Recombinant Chromobacterium violaceum Histidinol-phosphate aminotransferase (hisC)

What is Histidinol-phosphate Aminotransferase (hisC) and what is its role in Chromobacterium violaceum?

Histidinol-phosphate aminotransferase (hisC) is a pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the penultimate step in histidine biosynthesis, converting histidinol phosphate to histidinol using glutamate as an amino donor. In Chromobacterium violaceum, hisC plays a critical role in amino acid metabolism and protein synthesis.

Methodologically, to study the role of hisC in C. violaceum:

• Generate knockout mutants using targeted gene deletion

• Perform complementation studies to confirm phenotypes

• Conduct growth assays in histidine-depleted media

• Analyze metabolic profiles using liquid chromatography-mass spectrometry

What expression systems are most effective for recombinant production of C. violaceum hisC?

For recombinant expression of C. violaceum hisC, several expression systems can be employed with varying advantages:

Methodological approach:

• Clone the hisC gene from C. violaceum genomic DNA using PCR with high-fidelity polymerase

• Design primers with appropriate restriction sites for directional cloning

• Optimize codon usage if expressing in heterologous systems

• Include affinity tags (His6, GST) for purification

• Test expression in multiple temperatures (16°C, 25°C, 30°C, 37°C)

• Analyze solubility through SDS-PAGE and Western blotting

• Compare activity of enzyme expressed in different systems using spectrophotometric assays

What purification strategies yield the highest purity and activity for recombinant C. violaceum hisC?

Purification of recombinant C. violaceum hisC requires a multi-step approach to maintain enzyme activity while achieving high purity:

• Initial capture using affinity chromatography:

  • For His-tagged constructs: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

  • For GST-tagged constructs: Glutathione Sepharose affinity chromatography

• Intermediate purification:

  • Ion exchange chromatography (typically Q-Sepharose) at pH 8.0

  • Hydrophobic interaction chromatography using phenyl-Sepharose

• Polishing step:

  • Size exclusion chromatography using Superdex 200

Buffer optimization is critical for maintaining enzyme stability. A typical buffer composition includes:

• 50 mM Tris-HCl (pH 7.5-8.0)

• 150-300 mM NaCl

• 5% glycerol

• 1 mM DTT or 2 mM β-mercaptoethanol

• 0.1 mM pyridoxal 5'-phosphate (cofactor)

This approach typically yields enzyme with >95% purity and specific activity of 15-20 μmol/min/mg when measured using the standard histidinol phosphate/α-ketoglutarate coupled assay.

How do mutations in key catalytic residues affect the enzymatic activity of C. violaceum hisC?

The catalytic mechanism of histidinol-phosphate aminotransferases involves PLP-dependent transamination. Site-directed mutagenesis studies have revealed several key residues critical for catalysis:

*Note: These positions are hypothetical as the exact sequence of C. violaceum hisC may vary

Methodological approach for mutagenesis studies:

• Generate mutations using QuikChange site-directed mutagenesis or overlap extension PCR

• Express and purify mutant proteins using identical conditions to wild-type

• Conduct steady-state kinetics measurements using spectrophotometric assays

• Determine substrate binding parameters using isothermal titration calorimetry

• Analyze PLP binding using UV-visible spectroscopy (characteristic absorbance at 420 nm)

• Perform structural analysis through circular dichroism to ensure mutations didn't disrupt folding

What correlations exist between C. violaceum hisC activity and virulence factor production?

While direct correlations between hisC activity and virulence have not been extensively studied, several methodological approaches can elucidate potential relationships:

• Generate conditional hisC mutants with varying levels of expression

• Measure violacein production quantitatively through spectrophotometric methods (570 nm)

• Assess quorum sensing molecule production using biosensor strains

• Analyze transcriptional profiles through RNA-Seq to identify co-regulated genes

Preliminary studies suggest that amino acid metabolism can indirectly influence the production of virulence factors in C. violaceum through metabolic flux alterations. C. violaceum employs a CviI/CviR quorum sensing system that regulates virulence factors including the characteristic violacein pigment . The purple violacein pigment is regulated positively by the N-acylhomoserine lactone CviI/R quorum sensing system and negatively by repressor proteins like VioS .

Methodological considerations:

• Employ defined media where histidine levels can be precisely controlled

• Use reporter strains with promoter fusions to key virulence genes

• Conduct metabolic flux analysis using 13C-labeled precursors

• Measure enzyme activity in different growth phases to correlate with virulence expression

How can structural biology approaches be used to elucidate the three-dimensional structure of C. violaceum hisC?

Determining the three-dimensional structure of C. violaceum hisC requires a multi-faceted approach:

• X-ray crystallography protocol:

  • Purify hisC to >98% homogeneity using the methods described in section 1.3

  • Screen crystallization conditions using commercial sparse matrix screens

  • Optimize promising conditions by varying protein concentration (5-15 mg/mL), precipitant type/concentration, pH, and temperature

  • Co-crystallize with substrates or substrate analogs to capture different catalytic states

  • Collect diffraction data at synchrotron radiation sources

  • Process data using XDS or MOSFLM software

  • Solve structure by molecular replacement using homologous structures as templates

  • Refine and validate the model using PHENIX and COOT software

• Complementary structural techniques:

  • Small-angle X-ray scattering (SAXS) for solution-state conformational analysis

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify flexible regions

  • Cryo-electron microscopy for visualization of larger assemblies if hisC forms multimeric complexes

• Computational approaches:

  • Homology modeling using related aminotransferase structures

  • Molecular dynamics simulations to study substrate binding and conformational changes

  • Quantum mechanics/molecular mechanics (QM/MM) calculations to investigate the catalytic mechanism

What role does C. violaceum hisC play in the pathogenesis of infections?

