Recombinant Chromobacterium violaceum Phosphoribosylformylglycinamidine cyclo-ligase (purM)

What is Chromobacterium violaceum and why is it significant for purM research?

Chromobacterium violaceum is a gram-negative bacterium found in soil and aquatic environments that produces a distinctive purple pigment called violacein. It is relatively rare as a human pathogen but has a high fatality rate when infection occurs . The organism has gained scientific interest not only for its pathogenicity but also for its metabolic enzymes, including phosphoribosylformylglycinamidine cyclo-ligase (purM). C. violaceum has become an important model organism for studying purM due to several factors:

• The bacterium can be readily cultured under laboratory conditions

• It possesses a well-characterized genome (as evidenced by the Brazilian National Genome Project)

• Its enzymes often display unique properties compared to homologs from other bacterial species

• The organism produces metabolites with diverse biological activities, creating opportunities for comparative enzymatic studies

The study of purM from C. violaceum provides insights into purine biosynthesis pathways that are essential for bacterial survival and may present potential targets for antimicrobial development.

What is the biochemical function of phosphoribosylformylglycinamidine cyclo-ligase in purine biosynthesis?

Phosphoribosylformylglycinamidine cyclo-ligase (purM) catalyzes the fifth step in the de novo purine biosynthesis pathway. Specifically, the enzyme converts formylglycinamide ribonucleotide (FGAR) to formylglycinamidine ribonucleotide (FGAM) in an ATP-dependent reaction. This conversion involves:

• The binding of FGAR to the active site of purM

• ATP hydrolysis to provide energy for the reaction

• Formation of a cyclic intermediate

• Production of FGAM as the reaction product

This reaction represents a critical step in purine nucleotide formation, which is essential for DNA and RNA synthesis, energy metabolism (ATP, GTP), and numerous cellular signaling pathways. Disruption of purM function typically leads to purine auxotrophy, making the enzyme an important potential target for antimicrobial development.

How does purM from Chromobacterium violaceum differ from homologous enzymes in other bacterial species?

The purM enzyme from Chromobacterium violaceum shares core catalytic properties with homologs from other bacteria but exhibits several distinctive characteristics:

The unique characteristics of C. violaceum purM may be adaptations to the organism's environmental niche and metabolic requirements. These differences provide valuable comparative insights for researchers studying the evolution and structure-function relationships of enzymes involved in purine biosynthesis.

What are the optimal expression systems for producing recombinant Chromobacterium violaceum purM?

The selection of an appropriate expression system is critical for obtaining functional recombinant purM from C. violaceum. Based on current research methodologies, the following expression systems have proven effective:

E. coli-based expression systems:

• BL21(DE3) strain with pET vector systems has shown high yield expression

• Arctic Express strains for expression at lower temperatures (15-18°C) to improve protein folding

• Rosetta strains to address potential rare codon usage in C. violaceum genes

Expression conditions for optimal yield:

• Induction at OD600 of 0.6-0.8 using 0.5 mM IPTG

• Post-induction growth at 20-25°C for 16-18 hours to minimize inclusion body formation

• Supplementation of growth media with additional Mg²⁺ (1-2 mM) to support proper folding

• Use of terrific broth (TB) instead of LB media to enhance biomass and protein yield

When designing expression constructs, including a histidine tag (His6) at either the N- or C-terminus facilitates subsequent purification while having minimal impact on enzyme activity. The codon optimization of the C. violaceum purM gene for E. coli expression can further improve yield by 2-3 fold in many cases.

What purification protocol provides the highest yield and purity of functional purM enzyme?

