Recombinant Chromobacterium violaceum S-adenosylmethionine synthase (metK)

What is the primary function of S-adenosylmethionine synthase in Chromobacterium violaceum?

S-adenosylmethionine synthase (metK) in Chromobacterium violaceum catalyzes the formation of S-adenosylmethionine (AdoMet or SAM) from methionine and ATP. This enzyme (EC 2.5.1.6) is also known as methionine adenosyltransferase (MAT) . The reaction produces S-adenosylmethionine, which serves as the principal biological methyl donor in numerous cellular processes. In C. violaceum specifically, SAM plays a crucial role in quorum sensing pathways where it contributes to the conversion of fatty acids to AHLs (acyl-homoserine lactones) via the CviI synthase enzyme . This process ultimately influences the expression of the vioABCDE operon that governs violacein production, the characteristic purple pigment of this bacterium.

The significance of metK extends beyond basic metabolism, as methyl donation reactions are essential for numerous cellular processes including DNA methylation, protein modification, and various biosynthetic pathways that contribute to bacterial adaptation and virulence.

What are the optimal expression systems and conditions for producing recombinant C. violaceum metK?

The recombinant C. violaceum S-adenosylmethionine synthase can be successfully expressed in yeast expression systems, as indicated by the product information . When designing expression experiments, researchers should consider the following methodological approaches:

• Expression System Selection:

  • Yeast systems are documented to work effectively

  • E. coli systems may also be suitable using pET-based vectors with T7 promoters

  • Baculovirus-insect cell systems can be considered for higher yields of soluble protein

• Optimization Parameters:

  • Induction conditions: For IPTG-inducible systems, concentrations between 0.1-1.0 mM IPTG at OD600 of 0.6-0.8

  • Temperature: Lower post-induction temperatures (16-25°C) often improve solubility

  • Duration: 4-18 hours depending on temperature and expression system

• Fusion Tags:

  • Tag type will be determined during the manufacturing process

  • Common options include His6, GST, or MBP tags to facilitate purification

  • Consider TEV or Factor Xa protease cleavage sites if tag removal is necessary

For optimal results, pilot expressions should be conducted to determine the best combination of these parameters for your specific experimental context. Expression levels should be monitored via SDS-PAGE and Western blotting using antibodies against the target protein or fusion tag.

What purification strategies yield the highest purity and activity for recombinant metK?

Purification of recombinant C. violaceum S-adenosylmethionine synthase should follow a multi-step approach to achieve high purity (>85% as indicated in product specifications) :

Table 1: Recommended Purification Protocol

During purification, researchers should:

• Monitor enzyme activity at each step using a spectrophotometric assay measuring SAM formation

• Verify protein identity through mass spectrometry or Western blotting

• Assess purity by SDS-PAGE and determine final concentration using Bradford or BCA assays

After purification, the protein should be stored properly to maintain activity. As indicated in the product information, avoiding repeated freeze-thaw cycles is critical, and working aliquots can be stored at 4°C for up to one week . For long-term storage, adding glycerol to a final concentration of 50% and storing at -20°C/-80°C is recommended, with an expected shelf life of 6 months for liquid form and 12 months for lyophilized form .

How can researchers evaluate the catalytic activity of purified metK enzyme?

The catalytic activity of purified C. violaceum S-adenosylmethionine synthase can be evaluated through several complementary approaches:

• Spectrophotometric Coupled Assays:

  • The formation of SAM can be coupled to reactions that produce measurable spectroscopic changes

  • For example, coupling to 5'-methylthioadenosine nucleosidase and adenine deaminase reactions allows monitoring at 265 nm

  • Calculate enzyme activity in μmol of SAM produced per minute per mg protein

• HPLC-based Product Quantification:

  • Separate reaction components (methionine, ATP, SAM, and Pi) using reverse-phase HPLC

  • Monitor SAM formation at 254 nm using appropriate standards

  • This method provides direct quantification without interference from coupled reactions

• Isothermal Titration Calorimetry:

  • Measure heat released during the enzymatic reaction

  • Determine thermodynamic parameters (ΔH, ΔS, ΔG) and binding constants

  • Particularly useful for comparing wild-type and mutant variants

When designing activity assays, researchers should consider:

• The optimal reaction conditions (buffer composition, pH 7.5-8.0, Mg²⁺ concentration)

• Temperature dependency (typically assayed at 37°C)

• Substrate concentrations (methionine and ATP)

• The presence of potential inhibitors or activators

Control experiments should include heat-inactivated enzyme and reactions without key substrates to establish baseline activity levels.

What structural features distinguish C. violaceum metK from homologous enzymes in other bacteria?

