GO:0016836 hydro-lyase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0016836 (hydro-lyase activity) describes catalysis of carbon-oxygen bond cleavage by elimination of water, a fundamental reaction in metabolism and immunity.
• Key enzymes include IRG1 (ACOD1), fumarase (FH), carbonic anhydrases, and ENO1, each removing water to form essential products like itaconate, fumarate, and phosphoenolpyruvate [1,4,6].
• Dysregulation of hydro-lyases is linked to obesity, nonalcoholic steatohepatitis (NASH), stroke, and cancer, making them attractive therapeutic targets [2,3,4].
• CRISPR knockout, point mutation, and knock-in models enable precise dissection of hydro-lyase function in disease [1,3].
• Advanced methods such as metabolomics, isotope tracing, and activity assays are critical for validating hydro-lyase mechanisms [1,4].
• EDITGENE provides comprehensive CRISPR services to accelerate research on hydro-lyase activity and its role in human disease.
Description
Hydro-lyase activity (GO:0016836) is a molecular function defined as the catalysis of carbon-oxygen bond cleavage by elimination of water. This reaction is essential for diverse metabolic pathways, including the tricarboxylic acid (TCA) cycle, glycolysis, and immune responses [1,4]. Enzymes with hydro-lyase activity, such as fumarase and enolase, are highly conserved and play critical roles in cellular homeostasis [4,6]. Understanding their mechanisms and regulation is vital for uncovering disease pathologies and developing targeted therapies [2,3].
hydro-lyase activity At A Glance
| GO ID | GO:0016836 |
|---|---|
| GO term | hydro-lyase activity |
| Ontology | molecular_function |
| Synonym | none |
| Major function | Catalysis of carbon-oxygen bond cleavage by elimination of water |
| EC number | 4.2.1.- |
| Reaction type | Elimination of water (dehydration) |
| Common substrates | Citrate, fumarate, 2-phosphoglycerate, cis-aconitate |
| Representative enzymes | Fumarase (FH), enolase (ENO1), IRG1 (ACOD1), carbonic anhydrase |
What Is GO:0016836?
Hydro-lyase activity (GO:0016836) refers to the catalytic removal of a water molecule from a substrate, resulting in the cleavage of a carbon-oxygen bond and often the formation of a double bond. This class of enzymes includes dehydratases and hydro-lyases that act on various substrates, such as citrate, fumarate, and 2-phosphoglycerate [4,6].
Why Is hydro-lyase activity Important in Cell Biology?
Hydro-lyase activity is central to fundamental metabolic processes and immune defense. For example, IRG1 catalyzes the production of itaconate, a key immunometabolite with antimicrobial and anti-inflammatory properties. Fumarase deficiency leads to severe neurological disorders, while enolase is involved in glycolysis and cancer metabolism [4,6]. Targeting hydro-lyases offers therapeutic potential for metabolic diseases, cancer, and infections [2,3,5].
• Essential for the TCA cycle and energy production.
• Regulates immune responses through itaconate production by IRG1.
• Involved in glycolysis via enolase (ENO1).
• Linked to obesity and NASH through fumarase and KLF10-FH1 axis [3,4].
• Plays a role in neuroprotection after stroke via ENO1/KLF2 axis.
• Carbonic anhydrases (hydro-lyases) regulate pH and fluid balance.
• Potential targets for cancer therapy, e.g., pseudouridine synthase 1.
• Modulated by exercise and metabolic interventions [2,3].
• Subject to regulation by microRNAs such as miR-144.
• Can be studied using CRISPR-based gene editing for precise functional analysis [1,3].
Molecular Mechanism of hydro-lyase activity
Substrate Binding and Activation
In simple terms: The enzyme grabs its target molecule and gets it ready to lose water.
Hydro-lyases bind specific substrates, such as citrate or 2-phosphoglycerate, often requiring metal ions or specific residues for activation [1,6]. For example, IRG1 binds cis-aconitate to produce itaconate.
