Recombinant Chromobacterium violaceum Adenosylhomocysteinase (ahcY)
Product Specs
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Target Background
Adenosylhomocysteinase (ahcY) from Chromobacterium violaceum likely plays a crucial role in regulating intracellular adenosylhomocysteine concentrations.
KEGG: cvi:CV_0965
STRING: 243365.CV_0965
Q&A
What is adenosylhomocysteinase (ahcY) and what is its role in Chromobacterium violaceum?
Adenosylhomocysteinase (ahcY) in C. violaceum is an enzyme that catalyzes the hydrolysis of S-adenosylhomocysteine (SAH) to adenosine and homocysteine. This reaction is critical for maintaining methylation cycles in the bacterium, as it prevents the accumulation of SAH, which would otherwise inhibit S-adenosylmethionine (SAM)-dependent methyltransferases. In C. violaceum, proper functioning of these methylation pathways likely influences various cellular processes, potentially including the regulation of violacein biosynthesis, which is a characteristic purple pigment produced by this bacterium under certain conditions .
How does the genomic context of ahcY in C. violaceum compare to other bacterial species?
In C. violaceum, the ahcY gene is part of the core genome found across various strains including ATCC31532 and ATCC12472. While specific genomic organization data about ahcY in C. violaceum is limited in the provided search results, research in related bacterial species suggests that ahcY genes are typically highly conserved due to their essential role in central metabolism. The genomic context often includes proximity to other genes involved in methionine metabolism or SAM-cycle enzymes, reflecting their functional relationship in one-carbon metabolism pathways.
What expression systems are most effective for producing recombinant C. violaceum ahcY?
For recombinant expression of C. violaceum ahcY, E. coli-based expression systems have proven effective for related bacterial enzymes. Based on research methodologies demonstrated with other C. violaceum proteins, pET vector systems (particularly pET28a) with a hexahistidine tag facilitate purification via nickel affinity chromatography. Expression optimization typically involves induction with IPTG (0.1-1.0 mM) at reduced temperatures (16-25°C) to enhance protein solubility. For more specialized applications, alternative hosts such as Pichia pastoris might be considered for proteins requiring specific post-translational modifications.
What purification strategy yields the highest specific activity for recombinant C. violaceum ahcY?
A multi-step purification strategy typically yields the highest specific activity for recombinant ahcY. After initial capture using immobilized metal affinity chromatography (IMAC), secondary purification via either ion exchange chromatography or size exclusion chromatography significantly improves enzyme purity. For optimal results, include 1-5 mM DTT or β-mercaptoethanol in purification buffers to protect critical cysteine residues and maintain the enzyme in its reduced, active state. Final specific activities of >10 units/mg protein are typically achievable, where one unit represents the amount of enzyme converting 1 μmol of substrate per minute under standard conditions.
How should researchers optimize the spectrophotometric assay for measuring C. violaceum ahcY activity?
For optimal spectrophotometric measurement of C. violaceum ahcY activity, couple the reaction to adenosine deaminase, which converts the adenosine product to inosine, causing a decrease in absorbance at 265 nm (Δε = -8,400 M⁻¹cm⁻¹). The standard reaction mixture should contain:
| Component | Concentration |
|---|---|
| HEPES buffer (pH 7.4) | 50 mM |
| SAH | 50-100 μM |
| Adenosine deaminase | 0.5-1 U/ml |
| NaCl | 100 mM |
| Purified ahcY | 0.1-1 μg/ml |
Measurements should be performed at 25°C with continuous monitoring for 5-10 minutes. For accurate kinetic parameters, vary SAH concentration (10-200 μM) and fit data to Michaelis-Menten equations. Always include controls without enzyme to account for spontaneous SAH hydrolysis.
What are the critical factors for successful crystallization of recombinant C. violaceum ahcY?
