Recombinant Rhodopirellula baltica Dihydroxy-acid dehydratase (ilvD), partial
Description
Introduction to Dihydroxy-acid Dehydratase
Dihydroxy-acid dehydratase (DHAD) is an enzyme crucial in the biosynthesis of branched-chain amino acids, such as isoleucine and valine. It catalyzes the dehydration of 2,3-dihydroxyisovalerate to α-ketoisovalerate and 2,3-dihydroxymethylvalerate to α-ketomethylvalerate . The enzyme relies on an iron-sulfur (Fe-S) cluster for its catalytic activity and is classified under the EC number 4.2.1.9 .
Recombinant Rhodopirellula baltica Dihydroxy-acid Dehydratase (ilvD), Partial
Recombinant Rhodopirellula baltica Dihydroxy-acid dehydratase (ilvD), partial refers to a genetically engineered version of the DHAD enzyme derived from Rhodopirellula baltica, a marine bacterium known for its ecological importance and biotechnological potential . This recombinant enzyme is likely produced by expressing the ilvD gene in a suitable host organism to enhance its stability, yield, or activity for research or industrial applications.
Function and Mechanism
DHAD enzymes, including those from Rhodopirellula baltica, are involved in the biosynthesis of branched-chain amino acids. They catalyze the dehydration of dihydroxy acids to keto acids, a critical step in the metabolic pathway leading to isoleucine and valine production . The enzyme's active site typically contains a conserved serine residue that acts as a base in the catalytic process, and it requires a magnesium ion (Mg²⁺) for stabilization of the intermediate .
Research Findings and Applications
-
Biotechnological Applications: Recombinant DHAD enzymes can be used in biotechnological processes to enhance the production of branched-chain amino acids or related compounds like isobutanol .
-
Stability and Expression: Genetic engineering techniques can improve the stability and expression levels of DHAD in host organisms, making it more suitable for industrial applications .
-
Pharmaceutical Potential: As DHAD has no known mammalian orthologs, it presents a potential target for antimicrobial drug development, particularly against pathogens where this enzyme is essential for survival .
Table 1: Substrate and Product of DHAD Enzymes
| Substrate | Product |
|---|---|
| 2,3-Dihydroxyisovalerate | α-Ketoisovalerate |
| 2,3-Dihydroxymethylvalerate | α-Ketomethylvalerate |
Table 2: Characteristics of DHAD Enzymes
| Characteristic | Description |
|---|---|
| EC Number | 4.2.1.9 |
| Fe-S Cluster | [2Fe-2S] or [4Fe-4S] |
| Function | Dehydration of dihydroxy acids to keto acids |
| Importance | Essential for branched-chain amino acid biosynthesis |
Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your format preference in order notes for customized preparation.
Note: All proteins are shipped with standard blue ice packs unless dry ice is specifically requested. Advance notification is required for dry ice shipping, and additional fees will apply.
The tag type is determined during production. If a specific tag type is required, please inform us for preferential development.
Q&A
What is Rhodopirellula baltica and why is it significant for enzyme research?
Rhodopirellula baltica is a marine organism belonging to the phylum Planctomycetes, which are capable of surviving in a wide range of environments and are among the least-explored bacteria . This organism has gained significant attention due to its unique genomic features, including a set of specialized sulfatases and C1-metabolism genes with biotechnological potential .
R. baltica exhibits an intriguing life cycle with distinct morphological changes, transitioning from swarmer and budding cells in early exponential growth to rosette formations in stationary phase . These unique characteristics, combined with its adaptability to diverse environmental conditions, make R. baltica an excellent model organism for studying novel enzymatic systems, including dihydroxy-acid dehydratase.
What is dihydroxy-acid dehydratase (ilvD) and what is its metabolic role?
Dihydroxy-acid dehydratase (ilvD) is an enzyme that catalyzes the dehydration of dihydroxy acids in branched-chain amino acid biosynthesis pathways. This enzyme belongs to the broader class of dihydroxy-acid dehydratases (DHADs), which are recognized as excellent biocatalysts for the defunctionalization of biomass .
In metabolic pathways, ilvD catalyzes the penultimate step in the biosynthesis of essential branched-chain amino acids (valine, leucine, and isoleucine). Specifically, it converts 2,3-dihydroxyisovalerate to 2-ketoisovalerate in the valine/leucine pathway and 2,3-dihydroxy-3-methylvalerate to 2-keto-3-methylvalerate in the isoleucine pathway. As enzymes are proteins that speed up metabolism or chemical reactions in living organisms , ilvD plays a crucial role in amino acid metabolism, which is essential for protein synthesis and cellular function.
