Recombinant Chromobacterium violaceum Phosphopantetheine adenylyltransferase (coaD)

What is Phosphopantetheine adenylyltransferase (PPAT) and what role does it play in bacterial metabolism?

Phosphopantetheine adenylyltransferase (PPAT), encoded by the coaD gene, catalyzes the penultimate step in the coenzyme A (CoA) biosynthetic pathway. Specifically, PPAT transfers an adenylyl group from ATP to 4′-phosphopantetheine, forming dephospho-CoA and pyrophosphate. This reaction is critical in the five-step synthesis of CoA from pantothenate (vitamin B5). CoA serves as a vital cofactor that functions as an acyl group carrier or carbonyl-activating group in numerous essential biochemical transformations throughout bacterial metabolism . The essential nature of this pathway makes PPAT a potential target for antimicrobial development, as has been demonstrated in studies with Mycobacterium tuberculosis PPAT (MtbPPAT) .

How is the coaD gene organized in Chromobacterium violaceum compared to other bacterial species?

While the search results don't provide specific information about the genomic organization of coaD in C. violaceum, we can infer from studies in other bacteria that the coaD gene likely exists within the bacterial chromosome as part of the CoA biosynthetic pathway. In M. tuberculosis, the coaD gene was previously classified as non-essential based on transposon mutagenesis studies, but recent research using CRISPR interference has confirmed its essentiality for bacterial growth in vitro . Researchers studying C. violaceum coaD would need to examine its genomic context, potentially looking for conserved gene arrangements or regulatory elements that might be shared with other bacterial species. This comparative genomic approach could provide insights into potential regulatory mechanisms specific to C. violaceum.

What are the optimal conditions for expressing recombinant C. violaceum PPAT in heterologous systems?

For optimal expression of recombinant C. violaceum PPAT, researchers should consider established protocols for similar enzymes, such as those used for M. tuberculosis PPAT. A typical approach would involve:

• Cloning the C. violaceum coaD gene into an expression vector with an appropriate promoter (e.g., T7) and affinity tag (e.g., His-tag)

• Transforming into an E. coli expression strain such as BL21(DE3)

• Culturing in Luria-Bertani (LB) medium at 37°C until reaching mid-log phase (OD600 of 0.6-0.8)

• Inducing protein expression with IPTG (typically 0.5-1 mM)

• Lowering the temperature to 18-25°C for overnight expression to enhance protein solubility

Researchers should optimize these conditions specifically for C. violaceum PPAT, potentially testing different E. coli strains, induction temperatures (18°C, 25°C, 30°C), IPTG concentrations, and induction times to maximize yield of soluble protein. The choice of affinity tag and its position (N- or C-terminal) should also be considered based on structural information about PPAT enzymes to ensure tag placement doesn't interfere with enzymatic activity or hexamer formation.

What purification strategy yields the highest purity and activity for recombinant C. violaceum PPAT?

A multi-step purification strategy is recommended to obtain high-purity, active C. violaceum PPAT:

• Initial purification using affinity chromatography (if His-tagged, use Ni-NTA resin)

• Buffer optimization to include components that stabilize the hexameric structure (typically including divalent cations like Mg2+)

• Secondary purification step using size exclusion chromatography to isolate the hexameric form of the enzyme and remove aggregates

• Optional ion exchange chromatography step if higher purity is required

Throughout purification, buffer conditions should be optimized to maintain enzyme stability, potentially including:

• HEPES or Tris buffer (pH 7.5-8.0)

• NaCl (100-300 mM)

• MgCl2 (5-10 mM)

• Glycerol (10-20%)

• Reducing agent such as DTT or β-mercaptoethanol (1-5 mM)

Activity assays should be performed after each purification step to monitor retention of enzymatic function. For long-term storage, the purified enzyme can be flash-frozen in liquid nitrogen and stored at -80°C in small aliquots to avoid repeated freeze-thaw cycles.

How can researchers confirm the correct folding and oligomeric state of recombinant C. violaceum PPAT?

