GO:0050011 itaconyl-CoA hydratase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0050011 (itaconyl-CoA hydratase activity) catalyzes the reversible hydration of itaconyl-CoA to citramalyl-CoA, a central step in bacterial itaconate degradation.
• The enzyme is part of the itaconate degradation pathway that enables pathogens such as Pseudomonas aeruginosa, Mycobacterium tuberculosis, and Yersinia pestis to grow on itaconate, a host-derived antimicrobial metabolite.
• Itaconyl-CoA hydratase belongs to the enoyl-CoA hydratase/isomerase superfamily and typically forms a complex with citramalyl-CoA lyase to channel the product to acetyl-CoA and pyruvate.
• Loss of itaconyl-CoA hydratase activity impairs itaconate catabolism and reduces bacterial virulence in infection models.
• The enzyme is a promising target for anti-virulence drugs and for metabolic engineering of itaconate-derived bioproducts.
• Research on this activity uses structural biology, enzyme kinetics, bacterial genetics, and CRISPR-based knockouts to dissect its role in metabolism and pathogenesis.
Description
Itaconyl-CoA hydratase activity (GO:0050011) is a molecular function that catalyzes the reversible conversion of itaconyl-CoA to citramalyl-CoA, a key step in the bacterial degradation of itaconate. Itaconate is a metabolite produced by mammalian macrophages during infection and has antimicrobial properties; however, some bacterial pathogens have evolved pathways to catabolize itaconate, allowing them to survive and cause disease. The enzyme responsible for this activity is encoded within the itaconate degradation gene cluster found in species such as Pseudomonas aeruginosa, Mycobacterium tuberculosis, and Yersinia pestis. Understanding this activity is important for microbiology, infectious disease research, and metabolic engineering, as it represents a point of interaction between host immunity and bacterial metabolism. Recent structural and functional studies have revealed the architecture of the enzyme and its partnership with citramalyl-CoA lyase, providing a foundation for inhibitor design and biotechnological applications.
itaconyl-CoA hydratase activity At A Glance
| GO ID | GO:0050011 |
|---|---|
| GO term | itaconyl-CoA hydratase activity |
| Ontology | molecular_function |
| Synonym | citramalyl-CoA hydro-lyase activity; citramalyl-CoA hydro-lyase (itaconyl-CoA-forming); itaconyl coenzyme A hydratase activity |
| Definition | Catalysis of the reaction: citramalyl-CoA = itaconyl-CoA + H2O. |
| Major function | Reversible hydration/dehydration in itaconate degradation |
| Pathway context | Itaconate degradation pathway, often clustered with citramalyl-CoA lyase |
| Representative organisms | Pseudomonas aeruginosa, Mycobacterium tuberculosis, Yersinia pestis, Burkholderia xenovorans |
| EC number | 4.2.1.- (hydro-lyase) |
What Is GO:0050011?
According to the Gene Ontology, itaconyl-CoA hydratase activity (GO:0050011) is defined as the catalysis of the reaction: citramalyl-CoA = itaconyl-CoA + H2O. In other words, it is a hydro-lyase that removes a water molecule from citramalyl-CoA to form itaconyl-CoA, or adds water to itaconyl-CoA to form citramalyl-CoA. This activity is synonymous with citramalyl-CoA hydro-lyase, citramalyl-CoA hydro-lyase (itaconyl-CoA-forming), and itaconyl coenzyme A hydratase activity. The reaction is part of the itaconate degradation pathway and is typically coupled with a subsequent lyase step that cleaves citramalyl-CoA into acetyl-CoA and pyruvate.
Why Is itaconyl-CoA hydratase activity Important in Cell Biology?
Itaconyl-CoA hydratase activity is important because it enables bacterial pathogens to degrade itaconate, an antimicrobial metabolite produced by host immune cells, thereby promoting bacterial survival and virulence. This activity is a validated component of the itaconate degradation pathway in several clinically relevant pathogens, including Mycobacterium tuberculosis and Pseudomonas aeruginosa. Inhibiting this enzyme could disarm pathogens without directly killing them, potentially reducing the emergence of resistance. Additionally, the enzyme is of interest for metabolic engineering, as it can be used to convert itaconate into value-added chemicals or to improve itaconate production by manipulating its degradation. Thus, GO:0050011 sits at the intersection of infectious disease, immunometabolism, and synthetic biology.
• Enables bacterial growth on itaconate, a host-derived antimicrobial metabolite.
• Contributes to virulence of pathogens such as Yersinia pestis and Pseudomonas aeruginosa.