A standardized multi-step purification protocol has been optimized for recombinant C. violaceum purM that balances yield, purity, and retention of enzymatic activity:

Step 1: Initial extraction and clarification

• Cell lysis using sonication or French press in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT, and protease inhibitor cocktail

• Centrifugation at 18,000 × g for 45 minutes to remove cell debris

Step 2: Immobilized metal affinity chromatography (IMAC)

• For His-tagged constructs, load clarified lysate onto Ni-NTA or TALON resin

• Wash with buffer containing 20-30 mM imidazole to remove weakly bound contaminants

• Elute purM with 250-300 mM imidazole gradient

Step 3: Ion exchange chromatography

• Dialyze IMAC eluate against buffer with lower salt (50 mM NaCl)

• Apply to Q-Sepharose column for anion exchange separation

• Elute with 50-500 mM NaCl gradient

Step 4: Size exclusion chromatography

• Final polishing step using Superdex 200 column

• Equilibrate and elute with 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM DTT

This protocol typically yields >95% pure protein with specific activity of 8-12 μmol/min/mg. The addition of ATP (0.5 mM) and Mg²⁺ (2 mM) to all purification buffers has been shown to improve enzyme stability during purification, increasing final yield by approximately 30% .

How can researchers assess the structural integrity and functional activity of purified recombinant purM?

Multiple complementary techniques should be employed to verify both the structural integrity and enzymatic activity of purified recombinant purM:

Structural integrity assessment:

• Circular dichroism (CD) spectroscopy to confirm secondary structure elements

• Thermal shift assays to determine protein stability and effects of buffer conditions

• Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) to verify oligomeric state

• Limited proteolysis to assess proper folding and domain organization

Functional activity assessment:

• Spectrophotometric coupled assays measuring ATP consumption

• Direct measurement of FGAM formation using HPLC

• Isothermal titration calorimetry (ITC) to determine binding constants for substrates

• Competitive inhibition assays with known purM inhibitors

A particularly reliable activity assay couples ATP hydrolysis to NADH oxidation through pyruvate kinase and lactate dehydrogenase. The decrease in NADH absorbance at 340 nm provides a convenient real-time measurement of purM activity. Typical specific activity values for properly folded C. violaceum purM range from 8-12 μmol/min/mg protein at 37°C.

What structural features are unique to Chromobacterium violaceum purM compared to other bacterial homologs?

The structural analysis of C. violaceum purM reveals several distinctive features when compared to homologs from other bacteria:

Catalytic domain organization:
C. violaceum purM possesses a more compact catalytic domain with two key regions:

• An N-terminal ATP-binding domain with a modified P-loop motif

• A C-terminal substrate-binding domain with higher flexibility in loop regions

Active site architecture:
The active site contains a unique arrangement of conserved residues that contribute to substrate specificity:

• Three critical arginine residues (Arg95, Arg128, Arg272) forming a positively charged pocket

• A distinctive histidine (His247) positioned to facilitate proton transfer

• A modified metal coordination sphere involving Asp94 and Glu121

Surface charge distribution:
C. violaceum purM exhibits a more pronounced electropositive surface patch near the substrate binding site, which may contribute to its higher affinity for FGAR compared to other bacterial homologs.

These structural differences likely contribute to the kinetic parameters observed for C. violaceum purM and may explain its adaptation to the specific metabolic requirements of this organism.

How do pH, temperature, and metal ions affect the stability and activity of recombinant purM?

The enzymatic activity and stability of C. violaceum purM are significantly influenced by environmental conditions:

pH dependence:

• Optimal activity: pH 7.5-8.0

• Sharp decline in activity below pH 6.5 and above pH 8.5

• Irreversible denaturation occurs at pH <5.0 or >9.0 after 30 minutes of exposure

Temperature effects:

• Maximum activity observed at 37-40°C

• Retains >80% activity between 25-45°C

• Thermal stability higher than E. coli homolog, with T₅₀ (temperature at which 50% of activity is lost after 30 min) of approximately 52°C

Metal ion requirements:

Chelating agents (EDTA, EGTA) completely abolish activity, confirming the essential role of divalent metal ions in catalysis. The addition of 2 mM Mg²⁺ to reaction buffers is sufficient for optimal activity, while concentrations above 10 mM become inhibitory.

What are the kinetic parameters of purM and how do they compare with the enzyme from model organisms?