Water Elimination and Bond Cleavage
In simple terms: The enzyme removes a water molecule, breaking a carbon-oxygen bond.
The catalytic mechanism involves the abstraction of a proton and a hydroxyl group, leading to the formation of a double bond. Fumarase converts malate to fumarate by removing water.
Product Release and Enzyme Regeneration
In simple terms: The product is released, and the enzyme is ready for another round.
After product formation, the enzyme undergoes conformational changes to release the product and return to its active state. This step can be regulated by post-translational modifications or allosteric effectors [3,4].
Cofactors and Metal Dependence
In simple terms: Some hydro-lyases need metal helpers to work.
Carbonic anhydrases require a zinc ion for catalysis, while other hydro-lyases may depend on magnesium or iron. These cofactors stabilize the transition state and facilitate water elimination.
Regulation by Cellular Signals
In simple terms: The activity of these enzymes can be turned up or down by cellular signals.
Hydro-lyase activity is regulated by microRNAs (e.g., miR-144 targeting fumarase) and metabolic cues such as exercise, which modulates the KLF10-FH1 axis [3,4].
Key Genes Involved in GO:0016836 hydro-lyase activity
The following genes encode enzymes with hydro-lyase activity and are critical for various metabolic and immune pathways.
| Gene | Major Role | Research Relevance |
|---|---|---|
| IRG1 (ACOD1) | Catalyzes itaconate production from cis-aconitate | Immunometabolism, antimicrobial defense |
| FH (Fumarase) | Converts malate to fumarate in TCA cycle | Cancer, NASH, obesity [3,4] |
| ENO1 (Enolase 1) | Converts 2-phosphoglycerate to phosphoenolpyruvate in glycolysis | Stroke, neuroprotection, cancer |
| CA (Carbonic anhydrase) | Interconverts CO2 and bicarbonate | pH regulation, mitochondrial function |
| KLF10 | Transcription factor regulating Fh1 expression | Exercise-mediated NASH amelioration |
| KLF2 | Transcription factor downstream of ENO1 | Neuroprotection in stroke |
| PUS1 | Pseudouridine synthase, not a hydro-lyase but related to RNA modification | Cancer immunotherapy |
| FASN | Fatty acid synthase, lactylation affects lipid synthesis | Exercise-induced metabolic regulation |
| PAM | Peptide amidation enzyme, not a hydro-lyase | Peptide hormone processing |
| miR-144 | MicroRNA targeting fumarase | Obesity, NRF2 activation |
| NRF2 | Transcription factor activated upon fumarase inhibition | Oxidative stress response |
| ACOD1 | Alternative name for IRG1 | Itaconate production |
| FH1 | Fumarase 1 in mice | NASH, exercise |
| ENO1 | Enolase 1, moonlighting protein | Glycolysis, stroke |
| CA5A | Mitochondrial carbonic anhydrase | Mitochondrial metabolism |
| CA5B | Mitochondrial carbonic anhydrase | Mitochondrial metabolism |
| KLF10 | Kruppel-like factor 10 | NASH, exercise |
| KLF2 | Kruppel-like factor 2 | Stroke, neuroprotection |
How Is hydro-lyase activity Regulated?
Hydro-lyase activity is regulated at multiple levels. Transcriptional control includes KLF10-mediated expression of Fh1 in response to exercise. Post-transcriptional regulation by microRNAs, such as miR-144 targeting fumarase, modulates enzyme levels. Post-translational modifications, including lactylation of fatty acid synthase, can affect related metabolic pathways. Additionally, allosteric regulation by metabolites and cofactors fine-tunes enzyme activity [1,6].
hydro-lyase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| FH | NASH, obesity | Liver-specific KO mice, high-fat diet [3,4] |
| ENO1 | Stroke, neuroprotection | Middle cerebral artery occlusion (MCAO) mice |
| IRG1 | Immunometabolism, infections | LPS-stimulated macrophages, KO mice |
| CA5A/CA5B | Mitochondrial dysfunction | KO cell lines, metabolic assays |
| KLF10 | NASH | Exercise intervention in mice |
Hydro-lyase dysfunction in metabolic diseases
Dysregulation of hydro-lyases contributes to obesity and NASH. Hepatic miR-144 drives fumarase activity, leading to NRF2 activation and metabolic imbalance during obesity. The KLF10-FH1 axis promotes exercise-mediated amelioration of NASH, highlighting the therapeutic potential of targeting this pathway.
