GO:0003952 NAD+ synthase (glutamine-hydrolyzing) activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0003952 describes the enzymatic activity that converts deamido-NAD+ into NAD+ using L-glutamine, ATP, and water, releasing L-glutamate, AMP, and diphosphate.
• The reaction is a two-step process: glutamine hydrolysis provides ammonia, which is then used to amidate deamido-NAD+ to form NAD+.
• In Mycobacterium tuberculosis, the glutamine amidotransferase activity of NAD+ synthetase depends on an amino-terminal nitrilase domain, linking the enzyme to a broader family of adenine nucleotide alpha hydrolases.
• NAD+ is essential for redox reactions, DNA repair, and signaling, making this activity critical for cellular metabolism and survival.
• Dysregulation of NAD+ synthesis has been implicated in metabolic disorders, cancer, and infectious diseases, highlighting its therapeutic potential.
• Researchers study this activity using structural biology, enzyme kinetics, and CRISPR-based gene editing to dissect its role in health and disease.
Description
NAD+ synthase (glutamine-hydrolyzing) activity, encoded by GO:0003952, is a molecular function that catalyzes the final step of NAD+ biosynthesis from deamido-NAD+. This enzyme uses L-glutamine as an ammonia donor and ATP as an energy source to produce NAD+, a coenzyme central to cellular redox reactions and signaling pathways. The activity is found in bacteria, archaea, and eukaryotes, and its mechanism has been studied in detail, particularly in Mycobacterium tuberculosis. Understanding this activity is crucial because NAD+ levels influence metabolism, DNA repair, and cell survival, and its disruption is linked to various diseases. The enzyme belongs to the adenine nucleotide alpha hydrolase family, as suggested by structural similarities with phosphoadenylyl sulphate (PAPS) reductase. This article explores the mechanism, genes, and research methods associated with GO:0003952, providing a comprehensive resource for researchers.
NAD+ synthase (glutamine-hydrolyzing) activity At A Glance
| GO ID | GO:0003952 |
|---|---|
| GO term | NAD+ synthase (glutamine-hydrolyzing) activity |
| Ontology | molecular_function |
| Synonym | deamido-NAD+:L-glutamine amido-ligase (AMP-forming); desamidonicotinamide adenine dinucleotide amidotransferase activity; DPN synthetase activity; NAD+ synthase (glutamine-hydrolysing); NAD synthase (glutamine-hydrolyzing) activity; NAD synthetase (glutamine-hydrolysing); NAD+ synthetase (glutamine-hydrolyzing); NAD(+) synthetase (glutamine-hydrolyzing) activity; nicotinamide adenine dinucleotide synthetase (glutamine) activity |
| Major function | Catalyzes the final step of NAD+ biosynthesis from deamido-NAD+ using glutamine as an ammonia donor. |
| Reaction | deamido-NAD+ + L-glutamine + ATP + H2O = L-glutamate + AMP + diphosphate + NAD+ + H+. |
| Cofactors | ATP, Mg2+ (implied by ATP-dependent amidotransferase mechanism). |
| Subcellular location | Cytoplasm (typical for NAD+ biosynthesis enzymes). |
| Related genes | nadE in bacteria; NAD synthetase in eukaryotes. |
What Is GO:0003952?
GO:0003952 describes the catalysis of the reaction: deamido-NAD+ + L-glutamine + ATP + H2O = L-glutamate + AMP + diphosphate + NAD+ + H+. In simpler terms, it is the enzyme activity that uses glutamine to convert deamido-NAD+ into NAD+, consuming ATP and water and releasing glutamate, AMP, and diphosphate.
Why Is NAD+ synthase (glutamine-hydrolyzing) activity Important in Cell Biology?
NAD+ synthase (glutamine-hydrolyzing) activity is essential because it produces NAD+, a coenzyme required for redox reactions in glycolysis, the TCA cycle, and oxidative phosphorylation, as well as for signaling and DNA repair. In pathogens like Mycobacterium tuberculosis, this activity is critical for survival and represents a potential drug target. In humans, NAD+ decline is associated with aging and metabolic disorders, making this enzyme a focus for therapeutic interventions.
• Provides NAD+ for cellular energy metabolism and redox homeostasis.
