipids and play roles in cell recognition, signaling, and immune regulation. In humans, the enzyme responsible for this activity is encoded by the NANS gene, and its product is essential for producing N-acetylneuraminic acid (Neu5Ac), the predominant sialic acid in humans. Researchers study GO:0047444 to understand sialic acid biology, to dissect the catalytic mechanism of the enzyme, and to explore its involvement in developmental disorders and potential therapeutic applications.
N-acylneuraminate-9-phosphate synthase activity At A Glance
| GO ID | GO:0047444 |
|---|---|
| GO term | N-acylneuraminate-9-phosphate synthase activity |
| Ontology | molecular_function |
| Synonym | N-acetylneuraminate 9-phosphate synthase activity; sialic acid 9-phosphate synthetase activity; N-acetylneuraminic acid phosphate synthase activity |
| Major function | Catalyzes the synthesis of N-acylneuraminate 9-phosphate from N-acyl-D-mannosamine 6-phosphate and phosphoenolpyruvate |
| Reaction | H2O + phosphoenolpyruvate + N-acyl-D-mannosamine 6-phosphate = phosphate + N-acylneuraminate 9-phosphate |
| Human gene | NANS (N-acetylneuraminic acid synthase) |
| Pathway | Sialic acid biosynthesis (de novo pathway) |
| Disease association | NANS mutations cause severe neurodevelopmental disorder with brain and skeletal abnormalities |
What Is GO:0047444?
According to the Gene Ontology, GO:0047444 is defined as the catalysis of the reaction: H2O + phosphoenolpyruvate + N-acyl-D-mannosamine 6-phosphate = phosphate + N-acylneuraminate 9-phosphate. In simpler terms, it is an enzyme activity that uses phosphoenolpyruvate as a donor to add a three-carbon pyruvate moiety to N-acyl-D-mannosamine 6-phosphate, forming N-acylneuraminate 9-phosphate and releasing phosphate. This activity is synonymous with N-acetylneuraminate 9-phosphate synthase, sialic acid 9-phosphate synthetase, and related names.
Why Is N-acylneuraminate-9-phosphate synthase activity Important in Cell Biology?
GO:0047444 is essential for the biosynthesis of sialic acids, which are critical for many biological processes including cell-cell communication, immune response, and brain development. The enzyme activity is highly conserved across species, and its dysfunction leads to severe developmental defects in humans. Understanding this activity at the molecular level can inform therapeutic strategies for sialic acid-related disorders and provide insights into the evolution of sialic acid diversity.
• Sialic acids are essential components of glycoproteins and glycolipids involved in cell recognition and signaling.
• NANS-mediated synthesis of sialic acid is required for normal brain and skeletal development in humans.
• The enzyme activity is a target for understanding the evolution of sialic acid diversity, including Neu5Ac and KDN.
• Defects in sialic acid biosynthesis are linked to developmental disorders and may contribute to cancer progression.
• The activity can be studied using enzymatic assays, CRISPR models, and analytical techniques like mass spectrometry.
• Plant engineering with this activity enables production of CMP-sialic acid in Arabidopsis, demonstrating biotechnological potential.
• Mouse models lacking this activity show embryonic lethality, underscoring its importance.
• The enzyme's ability to utilize different N-acyl substrates has implications for understanding substrate specificity.
What Happens During N-acylneuraminate-9-phosphate synthase activity?
Substrate Binding and Catalysis
In simple terms: The enzyme grabs two molecules and joins them together.
The enzyme binds N-acyl-D-mannosamine 6-phosphate and phosphoenolpyruvate in its active site. The reaction proceeds through a mechanism where the enolpyruvate moiety of phosphoenolpyruvate is transferred to the sugar phosphate, forming a new carbon-carbon bond and releasing phosphate. This step is essential for generating the nine-carbon backbone of sialic acids.
Product Formation and Release
In simple terms: The enzyme releases the newly made molecule.
The product, N-acylneuraminate 9-phosphate, is released from the active site. This molecule is then dephosphorylated by a specific phosphatase to yield free N-acylneuraminic acid (e.g., Neu5Ac), which can be activated to CMP-sialic acid for incorporation into glycoconjugates.
Substrate Specificity and Isoforms
In simple terms: Different versions of the enzyme can make different sialic acids.
The human enzyme can utilize both N-acetyl-D-mannosamine 6-phosphate and N-glycolyl-D-mannosamine 6-phosphate, leading to Neu5Ac and Neu5Gc, respectively. It can also synthesize KDN-9-P from mannose 6-phosphate in some species, but the mouse enzyme lacks this activity. A single mutation in human NANS can abolish KDN-9-P synthase activity without affecting Neu5Ac-9-P synthase activity, indicating distinct catalytic requirements.
