Recombinant Chromobacterium violaceum ATP phosphoribosyltransferase (hisG)

What is the biochemical function of ATP phosphoribosyltransferase (HisG) in Chromobacterium violaceum?

ATP phosphoribosyltransferase (HisG) catalyzes the first step in the histidine biosynthetic pathway in C. violaceum. The enzyme mediates the condensation of ATP and 5′-phosphoribosyl-1-pyrophosphate (PRPP) to form N′-5′-phosphoribosyl-ATP (PR-ATP). This reaction requires Mg²⁺ ions as cofactors and is typically coupled with inorganic pyrophosphatase activity to drive the reaction toward product formation by hydrolyzing the emerging inorganic pyrophosphate (PPi) . The HisG-catalyzed reaction represents the entry point and a rate-limiting step in histidine biosynthesis, making it a crucial regulatory point for the entire pathway.

How does C. violaceum HisG differ structurally from HisG proteins in other bacterial species?

In prokaryotes, ATP phosphoribosyltransferase exists in two main structural forms: a homo-hexameric long form or a hetero-octameric short form . C. violaceum HisG belongs to the long-form category, which typically consists of three functional domains: an N-terminal catalytic domain, a central domain involved in oligomerization, and a C-terminal regulatory domain responsible for histidine-mediated feedback inhibition.

While maintaining the core catalytic machinery common across bacterial HisG proteins, C. violaceum HisG exhibits species-specific variations in the regulatory domain that influence its sensitivity to allosteric regulation. These structural differences can be exploited when designing experiments to study inhibition mechanisms or when developing species-selective inhibitors for potential antimicrobial applications.

What expression systems are most effective for producing recombinant C. violaceum HisG?

For recombinant expression of C. violaceum HisG, Escherichia coli-based expression systems are most commonly employed due to their high yield and relatively straightforward protocols. The methodology typically involves:

• Gene cloning: Amplification of the C. violaceum hisG gene using PCR with appropriate restriction sites, followed by insertion into an expression vector (pET system vectors are commonly used).

• Expression optimization: Best results are typically achieved using BL21(DE3) or Rosetta(DE3) E. coli strains grown at lower temperatures (16-20°C) after IPTG induction to improve protein solubility.

• Purification strategy: A two-step purification process incorporating:

  • Immobilized metal affinity chromatography (IMAC) using His-tagged HisG

  • Size exclusion chromatography to separate hexameric HisG from aggregates and other contaminants

The presence of Mg²⁺ ions (typically 5 mM MgCl₂) in all buffers is crucial for maintaining the structural integrity and activity of the purified enzyme.

How do the regulatory mechanisms of C. violaceum HisG compare with those of other bacterial species in the context of the histidine biosynthetic pathway?

The regulatory pattern in C. violaceum appears to integrate with broader metabolic networks, particularly considering that the histidine biosynthetic pathway represents a metabolic crossroad connecting purine de novo biosynthesis with nitrogen metabolism . Specifically:

• Histidine inhibition: Functions through binding to the C-terminal regulatory domain, inducing conformational changes that reduce catalytic activity.

• AMP inhibition: Suggests an additional layer of regulation tied to cellular energy status.

• Transcriptional regulation: Preliminary evidence indicates possible integration with the CviI/CviR quorum sensing system in C. violaceum, which regulates multiple virulence and metabolic functions in response to cell density .

This multi-layered regulation reflects the importance of precisely controlling histidine biosynthesis, which requires 31-41 ATP molecules per histidine molecule produced—a substantial energy investment for the bacterium .

What analytical methods are most effective for characterizing the kinetic parameters and inhibition profiles of recombinant C. violaceum HisG?

Several complementary approaches are recommended for comprehensive kinetic characterization of C. violaceum HisG:

• Continuous spectrophotometric assays: Monitoring pyrophosphate release using a coupled enzyme system with inorganic pyrophosphatase and a pyrophosphate detection reagent.

• Discontinuous HPLC-based assays: Quantifying PR-ATP formation directly, which offers higher specificity and is particularly useful for inhibition studies.

• Isothermal titration calorimetry (ITC): For precise determination of binding constants for substrates, products, and inhibitors.

• Surface plasmon resonance (SPR): For real-time analysis of binding interactions.

For inhibition studies, I recommend the following experimental design:

All reaction conditions should include 10 mM MgCl₂, pH 8.0, at 25°C, with rigorous controls for metal ion contamination which can significantly impact kinetic parameters.

How can structural biology approaches inform the design of selective inhibitors for C. violaceum HisG?