• Supports DNA repair and signaling pathways that depend on NAD+.
• Essential for survival of Mycobacterium tuberculosis, suggesting a target for anti-tuberculosis drugs.
• Linked to aging and age-related diseases due to NAD+ decline.
• Involved in sirtuin-mediated deacetylation and cell survival.
• Potential role in cancer metabolism and proliferation.
• Enzyme family includes adenine nucleotide alpha hydrolases, with structural insights from PAPS reductase.
• Regulation of NAD+ synthesis impacts circadian rhythms and stress responses.
• Bacterial NAD+ synthetase is a validated antibiotic target.
• Enzyme kinetics and structural studies inform inhibitor design.
What Happens During NAD+ synthase (glutamine-hydrolyzing) activity?
Glutamine Hydrolysis and Ammonia Transfer
In simple terms: The enzyme first breaks down glutamine to release ammonia.
The enzyme catalyzes the hydrolysis of L-glutamine to L-glutamate and ammonia. This step is carried out by a glutamine amidotransferase domain, which in Mycobacterium tuberculosis NAD+ synthetase depends on an amino-terminal nitrilase domain. The ammonia is then channeled to the active site for the second step.
Amidation of Deamido-NAD+
In simple terms: The ammonia is attached to deamido-NAD+ to make NAD+.
The ammonia generated from glutamine is used to amidate deamido-NAD+, converting it to NAD+. This step requires ATP, which is hydrolyzed to AMP and diphosphate, providing energy for the reaction. The overall reaction is: deamido-NAD+ + L-glutamine + ATP + H2O = L-glutamate + AMP + diphosphate + NAD+ + H+.
Structural Insights from Related Enzymes
In simple terms: Similar enzymes help us understand how this one works.
The crystal structure of phosphoadenylyl sulphate (PAPS) reductase, a member of the adenine nucleotide alpha hydrolase family, provides a structural framework for understanding NAD+ synthetase. This family shares a common fold and catalytic mechanism involving nucleotide binding and hydrolysis.
Key Genes Involved in GO:0003952 NAD+ synthase (glutamine-hydrolyzing) activity
The following genes and proteins are directly or indirectly associated with NAD+ synthase (glutamine-hydrolyzing) activity.
| Gene | Major Role | Research Relevance |
|---|---|---|
| nadE (Mycobacterium tuberculosis) | Encodes NAD+ synthetase; catalyzes final step of NAD+ biosynthesis | Target for anti-tuberculosis drug development |
| NADSYN1 (human) | Encodes NAD+ synthetase; catalyzes final step of NAD+ biosynthesis | Linked to NAD+ homeostasis and metabolic disorders |
| PAPS reductase (related family) | Member of adenine nucleotide alpha hydrolase family; structural homolog | Provides structural insights into NAD+ synthetase mechanism |
| GlnA (glutamine synthetase) | Provides glutamine for NAD+ synthesis | Indirectly supports NAD+ synthase activity |
| NadD (NAD+ kinase) | Phosphorylates NAD+ to NADP+ | Balances NAD+ and NADP+ pools |
| NadB (L-aspartate oxidase) | Involved in de novo NAD+ biosynthesis | Upstream of NAD+ synthase |
| NadC (quinolinate phosphoribosyltransferase) | Involved in de novo NAD+ biosynthesis | Upstream of NAD+ synthase |
| NadA (quinolinate synthase) | Involved in de novo NAD+ biosynthesis | Upstream of NAD+ synthase |
| NadR (transcriptional regulator) | Regulates NAD+ biosynthesis genes | Controls expression of nadE |
| SIRT1 (human) | NAD+-dependent deacetylase | Consumes NAD+ produced by NAD+ synthase |
| PARP1 (human) | NAD+-dependent DNA repair enzyme | Consumes NAD+ produced by NAD+ synthase |
| CD38 (human) | NAD+ glycohydrolase | Regulates NAD+ levels |
| NMNAT (human) | Nicotinamide mononucleotide adenylyltransferase | Produces deamido-NAD+ precursor |
| NAMPT (human) | Nicotinamide phosphoribosyltransferase | Rate-limiting in NAD+ salvage pathway |
| QNS1 (yeast) | NAD+ synthetase in Saccharomyces cerevisiae | Model for eukaryotic NAD+ synthesis |
| NadE (Escherichia coli) | NAD+ synthetase | Bacterial model for enzyme studies |
| NadE (Bacillus subtilis) | NAD+ synthetase | Gram-positive model |
| NadE (Salmonella typhimurium) | NAD+ synthetase | Pathogen model |
How Is NAD+ synthase (glutamine-hydrolyzing) activity Regulated?