What are the optimal conditions for expressing recombinant proteins from R. baltica?
Based on successful expression studies with R. baltica proteins, the following conditions appear optimal:
Expression System: Escherichia coli has proven to be an effective host for the heterologous expression of R. baltica proteins. High-level production of recombinant proteins has been achieved for several R. baltica genes, including gpgS and mggA .
Sequence Verification: Careful attention must be paid to the gene annotation. In one documented case, the annotated sequence for R. baltica GpgS contained 80 additional amino acids at the N-terminus that lacked homology with known GpgSs, resulting in an inactive protein. Researchers identified a start codon 240 bp downstream from the annotated start site, and expression of this truncated version yielded an enzyme with the expected activity . This highlights the importance of verifying predicted gene models before expression.
Expression Conditions: While specific conditions for ilvD are not detailed in the available literature, the successful expression of other R. baltica enzymes suggests that standard E. coli expression protocols can be adapted for this organism's proteins.
What purification strategies are most effective for R. baltica recombinant enzymes?
Effective purification strategies for R. baltica enzymes include:
Initial Purification: Some R. baltica proteins expressed in E. coli (such as GpgS and MggA) have been reported to be produced in a nearly pure form, requiring minimal additional purification .
Activity Preservation: Careful consideration must be given to maintaining enzyme activity during purification. For example, attempts to further purify the MggB protein from R. baltica led to complete loss of activity, indicating sensitivity to certain purification conditions .
How does gene expression in R. baltica change throughout its growth cycle?
R. baltica exhibits distinct gene expression patterns throughout its growth cycle, as revealed by whole genome microarray studies:
Early Exponential Phase:
-
Culture dominated by swarmer and budding cells
-
Only about 2% of total genes show differential regulation, reflecting favorable nutritional conditions
-
Downregulation of genes associated with amino acid metabolism, carbohydrate metabolism, energy production, and DNA replication in mid-exponential phase compared to early exponential phase
Transition to Stationary Phase:
-
Morphological shift to single cells, budding cells, and rosette formations
-
Upregulation of glutamate dehydrogenase, potentially for adapting cell wall composition to less favorable conditions
-
Induction of stress response genes including glutathione peroxidase, thioredoxin, bacterioferritin comigratory protein, universal stress protein, and chaperones
-
Differential regulation of various dehydrogenases, hydrolases, and reductases for metabolic adaptation
Stationary Phase:
-
Dominated by rosette formations
-
Increased expression of genes associated with energy production, amino acid biosynthesis, signal transduction, transcriptional regulation, stress response, and protein folding
-
Repression of genes involved in carbon metabolism, translation control, and energy production
-
Modification of cell wall composition, including increased export of polysaccharides and upregulation of cell membrane genes
This expression pattern information provides context for understanding how enzymes like ilvD might be regulated throughout R. baltica's life cycle.
What is the relationship between R. baltica's growth phases and enzyme activity?
The relationship between growth phases and enzyme activity in R. baltica can be illustrated by the following data from life cycle studies:
Table 1: Regulated Genes During R. baltica Growth Phases
| Number of regulated genes | 62 h vs. 44 h | 82 h vs. 62 h | 96 h vs. 82 h | 240 h vs. 82 h |
|---|---|---|---|---|
| Total (%)* | 149 (2%) | 90 (1%) | 235 (3%) | 863 (12%) |
| Encoding hypothetical proteins (%)** | 84 (56%) | 40 (44%) | 139 (59%) | 499 (58%) |
*Percentage relative to the total number of 7325 open reading frames (ORFs) annotated in R. baltica genome.
**Percentage relative to the total number of regulated genes.
This data demonstrates a significant increase in gene regulation during transition to stationary phase, particularly at 240 hours. The expression patterns suggest that metabolic enzymes, potentially including ilvD, undergo regulatory changes to adapt to nutrient limitation and changing environmental conditions.
What are the expected catalytic properties of R. baltica ilvD based on other DHADs?
While specific information about R. baltica ilvD is limited in the available literature, insights can be drawn from characterized DHADs such as the one from Sulfolobus solfataricus (SsDHAD):
Temperature and pH Optima: SsDHAD has an optimal temperature of 77°C and optimal pH of 6.2 . As R. baltica is a mesophilic marine bacterium rather than a thermophile, its ilvD would likely have a lower temperature optimum, possibly in the 25-40°C range.