Role in Sialic Acid Pathway
In simple terms: This is a key step in making sialic acid, which cells use for many functions.
The activity is part of the de novo sialic acid biosynthesis pathway. Following this step, the product is dephosphorylated and converted to CMP-sialic acid, which is the donor substrate for sialyltransferases that modify glycoproteins and glycolipids. This pathway is essential for providing sialic acids for cell surface decoration and signaling.
Key Genes Involved in GO:0047444 N-acylneuraminate-9-phosphate synthase activity
The following genes and proteins are directly involved in or regulate N-acylneuraminate-9-phosphate synthase activity and related sialic acid biosynthesis.
| Gene | Major Role | Research Relevance |
|---|---|---|
| NANS | Encodes the enzyme with N-acylneuraminate-9-phosphate synthase activity | Mutations cause severe neurodevelopmental disorder; target for functional studies |
| NANP | Encodes N-acylneuraminate-9-phosphatase, which dephosphorylates the product | Essential for completing sialic acid biosynthesis |
| GNE | Encodes UDP-GlcNAc 2-epimerase/ManNAc kinase, upstream of NANS | Mutations cause sialuria and hereditary inclusion body myopathy |
| CMAS | Encodes CMP-sialic acid synthetase, activating sialic acid | Required for sialylation of glycoconjugates |
| SLC35A1 | Encodes CMP-sialic acid transporter | Defects cause sialic acid transporter deficiency |
| ST3GAL1 | Sialyltransferase that adds sialic acid to glycoproteins | Downstream effector of sialic acid function |
| ST6GAL1 | Sialyltransferase that adds sialic acid to glycoproteins | Downstream effector of sialic acid function |
| NEU1 | Sialidase that removes sialic acid | Defects cause sialidosis |
| NEU2 | Cytosolic sialidase | Involved in sialic acid catabolism |
| NEU3 | Plasma membrane sialidase | Modulates cell signaling |
| NEU4 | Lysosomal/mitochondrial sialidase | Involved in ganglioside catabolism |
| KDNase | KDN aldolase, involved in KDN catabolism | Relevant for KDN biology |
| SLC17A5 | Sialin, transports sialic acid | Defects cause Salla disease |
| GFPT1 | Glutamine-fructose-6-phosphate transaminase, upstream of sialic acid pathway | Regulates flux into hexosamine pathway |
| PGM3 | Phosphoglucomutase 3, involved in sugar phosphate metabolism | Mutations cause immunodeficiency |
| UAP1 | UDP-N-acetylglucosamine pyrophosphorylase, upstream | Provides substrate for sialic acid pathway |
| NPL | N-acetylneuraminate pyruvate lyase, involved in sialic acid catabolism | Regulates sialic acid levels |
How Is N-acylneuraminate-9-phosphate synthase activity Regulated?
The expression and activity of N-acylneuraminate-9-phosphate synthase are regulated at multiple levels. Transcription of the NANS gene can be influenced by developmental cues and metabolic status, as suggested by its essential role in brain and skeletal development. The enzyme's activity may also be modulated by substrate availability, particularly the levels of N-acyl-D-mannosamine 6-phosphate, which are controlled by upstream enzymes such as GNE. Additionally, post-translational modifications or interactions with other proteins could affect its function, though specific regulatory mechanisms remain to be fully elucidated.
N-acylneuraminate-9-phosphate synthase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| NANS | Neurodevelopmental disorder with brain and skeletal abnormalities | Patient-derived iPSCs or NANS knockout mice |
| GNE | Sialuria, hereditary inclusion body myopathy | GNE knockout or knock-in cell lines |
| CMAS | Defects in sialylation | CMAS knockout cells |
| SLC35A1 | Sialic acid transporter deficiency | SLC35A1 knockout cells |
| NEU1 | Sialidosis | NEU1 knockout mice |
NANS Deficiency and Neurodevelopmental Disorder
Biallelic mutations in NANS cause a severe neurodevelopmental disorder characterized by developmental delay, intellectual disability, and skeletal abnormalities. This condition, sometimes referred to as NANS-CDG, results from impaired sialic acid synthesis, highlighting the critical role of GO:0047444 in human development.
