GO:0047131 saccharopine dehydrogenase (NAD+, L-glutamate-forming) activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0047131 describes the molecular function of saccharopine dehydrogenase (NAD+, L-glutamate-forming) activity, which catalyzes the reversible NAD+-dependent oxidative deamination of L-saccharopine to L-glutamate, allysine, H+ and NADH.
• This activity is a key component of the saccharopine pathway, the major route for lysine degradation in mammalian mitochondria and for lysine biosynthesis in some organisms.
• The enzyme belongs to the family of NAD-dependent amine dehydrogenases, as revealed by the first crystal structure of a related L-lysine 6-dehydrogenase.
• In mammals, the activity is part of the bifunctional enzyme aminoadipic semialdehyde synthase (AASS), which also harbors saccharopine dehydrogenase (NAD+, L-lysine-forming) activity.
• Defects in the saccharopine pathway are linked to metabolic disorders such as hyperlysinemia and saccharopinuria, and to neurological phenotypes.
• Studying GO:0047131 requires careful enzyme assays, often coupled to NADH detection, and can be optimized using continuous spectrophotometric methods.
Description
Saccharopine dehydrogenase (NAD+, L-glutamate-forming) activity, classified under GO:0047131, is a molecular function that catalyzes the reaction: L-saccharopine + H2O + NAD+ = L-glutamate + allysine + H+ + NADH. This activity is central to the saccharopine pathway, which in mammals operates in the mitochondrial matrix and represents the principal route for lysine catabolism. The enzyme is an NAD-dependent amine dehydrogenase, and its catalytic mechanism involves hydride transfer from the substrate to NAD+, forming NADH and releasing allysine and glutamate. The saccharopine pathway is not only important for lysine degradation but also for providing precursors for energy metabolism and for maintaining cellular redox balance. In microorganisms and plants, a related enzyme activity participates in lysine biosynthesis, highlighting the evolutionary conservation of this molecular function. The dual role of the pathway in catabolism and biosynthesis makes it a subject of intense research interest. From a biomedical perspective, understanding GO:0047131 is relevant to inherited metabolic disorders such as hyperlysinemia and saccharopinuria, which result from deficiencies in saccharopine pathway enzymes. Moreover, the enzyme's dependence on NAD+ links it to cellular energy status and oxidative stress responses. Researchers studying this activity employ a range of biochemical and genetic tools, including enzyme assays and CRISPR-based models, to dissect its physiological roles.
saccharopine dehydrogenase (NAD+, L-glutamate-forming) activity At A Glance
| GO ID | GO:0047131 |
|---|---|
| GO term | saccharopine dehydrogenase (NAD+, L-glutamate-forming) activity |
| Ontology | molecular_function |
| Synonym | aminoadipic semialdehyde synthase activity; saccharopin dehydrogenase activity; NAD+ oxidoreductase (L-2-aminoadipic-delta-semialdehyde and glutamate forming) |
| Major function | Catalyzes the NAD+-dependent oxidative deamination of L-saccharopine to L-glutamate and allysine |
| Reaction | L-saccharopine + H2O + NAD+ = L-glutamate + allysine + H+ + NADH |
| Cofactor | NAD+ (nicotinamide adenine dinucleotide) |
| Pathway | Saccharopine pathway (lysine degradation/biosynthesis) |
| EC number | 1.5.1.9 (related) |
What Is GO:0047131?
GO:0047131, saccharopine dehydrogenase (NAD+, L-glutamate-forming) activity, is defined as the catalysis of the reaction: L-saccharopine + H2O + NAD+ = L-glutamate + allysine + H+ + NADH. In other words, it is an oxidoreductase that uses NAD+ as an electron acceptor to convert L-saccharopine into L-glutamate and allysine, releasing NADH and a proton. This activity is synonymous with aminoadipic semialdehyde synthase activity and several other names reflecting its role in lysine metabolism.
Why Is saccharopine dehydrogenase (NAD+, L-glutamate-forming) activity Important in Cell Biology?
