GO:0061697 protein-glutaryllysine deglutarylase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0061697 defines the enzymatic activity that removes a glutaryl group from N(6)-glutaryl-L-lysine on proteins, using NAD+ and water to produce 2''-O-glutaryl-ADP-D-ribose, nicotinamide, and a free lysine.
• This deglutarylase activity is a key counter-regulatory mechanism for lysine glutarylation, a recently discovered post-translational modification.
• SIRT5 is the best-characterized enzyme with protein-glutaryllysine deglutarylase activity, and its catalytic action links mitochondrial metabolism to protein function.
• Glutarylation and deglutarylation influence diverse cellular processes, including mitochondrial quality control and neurodegenerative disease pathways.
• Dysregulation of deglutarylase activity has been implicated in cancer, metabolic disorders, and neurodegeneration, making it a potential therapeutic target.
• Studying GO:0061697 requires integrated approaches such as CRISPR knockout, point mutation, and proteomics to dissect its substrates and regulatory networks.
Description
Protein-glutaryllysine deglutarylase activity (GO:0061697) is a molecular function that reverses the post-translational modification known as lysine glutarylation. This enzymatic activity removes a glutaryl group from modified lysine residues on target proteins, thereby resetting their functional state. The reaction consumes NAD+ and water and releases 2''-O-glutaryl-ADP-D-ribose, nicotinamide, and an unmodified lysine. Since its discovery, this deglutarylase activity has emerged as a critical regulator of mitochondrial and cellular metabolism. Researchers study GO:0061697 to understand how dynamic changes in protein glutarylation control enzyme activity, protein stability, and signaling pathways. The importance of this activity is underscored by its evolutionary conservation and its links to human diseases such as cancer and neurodegeneration. This article provides a comprehensive overview of the mechanism, key genes, regulatory features, and experimental models used to investigate protein-glutaryllysine deglutarylase activity.
protein-glutaryllysine deglutarylase activity At A Glance
| GO ID | GO:0061697 |
|---|---|
| GO term | protein-glutaryllysine deglutarylase activity |
| Ontology | molecular_function |
| Synonym | none |
| Major function | Catalyzes the removal of a glutaryl group from N(6)-glutaryl-L-lysine residues on proteins, using NAD+ and water. |
| Reaction | N(6)-glutaryl-L-lysyl-[protein] + NAD+ + H2O = 2''-O-glutaryl-ADP-D-ribose + nicotinamide + L-lysyl-[protein]. |
| Cofactor | NAD+ |
| Substrate | N(6)-glutaryl-L-lysyl-[protein] |
| Product | L-lysyl-[protein], 2''-O-glutaryl-ADP-D-ribose, nicotinamide |
| Enzyme class | NAD+-dependent deacylase (sirtuin family) |
What Is GO:0061697?
Protein-glutaryllysine deglutarylase activity (GO:0061697) is defined as the catalysis of the reaction: N(6)-glutaryl-L-lysyl-[protein] + NAD+ + H2O = 2''-O-glutaryl-ADP-D-ribose + nicotinamide + L-lysyl-[protein]. In simpler terms, it is an enzymatic activity that removes a glutaryl chemical group from a lysine residue on a protein, using NAD+ as a cofactor and water as a reactant. This activity is a type of protein deglutarylase function and is part of the broader class of NAD+-dependent deacylases.
Why Is protein-glutaryllysine deglutarylase activity Important in Cell Biology?
Protein-glutaryllysine deglutarylase activity is important because it provides a dynamic switch for controlling protein function through the removal of glutaryl marks. This activity counteracts lysine glutarylation, a modification that can alter enzyme activity, protein-protein interactions, and subcellular localization. By regulating these marks, deglutarylases influence fundamental processes such as mitochondrial metabolism, oxidative stress responses, and cell survival. Moreover, the dysregulation of deglutarylase activity has been linked to cancer, neurodegeneration, and metabolic disorders, highlighting its potential as a therapeutic target. Understanding GO:0061697 is therefore essential for researchers aiming to manipulate protein function in disease models and to develop novel treatments.
• Regulates protein function by removing glutaryl groups from lysine residues.
• Counterbalances lysine glutarylation, a PTM linked to metabolic stress.