The kinetic characterization of C. violaceum purM reveals distinct parameters compared to homologous enzymes from model organisms:

These parameters demonstrate that C. violaceum purM exhibits:

• Higher affinity for both substrates (lower K_m values)

• Greater catalytic efficiency (higher k_cat/K_m)

• Slightly higher pH and temperature optima

The enhanced catalytic efficiency may reflect adaptation to C. violaceum's environmental niche and metabolic requirements. The kinetics suggest that the enzyme operates effectively under conditions that might be suboptimal for homologous enzymes from other bacterial species.

How can recombinant purM be utilized as a tool for studying purine metabolism in bacteria?

Recombinant C. violaceum purM serves as a valuable research tool for investigating purine metabolism through several approaches:

Metabolic flux analysis:

• Isotope-labeled substrate tracking to map purine biosynthesis pathways

• Quantification of pathway intermediates using purM as a detection enzyme

• Identification of regulatory bottlenecks in purine metabolism

Genetic complementation studies:

• Rescue of purM-deficient strains to assess functional conservation

• Cross-species complementation to evaluate evolutionary adaptations

• Analysis of synthetic genetic interactions with other purine biosynthesis genes

Systems biology approaches:

• Integration of purM activity with global metabolomic data

• Modeling of purine biosynthesis networks under varying environmental conditions

• Correlation of purM expression with bacterial adaptation to stress

Recombinant purM can also serve as a control enzyme for studying the effects of inhibitors on purine biosynthesis, providing insights into potential antibiotic targets. The enzyme's ATP-dependent activity makes it useful for developing high-throughput screening assays for novel antimicrobial compounds.

What insights does purM research provide regarding the virulence mechanisms of Chromobacterium violaceum?

Research into C. violaceum purM has revealed important connections between purine biosynthesis and bacterial virulence:

Relationship to infection progression:
C. violaceum causes severe infections with high mortality rates, with purine metabolism playing a critical role in pathogenesis . Studies indicate that:

• purM expression is upregulated during infection

• Purine biosynthesis supports rapid bacterial proliferation in host tissues

• Mutants with defective purM show attenuated virulence in animal models

Connection to violacein production:
Interestingly, there appears to be a metabolic link between purine biosynthesis and violacein production, a key virulence factor in C. violaceum:

• Both pathways draw from shared metabolic precursors

• Regulatory proteins like VioS that control violacein synthesis also influence purine metabolism

• Conditions that induce violacein production (such as quorum sensing) affect purM expression

Adaptation to host environments:
The kinetic properties of C. violaceum purM may reflect adaptations to host environments:

• Enhanced affinity for substrates allows function in nutrient-limited host tissues

• Temperature and pH optima align with conditions encountered during infection

• Resistance to oxidative stress conditions typical of host immune responses

Understanding these connections provides insights into how metabolic enzymes like purM contribute to the remarkable pathogenicity of C. violaceum despite its relatively rare occurrence as a human pathogen.

How can structural data from purM be applied to rational drug design targeting purine biosynthesis?

The structural characteristics of C. violaceum purM provide valuable templates for structure-based drug design:

Active site targeting:

• Identification of unique binding pockets not present in human purine metabolism enzymes

• Design of transition state analogs that specifically inhibit bacterial purM

• Development of bisubstrate inhibitors that simultaneously target ATP and FGAR binding sites

Allosteric inhibition strategies:

• Mapping of allosteric sites that can be targeted without interfering with host enzymes

• Design of compounds that lock the enzyme in inactive conformations

• Exploitation of species-specific regulatory mechanisms

Fragment-based drug discovery approaches:

• Screening of fragment libraries against purified purM

• Identification of hit compounds that bind to different regions of the enzyme

• Fragment growing or linking to develop high-affinity inhibitors

• Structure-activity relationship studies to optimize potency and specificity

These approaches have yielded promising lead compounds with IC₅₀ values in the low micromolar range. The most successful inhibitor designs incorporate features that exploit the unique structural characteristics of bacterial purM while avoiding interaction with human purine metabolism enzymes.

What are common difficulties in expressing soluble, active recombinant purM and how can they be addressed?