GO:0047131 is important because it represents a key enzymatic step in the saccharopine pathway, which is the major route for lysine catabolism in mammals and for lysine biosynthesis in certain microorganisms and plants. The activity regulates cellular levels of lysine and its metabolites, and its dysfunction is associated with metabolic disorders such as hyperlysinemia and saccharopinuria. Additionally, the enzyme's requirement for NAD+ connects it to cellular energy metabolism and redox homeostasis, making it a potential target for understanding metabolic diseases and for biotechnological applications.
• Catalyzes a key step in the saccharopine pathway for lysine degradation in mammals.
• Contributes to lysine biosynthesis in some bacteria and plants.
• Deficiency leads to hyperlysinemia and saccharopinuria, with neurological symptoms.
• Links lysine metabolism to NAD+/NADH redox balance.
• Provides precursors for energy production via acetyl-CoA and glutamate.
• Potential target for metabolic engineering of lysine production.
• Involved in mitochondrial metabolism and ammonia detoxification.
• Relevant to rare inherited metabolic disorders and newborn screening.
• Enzyme assays for this activity are used in diagnostics and research.
• Structural studies inform inhibitor design and mechanistic understanding.
Molecular Mechanism of saccharopine dehydrogenase (NAD+, L-glutamate-forming) activity
Substrate Binding and Specificity
In simple terms: The enzyme grabs its substrate, saccharopine, and holds it in place for reaction.
The enzyme binds L-saccharopine, a intermediate of the saccharopine pathway, in a specific active site that accommodates the dicarboxypropyl-lysine moiety. Structural studies of related NAD-dependent amine dehydrogenases reveal a conserved fold that positions the substrate for hydride transfer to NAD+. The binding is stereospecific, ensuring that only L-saccharopine is converted to L-glutamate and allysine.
Catalytic Mechanism and NAD+ Reduction
In simple terms: The enzyme removes hydrogen from saccharopine and transfers it to NAD+, making NADH.
The catalytic mechanism involves the oxidation of the substrate's secondary amine, with hydride transfer to the nicotinamide ring of NAD+, forming NADH and releasing a proton. This step is analogous to other NAD-dependent amine dehydrogenases, where a conserved lysine or tyrosine residue acts as a general base. The reaction is reversible, but in vivo the equilibrium favors glutamate and allysine formation in lysine degradation.
Cofactor Requirements and Regeneration
In simple terms: The enzyme needs NAD+ to work, and NADH is produced as a result.
The activity strictly requires NAD+ as an electron acceptor; NADP+ cannot substitute. The produced NADH must be reoxidized by the respiratory chain or other dehydrogenases to maintain flux through the pathway. In vitro assays often couple NADH production to a secondary reaction for continuous monitoring.
Regulation and Post-translational Modifications
In simple terms: The enzyme's activity can be turned up or down by cellular signals.
The saccharopine dehydrogenase activity is part of a bifunctional enzyme in mammals, and its activity can be regulated by substrate availability and redox state. Although direct post-translational modifications are not well characterized, the enzyme's dependence on NAD+ links its activity to cellular energy status. In some organisms, expression of the enzyme is regulated in response to lysine levels.
Key Genes Involved in GO:0047131 saccharopine dehydrogenase (NAD+, L-glutamate-forming) activity
The following genes and proteins are directly or functionally associated with saccharopine dehydrogenase (NAD+, L-glutamate-forming) activity and the saccharopine pathway.