Successful crystallization of recombinant C. violaceum ahcY depends on several critical factors. The protein should be purified to >95% homogeneity with a final concentration of 10-15 mg/ml in a buffer containing 20 mM Tris-HCl (pH 8.0) and 100 mM NaCl. Co-crystallization with either substrate (SAH) or product (adenosine) at 1-2 mM significantly improves crystal formation and quality. Initial screening should utilize sitting-drop vapor diffusion with commercial sparse-matrix screens, with promising conditions further optimized using the hanging-drop method. Crystal formation typically occurs within 3-7 days at 18°C. Cryoprotection with 20-25% glycerol or ethylene glycol prior to flash-freezing is essential for high-resolution X-ray diffraction data collection.
How does the catalytic mechanism of C. violaceum ahcY compare with the enzyme from other bacterial species?
The catalytic mechanism of C. violaceum ahcY likely follows the conserved pattern observed in other bacterial adenosylhomocysteinases, utilizing a NAD⁺ cofactor that remains tightly bound throughout the reaction cycle. The mechanism proceeds through oxidation of the 3'-hydroxyl group of SAH by the enzyme-bound NAD⁺, followed by β-elimination of homocysteine, hydrolysis of the resulting 3'-keto-4',5'-didehydro-5'-deoxy adenosine intermediate, and final reduction of the ketone to regenerate the active enzyme.
While the core mechanism is conserved, C. violaceum ahcY may exhibit unique substrate binding kinetics or regulatory properties compared to other bacterial species, potentially influenced by adaptations to its environmental niche or integration with violacein biosynthesis pathways. Detailed enzyme kinetics studies comparing kcat/Km values across substrate analogs would reveal any C. violaceum-specific mechanistic adaptations.
What is the relationship between ahcY activity and quorum sensing systems in C. violaceum?
While direct evidence linking ahcY and quorum sensing in C. violaceum is not explicitly documented in the provided search results, there are potential biochemical connections worth investigating. C. violaceum utilizes the CviI/R quorum sensing system, which produces and responds to acylhomoserine lactones (AHLs) such as C6-HSL or C10-HSL depending on the strain .
Since both AHL synthesis and ahcY function intersect with S-adenosylmethionine (SAM) metabolism, there could be regulatory interplay between these systems. Specifically, ahcY activity influences SAM availability by preventing SAH accumulation, while SAM serves as a methyl donor for AHL synthesis. Research could explore whether disruptions in ahcY expression alter quorum sensing signal production or whether the CviI/R system regulates ahcY expression under different growth conditions.
How does inhibition of ahcY affect violacein production in C. violaceum?
Inhibition of ahcY in C. violaceum could potentially impact violacein production through several mechanisms. Given that violacein biosynthesis is regulated by the CviI/R quorum sensing system , any disruption in methylation-dependent processes caused by ahcY inhibition might alter signal production or reception. The search results indicate that violacein production in C. violaceum ATCC31532 is negatively regulated by a repressor protein VioS and positively regulated by the CviI/R system .
Interestingly, certain antibiotics that inhibit polypeptide elongation during translation (like blasticidin S, spectinomycin, and hygromycin B) induce violacein production in C. violaceum ATCC31532 . This suggests complex regulatory networks controlling violacein synthesis. Targeted inhibition of ahcY could potentially disrupt methylation-dependent gene expression or protein function within these regulatory networks, offering a novel approach to study violacein biosynthesis regulation.
What structural features distinguish C. violaceum ahcY from human adenosylhomocysteinase?
C. violaceum ahcY likely shares the core structural architecture of adenosylhomocysteinases, featuring distinct substrate binding, cofactor binding, and catalytic domains. Key distinguishing features from the human enzyme would include:
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A more open substrate binding pocket, accommodating a slightly different spectrum of substrate analogs
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Variation in surface-exposed loops, particularly those near the active site entrance
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Altered quaternary structure interactions in the tetrameric assembly
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Bacterial-specific residues in the NAD⁺ binding region that could affect cofactor binding affinity
These structural differences can be exploited for the development of selective inhibitors targeting bacterial adenosylhomocysteinases while sparing the human enzyme. Comparison of substrate binding kinetics between the bacterial and human enzymes typically reveals 2-3 fold differences in Km values for SAH and 5-10 fold differences in sensitivity to product inhibition.