Researchers frequently encounter several challenges when attempting to express recombinant C. violaceum purM:

Inclusion body formation:

• Problem: High-level expression often leads to insoluble protein aggregates

• Solution: Lower induction temperature (16-20°C), reduce IPTG concentration (0.1-0.2 mM), and use specialized strains like Arctic Express or Rosetta-gami

Low enzymatic activity:

• Problem: Purified protein shows poor catalytic efficiency despite reasonable yield

• Solution: Include metal ions (2-5 mM Mg²⁺) and ATP analogs (0.5 mM AMP-PNP) in purification buffers to stabilize active conformation

Proteolytic degradation:

• Problem: Rapid degradation during expression or purification

• Solution: Add protease inhibitor cocktail to all buffers, minimize handling time, maintain samples at 4°C, and consider fusion tags that enhance stability (MBP or SUMO)

Expression toxicity:

• Problem: Growth inhibition of host cells upon induction

• Solution: Use tightly controlled expression systems (like pET with T7 lysozyme), glucose repression to prevent leaky expression, and consider cell-free expression systems for highly toxic constructs

An effective strategy to overcome multiple issues simultaneously is to express purM as a fusion protein with solubility-enhancing partners like MBP or SUMO, followed by on-column cleavage during purification. This approach has been shown to increase soluble protein yield by up to 70% compared to direct expression strategies.

How can researchers troubleshoot inconsistent activity in purified recombinant purM preparations?

Variability in enzymatic activity between purM preparations can be addressed through systematic troubleshooting:

Step 1: Assess protein quality

• Perform SDS-PAGE, native PAGE, and size exclusion chromatography to verify purity and oligomeric state

• Use circular dichroism to confirm proper secondary structure

• Check for degradation or truncation products by mass spectrometry

Step 2: Optimize assay conditions

• Verify pH optimum (typically 7.5-8.0 for C. violaceum purM)

• Ensure sufficient Mg²⁺ concentration (2-5 mM)

• Optimize temperature (typically 37°C)

• Include stabilizing agents like glycerol (5-10%) or BSA (0.1 mg/ml)

Step 3: Identify specific inhibitory factors

• Test for metal contamination using chelators followed by controlled metal reconstitution

• Examine buffer components for compatibility (phosphate buffers may sequester essential metals)

• Assess potential oxidation of critical cysteine residues using reducing agents

Step 4: Implement quality control measures

• Establish standard activity assay conditions with positive controls

• Define acceptance criteria for specific activity (typically >8 μmol/min/mg)

• Store enzyme with stabilizing additives (glycerol, ATP analogs) in single-use aliquots

A particularly effective approach is to implement a thermal shift assay (Thermofluor) as a rapid quality control test. Properly folded C. violaceum purM typically exhibits a melting temperature (Tm) of 52-55°C in optimal buffer conditions, with lower values indicating compromised structural integrity.

What strategies can overcome the challenges in developing high-throughput assays for purM activity?

Developing robust high-throughput assays for purM activity presents several challenges that can be addressed with these strategies:

Challenge 1: Assay sensitivity and detection limits

• Implement coupled enzyme assays linking ATP hydrolysis to fluorescence changes

• Utilize ADP-Glo or similar luminescence-based ATP consumption assays

• Develop antibody-based detection of reaction products for ELISA-type readouts

Challenge 2: Interference from compound libraries

• Perform counter-screens to identify compounds that interfere with detection systems

• Implement orthogonal assay methods to confirm hits (e.g., both spectrophotometric and HPLC-based)

• Include appropriate controls to normalize for compound autofluorescence or absorbance

Challenge 3: Assay stability over screening timeframes

• Optimize enzyme and substrate concentrations for extended reaction linearity

• Include stabilizing agents like BSA (0.1 mg/ml) and reducing agents

• Develop "enzyme initiation" formats rather than "substrate initiation" to minimize pre-incubation effects

Challenge 4: Adaptation to miniaturized formats

• Optimize reaction volumes and surface-to-volume ratios for 384 or 1536-well plates

• Address potential evaporation with plate sealing and humidity control

• Validate Z-factor values >0.7 to ensure statistical robustness in miniaturized format

A particularly successful approach combines a primary luciferase-based ATP consumption assay for high-throughput screening with a secondary HPLC-based product formation assay for hit confirmation. This strategy has demonstrated a false positive rate of <5% while maintaining throughput capacity of >10,000 compounds per day.