Role in neuroprotection and stroke
CircFndc3b mediates exercise-induced neuroprotection by mitigating microglial pyroptosis via the ENO1/KLF2 axis in stroke mice. This suggests that hydro-lyase ENO1 is involved in neuroinflammatory responses and could be a target for stroke therapy.
Hydro-lyases in cancer and immunity
IRG1 catalyzes itaconate production, linking metabolism to immunity and potentially influencing tumor progression. Pseudouridine synthase 1-targeted therapy activates antiviral immunity to boost cancer immunotherapy, though it is not a hydro-lyase, it highlights the broader context of metabolic enzymes in cancer.
From hydro-lyase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does IRG1 knockout affect itaconate production? | IRG1 KO mice or macrophages |
| Does fumarase deficiency alter NASH progression? | Liver-specific FH KO mice [3,4] |
| Can ENO1 overexpression protect against stroke? | ENO1 transgenic mice |
| Does miR-144 inhibition restore fumarase activity? | miR-144 KO mice |
| Does point mutation in carbonic anhydrase affect activity? | CA point-mutant cell lines |
| Does KLF10 knock-in enhance Fh1 expression? | KLF10 knock-in mice |
How to Study the hydro-lyase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Metabolomics | Substrate and product levels | Itaconate production by IRG1 |
| Isotope tracing | Flux through hydro-lyase reactions | TCA cycle activity |
| Enzyme activity assay | Catalytic rate | Fumarase and carbonic anhydrase [4,6] |
| CRISPR knockout screen | Gene essentiality for hydro-lyase function | Identify regulators |
| RNA-seq | Gene expression changes | Exercise-induced KLF10-FH1 |
| Proteomics | Protein abundance and modifications | Lactylation of FASN |
| Western blot | Protein levels | ENO1 expression in stroke |
| Immunohistochemistry | Tissue localization | Fumarase in liver |
Metabolomics and Isotope Tracing
Metabolomics coupled with 13C-isotope tracing is used to measure hydro-lyase activity by tracking substrate conversion and product formation, such as itaconate from cis-aconitate.
Enzymatic Activity Assays
In vitro assays with purified enzymes or cell lysates measure the rate of water elimination spectrophotometrically or by HPLC, as demonstrated for fumarase and carbonic anhydrase [4,6].
CRISPR Screening and Functional Genomics
Genome-wide CRISPR knockout screens identify genes required for hydro-lyase activity and related pathways, enabling discovery of novel regulators [1,3].
Transcriptomics and Proteomics
RNA-seq and proteomics reveal changes in hydro-lyase expression under conditions like exercise or obesity, as shown for KLF10-FH1 and miR-144 [3,4].
How CRISPR Can Be Used to Study GO:0016836 hydro-lyase activity
Knockout
CRISPR knockout of hydro-lyase genes (e.g., IRG1, FH) in cell lines or mice abolishes enzyme activity, enabling studies of metabolic and immune consequences [1,3].
Point Mutation
Introducing point mutations in catalytic residues (e.g., in carbonic anhydrase) via CRISPR base editing allows precise dissection of mechanism and cofactor dependence.
Knock-in
Knock-in of tagged or reporter versions of hydro-lyases (e.g., ENO1-GFP) facilitates localization and interaction studies in vivo.
Overexpression
CRISPR activation (CRISPRa) or transgenic overexpression of hydro-lyases (e.g., ENO1) can test gain-of-function effects in disease models like stroke.
