Recombinant Escherichia coli O127:H6 Glycine cleavage system H protein (gcvH)
Description
Genomic Context and Protein Characteristics
The gcvH gene is part of the gcvTHP operon in E. coli O127:H6 strain E2348/69, a model enteropathogenic E. coli (EPEC) isolate . This operon encodes four proteins:
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GcvT: Aminomethyltransferase (41 kDa)
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GcvH: Lipoic acid carrier (12.7 kDa)
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GcvP: Glycine dehydrogenase (104 kDa)
Key genomic features of gcvH in E. coli O127:H6:
Biochemical Properties of Recombinant GcvH
Recombinant GcvH is produced in E. coli expression systems, often fused with affinity tags (e.g., His-tag) for purification . Key attributes include:
Structural and functional motifs:
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Lipoic acid-binding domain: Critical for shuttling aminomethyl groups between GcvP and GcvT .
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Signal peptide: Predicted cleavage site for periplasmic localization .
Physicochemical properties:
| Property | Value | Source |
|---|---|---|
| Molecular weight | ~12.7 kDa | |
| Isoelectric point (pI) | Predicted 5.2 (via ExPASy) | – |
| Thermal stability | Retains activity up to 45°C |
Role in Glycine Metabolism
GcvH facilitates the transfer of aminomethyl groups during glycine degradation (Fig. 1). Disruption of gcvH in Sinorhizobium fredii USDA257—a close relative of E. coli—abolished glycine cleavage activity, impairing nitrogen fixation in soybean symbionts . In E. coli O127:H6, this system likely influences metabolic adaptability in host environments.
Host-Pathogen Interactions
While gcvH is not directly linked to virulence factors like the locus of enterocyte effacement (LEE), its role in central metabolism may indirectly affect EPEC’s survival in the gut . For example:
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Glycine cleavage provides one-carbon units for folate metabolism, crucial for DNA synthesis .
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Metabolic byproducts (e.g., ammonia) could modulate host epithelial responses .
Research Applications
Recombinant GcvH is utilized in:
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Enzyme kinetics studies: Assessing decarboxylation efficiency under varying glycine concentrations .
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Metabolic engineering: Modulating one-carbon flux in synthetic biology platforms .
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Antibiotic target exploration: Screening inhibitors of bacterial glycine metabolism .
Challenges and Future Directions
Product Specs
Target Background
KEGG: ecg:E2348C_3156
Q&A
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What is the experimental design for studying the glycine cleavage system H protein in Escherichia coli O127:H6?
To investigate the glycine cleavage system H protein (gcvH), researchers typically employ a combination of genetic, biochemical, and structural biology techniques. A common approach includes:
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Gene Cloning and Expression: The gcvH gene is cloned into an expression vector suitable for Escherichia coli, followed by transformation and selection of competent cells.
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Protein Purification: The expressed protein is purified using affinity chromatography, often utilizing tags such as His-tag or GST for effective isolation.
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Functional Assays: Enzymatic activity is assessed through in vitro assays that measure the rate of glycine decarboxylation and synthesis under varying substrate concentrations.
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Mutagenesis Studies: Site-directed mutagenesis is performed to identify critical residues involved in the protein's function, followed by kinetic analysis to evaluate the impact of these mutations on enzyme activity.
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Structural Analysis: Techniques such as X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy may be used to elucidate the three-dimensional structure of gcvH and its interactions with other components of the glycine cleavage system .
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How can data contradictions in glycine cleavage system research be analyzed?
Analyzing data contradictions involves several methodological steps:
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Re-evaluation of Experimental Conditions: Researchers should systematically review experimental conditions such as temperature, pH, and substrate concentrations that may have influenced results.
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Statistical Analysis: Employing robust statistical methods to determine the significance of observed differences can clarify whether discrepancies are due to random variation or genuine biological differences.
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Replication Studies: Conducting independent replication studies can help confirm findings. If results consistently diverge, this may indicate a need for reevaluation of hypotheses or methodologies.
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Meta-Analysis: Aggregating data from multiple studies can provide a broader perspective on the issue, helping to identify trends or patterns that may not be evident in individual studies.
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Collaboration with Other Laboratories: Engaging with other research groups can facilitate cross-validation of findings and promote sharing of techniques that may resolve conflicting data .
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What advanced techniques are used to investigate the lipoylation process in gcvH?
Advanced techniques for studying lipoylation in gcvH include:
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Molecular Dynamics Simulations: These simulations allow researchers to model the dynamic interactions between gcvH and other proteins in the glycine cleavage system, providing insights into conformational changes during lipoylation.
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Mass Spectrometry: This technique is employed to analyze post-translational modifications, including lipoylation status of gcvH. It can detect specific modifications and quantify their abundance under different conditions.
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Cryo-Electron Microscopy: This method offers high-resolution structural data on protein complexes, allowing visualization of gcvH in its native state within the glycine cleavage system.
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In Vivo Labeling Experiments: Using isotopically labeled precursors can help trace the incorporation of lipoic acid into gcvH within living organisms, providing context on its physiological relevance.
These techniques together enhance understanding of how lipoylation affects the function and regulation of gcvH within metabolic pathways .
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What roles does gcvH play beyond its function in glycine metabolism?
Recent studies suggest that gcvH may have additional roles beyond its established function in glycine metabolism:
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Developmental Processes: Research indicates that mutations in gcvH can lead to embryonic lethality in model organisms, suggesting its involvement in crucial developmental pathways beyond amino acid metabolism .
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Protein Interactions: gcvH has been implicated in interactions with other mitochondrial proteins involved in metabolic pathways, indicating a potential role as a regulatory hub within cellular metabolism.
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Lipoylation Mechanism: The ability of gcvH to participate in lipoylation processes for other enzymes suggests it could influence broader metabolic functions related to energy production and nutrient utilization .
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How does the structure of gcvH influence its enzymatic activity?
The structure of gcvH significantly impacts its enzymatic activity through several mechanisms:
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Active Site Configuration: The arrangement of amino acids at the active site determines substrate binding efficiency and catalytic turnover rates. Structural studies can reveal how specific residues facilitate substrate interaction and product release.
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Conformational Flexibility: The dynamic nature of gcvH allows it to adopt different conformations necessary for interacting with other components in the glycine cleavage system. This flexibility is crucial for its function as a molecular shuttle.
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Lipoyl Arm Dynamics: The presence of a lipoyl swinging arm attached to gcvH plays a key role in transferring intermediates between enzymes. Structural insights into how this arm moves during catalysis can elucidate mechanisms underlying its function .
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