Recombinant Chromobacterium violaceum D-amino acid dehydrogenase small subunit (dadA)
Description
Functional Role in D-Amino Acid Metabolism
DadA catalyzes the oxidative deamination of D-amino acids, contributing to nitrogen metabolism and energy production. Key functional insights include:
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Substrate Specificity: Homologous DadA enzymes (e.g., in Pseudomonas aeruginosa) exhibit broad activity across D-amino acids except D-Glu/D-Gln .
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Genetic Linkage: In Escherichia coli, the dadA gene is closely linked to dadR (regulator) and hemA (heme biosynthesis), with cotransduction frequencies exceeding 90% .
Recombinant Expression and Applications
Recombinant DadA from C. violaceum is produced in heterologous systems for:
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Mechanistic Studies: Structural analysis of substrate binding pockets and catalytic residues (e.g., Arg 64, Lys 269, Tyr 309 in homologous enzymes) .
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Biotechnological Optimization: Proteomic studies highlight challenges in recombinant protein stability, requiring cofactors like FMN for proper folding .
Research Implications
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Metabolic Engineering: DadA’s role in D-alanine catabolism provides insights into pathways for synthesizing noncanonical amino acids .
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Interspecies Interactions: While not directly linked to violacein biosynthesis, DadA’s regulatory neighbors (e.g., vioABCDE operon) suggest overlapping quorum-sensing mechanisms in C. violaceum .
Product Specs
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Target Background
Oxidative deamination of D-amino acids.
KEGG: cvi:CV_1914
STRING: 243365.CV_1914
Q&A
Basic Research Questions
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What are the optimal conditions for expressing recombinant C. violaceum dadA?
Based on available research data, the following expression conditions have proven effective:
The protein typically shows good expression in E. coli with >85% purity achievable using standard affinity chromatography techniques . Key considerations include the addition of appropriate cofactors during purification and maintaining reducing conditions to preserve enzyme activity.
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How does C. violaceum dadA compare to similar enzymes in other bacterial species?
D-amino acid dehydrogenase from C. violaceum shares significant structural and functional similarities with homologous enzymes in other bacterial species, though with distinct characteristics:
The E. coli K12 D-amino acid dehydrogenase is particularly well-characterized and shows inducibility by alanine and repressibility by glucose, similar to the C. violaceum enzyme . The broad substrate specificity and membrane localization patterns are conserved features among these homologs, suggesting evolutionary conservation of function.
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What is the relationship between Chromobacterium violaceum pathogenicity and its enzymes?
C. violaceum is predominantly an environmental bacterium found in soil and water in tropical regions, but it can act as an opportunistic pathogen with a high fatality rate in humans . While dadA itself has not been directly implicated in pathogenicity, understanding C. violaceum's enzymes provides insight into its virulence mechanisms:
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C. violaceum possesses Type III Secretion Systems (T3SSs) that play a pivotal role in host interactions and virulence
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The bacterium produces violacein, a purple pigment with antimicrobial properties that may contribute to its ecological competitive advantage and possibly its pathogenicity
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Quorum sensing systems regulate virulence factor production, including enzymes involved in bacterial communication and host interaction
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Infection typically manifests as skin lesions, sepsis, and liver abscesses, with the pathogen showing unexpected antibiotic resistance patterns
Research into C. violaceum's enzymes, including dadA, contributes to the broader understanding of its metabolism and potential virulence factors, which could inform therapeutic strategies against this rare but dangerous pathogen.
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