| Gene | Major Role | Research Relevance |
|---|---|---|
| AASS | Bifunctional enzyme with saccharopine dehydrogenase (NAD+, L-glutamate-forming) and saccharopine dehydrogenase (NAD+, L-lysine-forming) activities in mammals | Mutations cause hyperlysinemia; target for metabolic studies |
| LYS1 | Saccharopine dehydrogenase in yeast, involved in lysine biosynthesis | Model for enzyme mechanism and pathway regulation |
| LYS9 | Saccharopine dehydrogenase in yeast, catalyzes reverse reaction in lysine biosynthesis | Studied for cofactor specificity and kinetics |
| AASS (mouse) | Ortholog of human AASS, involved in lysine degradation | Knockout models for metabolic disorders |
| DHTKD1 | Component of the 2-oxoadipate dehydrogenase complex, downstream of saccharopine pathway | Linked to Charcot-Marie-Tooth disease; interacts with pathway |
| PYCR1 | Proline synthesis, indirectly linked to glutamate metabolism | Potential crosstalk with saccharopine pathway |
| GLUD1 | Glutamate dehydrogenase, balances glutamate/alpha-ketoglutarate | Affects flux through saccharopine pathway |
| SLC25A21 | Mitochondrial oxodicarboxylate carrier, transports 2-oxoadipate | Relevant to saccharopine pathway metabolite transport |
| ACADM | Medium-chain acyl-CoA dehydrogenase, fatty acid oxidation | Indirectly linked via acetyl-CoA from lysine degradation |
| HADHA | Mitochondrial trifunctional protein, fatty acid oxidation | Energy metabolism crosstalk |
| SIRT3 | Mitochondrial deacetylase, regulates metabolic enzymes | Potential regulator of lysine degradation enzymes |
| PPARGC1A | PGC-1alpha, mitochondrial biogenesis regulator | May influence expression of saccharopine pathway genes |
| MYC | Oncogene, regulates glutamine metabolism | May affect NAD+ availability and pathway flux |
| HIF1A | Hypoxia-inducible factor, regulates metabolic adaptation | Links oxygen availability to NAD+ metabolism |
| SLC7A1 | Cationic amino acid transporter, imports lysine | Affects substrate supply for saccharopine pathway |
| SLC3A2 | Heavy chain of amino acid transporter, lysine transport | Modulates intracellular lysine levels |
| SLC7A5 | L-type amino acid transporter, transports large neutral amino acids | Indirectly affects lysine availability |
| SLC7A11 | Cystine/glutamate antiporter, affects glutamate levels | Crosstalk with glutamate produced by saccharopine dehydrogenase |
How Is saccharopine dehydrogenase (NAD+, L-glutamate-forming) activity Regulated?
The saccharopine dehydrogenase (NAD+, L-glutamate-forming) activity is regulated primarily by substrate availability and the cellular redox state, as it depends on NAD+ as a cofactor. In mammals, the bifunctional enzyme AASS is expressed in a tissue-specific manner, with highest levels in liver and kidney, reflecting the organ-specific demands for lysine degradation. Hormonal and nutritional signals, such as glucagon and high-protein diets, can induce the saccharopine pathway, likely through transcriptional upregulation of AASS. Additionally, the activity may be influenced by mitochondrial energy status, as NAD+ regeneration is tied to oxidative phosphorylation. Post-translational modifications have not been extensively characterized, but the enzyme contains potential phosphorylation sites that could modulate its activity.
saccharopine dehydrogenase (NAD+, L-glutamate-forming) activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| AASS | Hyperlysinemia, saccharopinuria | AASS knockout mice, patient-derived fibroblasts |
| DHTKD1 | Charcot-Marie-Tooth disease type 2Q | DHTKD1 knockout mice, neuronal cell lines |
| ALDH7A1 | Pyridoxine-dependent epilepsy | ALDH7A1 knockout mice, patient iPSC-derived neurons |
| SLC25A21 | Mitochondrial oxodicarboxylate carrier deficiency | SLC25A21 knockout cells, mitochondrial assays |
| GLUD1 | Hyperinsulinism-hyperammonemia syndrome | GLUD1 transgenic mice, pancreatic islets |
Hyperlysinemia and Saccharopinuria
Deficiency of saccharopine dehydrogenase (NAD+, L-glutamate-forming) activity, often due to mutations in the AASS gene, leads to hyperlysinemia and saccharopinuria, characterized by elevated lysine and saccharopine in blood and urine. Patients may present with neurological symptoms, including intellectual disability and seizures, although some cases are asymptomatic. The accumulation of saccharopine is thought to be neurotoxic, but the exact mechanisms remain under investigation.
Neurological Disorders
Dysregulation of lysine metabolism has been implicated in neurological disorders such as pyridoxine-dependent epilepsy, where mutations in antiquitin (ALDH7A1) lead to accumulation of alpha-aminoadipic semialdehyde, a metabolite downstream of saccharopine dehydrogenase. The saccharopine pathway also interacts with glutamate metabolism, and imbalances may contribute to excitotoxicity. Further research is needed to clarify the role of GO:0047131 in these conditions.