• Represents a potential target for anti-virulence therapeutics.
• Involved in the metabolism of itaconate, a key immunometabolite with anti-inflammatory properties.
• Provides a model for studying enzyme evolution and metabolic pathway assembly.
• Useful for metabolic engineering of itaconate-derived products.
• Structural insights facilitate inhibitor design.
• Coupled with citramalyl-CoA lyase for efficient substrate channeling.
• Relevant to understanding host-pathogen metabolic interactions.
• Can be studied using CRISPR knockouts to link genotype to phenotype.
What Happens During itaconyl-CoA hydratase activity?
Substrate binding and hydration
In simple terms: The enzyme grabs itaconyl-CoA and adds water to it.
Itaconyl-CoA hydratase binds its substrate, itaconyl-CoA, and catalyzes the addition of a water molecule across the double bond, forming citramalyl-CoA. This step is reversible and belongs to the enoyl-CoA hydratase family of reactions.
Product release and channeling
In simple terms: The product is handed off to the next enzyme in the pathway.
The product, citramalyl-CoA, is released and can be further processed by citramalyl-CoA lyase, which cleaves it into acetyl-CoA and pyruvate. In some bacteria, the two enzymes form a complex to facilitate substrate channeling.
Role in itaconate degradation pathway
In simple terms: This step is part of a chain that lets bacteria eat itaconate.
The itaconate degradation pathway converts itaconate to acetyl-CoA and pyruvate via itaconyl-CoA and citramalyl-CoA intermediates. Itaconyl-CoA hydratase is essential for this pathway, and its activity is often encoded in a gene cluster with other pathway enzymes.
Physiological significance in pathogens
In simple terms: This activity helps bacteria survive in the host.
By degrading itaconate, pathogens can overcome its antimicrobial effects and colonize host tissues. Mutants lacking itaconyl-CoA hydratase activity show reduced virulence in animal models.
Key Genes Involved in GO:0050011 itaconyl-CoA hydratase activity
The following genes and proteins are directly or functionally associated with itaconyl-CoA hydratase activity (GO:0050011) based on published literature.
| Gene | Major Role | Research Relevance |
|---|---|---|
| ichA (PA0883) | Encodes itaconyl-CoA hydratase in Pseudomonas aeruginosa | Structural and functional studies; virulence |
| ichB (PA0884) | Encodes citramalyl-CoA lyase in P. aeruginosa | Partners with IchA for itaconate degradation |
| icl1 (Rv2498c) | Itaconyl-CoA hydratase in Mycobacterium tuberculosis | Essential for itaconate catabolism and survival |
| icl2 (Rv2499c) | Citramalyl-CoA lyase in M. tuberculosis | Works with Icl1 in the itaconate degradation pathway |
| ichA (Yp) | Itaconyl-CoA hydratase in Yersinia pestis | Required for virulence in plague |
| ichB (Yp) | Citramalyl-CoA lyase in Y. pestis | Part of the itaconate degradation operon |
| mch (Bxe) | Mesaconase/class I fumarase in Burkholderia xenovorans | Related activity for mesaconate utilization |
| ichA (P. putida) | Itaconyl-CoA hydratase in Pseudomonas putida | Model for itaconate degradation gene cluster |
| ichB (P. putida) | Citramalyl-CoA lyase in P. putida | Methylsuccinate utilization |
| tesB | Thioesterase involved in itaconate production | Engineering target for itaconate yield |
| paaZ | Aldehyde dehydrogenase in phenylacetate pathway | May interact with itaconate metabolism |
| gabD | Succinate-semialdehyde dehydrogenase | Potential bypass in itaconate degradation |
| acuA | Acetate utilization | Linked to acetyl-CoA production from itaconate |
| pta | Phosphotransacetylase | Acetyl-CoA metabolism |
| ackA | Acetate kinase | Acetyl-CoA metabolism |
| glcB | Malate synthase | Glyoxylate shunt, may affect itaconate metabolism |
| icl (aceA) | Isocitrate lyase | Glyoxylate shunt, linked to itaconate |
| ms (aceB) | Malate synthase | Glyoxylate shunt |
How Is itaconyl-CoA hydratase activity Regulated?