Confirming the correct folding and oligomeric state of recombinant C. violaceum PPAT is crucial for ensuring functional studies accurately reflect the enzyme's native properties. Several complementary techniques should be employed:

• Size Exclusion Chromatography (SEC): PPAT typically forms a hexamer, which should elute at the expected molecular weight (~90-120 kDa depending on the exact size of the monomer). The elution profile should show a predominant peak corresponding to the hexamer.

• Dynamic Light Scattering (DLS): This can provide information about the size distribution and potential aggregation of the purified protein.

• Circular Dichroism (CD): To assess secondary structure content and compare with known PPAT structures from other organisms.

• Thermal Shift Assay: To evaluate protein stability and the effects of different buffer conditions.

• Native PAGE: To visualize the oligomeric state under non-denaturing conditions.

• Enzymatic Activity Assays: A functionally active enzyme suggests proper folding and oligomerization.

• Analytical Ultracentrifugation: For detailed analysis of the oligomeric state and potential equilibrium between different states.

For definitive confirmation, researchers should aim to obtain crystal structures of C. violaceum PPAT, following approaches similar to those used for the MtbPPAT structural studies .

What assays are available to measure the enzymatic activity of C. violaceum PPAT?

Several assays can be adapted to measure C. violaceum PPAT activity, based on methods established for PPAT enzymes from other organisms:

• Coupled Enzymatic Assay: This assay links PPAT activity to the oxidation of NADH, which can be monitored spectrophotometrically at 340 nm. The pyrophosphate released during the PPAT reaction is used as a substrate by auxiliary enzymes (pyrophosphatase, phosphorylase, phosphoglucomutase, and glucose-6-phosphate dehydrogenase) ultimately leading to NADH oxidation.

• Radioactive Assay: Using [α-32P]ATP or [γ-32P]ATP as a substrate and measuring the transfer of radioactive adenylyl group to 4′-phosphopantetheine.

• HPLC-based Assay: Monitoring the formation of dephospho-CoA directly by HPLC separation and UV detection.

• Malachite Green Assay: Detecting the release of inorganic pyrophosphate (after conversion to phosphate) through color development with malachite green.

• Isothermal Titration Calorimetry (ITC): Measuring the heat released during the enzymatic reaction to determine kinetic parameters.

Each assay has advantages and limitations in terms of sensitivity, throughput, and equipment requirements. Researchers should select the most appropriate method based on their specific experimental needs and available instrumentation.

How do the kinetic parameters of C. violaceum PPAT compare to those from other bacterial species?

While specific kinetic parameters for C. violaceum PPAT are not provided in the search results, researchers can anticipate similarities with PPAT enzymes from related bacteria. A comparative analysis should include determination of:

• Km values for both substrates (4′-phosphopantetheine and ATP)

• kcat values under standardized conditions

• Substrate specificity profiles

• pH and temperature optima

• Requirements for divalent cations (typically Mg2+)

• Sensitivity to product inhibition

The experimental design should include direct comparisons with PPAT from E. coli (the most well-characterized bacterial PPAT) and other bacteria. This data can be presented in a comprehensive table format:

Such comparative analysis would provide insights into evolutionary conservation and potential specialized adaptations of C. violaceum PPAT.

What factors influence the catalytic efficiency of recombinant C. violaceum PPAT?

Multiple factors can influence the catalytic efficiency of recombinant C. violaceum PPAT, including:

• Buffer composition: Optimal pH, ionic strength, and presence of stabilizing agents can significantly affect enzyme activity. Most PPAT enzymes function optimally around pH 7.5-8.0.

• Divalent cation concentration: Mg2+ typically serves as a cofactor, with optimal concentrations around 5-10 mM. Other divalent cations (Mn2+, Co2+) may substitute with varying efficiencies.

• Temperature: While physiological temperature is likely optimal, stability at different temperatures should be characterized.

• Substrate concentrations: Potential substrate inhibition at high concentrations should be investigated.

• Allosteric regulation: Many metabolic enzymes are subject to allosteric regulation by pathway intermediates or end products. Researchers should test whether CoA or other metabolites modulate C. violaceum PPAT activity.

• Post-translational modifications: If present, these could affect activity and should be characterized.