Sialic Acid Metabolism in Cancer
Altered sialylation is a hallmark of cancer, and changes in sialic acid biosynthesis enzymes, including NANS, have been observed in various malignancies. Hypersialylation can promote tumor cell survival, immune evasion, and metastasis, making the pathway a potential therapeutic target.
Sialic Acid in Immune Regulation
Sialic acids are involved in immune cell recognition and signaling. Defects in sialic acid biosynthesis can lead to immune dysregulation, and pathogens often exploit sialic acids for entry and immune evasion. Understanding GO:0047444 helps clarify these processes.
From N-acylneuraminate-9-phosphate synthase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| What is the effect of NANS loss on sialic acid levels? | NANS knockout cell lines (e.g., HEK293) |
| How does a specific point mutation affect enzyme activity? | Point-mutant NANS knock-in cells |
| Can wild-type NANS rescue developmental defects? | Knock-in mice expressing human NANS |
| Where is NANS localized in the cell? | Tagged knock-in of NANS with fluorescent protein |
| What is the impact of NANS overexpression on sialylation? | NANS overexpression cell lines |
| Can plant cells produce sialic acid with NANS? | Arabidopsis expressing NANS and other pathway genes |
How to Study the N-acylneuraminate-9-phosphate synthase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Enzymatic assay | N-acylneuraminate-9-phosphate synthase activity | Characterization of wild-type and mutant enzymes |
| Mass spectrometry | Sialic acid species and quantity | Global sialylation analysis |
| Lectin staining | Cell surface sialylation | Flow cytometry or microscopy |
| CRISPR-Cas9 knockout | Loss of gene function | Studying effects of NANS deletion |
| CRISPR-Cas9 knock-in | Introduction of specific mutations | Modeling patient mutations |
| RNA-seq | Gene expression changes | Transcriptional response to NANS loss |
| Western blot | Protein expression levels | Validation of knockout or overexpression |
| Immunofluorescence | Subcellular localization | Determining organelle distribution |
Enzymatic Activity Assays
Direct measurement of N-acylneuraminate-9-phosphate synthase activity can be performed using radiolabeled substrates or by coupling the reaction to downstream enzymes and detecting products via colorimetric or fluorometric methods. These assays are essential for characterizing wild-type and mutant enzymes.
Mass Spectrometry and Glycan Analysis
Mass spectrometry-based glycomics can quantify sialic acid species and their linkages on glycoproteins and glycolipids. This approach is powerful for assessing the impact of NANS mutations or knockout on global sialylation patterns.
Lectin Staining and Flow Cytometry
Sialic acid-specific lectins, such as Sambucus nigra agglutinin (SNA) and Maackia amurensis lectin (MAL), can be used to detect cell surface sialylation by flow cytometry or microscopy. This provides a rapid readout of sialic acid levels in cells with modified NANS activity.
CRISPR-Cas9 Genome Editing
CRISPR-Cas9 can be used to generate NANS knockout, knock-in, or point-mutant cell lines and animal models. These models are invaluable for studying the physiological consequences of altered N-acylneuraminate-9-phosphate synthase activity.
How CRISPR Can Be Used to Study GO:0047444 N-acylneuraminate-9-phosphate synthase activity
Knockout
CRISPR-Cas9 knockout of NANS in cell lines such as HEK293 or HeLa results in loss of N-acylneuraminate-9-phosphate synthase activity, leading to reduced sialic acid levels and altered cell surface sialylation. These models are useful for studying the consequences of sialic acid deficiency and for validating the role of the enzyme in cellular processes.
Point Mutation
Introducing specific point mutations into the NANS gene via CRISPR-Cas9 homology-directed repair allows researchers to dissect the catalytic mechanism and substrate specificity. For example, a single mutation can eliminate KDN-9-P synthase activity while preserving Neu5Ac-9-P synthase activity, providing insights into distinct catalytic requirements.
Knock-in
Knock-in of tagged versions of NANS (e.g., GFP or FLAG) enables visualization and purification of the enzyme for biochemical studies. Additionally, knock-in of patient-specific mutations can model NANS deficiency in cell lines or animal models, facilitating the study of disease mechanisms and potential therapies.
Overexpression
Overexpression of wild-type or mutant NANS in cells can increase sialic acid production and alter sialylation patterns. This approach is useful for producing sialylated glycoproteins for biotechnological applications and for studying the effects of elevated sialic acid levels on cell behavior.
How EDITGENE Supports N-acylneuraminate-9-phosphate synthase activity Research
Researchers studying N-acylneuraminate-9-phosphate synthase activity-related genes often need to determine whether a candidate gene is causally involved in sialic acid biosynthesis, developmental disorders, or cancer. EDITGENE provides comprehensive CRISPR-based services to generate precisely engineered cell and animal models, enabling functional validation and mechanistic studies.