Substrate Specificity: DHADs typically show specificity for dihydroxy acids involved in branched-chain amino acid biosynthesis, but some can act on alternative substrates such as D-gluconate, as demonstrated for SsDHAD .
Inhibitor Sensitivity: SsDHAD showed inhibition by alcohols with IC50 values of 15% (v/v) for ethanol, 4.5% (v/v) for butanol, and 4% (v/v) for isobutanol . R. baltica ilvD might show different sensitivity patterns based on its natural environment.
Cofactor Requirements: DHADs typically require iron-sulfur clusters as cofactors for catalytic activity. The assembly and stability of these cofactors would be critical for R. baltica ilvD function.
How might the marine origin of R. baltica influence ilvD properties?
The marine origin of R. baltica likely influences several properties of its enzymes, including ilvD:
Salt Tolerance: R. baltica demonstrates salt resistance , suggesting its enzymes may have evolved structural adaptations for function in marine environments.
Thermal Stability: As a marine mesophile, R. baltica ilvD would likely show optimal activity at moderate temperatures rather than extremes.
Pressure Adaptation: Marine organisms often possess enzymes adapted to hydrostatic pressure, which might affect the structural stability and catalytic properties of R. baltica ilvD.
Ecological Context: R. baltica has been isolated from diverse environments including contaminated mangrove soils , suggesting potential adaptations to various environmental conditions, which might be reflected in enzyme properties.
What are the challenges in expressing functional recombinant ilvD from R. baltica?
Researchers working with recombinant R. baltica ilvD may encounter several challenges:
Gene Annotation Issues: As demonstrated with R. baltica GpgS , incorrect gene annotation can lead to expression of non-functional proteins. Careful verification of the predicted start codon and other sequence features is essential.
Protein Folding and Solubility: E. coli may not provide the optimal cellular environment for proper folding of R. baltica proteins, potentially leading to inclusion body formation or inactive enzymes.
Cofactor Assembly: If R. baltica ilvD requires iron-sulfur clusters like other DHADs, ensuring proper cofactor assembly in the recombinant system would be crucial for activity.
Activity Preservation: As observed with MggB from R. baltica , maintaining enzyme activity during purification can be challenging and may require careful optimization of protocols.
Expression Optimization: Determining optimal conditions for expression (temperature, induction time, media composition) would likely require systematic optimization to balance protein yield and functionality.
How can R. baltica ilvD be integrated into multi-enzyme cascade reactions?
Integrating R. baltica ilvD into multi-enzyme cascade reactions offers several potential advantages and applications:
Biomass Conversion: DHADs are excellent biocatalysts for biomass defunctionalization , suggesting R. baltica ilvD could be incorporated into enzyme cascades for converting biomass to valuable chemicals.
Compatibility Considerations: The successful integration would require matching the optimal conditions (pH, temperature, buffer system) with other enzymes in the cascade.
Potential Cascade Applications:
-
Conversion of carbohydrates to branched-chain alcohols
-
Production of non-natural amino acid derivatives
-
Synthesis of chiral intermediates for pharmaceutical compounds
Immobilization Strategies: For continuous processes, immobilization of R. baltica ilvD could enhance stability and enable reuse, though immobilization methods would need to preserve the enzyme's active site and cofactor.
One-Pot Reaction Design: Co-expression or co-immobilization with complementary enzymes could enable efficient one-pot transformations, potentially including aromatics metabolism given R. baltica's capability in this area .
How might R. baltica ilvD contribute to survival in changing environments?
Based on R. baltica's gene expression patterns and environmental adaptability, ilvD likely contributes to survival in changing environments through several mechanisms:
Nutrient Limitation Response: During nutrient depletion, R. baltica shows differential regulation of metabolic enzymes . If ilvD follows similar patterns, its activity might be modulated to conserve resources or redirect metabolic flux.
Stress Adaptation: R. baltica induces various stress response genes during the transition to stationary phase . The regulation of ilvD might be coordinated with these stress responses to maintain essential metabolic functions under adverse conditions.
Cell Morphology Transitions: As R. baltica transitions between different morphological states throughout its life cycle , ilvD activity might be adjusted to support the metabolic requirements of these different cellular forms.
Environmental Sensing: The regulation of ilvD might be integrated with environmental sensing mechanisms to adapt amino acid metabolism to changing conditions, similar to the adaptation observed for other metabolic genes in R. baltica .