• Oligomeric state: The hexameric structure typical of PPAT enzymes is often critical for full activity. Conditions that disrupt hexamer formation may reduce catalytic efficiency.

Researchers should systematically investigate these factors to establish optimal conditions for enzymatic assays and to understand the physiological regulation of PPAT activity in C. violaceum.

What approaches can be used to determine the three-dimensional structure of C. violaceum PPAT?

To determine the three-dimensional structure of C. violaceum PPAT, researchers should consider the following approaches:

• X-ray Crystallography: The most definitive method, following these steps:

  • Screening for crystallization conditions using commercial kits

  • Optimization of promising conditions to obtain diffraction-quality crystals

  • Data collection at synchrotron facilities

  • Structure determination using molecular replacement with known PPAT structures as templates

  • Model building, refinement, and validation

• Cryo-Electron Microscopy (cryo-EM): Particularly useful for visualizing the hexameric assembly:

  • Sample preparation on EM grids

  • Data collection using state-of-the-art cryo-EM facilities

  • Image processing and 3D reconstruction

  • Model building and refinement

• Nuclear Magnetic Resonance (NMR): More challenging due to the size of the hexamer but potentially useful for studying dynamics:

  • Isotopic labeling (15N, 13C) of the recombinant protein

  • Collection of multidimensional NMR spectra

  • Resonance assignment and structure calculation

• Homology Modeling: As an initial approach:

  • Using existing structures of PPAT from other organisms as templates

  • Validation using biochemical and biophysical experiments

  • Refinement based on experimental data

The approach used for MtbPPAT structure determination, which involved X-ray crystallography of apo-enzyme and enzyme-fragment complexes, provides a valuable template for similar studies with C. violaceum PPAT .

How can fragment-based drug discovery be applied to identify inhibitors of C. violaceum PPAT?

Fragment-based drug discovery (FBDD) can be a powerful approach for identifying inhibitors of C. violaceum PPAT, as demonstrated by the successful application to MtbPPAT . A comprehensive FBDD strategy would include:

• Fragment Library Screening:

  • Assembling a diverse fragment library (typically 500-2000 compounds)

  • Primary screening using biophysical methods such as thermal shift assays, surface plasmon resonance (SPR), or NMR

  • Confirmation of binding using multiple orthogonal techniques

• Structural Characterization:

  • Co-crystallization of PPAT with promising fragments

  • Structure determination to identify binding modes and interaction patterns

  • Classification of fragments into series based on binding site and chemical scaffold

• Fragment Elaboration/Linking:

  • For C. violaceum PPAT, researchers could follow the approach used for MtbPPAT, where three series of fragments binding to distinct regions of the active site were identified

  • Structure-guided design to link fragments occupying different sub-pockets

  • Iterative optimization to improve potency while maintaining favorable physicochemical properties

• Validation of Inhibitor Specificity:

  • Enzymatic assays to confirm inhibitory activity

  • Selectivity testing against human PPAT and other related enzymes

  • Cellular assays to demonstrate on-target activity in C. violaceum

The success story with MtbPPAT, where fragment linking led to an inhibitor with KD <20 μM and on-target anti-Mtb activity , provides a valuable precedent for similar work with C. violaceum PPAT.

What are the critical structural features of the C. violaceum PPAT active site that can be exploited for selective inhibitor design?

While specific structural information about C. violaceum PPAT is not provided in the search results, insights can be drawn from the MtbPPAT structure and other bacterial PPAT enzymes. Critical structural features likely include:

• Substrate Binding Pockets:

  • The 4′-phosphopantetheine binding site, which in MtbPPAT interacts with the benzophenone fragment through residues like Val73, Gly71, Val74, and Asn105

  • The ATP binding site, which in MtbPPAT accommodates indole and pyrazole fragments

  • The interface between these two binding sites, which presents opportunities for fragment linking

• Conserved vs. Variable Regions:

  • Identifying residues that are conserved across bacterial PPATs but differ from human PPAT

  • Targeting variable regions can enhance selectivity for C. violaceum PPAT

• Allosteric Sites:

  • Potential regulatory sites beyond the active site that could be targeted

  • Interfaces between monomers in the hexameric assembly

• Water-Mediated Interactions:

  • As observed in MtbPPAT, water molecules often play important roles in ligand binding

  • These can be exploited or displaced in inhibitor design

• Conformational Changes:

  • Understanding how substrate binding induces conformational changes

  • Potential for designing inhibitors that lock the enzyme in an inactive conformation

Researchers should perform detailed structural comparisons between C. violaceum PPAT and human PPAT to identify differences that can be exploited for selective inhibitor design, similar to the approach that has been successful for MtbPPAT inhibitor development.