• Influences mitochondrial quality control and energy metabolism.
• Implicated in cancer through modulation of oncogenic signaling.
• Associated with neurodegenerative diseases such as Alzheimer's and Parkinson's.
• Provides a mechanism for cellular adaptation to metabolic changes.
• Potential target for small-molecule modulators of sirtuin activity.
• Essential for understanding the interplay between acylation and cellular signaling.
• Enables researchers to study dynamic PTM crosstalk using CRISPR models.
• Contributes to the emerging field of acylspermidine metabolism.
What Happens During protein-glutaryllysine deglutarylase activity?
Substrate Recognition and Binding
In simple terms: The enzyme first finds and grabs onto a protein that has a glutaryl tag on one of its lysine residues.
The deglutarylase enzyme recognizes N(6)-glutaryl-L-lysyl-[protein] substrates through a specific binding pocket that accommodates the glutaryl moiety. This recognition is mediated by conserved amino acid residues within the catalytic domain of sirtuin enzymes, particularly SIRT5, which exhibits high specificity for glutaryl-lysine. The binding step is essential for positioning the substrate for subsequent catalysis.
NAD+ Binding and Catalysis
In simple terms: The enzyme uses NAD+ as a tool to cut off the glutaryl group, producing several byproducts.
Upon substrate binding, the enzyme binds NAD+ and catalyzes a reaction that cleaves the glutaryl-lysine bond. This reaction proceeds through the formation of an ADP-ribose intermediate, releasing nicotinamide and generating 2''-O-glutaryl-ADP-D-ribose as a product. The catalytic mechanism involves a conserved histidine residue that acts as a general base, facilitating nucleophilic attack by water.
Product Release and Protein Reset
In simple terms: After the glutaryl group is removed, the protein is left with a normal lysine, and the enzyme lets go of all products.
Following catalysis, the enzyme releases the deglutarylated protein (L-lysyl-[protein]), along with 2''-O-glutaryl-ADP-D-ribose and nicotinamide. The removal of the glutaryl group restores the original lysine residue, which can then participate in other post-translational modifications or protein functions. This step completes the catalytic cycle and allows the enzyme to act on additional substrates.
Regulation by Cellular Metabolites
In simple terms: The enzyme's activity can be turned up or down by molecules in the cell, such as NAD+ levels or other metabolites.
The deglutarylase activity is regulated by the availability of NAD+, which is a cofactor for the reaction. Additionally, recent studies have shown that reduced nicotinic acid riboside (NARH) can differentially regulate SIRT5 activity, suggesting that cellular metabolites can modulate deglutarylase function. This regulation ensures that deglutarylation is responsive to the metabolic state of the cell.
Key Genes Involved in GO:0061697 protein-glutaryllysine deglutarylase activity
The following genes and proteins are central to the study of protein-glutaryllysine deglutarylase activity, either as enzymes that catalyze the reaction or as substrates and regulators.
| Gene | Major Role | Research Relevance |
|---|---|---|
| SIRT5 | Primary enzyme with protein-glutaryllysine deglutarylase activity; removes glutaryl groups from lysine residues | Key target for studying deglutarylation in metabolism, cancer, and neurodegeneration |
| GCDH | Glutaryl-CoA dehydrogenase; involved in glutaryl-CoA metabolism and indirectly affects glutarylation levels | Mutations cause glutaric acidemia type I; used to study glutarylation stress |
| AKT1 | Kinase whose glutarylation is regulated by GCDH and SIRT5; deglutarylation modulates oncogenic signaling | Model to study crosstalk between deglutarylase activity and cancer pathways |
| SIRT1 | NAD+-dependent deacetylase with broader deacylase activity; may contribute to deglutarylation | Studied for overlapping substrate specificity with SIRT5 |
| SIRT3 | Mitochondrial deacetylase; can remove other acyl groups but not glutaryl efficiently | Used as a comparison to understand SIRT5 specificity |