Structural biology approaches offer powerful insights for rational inhibitor design targeting C. violaceum HisG. X-ray crystallography and cryogenic electron microscopy (cryoEM) have proven particularly valuable for characterizing the three-dimensional structures of histidine biosynthetic pathway enzymes .

A comprehensive inhibitor design strategy should include:

• Structure determination: Obtain high-resolution structures of C. violaceum HisG in apo form and in complex with:

  • Natural substrates (ATP, PRPP)

  • Reaction intermediates

  • Natural inhibitors (histidine, AMP)

• Computational approaches:

  • Virtual screening campaigns targeting identified binding hot-spots

  • Molecular dynamics simulations to identify transient binding pockets

  • Quantum mechanical/molecular mechanical (QM/MM) calculations to explore the reaction mechanism

• Fragment-based drug discovery:

  • Screening fragment libraries against purified HisG

  • X-ray crystallography to confirm binding modes

  • Structure-guided fragment growing and linking

I recommend focusing on exploiting structural differences between bacterial HisG and human phosphoribosyltransferases to ensure inhibitor selectivity. The results from such analyses can identify candidate molecules and linkers for the development of specific inhibitors , potentially leading to new antimicrobial compounds targeting the histidine biosynthetic pathway in C. violaceum.

What are the optimal conditions for measuring the enzymatic activity of recombinant C. violaceum HisG?

The optimal conditions for measuring C. violaceum HisG enzymatic activity involve carefully controlled buffer composition and reaction parameters:

• Standard reaction buffer:

  • 50 mM Tris-HCl, pH 8.0

  • 10 mM MgCl₂ (critical for activity)

  • 1 mM DTT (to maintain reduced cysteines)

  • 100 mM KCl (for ionic strength)

• Substrate concentrations:

  • ATP: 0.1-5 mM (K_m typically around 0.2-0.5 mM)

  • PRPP: 0.05-2 mM (K_m typically around 0.1-0.3 mM)

• Enzyme concentration:

  • 50-200 nM purified HisG (adjust based on specific activity)

• Temperature and time:

  • 25-30°C for 5-15 minutes (reaction remains linear)

• Coupling system:

  • Inorganic pyrophosphatase (0.1 U/ml) to drive the reaction forward

  • Appropriate detection method for phosphate or PR-ATP

For accurate measurements, it's essential to include controls for substrate degradation and to account for any background activity. The reaction should be coupled with inorganic pyrophosphatase to direct it toward product formation by hydrolyzing the emerging inorganic pyrophosphate , as accumulation of pyrophosphate can inhibit the forward reaction.

What strategies can overcome common challenges in the expression and purification of active C. violaceum HisG?

Several challenges commonly arise when working with recombinant C. violaceum HisG, including protein insolubility, poor activity, and oligomerization issues. The following strategies can help overcome these challenges:

• Addressing insolubility:

  • Lower induction temperature (16-18°C) during expression

  • Reduce IPTG concentration to 0.1-0.2 mM

  • Co-express with molecular chaperones (GroEL/GroES system)

  • Use solubility-enhancing fusion tags (SUMO or MBP) instead of simple His-tags

• Improving enzyme activity:

  • Include Mg²⁺ in all purification buffers

  • Add ATP (0.1-0.5 mM) during purification to stabilize the active site

  • Maintain reducing conditions (5 mM DTT or 2 mM β-mercaptoethanol)

  • Avoid freeze-thaw cycles; store at -80°C in single-use aliquots with 20% glycerol

• Ensuring proper oligomerization:

  • Use multi-angle light scattering (MALS) to confirm hexameric assembly

  • Include size exclusion chromatography as a final purification step

  • Optimize buffer conditions (150-300 mM NaCl, pH 7.5-8.0) to prevent aggregation

If expression in E. coli remains problematic, consider alternative expression systems such as Pseudomonas-based systems, which might provide a more compatible folding environment for a protein from C. violaceum.

How can site-directed mutagenesis be applied to investigate structure-function relationships in C. violaceum HisG?

Site-directed mutagenesis offers a powerful approach to investigate structure-function relationships in C. violaceum HisG. A methodical approach should include:

• Target selection based on:

  • Sequence conservation analysis across bacterial HisG proteins

  • Structural information identifying catalytic and regulatory residues

  • Molecular dynamics predictions of residues involved in conformational changes

• Recommended mutation types:

  • Conservative substitutions (e.g., Asp→Glu) to probe specific chemical properties

  • Alanine scanning of binding pockets to identify essential residues

  • Introduction of cysteine pairs for disulfide crosslinking studies of dynamics

• Experimental design for functional assessment:

  • Kinetic analysis comparing K_m, k_cat, and K_i values to wild-type

  • Thermal stability assays to detect structural perturbations

  • Oligomerization state analysis via analytical ultracentrifugation

A systematic mutation strategy should focus on:

For comprehensive structure-function analysis, combine mutagenesis with structural studies using X-ray crystallography to visualize the consequences of mutations on protein conformation and ligand binding.