The expression of itaconyl-CoA hydratase is often regulated as part of the itaconate degradation gene cluster, which is induced in the presence of itaconate. In Pseudomonas aeruginosa, the cluster is controlled by a LysR-type transcriptional regulator, and its expression is upregulated when itaconate is available. In Mycobacterium tuberculosis, the itaconate degradation genes are induced during infection, likely in response to host-derived itaconate. Additionally, the activity may be regulated by substrate availability and by the redox state of the cell, as the reaction involves a hydration step.
itaconyl-CoA hydratase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| ichA (Yersinia pestis) | Plague virulence | Mouse infection model, KO mutants |
| ichA (Pseudomonas aeruginosa) | Chronic wound and lung infections | Murine pneumonia model, KO mutants |
| icl1 (Mycobacterium tuberculosis) | Tuberculosis | Macrophage infection, KO mutants |
| ichA (Burkholderia xenovorans) | Environmental persistence | Growth on mesaconate, KO mutants |
| tesB (E. coli) | Itaconate production | Metabolic engineering, overexpression |
Bacterial infections and virulence
Itaconyl-CoA hydratase activity is critical for the virulence of several bacterial pathogens. In Yersinia pestis, deletion of the itaconate degradation genes reduces virulence in a mouse model of plague. Similarly, in Pseudomonas aeruginosa, the enzyme contributes to itaconate catabolism and may support chronic infections. Mycobacterium tuberculosis also relies on this activity for survival in macrophages.
Host-pathogen metabolic interactions
Itaconate is produced by macrophages as part of the immune response, and its degradation by bacterial itaconyl-CoA hydratase represents a metabolic arms race. Understanding this interaction may reveal new therapeutic strategies that target bacterial metabolism without affecting host cells.
Metabolic engineering and biotechnology
The enzyme is relevant to the production of itaconate and its derivatives. Engineering thioesterases to drive itaconate production via its degradation scheme has been explored, highlighting the biotechnological potential of manipulating this activity.
From itaconyl-CoA hydratase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of itaconyl-CoA hydratase reduce virulence? | Knockout mutant in Yersinia pestis or Pseudomonas aeruginosa |
| What is the catalytic mechanism? | Point mutations in active-site residues, kinetic assays |
| Can the enzyme be used for itaconate production? | Overexpression in E. coli with thioesterase engineering |
| How is the gene cluster regulated? | Transcriptional reporter fusions, knock-in of promoters |
| Does the enzyme interact with citramalyl-CoA lyase? | Tagged knock-in for co-immunoprecipitation |
| Is the activity essential for growth on itaconate? | Knockout and growth phenotyping |
How to Study the itaconyl-CoA hydratase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| UV spectrophotometry | Itaconyl-CoA consumption | Enzyme kinetics |
| Coupled enzyme assay | Citramalyl-CoA formation | Activity screening |
| X-ray crystallography | Three-dimensional structure | Active site mapping |
| CRISPR knockout | Gene function in vivo | Virulence studies |
| Metabolomics | Intracellular metabolite levels | Pathway flux |
| qRT-PCR | Gene expression | Regulation studies |
| Western blot | Protein levels | Expression analysis |
| Isothermal titration calorimetry | Binding affinity | Substrate specificity |
Enzyme kinetics and activity assays
Itaconyl-CoA hydratase activity can be measured spectrophotometrically by monitoring the decrease in absorbance of itaconyl-CoA at 260 nm or by coupled assays with citramalyl-CoA lyase. These assays are used to determine kinetic parameters and to test inhibitors.
Structural biology
X-ray crystallography and cryo-EM have been used to solve the structure of itaconyl-CoA hydratase from Pseudomonas aeruginosa, revealing the active site and substrate binding mode. These structures guide mutagenesis and inhibitor design.
Bacterial genetics and CRISPR
CRISPR-Cas9 or homologous recombination can be used to generate knockout mutants in the itaconyl-CoA hydratase gene to study its role in itaconate degradation and virulence. Complementation with wild-type or mutant alleles allows structure-function analysis.
Metabolomics and flux analysis
Metabolomic profiling of bacterial cultures grown on itaconate can reveal the accumulation or depletion of pathway intermediates, confirming the role of the enzyme. Isotope tracing can quantify flux through the pathway.
How CRISPR Can Be Used to Study GO:0050011 itaconyl-CoA hydratase activity
Knockout
CRISPR knockout of the itaconyl-CoA hydratase gene (e.g., ichA) in Pseudomonas aeruginosa or Yersinia pestis abolishes itaconate degradation and attenuates virulence in infection models. These mutants are valuable for identifying the enzyme's contribution to pathogenesis.
Point Mutation
Point mutations in catalytic residues (e.g., the conserved glutamate) can be introduced via CRISPR to dissect the mechanism and to test whether hydratase activity is essential for function. Such mutants can be complemented with wild-type enzyme to confirm specificity.