While the search results don't provide specific structural information for C. violaceum metK, comparative analysis with homologous enzymes suggests several distinguishing features:

The full sequence information for C. violaceum metK provided in the product datasheet can be analyzed for unique structural elements through:

• Sequence Alignment Analysis:

  • Align C. violaceum metK with homologs from other bacteria, particularly pathogenic species

  • Identify conserved catalytic residues and C. violaceum-specific substitutions

  • Secondary structure prediction tools can identify potential unique structural elements

• Homology Modeling:

  • Generate 3D structural models based on crystal structures of homologous proteins

  • Evaluate the geometry of active sites and potential allosteric sites

  • Analyze surface electrostatics and potential protein-protein interaction interfaces

• Experimental Structure Determination:

  • X-ray crystallography or cryo-EM studies to determine the actual structure

  • Conduct ligand binding studies using differential scanning fluorimetry or surface plasmon resonance

The sequence provided in the product information contains regions predicted to be involved in methionine binding (GFDFRGC) and ATP binding (GLDLNQGAG), which may have subtle variations from homologs that influence substrate specificity or catalytic efficiency. These variations could potentially be exploited for species-specific inhibitor design.

How does metK contribute to C. violaceum pathogenicity and virulence mechanisms?

S-adenosylmethionine synthase (metK) contributes significantly to C. violaceum pathogenicity through several interconnected mechanisms:

• Quorum Sensing Regulation:

  • SAM produced by metK serves as a precursor for acyl-homoserine lactone (AHL) synthesis

  • The CviI synthase enzyme utilizes SAM for converting fatty acids to AHLs

  • AHLs form complexes with CviR that stimulate the vioABCDE operon, regulating violacein production

  • This quorum sensing system coordinates population-dependent expression of virulence factors

• Methylation of Virulence Factors:

  • SAM-dependent methyltransferases modify numerous cellular components

  • Methylation of cell surface components can affect host recognition and immune evasion

  • DNA methylation may regulate expression of virulence genes

• Connection to Iron Acquisition Systems:

  • C. violaceum produces catecholate-type siderophores chromobactin and viobactin

  • While not directly demonstrated in the search results, SAM-dependent methylation likely influences siderophore biosynthesis pathways

  • Iron acquisition is critical for bacterial pathogenesis and establishment of infection

C. violaceum infections cause severe complications including bacterial hemophagocytic syndrome, brain abscess, chronic cellulitis, conjunctivitis, chronic granulomatosis, and other life-threatening conditions . The high lethality rate despite infrequent pathogenicity in humans may be partly attributed to the regulatory roles of metK in virulence factor production.

What is the relationship between metK activity and siderophore production in C. violaceum?

While the search results don't directly address the relationship between metK and siderophore production, we can infer potential connections based on known biochemical pathways:

• Biosynthetic Connection:

  • C. violaceum produces two catecholate-type siderophores: chromobactin and viobactin

  • These siderophores are synthesized by non-ribosomal peptide synthetase (NRPS) enzymes CbaF and VbaF respectively

  • SAM, produced by metK, may participate in methylation steps during siderophore biosynthesis

• Regulatory Interaction:

  • The ChuP protein regulates siderophore production, particularly the synthesis and/or uptake of viobactin

  • Mutation of chuP alters the levels of viobactin siderophore

  • SAM-dependent methylation could potentially affect the expression or activity of ChuP or other regulatory proteins

• Experimental Investigation:
  To investigate this relationship, researchers could:

  • Generate metK knockdown strains and measure siderophore production using the chrome azurol S (CAS) plate assay

  • Analyze siderophore halos that develop after incubation for 24 hours at 37°C using Image J software for quantification

  • Combine mutations in metK with mutations in cbaF or vbaF to understand the specific siderophore pathways affected

The regulation of siderophore production in C. violaceum appears to be complex, involving multiple systems including the ChuPRSTUV operon that encodes a heme uptake system . Further research is needed to clarify the specific role of metK in this regulatory network.

How can recombinant metK be utilized in drug discovery targeting C. violaceum infections?

Recombinant S-adenosylmethionine synthase (metK) from C. violaceum offers several strategic applications in drug discovery:

• High-Throughput Inhibitor Screening:

  • Establish in vitro enzyme assays using purified recombinant metK

  • Screen chemical libraries for compounds that selectively inhibit C. violaceum metK

  • Prioritize compounds that show selectivity over human MAT enzymes

• Structure-Based Drug Design:

  • Use the amino acid sequence provided to generate homology models

  • Identify unique binding pockets that differ from human homologs

  • Design inhibitors that exploit structural differences at the active site or allosteric sites

• Whole-Cell Testing Platform:

  • Develop reporter systems in C. violaceum where metK inhibition correlates with measurable outputs

  • Test promising compounds for their ability to reduce violacein production or siderophore activity

  • Monitor effects on quorum sensing pathways which are linked to metK activity through SAM production

• Target Validation Studies:

  • Create conditional knockdown strains of metK in C. violaceum

  • Confirm the essentiality of metK for virulence in infection models

  • Determine whether partial inhibition is sufficient to attenuate pathogenicity

• Combination Therapy Approaches:

  • Test metK inhibitors in combination with:

    • Iron chelators (to enhance the effect of siderophore disruption)

    • Quorum sensing inhibitors (to synergistically target virulence pathways)

    • Conventional antibiotics (to potentially restore sensitivity)

The development of drugs targeting metK is particularly promising given C. violaceum's genome has a broad but incomplete array of ORFs coding for mammalian pathogenicity-associated proteins, which may explain its high lethality rate but infrequent pathogenicity in humans .