How does the violacein pigment production in C. violaceum interact with histidine biosynthesis and HisG activity?

The relationship between violacein pigment production and histidine biosynthesis in C. violaceum represents an intriguing area of research at the intersection of metabolism and virulence. Violacein, the purple pigment expressed by C. violaceum, has been implicated in conferring virulence and possessing antibiotic-inhibiting properties .

Current evidence suggests several potential interactions between these pathways:

• Metabolic cross-talk: Both pathways compete for common precursors and energy resources. The violacein biosynthetic pathway requires tryptophan as a precursor, while histidine biosynthesis requires significant ATP input (31-41 ATP molecules per histidine molecule) .

• Regulatory overlap: The quorum sensing system CviI/CviR in C. violaceum regulates violacein production and may also influence expression of histidine biosynthetic genes including hisG, suggesting coordinated regulation of these pathways.

• Physiological role: Bacteria with dark violet color (high violacein production) show resistance to various antibiotics , suggesting that violacein production may be coordinated with other cellular stress responses, potentially including amino acid biosynthesis regulation.

To investigate these interactions experimentally, I recommend:

• Metabolic flux analysis comparing wild-type and hisG mutant strains

• Transcriptomics to identify co-regulation patterns between violacein and histidine biosynthetic genes

• Biochemical assays to test whether violacein or its precursors directly affect HisG activity

Understanding this relationship could provide insights into bacterial physiology and potentially reveal new strategies for antimicrobial development targeting C. violaceum.

What are the implications of HisG structure and function for developing antimicrobials against C. violaceum infections?

The critical role of HisG in histidine biosynthesis makes it an attractive target for antimicrobial development against C. violaceum, particularly given its involvement in potentially fatal human infections . Several factors make C. violaceum HisG particularly suitable as a drug target:

• Essentiality: As the first enzyme in the histidine biosynthetic pathway, inhibition of HisG would block the production of a critical amino acid.

• No human homolog: Humans lack the histidine biosynthetic pathway, obtaining histidine through diet, thus reducing potential off-target effects.

• Structural uniqueness: The hexameric structure of bacterial HisG provides multiple allosteric sites that can be targeted by inhibitors.

• Accessibility: The requirement for Mg²⁺ and ATP provides opportunities for designing competitive inhibitors with favorable pharmacokinetic properties.

Promising approaches for inhibitor development include:

• Structure-based design: Using the crystal structures of C. violaceum HisG with its substrates and product to identify ligand-binding hot-spots .

• Fragment-based screening: Identifying small molecular building blocks that bind to different sites on HisG and can be linked to create high-affinity inhibitors.

• Natural product derivatives: Investigating whether violacein derivatives or other C. violaceum metabolites may serve as starting points for inhibitor design.

• Multi-targeting approaches: Developing compounds that simultaneously inhibit HisG and other enzymes in the histidine biosynthetic pathway for synergistic effects.

The development of such inhibitors could address the challenge posed by C. violaceum's resistance to many conventional antibiotics, including vancomycin, ampicillin, and linezolid .

How can comparative genomics and phylogenetic analysis of hisG genes inform evolutionary studies of Chromobacterium species?

Comparative genomics and phylogenetic analysis of hisG genes provide valuable insights into the evolutionary history of Chromobacterium species and their adaptation to various ecological niches. This approach can reveal patterns of gene conservation, horizontal gene transfer, and selective pressures acting on histidine biosynthesis.

Methodological approach for evolutionary analysis of C. violaceum hisG:

• Sequence similarity networks (SSN) construction:

  • Collect hisG sequences from diverse bacterial species

  • Generate sequence similarity networks at different thresholds

  • Identify clusters that correspond to taxonomic or functional groups

• Phylogenetic tree construction:

  • Multiple sequence alignment of hisG coding sequences

  • Selection of appropriate evolutionary models

  • Maximum likelihood or Bayesian inference analysis

• Molecular evolution analysis:

  • Calculate dN/dS ratios to identify sites under positive selection

  • Perform ancestral sequence reconstruction

  • Analyze coevolution patterns with other histidine biosynthetic genes

• Comparative genomics:

  • Analyze synteny of hisG and surrounding genomic regions

  • Identify potential horizontal gene transfer events

  • Compare with the organization of histidine biosynthetic genes in other species