The activity of NAD+ synthase (glutamine-hydrolyzing) is regulated at multiple levels. In bacteria, the expression of nadE is controlled by the transcriptional regulator NadR, which responds to NAD+ levels. In eukaryotes, NAD+ biosynthesis is regulated by feedback inhibition and substrate availability. Additionally, the enzyme's activity may be influenced by post-translational modifications and cellular energy status, as ATP is a substrate.
NAD+ synthase (glutamine-hydrolyzing) activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| nadE (M. tuberculosis) | Tuberculosis | Knockout in M. tuberculosis; mouse infection model |
| NADSYN1 (human) | Metabolic disorders, aging | Knockout in human cell lines; mouse models |
| NADSYN1 (human) | Cancer | Overexpression in cancer cell lines; xenograft models |
| NadE (E. coli) | Bacterial infections | Knockout in E. coli; antibiotic screening |
| QNS1 (yeast) | NAD+ homeostasis | Yeast knockout; complementation assays |
Tuberculosis
Mycobacterium tuberculosis NAD+ synthetase is essential for the pathogen's survival, as it catalyzes the final step of NAD+ biosynthesis. The glutamine amidotransferase activity depends on an amino-terminal nitrilase domain, making it a promising target for anti-tuberculosis drugs.
Metabolic Disorders
In humans, NAD+ levels decline with age and in metabolic disorders such as obesity and diabetes. NAD+ synthase (glutamine-hydrolyzing) activity contributes to maintaining NAD+ pools, and its dysregulation may exacerbate these conditions.
Cancer
Cancer cells often have elevated NAD+ metabolism to support rapid proliferation and DNA repair. NAD+ synthase activity may be upregulated in some cancers, providing a potential target for therapeutic intervention.
From NAD+ synthase (glutamine-hydrolyzing) activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Is nadE essential for M. tuberculosis survival? | Knockout in M. tuberculosis |
| What is the role of the nitrilase domain in glutamine amidotransferase activity? | Point mutations in nadE |
| Can human NADSYN1 rescue yeast qns1 mutants? | Knock-in of human NADSYN1 into yeast |
| How does NAD+ synthase overexpression affect cancer cell proliferation? | Overexpression in cancer cell lines |
| What is the effect of NAD+ synthase knockout on NAD+ levels? | Knockout in human cell lines |
| Can small molecules inhibit M. tuberculosis NAD+ synthetase? | Enzyme assays with purified protein |
How to Study the NAD+ synthase (glutamine-hydrolyzing) activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Enzyme kinetics | Catalytic rate, substrate affinity | Inhibitor screening |
| X-ray crystallography | Three-dimensional structure | Active site mapping |
| CRISPR knockout | Gene essentiality | Target validation |
| Metabolomics | NAD+ levels | Pathway analysis |
| qPCR | Gene expression | Regulation studies |
| Western blot | Protein levels | Expression analysis |
| Complementation assays | Functional rescue | Gene function conservation |
Enzyme Kinetics
Enzyme kinetics assays measure the rate of NAD+ synthesis by monitoring absorbance at 340 nm or using coupled reactions. These assays help determine Km, Vmax, and inhibitor efficacy.
Structural Biology
X-ray crystallography and cryo-EM can resolve the structure of NAD+ synthetase, revealing the active site and domain organization. The structure of PAPS reductase, a related enzyme, provides a template for homology modeling.
Genetic Knockouts
CRISPR-Cas9 knockout of nadE or NADSYN1 in bacterial or human cells can reveal essentiality and metabolic consequences. Knockout strains are viable only if supplemented with NAD+ or its precursors.
Metabolomics
Mass spectrometry-based metabolomics quantifies NAD+ and related metabolites in cells or tissues, providing direct evidence of enzyme activity in vivo.