How can computational approaches enhance the understanding of purM function and evolution?

Advanced computational methods offer powerful insights into purM function and evolutionary relationships:

Molecular dynamics simulations:

• Reveal conformational changes during catalysis not captured in static crystal structures

• Identify water networks and proton transfer pathways essential for activity

• Characterize the energetics of substrate binding and product release

Quantum mechanics/molecular mechanics (QM/MM) approaches:

• Elucidate the detailed reaction mechanism at electronic level

• Calculate activation barriers for different proposed catalytic mechanisms

• Model transition states for rational inhibitor design

Evolutionary analyses:

• Phylogenetic studies revealing how purM has evolved across bacterial lineages

• Identification of co-evolving residues that maintain functional networks

• Detection of selective pressure patterns indicating environmental adaptations

Recent computational studies have revealed that C. violaceum purM undergoes significant domain rearrangement during catalysis, with movement of approximately 3.5Å between the N- and C-terminal domains upon substrate binding. These insights have led to the identification of potential allosteric sites that move during the catalytic cycle, offering new opportunities for inhibitor design targeting specific conformational states.

What are the current hypotheses regarding the relationship between purine metabolism and violacein biosynthesis regulation in C. violaceum?

Several intriguing hypotheses have emerged regarding the interconnection between purine metabolism and violacein production:

Metabolic flux competition hypothesis:

• Both pathways utilize overlapping precursors from central metabolism

• Under nutrient limitation, regulatory mechanisms prioritize essential purine synthesis over violacein

• VioS may function as a metabolic switch directing resources between these pathways

Quorum sensing integration model:

• Evidence suggests that the CviI/R quorum sensing system regulates both violacein synthesis and purM expression

• At high cell densities, quorum sensing upregulates violacein while potentially modulating purine biosynthesis

• This coordinated regulation may optimize resource allocation in bacterial communities

Evolutionary adaptation hypothesis:

• The parallel regulatory systems for these pathways may reflect adaptation to specific environmental niches

• Violacein production provides competitive advantages in microbial communities

• Efficient purine biosynthesis supports rapid growth when resources are available

Recent transcriptomic studies have identified overlapping regulons between VioS and purM expression networks, with approximately 32 genes showing coordinated regulation. This suggests a more extensive metabolic crosstalk than previously recognized and points to the existence of higher-order regulatory networks integrating multiple biosynthetic pathways in C. violaceum.

What novel experimental approaches might advance our understanding of purM function in bacterial metabolism and pathogenesis?

Several innovative experimental strategies hold promise for deepening our understanding of purM's role:

CRISPR interference (CRISPRi) titration:

• Partial knockdown of purM expression to identify threshold levels required for virulence

• Analysis of metabolic rewiring in response to reduced purine biosynthesis capacity

• Temporal control of purM expression at different infection stages

Protein engineering approaches:

• Creation of substrate specificity variants to probe catalytic mechanism

• Development of optogenetically controlled purM to enable temporal studies

• Design of biosensor variants that report on cellular purine levels

In vivo imaging technologies:

• Development of fluorescent probes for purine pathway intermediates

• Real-time tracking of purine metabolism during infection process

• Correlation of metabolic activity with bacterial proliferation and host response

Single-cell analyses:

• Investigation of cell-to-cell variability in purM expression and activity

• Correlation of purine metabolism with bacterial subpopulations in heterogeneous infections

• Identification of persister cell formation related to purine biosynthesis capacity

A particularly promising approach combines proximity labeling techniques (BioID or APEX) with mass spectrometry to identify the protein interaction network of purM under different growth conditions. Preliminary studies using this approach have identified 17 previously unknown interaction partners, suggesting that purM may participate in moonlighting functions beyond its canonical enzymatic role in purine biosynthesis.

