Recombinant Gloeobacter violaceus Dihydroxy-acid dehydratase (ilvD), partial
Description
Introduction to Recombinant Gloeobacter violaceus Dihydroxy-acid Dehydratase (ilvD), Partial
Dihydroxy-acid dehydratase (DHAD), also known as acetohydroxy acid dehydratase, is an enzyme that catalyzes the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate and 2,3-dihydroxymethylvalerate to α-ketomethylvalerate . It is classified as E.C. 4.2.1.9 and is a crucial component of the biosynthetic pathways that produce valine, isoleucine, leucine, and pantothenic acid (vitamin B5) . The enzyme is present in various organisms, including bacteria, fungi, and plants, but not humans .
Gloeobacter violaceus is a cyanobacterium known for lacking thylakoids and having a unique phycobilisome morphology, believed to be primitive . The "partial" designation typically indicates that the enzyme is either a fragment of the complete protein or that it has undergone some modification or alteration from its native state.
Function and Mechanism
DHAD enzymes are essential for synthesizing branched-chain amino acids. They facilitate the dehydration of 2,3-dihydroxy-3-methylvalerate and 2,3-dihydroxyisovalerate, producing corresponding ketoacids like 2-keto-3-methylvalerate and 2-ketoisovalerate .
Importance of ilvD in Microorganisms
The ilvD gene encodes DHAD, which is vital for synthesizing branched-chain amino acids. Disruption or mutation of ilvD can lead to auxotrophy, where the organism cannot produce specific amino acids and requires them for growth . For example, in Aspergillus fumigatus, deletion of AfIlv3A (a DHAD gene) results in a strain that requires isoleucine and valine supplementation for growth .
Expression and Purification
Recombinant DHADs are often expressed in heterologous systems like Escherichia coli to produce sufficient quantities for research . For example, the gloeorhodopsin gene from G. violaceus PCC7421 was expressed in E. coli . Similarly, a recombinant AfIlv3A protein from Aspergillus fumigatus was expressed in E. coli and purified, showing DHAD activity in in vitro assays .
Example of Expression and Purification of Gloeorhodopsins
| Step | Description |
|---|---|
| Induction | Gloeorhodopsins were expressed under the lacUV5 promoter in E. coli strain UT5600. Cells were induced with 1 mM IPTG for 6 h at 35°C, and all-trans retinal was added to a final concentration of 5∼10 µM. |
| Membrane Solubilization | Membranes were solubilized with 1% n-Dodecyl-β-D-Maltopyranoside (DDM). |
| Purification | Purification was performed using Ni2+-NTA agarose (Qiagen). |
| Final Sample Composition | Purified samples contained 0.02% DDM. |
Biochemical Assays
Activity assays are crucial for confirming the function of DHAD. For instance, recombinant AfIlv3A from A. fumigatus displayed DHAD activity with L-threonate, yielding a specific activity of 18 µmol min-1 mg-1 . Michaelis-Menten kinetics were observed, with a Km for threonate of 10.1 mM .
Relevance to Industrial Microbiology
Bacterial DHADs with a [2Fe-2S] cluster can be expressed as heterologous proteins in bacteria and yeast cells, providing DHAD activity for converting 2,3-dihydroxyisovalerate to α-ketoisovalerate or 2,3-dihydroxymethylvalerate to α-ketomethylvalerate . This is relevant in synthesizing isobutanol and other compounds through pathways involving bacterial [2Fe-2S] DHAD activity .
Role in Virulence
In pathogenic microorganisms like Aspergillus fumigatus, DHAD plays a role in virulence . Mutants lacking AfIlv3A exhibit reduced virulence in murine infection models, highlighting the importance of branched-chain amino acid biosynthesis in fungal infections .
Product Specs
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Q&A
Given the lack of specific information on "Recombinant Gloeobacter violaceus Dihydroxy-acid dehydratase (ilvD), partial" in the search results, I will create a general FAQ collection that addresses common research scenarios related to recombinant enzymes, focusing on experimental design, data analysis, and methodological considerations. This approach will provide a framework for researchers working with similar enzymes.
Purification Strategies for Recombinant Proteins
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Q: What are effective methods for purifying recombinant dihydroxy-acid dehydratase from bacterial lysates?
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A: Common purification strategies include affinity chromatography (e.g., His-tag), size exclusion chromatography, and ion exchange chromatography. Choose methods based on the enzyme's properties and the presence of tags.
Enzyme Activity Assays
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Q: How can I measure the activity of recombinant dihydroxy-acid dehydratase?
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A: Use spectrophotometric assays to monitor substrate conversion or product formation. For dihydroxy-acid dehydratase, measure the conversion of dihydroxy-acids to α-keto-acids. Consider using coupled assays to enhance sensitivity.
Data Analysis and Interpretation
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Q: How do I analyze and interpret kinetic data from enzyme assays to understand the enzyme's efficiency?
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A: Use Michaelis-Menten kinetics to determine and . These parameters help assess enzyme efficiency and substrate affinity. Consider using software like GraphPad Prism for data fitting.
Addressing Data Contradictions
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Q: What steps should I take if my experimental results contradict previously published data on a similar enzyme?
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A: Re-evaluate experimental conditions, enzyme preparation, and assay methods. Consider factors like enzyme purity, buffer composition, and temperature. Repeat experiments with controlled variables to confirm findings.
Advanced Research Questions: Structural Insights
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Q: How can structural biology techniques help in understanding the mechanism of dihydroxy-acid dehydratase?
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A: Use X-ray crystallography or cryo-EM to determine the enzyme's structure. This can reveal substrate binding sites and catalytic mechanisms, providing insights into enzyme specificity and efficiency.
Collaborative Research and Interdisciplinary Approaches
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Q: How can interdisciplinary collaboration enhance research on recombinant enzymes like dihydroxy-acid dehydratase?
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A: Collaborate with biochemists, structural biologists, and computational biologists to integrate experimental and theoretical approaches. This can lead to a more comprehensive understanding of enzyme function and potential applications.
Example Data Table: Enzyme Activity Assay
| Substrate Concentration (mM) | Activity (μmol/min/mg) |
|---|---|
| 0.1 | 10 |
| 0.5 | 20 |
| 1.0 | 30 |
| 2.0 | 35 |
This table illustrates how enzyme activity can be measured at different substrate concentrations, helping to determine kinetic parameters.
Detailed Research Findings
While specific research findings on "Recombinant Gloeobacter violaceus Dihydroxy-acid dehydratase (ilvD), partial" are not available, general research in recombinant enzymes often focuses on optimizing expression conditions, improving purification protocols, and characterizing enzyme kinetics. Advanced studies may involve structural analysis to elucidate the enzyme's mechanism of action.