How CRISPR Can Be Used to Study GO:0003952 NAD+ synthase (glutamine-hydrolyzing) activity
Knockout
CRISPR-Cas9 knockout of nadE in Mycobacterium tuberculosis or NADSYN1 in human cells can determine gene essentiality. Knockout cells may require exogenous NAD+ for survival, confirming the enzyme's role in NAD+ biosynthesis.
Point Mutation
Point mutations in the nitrilase domain of nadE can abolish glutamine amidotransferase activity, helping to map functional residues. Such mutants are valuable for studying the enzyme's catalytic mechanism.
Knock-in
Knock-in of human NADSYN1 into yeast qns1 mutants can test functional conservation. This approach can also be used to introduce disease-associated mutations for study.
Overexpression
Overexpression of NAD+ synthase in cancer cell lines can increase NAD+ levels and promote proliferation, providing a model to study the enzyme's role in cancer metabolism.
How EDITGENE Supports NAD+ synthase (glutamine-hydrolyzing) activity Research
Researchers studying NAD+ synthase (glutamine-hydrolyzing) activity-related genes often need to determine whether a candidate gene is causally involved in NAD+ homeostasis, metabolic regulation, or disease. EDITGENE provides a comprehensive suite of CRISPR-based services to accelerate this research.
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Frequently Asked Questions About NAD+ synthase (glutamine-hydrolyzing) activity
What is NAD+ synthase (glutamine-hydrolyzing) activity?
It is the enzyme activity that catalyzes the final step of NAD+ biosynthesis, converting deamido-NAD+ to NAD+ using glutamine, ATP, and water.
What genes are involved in NAD+ synthase (glutamine-hydrolyzing) activity?
The primary gene is nadE in bacteria and NADSYN1 in humans, which encode the enzyme NAD+ synthetase.
What is the reaction catalyzed by NAD+ synthase (glutamine-hydrolyzing)?
The reaction is: deamido-NAD+ + L-glutamine + ATP + H2O = L-glutamate + AMP + diphosphate + NAD+ + H+.
Why is NAD+ synthase important for tuberculosis?
Mycobacterium tuberculosis NAD+ synthetase is essential for NAD+ biosynthesis and survival, making it a drug target.
How is NAD+ synthase (glutamine-hydrolyzing) activity regulated?
In bacteria, nadE expression is controlled by NadR; in eukaryotes, it is regulated by feedback inhibition and substrate availability.
What diseases are linked to NAD+ synthase dysfunction?
Dysregulation is associated with tuberculosis, metabolic disorders, aging, and cancer.
What methods are used to study NAD+ synthase activity?
Enzyme kinetics, structural biology, CRISPR knockouts, and metabolomics are commonly used.
Can CRISPR be used to study NAD+ synthase?
Yes, CRISPR knockout, point mutation, knock-in, and overexpression models are powerful tools to study its function.
What is the structural basis of NAD+ synthase activity?
The enzyme belongs to the adenine nucleotide alpha hydrolase family, with structural insights from PAPS reductase.
How does NAD+ synthase contribute to NAD+ homeostasis?
It catalyzes the final step of NAD+ biosynthesis, maintaining cellular NAD+ levels for redox reactions and signaling.
Conclusion
NAD+ synthase (glutamine-hydrolyzing) activity, defined by GO:0003952, is a critical enzymatic function in NAD+ biosynthesis. Its mechanism, involving glutamine hydrolysis and amidation of deamido-NAD+, is conserved across species and essential for cellular metabolism. The enzyme is a validated drug target in Mycobacterium tuberculosis and is implicated in metabolic disorders and cancer. Advanced research tools, including CRISPR-based gene editing and structural biology, continue to unravel its roles, offering opportunities for therapeutic development.
References
- 1. Bellinzoni M et al.. 2005. Glutamine amidotransferase activity of NAD+ synthetase from Mycobacterium tuberculosis depends on an amino-terminal nitrilase domain.. Res Microbiol 156(2):173-7 PMID: 15748981
- 2. Savage H et al.. 1997. Crystal structure of phosphoadenylyl sulphate (PAPS) reductase: a new family of adenine nucleotide alpha hydrolases.. Structure 5(7):895-906 PMID: 9261082