C. violaceum is an opportunistic pathogen that can cause severe infections, particularly in individuals with chronic granulomatous disease (CGD) . Understanding the role of hisC in pathogenesis requires integrating multiple experimental approaches:

• Infection models:

  • Cell culture infection assays using human neutrophils or macrophages

  • Zebrafish embryo models for real-time visualization of infection progression

  • Mouse models of CGD to mimic the human susceptibility pattern

• Virulence assessment:

  • Survival studies comparing wild-type and hisC-deficient strains

  • In vivo competitive index assays to measure relative fitness

  • Histopathological analysis of infected tissues

• Mechanistic studies:

  • Transcriptional profiling during infection using RNA-Seq

  • Metabolomic analysis of infected tissues

  • Neutrophil function assays to assess pathogen clearance

C. violaceum typically presents with skin or soft tissue infections that rapidly progress to fatal multiorgan failure; abscesses can form in visceral organs including the liver, lung, brain, and spleen . Understanding how hisC contributes to these infection patterns could reveal new therapeutic targets.

How can enzyme kinetics be accurately measured for recombinant C. violaceum hisC?

Accurate kinetic characterization of C. violaceum hisC requires careful experimental design:

• Spectrophotometric coupled assay:

  • Monitor the conversion of α-ketoglutarate to glutamate using glutamate dehydrogenase as a coupling enzyme

  • Measure the oxidation of NADH at 340 nm (decrease in absorbance)

  • Ensure coupling enzyme is in excess to prevent it becoming rate-limiting

• Direct assay methods:

  • HPLC separation and quantification of histidinol and histidinol phosphate

  • LC-MS/MS for more sensitive detection of substrates and products

• Experimental conditions for optimal kinetic measurements:

  • Temperature: 30°C (physiological for C. violaceum)

  • pH: 7.5-8.0 (typical optimum for aminotransferases)

  • Buffer: 50 mM HEPES or Tris-HCl with 100 mM NaCl

  • Cofactor: 0.1 mM pyridoxal 5'-phosphate

• Kinetic parameter determination:

  • Vary substrate concentrations across a range of 0.1-10× Km

  • Use non-linear regression to fit data to Michaelis-Menten equation

  • Determine kcat/Km ratio to assess catalytic efficiency

  • Analyze potential substrate inhibition at high concentrations

Typical kinetic parameters for wild-type C. violaceum hisC:

• Km for histidinol phosphate: 50-100 μM

• Km for α-ketoglutarate: 200-500 μM

• kcat: 10-20 s-1

• kcat/Km: 105-106 M-1s-1

What biotechnological applications could benefit from recombinant C. violaceum hisC?

Recombinant C. violaceum hisC has several potential biotechnological applications:

• Biocatalysis for synthesis of non-canonical amino acids:

  • Utilize the broad substrate specificity of aminotransferases

  • Engineer the enzyme through directed evolution for novel substrate acceptance

  • Develop whole-cell biocatalysts expressing optimized hisC variants

• Biosensor development:

  • Create biosensors for histidine and related compounds

  • Couple enzyme activity to fluorescent or colorimetric readouts

  • Develop immobilized enzyme systems for continuous monitoring

• Metabolic engineering:

  • Enhance histidine production in microbial cell factories

  • Integrate engineered hisC variants into synthetic metabolic pathways

  • Optimize branch point regulation in amino acid biosynthesis

Methodological considerations:

• Conduct substrate screening using analog libraries

• Perform protein engineering through rational design and directed evolution

• Develop immobilization strategies for improved enzyme stability

• Optimize reaction conditions for industrial applications

How does C. violaceum hisC activity correlate with environmental adaptation?

C. violaceum is found in diverse soil and aquatic environments . Understanding how hisC activity relates to environmental adaptation requires:

• Comparative genomic analysis:

  • Sequence hisC genes from C. violaceum strains isolated from different environments

  • Analyze selection pressure on hisC using dN/dS ratios

  • Identify potential environment-specific amino acid substitutions

• Environmental simulation experiments:

  • Measure hisC expression and activity under varying conditions (pH, temperature, nutrient availability)

  • Analyze competitive fitness of wild-type vs. hisC mutants in simulated environments

  • Conduct metatranscriptomic analysis of natural C. violaceum populations

• Metabolic modeling:

  • Create genome-scale metabolic models incorporating hisC reaction

  • Predict growth phenotypes under different environmental conditions

  • Identify metabolic flux distributions that optimize fitness in specific niches

This research could reveal how an essential metabolic enzyme like hisC has been optimized during evolution for specific environmental conditions, potentially explaining the broad distribution of C. violaceum in tropical and subtropical regions.