Knock-in
Knock-in of epitope tags (e.g., FLAG or HA) at the endogenous locus allows for protein purification and interaction studies, such as co-immunoprecipitation with citramalyl-CoA lyase. This approach preserves native regulation.
Overexpression
Overexpression of itaconyl-CoA hydratase in E. coli or other hosts can be used for biochemical characterization, structural studies, and metabolic engineering to enhance itaconate degradation or production.
How EDITGENE Supports itaconyl-CoA hydratase activity Research
Researchers studying itaconyl-CoA hydratase activity-related genes often need to determine whether a candidate gene is causally involved in itaconate metabolism, bacterial virulence, or metabolic engineering. EDITGENE provides a comprehensive suite of CRISPR 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 itaconyl-CoA hydratase activity research.
Frequently Asked Questions About itaconyl-CoA hydratase activity
What is itaconyl-CoA hydratase activity?
It is a molecular function (GO:0050011) that catalyzes the reversible conversion of itaconyl-CoA to citramalyl-CoA, a step in bacterial itaconate degradation.
What genes are involved in itaconyl-CoA hydratase activity?
Genes include ichA in Pseudomonas aeruginosa and Yersinia pestis, and icl1 in Mycobacterium tuberculosis, often clustered with citramalyl-CoA lyase genes.
Which organisms have itaconyl-CoA hydratase?
It is found in bacteria such as Pseudomonas aeruginosa, Mycobacterium tuberculosis, Yersinia pestis, and Burkholderia xenovorans.
What is the reaction catalyzed by itaconyl-CoA hydratase?
The enzyme catalyzes citramalyl-CoA = itaconyl-CoA + H2O, a reversible hydration/dehydration.
Why is itaconyl-CoA hydratase important for infection?
It allows pathogens to degrade host-derived itaconate, an antimicrobial metabolite, thereby promoting survival and virulence.
How can I study itaconyl-CoA hydratase activity?
You can use enzyme assays, structural biology, CRISPR knockouts, and metabolomics to study its function and regulation.
Is itaconyl-CoA hydratase a drug target?
Yes, it is considered a potential anti-virulence target because inhibiting it could disarm pathogens without killing them.
What is the relationship between itaconyl-CoA hydratase and citramalyl-CoA lyase?
They are sequential enzymes in the itaconate degradation pathway; the hydratase produces citramalyl-CoA, which the lyase cleaves into acetyl-CoA and pyruvate.
Can itaconyl-CoA hydratase be used in biotechnology?
Yes, it can be engineered to manipulate itaconate production or degradation for bioproduct synthesis.
What model systems are used to study itaconyl-CoA hydratase?
Common models include Pseudomonas aeruginosa, Mycobacterium tuberculosis, Yersinia pestis, and Burkholderia xenovorans, using genetic knockouts and biochemical assays.
Conclusion
Itaconyl-CoA hydratase activity (GO:0050011) is a key enzymatic step in bacterial itaconate degradation, with significant implications for host-pathogen interactions, infectious disease, and metabolic engineering. Structural and genetic studies have elucidated its mechanism and its partnership with citramalyl-CoA lyase, providing a foundation for inhibitor development and biotechnological applications. Continued research using CRISPR and other advanced tools will further illuminate its roles and potential.
References
- 1. Huang Q et al.. 2024. Structural and functional characterization of itaconyl-CoA hydratase and citramalyl-CoA lyase involved in itaconate metabolism of Pseudomonas aeruginosa.. Structure 32(7):941-952.e3 PMID: 38677288
- 2. Priya M et al.. 2025. Itaconate mechanism of action and dissimilation in Mycobacterium tuberculosis.. Proc Natl Acad Sci U S A 122(4):e2423114122 PMID: 39841148
- 3. Kronen M et al.. 2015. Mesaconase Activity of Class I Fumarase Contributes to Mesaconate Utilization by Burkholderia xenovorans.. Appl Environ Microbiol 81(16):5632-8 PMID: 26070669
- 4. Gonner L et al.. 2025. Pseudomonadal itaconate degradation gene cluster encodes enzymes for methylsuccinate utilization.. Commun Biol 8(1):1099 PMID: 40707647
- 5. Wang RS et al.. 2024. Engineering thioesterase as a driving force for novel itaconate production via its degradation scheme.. Metab Eng Commun 19:e00246 PMID: 39224858
- 6. Sasikaran J et al.. 2014. Bacterial itaconate degradation promotes pathogenicity.. Nat Chem Biol 10(5):371-7 PMID: 24657929