What is the biological significance of PPAT in C. violaceum metabolism and potential pathogenicity?

The biological significance of PPAT in C. violaceum likely extends across multiple aspects of bacterial physiology and potential pathogenicity:

Understanding these connections requires further research, particularly studies examining the effects of coaD knockdown or inhibition on C. violaceum physiology and virulence.

How can CRISPR interference be used to validate C. violaceum PPAT as a potential antimicrobial target?

CRISPR interference (CRISPRi) can be a powerful approach to validate C. violaceum PPAT as a potential antimicrobial target, similar to the strategy used for MtbPPAT validation :

• CRISPRi System Construction:

  • Design a plasmid system expressing dCas9 (catalytically dead Cas9) under an inducible promoter (e.g., tetracycline-inducible)

  • Design sgRNAs targeting the coaD gene promoter or early coding sequence

  • Construct a control system with sgRNAs targeting unrelated genes

• Generation of C. violaceum CRISPRi Strains:

  • Transform C. violaceum with the CRISPRi system

  • Select transformants and verify construct integration

  • Optimize induction conditions for dCas9 expression

• Phenotypic Characterization:

  • Assess growth phenotypes under various inducer concentrations

  • Quantify coaD expression levels using RT-qPCR to confirm knockdown

  • Evaluate physiological changes, including potential effects on violacein production

• Target Validation Experiments:

  • Perform checkerboard assays with potential PPAT inhibitors and CRISPRi induction

  • A true on-target inhibitor should show enhanced potency when combined with coaD knockdown

  • Include control compounds (e.g., antibiotics with different mechanisms) to confirm specificity

• Chemical Rescue Experiments:

  • Attempt to rescue growth inhibition by supplementing with CoA pathway intermediates

  • This can provide further evidence for the specific role of PPAT in observed phenotypes

This approach, modeled after the successful MtbPPAT validation , would provide compelling evidence for whether C. violaceum PPAT represents a viable antimicrobial target.

What are the potential applications of recombinant C. violaceum PPAT in biotechnology and drug discovery?

Recombinant C. violaceum PPAT offers several potential applications in biotechnology and drug discovery:

• Antimicrobial Development:

  • Serving as a target for developing novel antibacterials with activity against C. violaceum

  • Providing a model system for targeting PPAT in other pathogenic bacteria

  • Enabling high-throughput screening assays for inhibitor discovery

• Biosynthetic Applications:

  • Enzymatic production of CoA and its derivatives for research purposes

  • Potential incorporation into synthetic biology pathways requiring CoA-dependent reactions

  • Development of PPAT variants with altered substrate specificity through protein engineering

• Structural Biology Platform:

  • Contributing to comparative structural analysis of PPAT enzymes across different species

  • Advancing understanding of structure-function relationships in adenylyltransferases

  • Serving as a model system for studying hexameric enzyme assemblies

• Biotransformation Applications:

  • Potential use in enzymatic synthesis of pantetheine analogs

  • Development of biocatalytic routes to CoA derivatives

• Fundamental Research:

  • Investigating the evolution of CoA biosynthesis across bacterial species

  • Exploring potential regulatory interactions between primary metabolism and specialized metabolite production in C. violaceum

  • Studying the relationship between metabolic enzymes and quorum sensing systems

These applications highlight the multifaceted value of research on recombinant C. violaceum PPAT beyond its primary role in bacterial metabolism.

How does C. violaceum PPAT compare structurally and functionally with PPAT enzymes from other bacterial species?