What experimental approaches can reveal the interplay between metK and other metabolic pathways in C. violaceum?

Investigating the metabolic interconnections of metK requires multi-faceted experimental approaches:

• Systems Biology Analysis:

  • Transcriptomics: Compare RNA-seq profiles of wild-type and metK-modulated strains under various conditions

  • Proteomics: Use mass spectrometry to identify differentially expressed proteins and post-translational modifications

  • Metabolomics: Measure changes in SAM, methionine, and downstream metabolites

• Genetic Interaction Studies:

  • Generate double mutants combining metK modulation with mutations in:

    • Quorum sensing genes (cviI, cviR)

    • Siderophore biosynthesis genes (cbaF, vbaF)

    • Heme utilization genes (chuP, chuR)

  • Analyze growth phenotypes, violacein production, and virulence factor expression

• Biochemical Pathway Analysis:

  • Track isotopically labeled metabolites (¹³C-methionine) to map flux through SAM-dependent pathways

  • Measure activities of key enzymes in methionine recycling pathways

  • Characterize the methylome (all methylated substrates) dependent on SAM availability

• Computational Modeling:

  • Develop metabolic flux models incorporating metK-dependent reactions

  • Predict metabolic bottlenecks and potential compensatory pathways

  • Identify synthetic lethal interactions that could be exploited therapeutically

Table 2: Experimental Design for Metabolic Integration Studies

The regulatory protein ChuP, which connects heme and siderophore utilization in C. violaceum , may serve as an excellent starting point for studying these metabolic interconnections, as it demonstrates how regulatory systems can integrate multiple iron acquisition pathways.

What are common challenges in working with recombinant metK and how can they be addressed?

Researchers working with recombinant C. violaceum S-adenosylmethionine synthase frequently encounter several technical challenges:

• Protein Solubility Issues:

  • Challenge: Formation of inclusion bodies during overexpression

  • Solution: Express at lower temperatures (16-20°C), use solubility-enhancing fusion tags (MBP, SUMO), or optimize buffer conditions with stabilizing additives (glycerol, arginine)

• Enzyme Stability:

  • Challenge: Loss of activity during storage

  • Solution: Follow recommended storage guidelines: avoid repeated freeze-thaw cycles, store working aliquots at 4°C for up to one week only, and use 50% glycerol for long-term storage at -20°C/-80°C

• Substrate Availability:

  • Challenge: ATP degradation during activity assays

  • Solution: Prepare fresh ATP solutions, add phosphatase inhibitors, and include an ATP-regenerating system (phosphoenolpyruvate and pyruvate kinase)

• Interference in Assays:

  • Challenge: Background activity from endogenous enzymes

  • Solution: Use highly purified enzyme preparations (>85% purity as specified) , include appropriate controls, and design specific assay conditions that minimize interference

• Protein Quantification:

  • Challenge: Inaccurate determination of enzyme concentration

  • Solution: Use multiple quantification methods (Bradford, BCA, and A280 measurements) and calibrate with appropriate protein standards

For reconstitution of lyophilized protein, researchers should follow the specific protocol: briefly centrifuge the vial prior to opening, reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL, and add glycerol (final concentration 50%) for long-term storage .

How can researchers distinguish between specific metK effects and broader metabolic perturbations in experimental systems?

Differentiating direct metK effects from secondary metabolic perturbations requires careful experimental design:

• Complementation Strategies:

  • Challenge: Determining if observed phenotypes are directly due to metK alterations

  • Solution: Create genetic complementation strains restoring wild-type metK function in mutant backgrounds

  • Approach: Similar to methods used for chuP complementation in research on heme utilization , where complementation fully restored growth in the presence of heme and hemoglobin

• Targeted Metabolite Supplementation:

  • Challenge: Separating methionine cycle disruption from SAM depletion effects

  • Solution: Supplement growth media with key metabolites (methionine, SAM, spermidine) to bypass specific blocks

  • Analysis: Compare phenotypic rescue patterns to map the primary metabolic block

• Time-Course Analysis:

  • Challenge: Distinguishing primary from secondary effects

  • Solution: Monitor transcriptomic, proteomic, and metabolomic changes at multiple time points following metK perturbation

  • Interpretation: Primary effects typically manifest earlier than downstream consequences

• Enzyme Activity Variants:

  • Challenge: Generating targeted enzyme function alterations

  • Solution: Create point mutations that affect specific aspects of metK function (e.g., catalytic efficiency, substrate binding, protein interactions)

  • Analysis: Compare phenotypic profiles of different variants to dissect functional domains

• Controlled Expression Systems:

  • Challenge: Avoiding developmental compensation

  • Solution: Use inducible promoters to modulate metK expression at specific experimental timepoints

  • Approach: Similar to expression assays for the chu operon described in the research literature , where treatment conditions were carefully controlled

When evaluating experimental results, researchers should implement appropriate statistical analyses and include multiple biological and technical replicates, as demonstrated in studies of the ChuP regulatory protein where experiments were performed in three biological replicates .