Metabolic and Mitochondrial Diseases
The saccharopine pathway is mitochondrial, and its dysfunction may impact mitochondrial energy metabolism. Conditions such as Charcot-Marie-Tooth disease type 2Q, caused by mutations in DHTKD1, which acts downstream of saccharopine dehydrogenase, highlight the importance of this pathway in mitochondrial homeostasis. Understanding the regulation of GO:0047131 could provide insights into these disorders.
From saccharopine dehydrogenase (NAD+, L-glutamate-forming) activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| What is the effect of AASS loss on lysine metabolism? | AASS knockout cell line (e.g., HepG2) or mouse model |
| How does a point mutation in the active site affect enzyme kinetics? | Point-mutation knock-in of AASS in HEK293 cells |
| Can we tag the enzyme to study its localization? | Knock-in of fluorescent tag (e.g., GFP) at the AASS locus |
| What is the impact of AASS overexpression on lysine degradation? | Overexpression of AASS in liver cells |
| Which genes interact with the saccharopine pathway? | CRISPR library screening in cells with metabolic readout |
| How does NAD+ availability regulate enzyme activity? | Knockout of NAD+ salvage genes (e.g., NAMPT) in cells |
How to Study the saccharopine dehydrogenase (NAD+, L-glutamate-forming) activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| NADH-coupled enzyme assay | Saccharopine dehydrogenase activity | Kinetic characterization, inhibitor screening |
| LC-MS/MS metabolomics | Lysine, saccharopine, and related metabolites | Diagnosis of metabolic disorders, flux analysis |
| 13C-lysine tracing | Metabolic flux through saccharopine pathway | Quantifying pathway contribution to energy metabolism |
| CRISPR-Cas9 knockout | Loss-of-function phenotypes | Studying gene function in cell models |
| CRISPR point mutation | Effect of specific amino acid changes | Dissecting catalytic residues |
| Knock-in tagging | Protein localization and interactions | Live-cell imaging, immunoprecipitation |
| RNA-seq | Transcriptional changes upon pathway perturbation | Identifying regulatory networks |
| Proteomics | Protein expression and modifications | Discovering post-translational regulation |
Enzyme Activity Assays
Direct measurement of saccharopine dehydrogenase (NAD+, L-glutamate-forming) activity is typically performed by monitoring NADH production at 340 nm in the presence of L-saccharopine and NAD+. Coupled assays using diaphorase or lactate dehydrogenase can enhance sensitivity and allow continuous monitoring. These assays are essential for characterizing enzyme kinetics and for diagnosing enzyme deficiencies in patient samples.
Metabolomics and Flux Analysis
Quantification of lysine, saccharopine, and downstream metabolites by LC-MS/MS provides a readout of pathway flux. Stable isotope tracing with 13C-lysine can reveal the contribution of the saccharopine pathway to central metabolism. These methods are useful for studying the impact of genetic perturbations on the pathway.
CRISPR-Cas9 Genome Editing
CRISPR-Cas9 can be used to generate knockout, point mutation, or knock-in models to study the function of AASS and related genes. For example, knockout of AASS in cell lines leads to accumulation of saccharopine, confirming the role of the enzyme in lysine degradation. These models enable precise dissection of the pathway's physiological roles.
Structural Biology
X-ray crystallography and cryo-EM can determine the three-dimensional structure of the enzyme, revealing substrate binding and catalytic residues. The first crystal structure of a related L-lysine 6-dehydrogenase provided insights into the NAD-dependent amine dehydrogenase family. Such studies guide inhibitor design and mechanistic understanding.
How CRISPR Can Be Used to Study GO:0047131 saccharopine dehydrogenase (NAD+, L-glutamate-forming) activity
Knockout
CRISPR-Cas9 knockout of AASS or related genes in cell lines (e.g., HepG2, HEK293) can abolish saccharopine dehydrogenase activity, leading to accumulation of saccharopine and lysine. These models are valuable for studying the metabolic consequences of enzyme deficiency and for validating drug targets.
Point Mutation
Introducing specific point mutations in the AASS gene via CRISPR can mimic patient mutations or probe catalytic residues. For example, mutating the NAD+ binding site can reveal its role in catalysis. Such models help establish genotype-phenotype relationships.