How EDITGENE Supports hydro-lyase activity Research
Researchers studying hydro-lyase activity-related genes often need to determine whether a candidate gene is causally involved in metabolic or immune pathways. EDITGENE provides end-to-end CRISPR solutions to generate precisely engineered cell and animal models, accelerating functional validation and therapeutic development.
Contact EDITGENE today to design your custom CRISPR model for hydro-lyase activity research.
Frequently Asked Questions About hydro-lyase activity
What is hydro-lyase activity?
Hydro-lyase activity (GO:0016836) is the catalysis of carbon-oxygen bond cleavage by elimination of water, a fundamental reaction in metabolism.
What genes are involved in hydro-lyase activity?
Key genes include IRG1 (ACOD1), FH (fumarase), ENO1 (enolase), and carbonic anhydrases (CA5A, CA5B) [1,4,6].
How is hydro-lyase activity regulated?
It is regulated transcriptionally (e.g., KLF10), post-transcriptionally (miR-144), and by post-translational modifications [3,4,7].
What diseases are associated with hydro-lyase dysfunction?
Diseases include NASH, obesity, stroke, and cancer [2,3,4,5].
What methods are used to study hydro-lyase activity?
Metabolomics, isotope tracing, enzyme assays, CRISPR screens, and omics approaches [1,4,6].
Can CRISPR be used to study hydro-lyase genes?
Yes, CRISPR knockout, point mutation, knock-in, and overexpression models are widely used [1,3].
What is the role of IRG1 in immunity?
IRG1 catalyzes itaconate production, which has antimicrobial and anti-inflammatory effects.
How does fumarase relate to NASH?
The KLF10-FH1 axis promotes exercise-mediated amelioration of NASH, and miR-144 drives fumarase activity in obesity [3,4].
What is the ENO1/KLF2 axis?
It mediates exercise-induced neuroprotection by mitigating microglial pyroptosis in stroke.
What services does EDITGENE offer for hydro-lyase research?
EDITGENE provides CRISPR knockout, point mutation, knock-in, overexpression, library screening, and bioinformatics services [1,3].
Conclusion
Hydro-lyase activity (GO:0016836) is a fundamental molecular function with critical roles in metabolism, immunity, and disease. Understanding its mechanisms and regulation offers insights into conditions such as NASH, obesity, stroke, and cancer. Advanced CRISPR tools and multi-omics approaches are essential for dissecting these pathways and developing targeted therapies.
References
- 1. Michelucci A et al.. 2013. Immune-responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production.. Proc Natl Acad Sci U S A 110(19):7820-5 PMID: 23610393
- 2. Zhao Y et al.. 2025. CircFndc3b Mediates Exercise-Induced Neuroprotection by Mitigating Microglial/Macrophage Pyroptosis via the ENO1/KLF2 Axis in Stroke Mice.. Adv Sci (Weinh) 12(1):e2403818 PMID: 39467260
- 3. Luo HY et al.. 2024. Hepatic Klf10-Fh1 axis promotes exercise-mediated amelioration of NASH in mice.. Metabolism 155:155916 PMID: 38615945
- 4. Azzimato V et al.. 2021. Hepatic miR-144 Drives Fumarase Activity Preventing NRF2 Activation During Obesity.. Gastroenterology 161(6):1982-1997.e11 PMID: 34425095
- 5. Wang F et al.. 2025. Pseudouridine synthase 1-targeted therapy activates antiviral immunity to boost cancer immunotherapy.. Cell Rep 44(9):116233 PMID: 40911416
- 6. Dodgson SJ et al.. 1980. Mitochondrial carbonic anhydrase.. Proc Natl Acad Sci U S A 77(9):5562-6 PMID: 6776540
- 7. Chen X et al.. 2023. High-intensity interval training induces lactylation of fatty acid synthase to inhibit lipid synthesis.. BMC Biol 21(1):196 PMID: 37726733