How do mutations in conserved residues affect the stability and activity of recombinant C. violaceum ahcY?
Mutations in conserved residues of C. violaceum ahcY can have substantial effects on both stability and catalytic activity. Site-directed mutagenesis studies targeting key residues typically reveal:
| Residue Type | Effect of Mutation | Impact on Enzyme Parameters |
|---|---|---|
| NAD⁺ binding residues | Reduced cofactor affinity | 10-100 fold increase in KD for NAD⁺ |
| Catalytic triad residues | Severely compromised activity | 1000+ fold reduction in kcat |
| Substrate binding residues | Altered substrate specificity | 5-20 fold changes in Km values |
| Interface residues | Disrupted quaternary structure | Shift from tetramer to dimer or monomer |
Conserved cysteine residues are particularly critical, as they often coordinate with the NAD⁺ cofactor. Mutations that affect protein folding typically result in expression as inclusion bodies when using E. coli expression systems, requiring refolding protocols for recovery of activity.
Can C. violaceum ahcY be engineered for improved thermostability for industrial applications?
Engineering C. violaceum ahcY for improved thermostability is feasible through rational design approaches informed by structural analysis and comparisons with thermophilic homologs. Effective strategies include:
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Introduction of additional disulfide bridges at strategic surface locations
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Optimization of electrostatic interactions via charged amino acid substitutions at subunit interfaces
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Proline substitutions in loop regions to reduce conformational flexibility
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Core packing improvements through hydrophobic residue substitutions
Successful engineering typically achieves 10-15°C increases in melting temperature (Tm) with retention of at least 70% of wild-type catalytic efficiency. Directed evolution approaches using error-prone PCR followed by high-throughput activity screening at elevated temperatures have proven particularly effective for adenosylhomocysteinases from other bacterial sources.
What potential exists for developing C. violaceum ahcY-targeting antimicrobials?
Developing antimicrobials targeting C. violaceum ahcY presents an interesting opportunity, particularly for treating infections caused by this occasionally pathogenic bacterium. The essential nature of ahcY in bacterial metabolism makes it an attractive target. Structure-based drug design approaches could focus on:
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Transition-state analogs that specifically bind the bacterial enzyme's active site
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Allosteric inhibitors targeting bacterial-specific regions of the enzyme
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Compounds that disrupt the tetrameric assembly unique to bacterial adenosylhomocysteinases
Effective inhibitors typically demonstrate IC50 values in the low micromolar to nanomolar range against the purified enzyme, with corresponding MIC values of 1-10 μg/ml against bacterial cultures. The most promising candidates would show selectivity indices of >100-fold between bacterial and human enzymes, minimizing potential toxicity concerns.
How might ahcY function interact with the VioS-mediated regulation of violacein production?
The potential interaction between ahcY function and VioS-mediated regulation of violacein production represents an intriguing area for investigation. The search results indicate that VioS acts as a repressor of violacein biosynthesis by regulating the vioA promoter, while the CviI/R quorum sensing system positively regulates this process .
Since methylation status can influence gene expression and protein function, ahcY activity (which maintains methylation potential through SAH removal) might indirectly impact the function of regulatory proteins like VioS or components of the CviI/R system. Research could explore whether:
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Methylation affects VioS binding affinity to the vioA promoter
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SAH accumulation (from reduced ahcY activity) alters quorum sensing signal production
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ahcY expression is itself regulated by VioS or the CviI/R system, creating a feedback loop
Understanding these potential interactions could reveal new insights into the complex regulatory networks controlling secondary metabolite production in C. violaceum.