| SIRT4 | Mitochondrial sirtuin with weak deglutarylase activity | Investigated for its role in metabolic regulation |
| SIRT6 | Nuclear sirtuin with deacylase activity on other modifications | Explored for potential deglutarylase function |
| SIRT7 | Nuclear sirtuin; primarily deacetylase | Studied for substrate overlap |
| GLS | Glutaminase; affects glutamate and glutaryl-CoA pools | Linked to glutarylation changes in brain metabolism |
| DLST | Dihydrolipoamide succinyltransferase; component of 2-oxoadipate dehydrogenase complex | Inhibition leads to protein glutarylation in brain |
| DLD | Dihydrolipoamide dehydrogenase; part of 2-oxoadipate dehydrogenase complex | Involved in glutaryl-CoA production and glutarylation |
| IDH2 | Isocitrate dehydrogenase 2; mitochondrial metabolic enzyme | Potential substrate for glutarylation and deglutarylation |
| SDHA | Succinate dehydrogenase complex subunit A; mitochondrial enzyme | Model substrate for studying glutarylation dynamics |
| ATP5A1 | ATP synthase subunit; mitochondrial protein | Commonly used as a substrate in deglutarylase assays |
| HSPD1 | Heat shock protein 60; mitochondrial chaperone | Studied for glutarylation-mediated functional changes |
| PDHA1 | Pyruvate dehydrogenase E1 subunit alpha | Glutarylation affects its activity; deglutarylation restores function |
| OGDH | 2-oxoglutarate dehydrogenase; mitochondrial enzyme | Inhibition alters glutarylation patterns |
| GOT2 | Glutamate oxaloacetate transaminase 2; mitochondrial enzyme | Linked to glutarylation in brain metabolism |
How Is protein-glutaryllysine deglutarylase activity Regulated?
Protein-glutaryllysine deglutarylase activity is regulated at multiple levels. The availability of NAD+ directly controls the catalytic rate, as it is a required cofactor. Cellular metabolites such as reduced nicotinic acid riboside (NARH) can differentially modulate SIRT5 activity, providing a layer of metabolic regulation. Additionally, the expression levels of SIRT5 and other sirtuins are subject to transcriptional and post-translational control. The activity can also be influenced by the presence of other post-translational modifications on the enzyme itself, such as phosphorylation or acetylation. Furthermore, the substrate specificity and catalytic efficiency of deglutarylases are determined by the local protein environment and the availability of glutarylated substrates.
protein-glutaryllysine deglutarylase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| SIRT5 | Cancer, neurodegeneration, metabolic disorders | SIRT5 knockout and overexpression cell lines; xenograft models |
| GCDH | Glutaric acidemia type I | Gcdh-/- mouse models; patient-derived fibroblasts |
| AKT1 | Oncogenic signaling | AKT1 mutant knock-in cells; cancer cell lines |
| DLST | Neurodegeneration linked to glutarylation | DLST inhibition in neuronal cultures |
| IDH2 | Cancer metabolism | IDH2 mutant knock-in models |
Cancer
Dysregulation of protein-glutaryllysine deglutarylase activity has been implicated in cancer. For example, the glutarylation status of AKT1, regulated by GCDH and SIRT5, suppresses oncogenic signaling. Loss of SIRT5-mediated deglutarylation can lead to hyperglutarylation of metabolic enzymes, promoting tumor growth in certain contexts. Therefore, targeting deglutarylase activity is being explored as a therapeutic strategy in cancers with altered mitochondrial metabolism.
Neurodegenerative Diseases
Sirtuins, including SIRT5, are at the crossroads between mitochondrial quality control and neurodegenerative diseases. Impaired deglutarylase activity may contribute to the accumulation of glutarylated proteins, leading to mitochondrial dysfunction and neuronal death. Studies in models of Alzheimer's and Parkinson's diseases suggest that modulating deglutarylase activity could be neuroprotective.
Metabolic Disorders
Glutarylation and deglutarylation are tightly linked to metabolic pathways. Inhibition of 2-oxoadipate dehydrogenase, as seen in glutaric acidemia type I, leads to increased protein glutarylation in the brain. This suggests that deglutarylase activity is crucial for maintaining metabolic homeostasis, and its impairment may contribute to metabolic disorders.