A comprehensive comparative analysis of C. violaceum PPAT with other bacterial PPAT enzymes would reveal evolutionary relationships and functional specializations:

• Sequence Conservation Analysis:

  • Multiple sequence alignment of PPAT sequences from diverse bacteria

  • Identification of highly conserved residues, likely essential for catalysis or structural integrity

  • Analysis of C. violaceum-specific sequence features

• Structural Comparison:

  • Superposition of available PPAT structures (or homology models)

  • Analysis of active site architecture conservation

  • Examination of oligomerization interfaces

  • Identification of conformational differences that might impact catalysis or regulation

• Functional Comparison:

  • Substrate specificity profiles across different bacterial PPATs

  • Catalytic efficiency parameters (kcat/Km)

  • Allosteric regulation mechanisms

  • Temperature and pH optima reflecting environmental adaptations

• Evolutionary Context:

  • Phylogenetic analysis to place C. violaceum PPAT in evolutionary context

  • Correlation of PPAT properties with bacterial lifestyle and habitat

  • Identification of potential horizontal gene transfer events

While specific data on C. violaceum PPAT is not provided in the search results, this comparative approach would generate valuable insights into the evolution and specialization of this essential enzyme across bacterial species.

Is there evidence for potential interactions between PPAT and quorum sensing regulation in C. violaceum?

• Transcriptional Analysis:

  • Examining coaD expression levels under conditions of activated or repressed quorum sensing

  • Analyzing promoter regions for potential binding sites for QS regulators like CviR

  • RNA-seq experiments comparing wild-type C. violaceum with QS mutants (cviI/cviR mutants)

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation experiments to detect potential physical interactions between PPAT and QS components

  • Bacterial two-hybrid or pull-down assays to screen for protein partners

  • Proximity labeling approaches to identify the PPAT interactome in vivo

• Metabolic Connections:

  • Investigating whether CoA-dependent pathways contribute to the synthesis of AHL signal molecules

  • Examining whether QS-regulated phenotypes like violacein production indirectly impact CoA metabolism

• Regulatory Network Analysis:

  • Constructing regulatory networks to identify potential shared regulators between coaD and QS genes

  • Computational prediction of regulatory motifs in coaD and QS gene promoters

• Mutant Phenotype Analysis:

  • Characterizing the effects of coaD knockdown on QS-regulated phenotypes

  • Examining whether QS mutants show altered sensitivity to PPAT inhibitors

While C. violaceum employs sophisticated QS systems including the CviI/R system and the VioS repressor to regulate various phenotypes , definitive connections to PPAT require targeted experimental investigation.

How might environmental factors influence the expression and activity of PPAT in C. violaceum?

Environmental factors likely influence PPAT expression and activity in C. violaceum, reflecting the bacterium's adaptation to diverse ecological niches:

• Nutrient Availability:

  • CoA requirements may vary depending on available carbon sources

  • PPAT expression could be regulated in response to pantothenate availability

  • Metabolic shifts under nutrient limitation might impact CoA utilization

• Temperature:

  • C. violaceum inhabits tropical and subtropical regions

  • Temperature fluctuations could affect PPAT expression or enzymatic activity

  • Thermal stability of the enzyme might reflect environmental adaptation

• pH:

  • Environmental pH variations in soil and water habitats

  • Potential pH-dependent regulation of enzyme activity or expression

  • Adaptation of the active site to function optimally at environmentally relevant pH

• Oxygen Levels:

  • Oxygen availability in soil and water microenvironments

  • Potential links between aerobic/anaerobic metabolism and CoA requirements

  • Redox-sensitive regulation of enzyme activity

• Population Density:

  • QS systems respond to bacterial population density

  • If PPAT is influenced by QS regulation, its expression might vary with population density

  • Competitive interactions in mixed microbial communities could indirectly impact CoA metabolism

• Host-Associated Factors:

  • During occasional host infection, host-derived signals might influence metabolism

  • Immune responses might create selective pressures on essential metabolic pathways

Experimental approaches to investigate these influences could include transcriptomic and proteomic analyses under various environmental conditions, reporter gene assays for coaD expression, and enzymatic activity measurements across a range of conditions mimicking C. violaceum's natural habitats.