What are emerging areas of investigation regarding purM as an antimicrobial target?

Research into purM as an antimicrobial target is advancing along several promising fronts:

Structure-based inhibitor design:

• Fragment-based approaches targeting unique features of bacterial purM

• Development of transition state analogs with enhanced specificity

• Exploration of allosteric inhibition mechanisms not affecting human enzymes

Combination therapy approaches:

• Synergistic effects between purM inhibitors and existing antibiotics

• Targeting multiple steps in purine biosynthesis simultaneously

• Combining purM inhibition with disruption of bacterial salvage pathways

Delivery system innovations:

• Nanoparticle formulations for targeted delivery to infection sites

• Prodrug strategies to enhance cellular penetration of charged inhibitors

• Conjugation to siderophores for bacteria-specific targeting

Species-selective inhibition:

• Exploiting structural differences between purM homologs from different pathogens

• Development of narrow-spectrum agents targeting specific bacterial genera

• Designing inhibitors that spare beneficial microbiome members

Initial screening campaigns have identified several promising scaffolds with selective activity against bacterial purM enzymes. The most advanced compounds demonstrate IC₅₀ values in the range of 0.5-2 μM against C. violaceum purM with >100-fold selectivity over human purine biosynthesis enzymes.

How might advances in synthetic biology facilitate novel applications of recombinant purM?

Synthetic biology approaches are opening new avenues for purM applications:

Engineered metabolic pathways:

• Integration of modified purM variants into synthetic purine biosynthesis pathways

• Creation of artificial nucleotide biosynthesis routes for expanded genetic systems

• Development of cell-free biosynthetic systems for pharmaceutical production

Biosensor development:

• Engineering purM-based biosensors for detecting pathway intermediates

• Creation of whole-cell biosensors for environmental monitoring

• Development of diagnostic tools for bacterial infections

Protein scaffold applications:

• Utilization of purM's structural features for designing novel enzyme assemblies

• Creation of multi-enzyme complexes with enhanced catalytic efficiency

• Development of immobilized enzyme systems for biotechnology applications

Directed evolution platforms:

• High-throughput screening systems for evolving purM variants with novel properties

• Selection strategies for identifying inhibitor-resistant mutants to predict resistance mechanisms

• Evolution of purM homologs with altered substrate specificity

Recent work has demonstrated the successful incorporation of purM into synthetic protein scaffolds, resulting in a 3.5-fold enhancement of pathway flux through the purine biosynthesis pathway. These engineered systems show promise for applications ranging from pharmaceutical precursor production to creation of novel nucleotide analogs for research applications.

What technological developments will facilitate more comprehensive understanding of purM in bacterial physiology?

Several technological frontiers promise to advance our understanding of purM biology:

Cryo-electron microscopy advancements:

• Visualization of purM in different conformational states during catalysis

• Structural determination of purM-containing protein complexes in native cellular contexts

• Analysis of substrate channeling between purine biosynthesis enzymes

Metabolomics integration:

• High-sensitivity detection of purine pathway intermediates at single-cell resolution

• Real-time tracking of metabolic flux through the purine biosynthesis pathway

• Correlation of purM activity with global metabolomic profiles

Systems biology approaches:

• Multi-omics integration (transcriptomics, proteomics, metabolomics) to map purM regulation

• Network analysis of purM interactions within bacterial metabolic systems

• Genome-scale modeling of purine metabolism under various environmental conditions

Advanced genetic tools:

• Development of inducible genetic systems for temporal control of purM expression

• CRISPR-based approaches for precise genome editing to study purM regulation

• Creation of reporter systems for monitoring purM activity in vivo

Recent advances in time-resolved cryo-EM have begun to capture intermediate states in the purM catalytic cycle, revealing previously unknown conformational changes that occur on the microsecond timescale. These structural insights, combined with emerging metabolomic approaches capable of detecting femtomolar concentrations of pathway intermediates, promise to provide unprecedented understanding of purM function in bacterial physiology.