From protein-glutaryllysine deglutarylase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does SIRT5 knockout increase global protein glutarylation? | SIRT5 KO cell lines (e.g., HEK293T, HeLa) |
| Does a point mutation in SIRT5 catalytic domain abolish deglutarylase activity? | SIRT5 point-mutant knock-in cells (e.g., H158A) |
| Can a tagged SIRT5 be used to identify interacting substrates? | Knock-in of FLAG- or HA-tagged SIRT5 |
| Does overexpression of SIRT5 reduce glutarylation of metabolic enzymes? | SIRT5 overexpression stable cell lines |
| What is the effect of NARH on SIRT5 activity? | Cells treated with NARH and assayed for deglutarylase activity |
| Does GCDH deficiency alter AKT1 glutarylation? | GCDH knockout cells and AKT1 glutarylation assays |
How to Study the protein-glutaryllysine deglutarylase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Mass spectrometry | Global glutarylation sites and stoichiometry | Identifying substrates of SIRT5 |
| Western blot | Global glutarylation levels | Validating changes in deglutarylase activity |
| In vitro activity assay | Enzymatic deglutarylase activity | Testing inhibitors or mutants |
| CRISPR knockout screen | Genes affecting glutarylation | Discovering regulators |
| Co-immunoprecipitation | Protein-protein interactions | Finding substrate-enzyme complexes |
| Immunofluorescence | Subcellular localization of glutarylated proteins | Assessing mitochondrial glutarylation |
| NAD+ quantification | Cellular NAD+ levels | Linking metabolism to deglutarylase activity |
| Site-directed mutagenesis | Specific residue function | Mapping catalytic residues |
Proteomics for Global Glutarylation Profiling
Mass spectrometry-based proteomics using glutaryl-lysine-specific enrichment can identify substrates and quantify changes in deglutarylase activity. This method allows researchers to map glutarylation sites across the proteome and assess the impact of SIRT5 manipulation.
Western Blotting with Pan-Glutaryllysine Antibodies
Immunoblotting with anti-glutaryllysine antibodies provides a rapid readout of global glutarylation levels, which inversely correlate with deglutarylase activity. This approach is commonly used to validate findings from genetic or pharmacological perturbations.
Enzymatic Activity Assays
In vitro deglutarylase assays using recombinant enzymes and fluorogenic or radioactive substrates can directly measure catalytic activity. These assays are essential for determining kinetic parameters and testing inhibitors.
CRISPR-Based Genetic Screens
Genome-wide CRISPR knockout screens can identify genes that regulate deglutarylase activity or are required for its function. Such screens have the potential to uncover novel regulators and disease-relevant pathways.
How CRISPR Can Be Used to Study GO:0061697 protein-glutaryllysine deglutarylase activity
Knockout
CRISPR knockout of SIRT5 or other candidate deglutarylases allows researchers to assess the loss of protein-glutaryllysine deglutarylase activity and its downstream effects on cellular metabolism and protein function. Knockout cell lines can be used to identify endogenous substrates and to study compensatory mechanisms.
Point Mutation
Introducing point mutations in the catalytic domain of SIRT5 (e.g., H158A) via CRISPR knock-in can abolish deglutarylase activity without affecting protein expression, enabling precise structure-function studies. Such models are valuable for distinguishing catalytic activity from scaffolding functions.
Knock-in
Knock-in of epitope tags (e.g., FLAG, HA) or fluorescent proteins at the endogenous SIRT5 locus facilitates affinity purification and imaging of the enzyme in its native context. This approach helps identify interacting partners and track subcellular localization.
Overexpression
CRISPR activation or lentiviral overexpression of SIRT5 can increase deglutarylase activity, allowing researchers to study the effects of enhanced deglutarylation on cellular phenotypes. Overexpression models are useful for testing whether increased activity is protective or detrimental in disease contexts.
How EDITGENE Supports protein-glutaryllysine deglutarylase activity Research
Researchers studying protein-glutaryllysine deglutarylase activity-related genes often need to determine whether a candidate gene is causally involved in a specific phenotype. EDITGENE provides a comprehensive suite of CRISPR-based services to enable such investigations, from gene knockout to precise point mutations and knock-in models.
Contact EDITGENE today to design your custom CRISPR model for protein-glutaryllysine deglutarylase activity research.
Frequently Asked Questions About protein-glutaryllysine deglutarylase activity
What is protein-glutaryllysine deglutarylase activity?