Contact EDITGENE today to design your custom CRISPR model for N-acylneuraminate-9-phosphate synthase activity research.
Frequently Asked Questions About N-acylneuraminate-9-phosphate synthase activity
What is N-acylneuraminate-9-phosphate synthase activity?
It is an enzyme activity (GO:0047444) that catalyzes the formation of N-acylneuraminate 9-phosphate from N-acyl-D-mannosamine 6-phosphate and phosphoenolpyruvate, a key step in sialic acid biosynthesis.
What genes are involved in N-acylneuraminate-9-phosphate synthase activity?
The primary gene is NANS, which encodes the enzyme in humans. Other genes in the pathway include GNE, NANP, CMAS, and sialyltransferases.
What diseases are associated with NANS mutations?
Biallelic mutations in NANS cause a severe neurodevelopmental disorder with brain and skeletal abnormalities, sometimes called NANS-CDG.
How is N-acylneuraminate-9-phosphate synthase activity measured?
It can be measured using enzymatic assays with radiolabeled substrates or coupled reactions, as well as by assessing downstream sialic acid levels via mass spectrometry or lectin staining.
Can NANS synthesize KDN-9-P?
Yes, the human enzyme can synthesize KDN-9-P from mannose 6-phosphate, but the mouse enzyme lacks this activity. A single mutation can eliminate KDN-9-P synthase activity without affecting Neu5Ac-9-P synthase activity.
What is the role of sialic acid in the body?
Sialic acids are essential for cell-cell communication, immune regulation, and brain development. They decorate cell surface glycoproteins and glycolipids.
How can CRISPR be used to study NANS?
CRISPR-Cas9 can create NANS knockout, knock-in, or point-mutant cell lines and animal models to study the effects of altered enzyme activity on sialylation and development.
Is N-acylneuraminate-9-phosphate synthase activity conserved across species?
Yes, the activity is found in bacteria, animals, and plants, though substrate specificity and regulation may differ.
What are the synonyms for N-acylneuraminate-9-phosphate synthase activity?
Synonyms include N-acetylneuraminate 9-phosphate synthase activity, sialic acid 9-phosphate synthetase activity, and N-acetylneuraminic acid phosphate synthase activity.
What model systems are used to study NANS deficiency?
Patient-derived iPSCs, NANS knockout mice, and cell lines with patient mutations are commonly used to model NANS deficiency and study disease mechanisms.
Conclusion
N-acylneuraminate-9-phosphate synthase activity (GO:0047444) is a fundamental enzymatic step in sialic acid biosynthesis, with critical roles in human development and disease. The NANS gene encoding this activity is essential for brain and skeletal development, and its dysfunction leads to severe neurodevelopmental disorders. Ongoing research using CRISPR models and advanced analytical techniques continues to unravel the molecular details of this activity and its broader implications in health and disease.
References
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- 2. Nakata D et al.. 2000. Molecular cloning and expression of the mouse N-acetylneuraminic acid 9-phosphate synthase which does not have deaminoneuraminic acid (KDN) 9-phosphate synthase activity.. Biochem Biophys Res Commun 273(2):642-8 PMID: 10873658
- 3. Chen H et al.. 2002. Purification and characterization of N-acetylneuraminic acid-9-phosphate synthase from rat liver.. Glycobiology 12(2):65-71 PMID: 11886839
- 4. Reaves ML et al.. 2008. Replacement of the antifreeze-like domain of human N-acetylneuraminic acid phosphate synthase with the mouse antifreeze-like domain impacts both N-acetylneuraminic acid 9-phosphate synthase and 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid 9-phosphate synthase activities.. BMB Rep 41(1):72-8 PMID: 18304454
- 5. Lawrence SM et al.. 2000. Cloning and expression of the human N-acetylneuraminic acid phosphate synthase gene with 2-keto-3-deoxy-D-glycero- D-galacto-nononic acid biosynthetic ability.. J Biol Chem 275(23):17869-77 PMID: 10749855
- 6. Castilho A et al.. 2008. Construction of a functional CMP-sialic acid biosynthesis pathway in Arabidopsis.. Plant Physiol 147(1):331-9 PMID: 18326787
- 7. van Karnebeek CD et al.. 2016. NANS-mediated synthesis of sialic acid is required for brain and skeletal development.. Nat Genet 48(7):777-84 PMID: 27213289