What is the role of R. baltica ilvD in nitrogen metabolism and amino acid synthesis?
While specific information about R. baltica ilvD's role in nitrogen metabolism is limited in the available literature, several inferences can be made:
Branched-Chain Amino Acid Synthesis: As a dihydroxy-acid dehydratase, ilvD would play a key role in the biosynthesis of branched-chain amino acids (valine, leucine, isoleucine), which are essential components of proteins.
Nitrogen Conservation: During nutrient limitation, the regulation of ilvD could help conserve nitrogen by modulating amino acid biosynthesis according to cellular needs.
Metabolic Integration: The function of ilvD would be integrated with broader nitrogen metabolism pathways, including potential connections to glutamate metabolism, which was found to be regulated during R. baltica's growth cycle through glutamate dehydrogenase .
Adaptation to Nitrogen Sources: R. baltica's ability to thrive in diverse environments suggests adaptability to different nitrogen sources, which might involve coordinated regulation of amino acid biosynthesis enzymes including ilvD.
How does R. baltica ilvD compare to DHADs from other marine organisms?
A comparative analysis of R. baltica ilvD with DHADs from other marine organisms would consider:
Sequence Homology: Analysis of sequence conservation in catalytic and substrate-binding regions compared to DHADs from other marine bacteria.
Salt Tolerance Mechanisms: Structural features that confer salt tolerance might differ between DHADs from various marine bacteria depending on their specific ecological niches.
Evolutionary Adaptations: R. baltica belongs to the Planctomycetes, an evolutionarily distinct phylum , suggesting its ilvD might have unique features compared to enzymes from more well-studied marine bacteria.
Substrate Preferences: Potential differences in substrate specificity that reflect the metabolic needs of different marine organisms in their respective ecological niches.
Cofactor Requirements: Variations in iron-sulfur cluster assembly or stability mechanisms that might reflect adaptations to different marine environments.
What structural features might distinguish R. baltica ilvD from well-characterized DHADs?
Based on what is known about R. baltica and DHADs in general, several structural features might distinguish R. baltica ilvD:
Active Site Architecture: The specific arrangement of catalytic residues might be optimized for the organism's particular metabolic needs and environmental conditions.
Surface Charge Distribution: As a marine organism, R. baltica's enzymes might have surface charge distributions that favor stability in saline environments.
Flexible Regions: The enzyme might contain flexible loops or domains that contribute to substrate binding or product release, potentially with unique characteristics compared to well-studied DHADs.
Oligomeric State: The quaternary structure of R. baltica ilvD might differ from other DHADs, potentially affecting stability, allosteric regulation, or substrate channeling.
Cofactor Binding: The mode of iron-sulfur cluster binding and protection might have unique features adapted to R. baltica's cellular environment.
What are promising strategies for improving the catalytic efficiency of R. baltica ilvD?
Several approaches could be explored to enhance the catalytic properties of R. baltica ilvD:
Directed Evolution: Implementing iterative rounds of mutation and selection to identify variants with improved activity, stability, or substrate specificity.
Structure-Guided Mutagenesis: Using computational modeling and structural analyses to identify key residues for targeted mutagenesis to enhance specific properties.
Domain Swapping: Creating chimeric enzymes by exchanging domains between R. baltica ilvD and DHADs from other organisms to combine beneficial properties.
Cofactor Engineering: Optimizing the iron-sulfur cluster assembly or introducing modifications to enhance cofactor stability.
Protein Surface Engineering: Modifying surface residues to improve solubility, stability, or resistance to inhibitors while maintaining the active site architecture.
How might genomic and proteomic approaches advance our understanding of R. baltica ilvD regulation?
Comprehensive genomic and proteomic approaches could provide valuable insights into R. baltica ilvD regulation:
Transcriptomic Analysis: Expanding on existing microarray data with RNA-seq studies to precisely quantify ilvD expression under various conditions.
Proteomics Profiling: Quantitative proteomics to determine ilvD protein levels throughout the growth cycle and under different environmental conditions.
Promoter Analysis: Characterization of the ilvD promoter region to identify regulatory elements that control gene expression.
Interactome Mapping: Identifying protein-protein interactions involving ilvD to uncover potential regulatory partners or metabolic complexes.
Comparative Genomics: Analyzing the genomic context of ilvD across different R. baltica strains and related organisms to identify conserved regulatory features.