It is an enzymatic activity that removes a glutaryl group from lysine residues on proteins, using NAD+ and water, as defined by GO:0061697.
What genes are involved in protein-glutaryllysine deglutarylase activity?
The primary gene is SIRT5, which encodes a sirtuin with robust deglutarylase activity; other sirtuins and metabolic genes such as GCDH and AKT1 also play roles.
What is the reaction catalyzed by protein-glutaryllysine deglutarylase?
The reaction is: N(6)-glutaryl-L-lysyl-[protein] + NAD+ + H2O = 2''-O-glutaryl-ADP-D-ribose + nicotinamide + L-lysyl-[protein].
Which enzyme has protein-glutaryllysine deglutarylase activity?
SIRT5 is the best-characterized enzyme with this activity, although other sirtuins may have overlapping functions.
How is protein-glutaryllysine deglutarylase activity regulated?
It is regulated by NAD+ availability, cellular metabolites such as NARH, and post-translational modifications of the enzyme itself.
What diseases are associated with protein-glutaryllysine deglutarylase activity?
Dysregulation has been linked to cancer, neurodegenerative diseases, and metabolic disorders such as glutaric acidemia type I.
How can I study protein-glutaryllysine deglutarylase activity in the lab?
Common methods include Western blotting with anti-glutaryllysine antibodies, mass spectrometry, in vitro activity assays, and CRISPR-based genetic screens.
What is the role of SIRT5 in deglutarylation?
SIRT5 catalyzes the removal of glutaryl groups from lysine residues, thereby reversing lysine glutarylation and modulating protein function.
Can CRISPR be used to study protein-glutaryllysine deglutarylase activity?
Yes, CRISPR knockout, point mutation, knock-in, and overexpression models are powerful tools to dissect the function of SIRT5 and related genes.
What are the potential therapeutic implications of targeting deglutarylase activity?
Modulating deglutarylase activity could offer new strategies for treating cancer, neurodegeneration, and metabolic diseases, though further research is needed.
Conclusion
Protein-glutaryllysine deglutarylase activity (GO:0061697) is a fundamental enzymatic function that regulates protein glutarylation, a key post-translational modification in mitochondrial and cellular metabolism. SIRT5 is the primary enzyme responsible for this activity, and its dysregulation is linked to cancer, neurodegeneration, and metabolic disorders. Understanding the mechanism, regulation, and disease relevance of this activity requires integrated approaches, including CRISPR-based models and advanced proteomics. EDITGENE provides comprehensive services to support researchers in this rapidly evolving field.
References
- 1. Zhang B et al.. 2024. Acylspermidines are conserved mitochondrial sirtuin-dependent metabolites.. Nat Chem Biol 20(7):812-822 PMID: 38167917
- 2. Bai L et al.. 2026. AKT1 glutarylation regulated by GCDH and SIRT5 suppresses oncogenic signaling.. Cell Rep 45(6):117394 PMID: 42207644
- 3. Hamza A et al.. 2025. Differential Regulation of SIRT5 Activity by Reduced Nicotinic Acid Riboside (NARH).. bioRxiv PMID: 40791482
- 4. Yang L et al.. 2017. Sirtuin 5: a review of structure, known inhibitors and clues for developing new inhibitors.. Sci China Life Sci 60(3):249-256 PMID: 27858336
- 5. Xu H et al.. 2023. Sirtuins at the Crossroads between Mitochondrial Quality Control and Neurodegenerative Diseases: Structure, Regulation, Modifications, and Modulators.. Aging Dis 14(3):794-824 PMID: 37191431
- 6. Boyko AI et al.. 2022. Delayed Impact of 2-Oxoadipate Dehydrogenase Inhibition on the Rat Brain Metabolism Is Linked to Protein Glutarylation.. Front Med (Lausanne) 9:896263 PMID: 35721081
- 7. Tan M et al.. 2014. Lysine glutarylation is a protein posttranslational modification regulated by SIRT5.. Cell Metab 19(4):605-17 PMID: 24703693
- 8. Hamza A et al.. 2026. Differential regulation of SIRT5 activity by reduced nicotinic acid riboside (NARH).. RSC Chem Biol 7(2):286-297 PMID: 41409579