Knock-in
Knock-in of epitope tags (e.g., FLAG, GFP) at the endogenous AASS locus allows for tracking protein expression, localization, and interactions without overexpression artifacts. This approach is useful for studying the enzyme's mitochondrial targeting and dynamics.
Overexpression
CRISPR activation (CRISPRa) or lentiviral overexpression of AASS can increase saccharopine dehydrogenase activity, enabling studies of pathway flux and metabolic remodeling. Overexpression models are also used to screen for substrates or inhibitors.
How EDITGENE Supports saccharopine dehydrogenase (NAD+, L-glutamate-forming) activity Research
Researchers studying saccharopine dehydrogenase (NAD+, L-glutamate-forming) activity-related genes often need to determine whether a candidate gene is causally involved in lysine metabolism, metabolic disorders, or mitochondrial function. EDITGENE provides a comprehensive suite of CRISPR-based services to accelerate this research, from gene knockout to precise point mutations and knock-in models.
Contact EDITGENE today to design your custom CRISPR model for saccharopine dehydrogenase (NAD+, L-glutamate-forming) activity research.
Frequently Asked Questions About saccharopine dehydrogenase (NAD+, L-glutamate-forming) activity
What is saccharopine dehydrogenase (NAD+, L-glutamate-forming) activity?
It is an enzymatic activity (GO:0047131) that catalyzes the NAD+-dependent conversion of L-saccharopine to L-glutamate and allysine, a key step in lysine degradation.
What genes are involved in saccharopine dehydrogenase (NAD+, L-glutamate-forming) activity?
The main gene is AASS in humans, which encodes a bifunctional enzyme with this activity. In yeast, LYS1 and LYS9 are involved.
What is the reaction catalyzed by GO:0047131?
The reaction is: L-saccharopine + H2O + NAD+ = L-glutamate + allysine + H+ + NADH.
What diseases are associated with saccharopine dehydrogenase deficiency?
Deficiency causes hyperlysinemia and saccharopinuria, which can present with neurological symptoms.
How is saccharopine dehydrogenase activity measured?
It is typically measured by monitoring NADH production at 340 nm using a coupled assay with L-saccharopine and NAD+.
What is the role of NAD+ in this enzyme activity?
NAD+ acts as an electron acceptor, being reduced to NADH during the oxidation of saccharopine.
Is saccharopine dehydrogenase involved in lysine biosynthesis?
In some organisms, a related enzyme activity participates in lysine biosynthesis, but in mammals it functions in lysine degradation.
What is the cellular location of saccharopine dehydrogenase (NAD+, L-glutamate-forming) activity?
In mammals, the activity is localized in the mitochondrial matrix as part of the bifunctional AASS enzyme.
Can CRISPR be used to study saccharopine dehydrogenase?
Yes, CRISPR-Cas9 can generate knockout, point mutation, or knock-in models to study the function of AASS and related genes.
What are the synonyms for GO:0047131?
Synonyms include aminoadipic semialdehyde synthase activity, saccharopin dehydrogenase activity, and NAD+ oxidoreductase (L-2-aminoadipic-delta-semialdehyde and glutamate forming).
Conclusion
GO:0047131, saccharopine dehydrogenase (NAD+, L-glutamate-forming) activity, is a critical enzymatic function in the saccharopine pathway, bridging lysine catabolism and NAD+ metabolism. Its dysfunction is linked to metabolic disorders, and ongoing research continues to uncover its roles in health and disease. Advanced CRISPR models and biochemical assays are essential tools for dissecting this activity and developing therapeutic strategies.
References
- 1. Yoneda K et al.. 2010. First crystal structure of L-lysine 6-dehydrogenase as an NAD-dependent amine dehydrogenase.. J Biol Chem 285(11):8444-53 PMID: 20056607
- 2. Noda C et al.. 1978. Purification and properties of L-lysine-alpha-ketoglutarate reductase from rat liver mitochondria.. Biochim Biophys Acta 525(2):307-13 PMID: 687635
- 3. Peverelli MG et al.. 2015. An optimized coupled assay for quantifying diaminopimelate decarboxylase activity.. Biochimie 115:78-85 PMID: 25986217
