Recombinant Nocardia farcinica Phosphoribosyl-ATP pyrophosphatase (hisE)

What is Nocardia farcinica and why is it significant in research?

Nocardia farcinica is a filamentous-growing Gram-positive soil saprophyte belonging to the family Actinomycetales, which also includes clinically and industrially important genera such as Mycobacterium, Streptomyces, Corynebacterium, and Rhodococcus. The significance of N. farcinica in research stems from its dual nature as both a pathogen and an organism with remarkable metabolic versatility .

N. farcinica IFM 10152, a clinical isolate, has been fully sequenced, revealing a genome consisting of a single circular chromosome of 6,021,225 bp with an average G+C content of 70.8% and two plasmids (pNF1 and pNF2) of 184,027 bp and 87,093 bp, respectively . This genomic data has provided researchers with valuable insights into the bacterium's pathogenicity mechanisms, multidrug resistance properties, and metabolic capabilities.

As a pathogen, N. farcinica causes nocardiosis, a disease affecting the lungs, central nervous system, brain, and cutaneous tissues in humans and animals. The incidence of nocardiosis is rising, with approximately 109-136 new cases annually in Japan and 500-1,000 in the United States . The bacterium's intrinsic resistance to multiple antibiotics makes treatment particularly challenging, highlighting the importance of understanding its biological mechanisms for developing effective therapeutic strategies.

What is Phosphoribosyl-ATP pyrophosphatase (hisE) and its role in metabolism?

Phosphoribosyl-ATP pyrophosphatase, encoded by the hisE gene, functions as the second enzyme in the histidine biosynthetic pathway. This critical enzyme irreversibly hydrolyzes phosphoribosyl-ATP to generate products necessary for histidine biosynthesis . The enzyme plays a vital role in the metabolic network of N. farcinica and other bacteria, controlling the flow of substrates through the histidine pathway.

The histidine biosynthesis pathway is essential for bacterial survival in environments where this amino acid is not readily available. Understanding the structure and function of hisE provides insights into bacterial metabolism and potential targets for antimicrobial development, particularly given the increasing resistance of N. farcinica to conventional antibiotics.

What are the best methods for cloning and expressing recombinant hisE from N. farcinica?

Effective cloning and expression of recombinant hisE from N. farcinica requires careful consideration of several methodological factors:

Gene isolation strategies:

• PCR amplification using primers designed based on the published genome sequence of N. farcinica IFM 10152

• Colony PCR screening for rapid identification of positive clones

• Restriction enzyme digestion and ligation into appropriate expression vectors

Expression systems optimization:
Considering the high G+C content (70.8%) of the N. farcinica genome , researchers should:

• Utilize expression hosts adapted for high G+C content genes (e.g., Streptomyces or modified E. coli strains)

• Optimize codon usage for the selected expression host

• Consider using inducible promoter systems to control expression levels

Purification approach:

• Fusion tags selection (His-tag, GST, MBP) for affinity purification

• Development of a purification protocol typically involving:

  • Cell lysis under optimized buffer conditions

  • Primary capture using affinity chromatography

  • Secondary purification using ion exchange or size exclusion chromatography

  • Quality assessment using SDS-PAGE and activity assays

When implementing these methods, researchers should monitor enzyme activity throughout the purification process to ensure the recombinant protein maintains its catalytic properties. The high G+C content of N. farcinica genes may necessitate optimization of PCR conditions, including the use of DMSO or specialized polymerases designed for GC-rich templates.

How should experimental design be structured for studying recombinant hisE activity?

A robust experimental design for studying recombinant hisE activity should follow these systematic steps:

Research question formulation:
Clearly define the specific aspects of hisE activity you aim to investigate, such as kinetic parameters, substrate specificity, or effects of environmental conditions .

Hypothesis development:
Create precise, testable hypotheses about expected enzyme behavior under various conditions .

Variable identification:

• Independent variables: substrate concentration, pH, temperature, buffer composition

• Dependent variables: reaction rate, product formation, enzyme stability

• Control variables: enzyme concentration, reaction time, equipment calibration

Experimental design selection:
Choose appropriate experimental designs based on your research questions:

• Factorial designs for studying interactions between multiple factors affecting enzyme activity

• Response surface methodology for optimizing reaction conditions

• Randomized block designs to control for batch-to-batch variation in enzyme preparations

Sample size calculation:
Determine appropriate replicate numbers to ensure statistical validity, considering:

• Expected magnitude of effects

• Inherent variability in enzyme assays

• Desired significance level and statistical power

Data collection protocol:
Develop standardized protocols for enzyme activity measurements, including:

• Spectrophotometric assays for product formation or substrate depletion

• Controls for spontaneous hydrolysis of phosphoribosyl-ATP

• Time-course measurements to establish reaction linearity

Analysis methods:
Select appropriate statistical methods based on your experimental design:

• ANOVA for comparing multiple conditions

• Regression analysis for establishing kinetic parameters

• Non-linear modeling for complex enzyme kinetics

Quality control measures:

• Internal standards and reference enzymes

• Technical and biological replicates

• Blinded sample analysis when appropriate

This structured approach ensures methodological rigor and reproducibility in investigating the biochemical properties of recombinant hisE from N. farcinica.

How does recombination analysis inform our understanding of hisE gene evolution in Nocardia species?

Recombination analysis provides critical insights into the evolutionary history and genetic diversity of the hisE gene across Nocardia species. Researchers investigating this aspect should consider the following methodological approaches:

Comparative sequence analysis:

• Align hisE sequences from multiple Nocardia species and related actinomycetes

• Identify conserved regions indicating functional importance

• Detect variable regions that might contribute to species-specific adaptations

Recombination detection:
Apply specialized algorithms and statistical methods to identify potential recombination events in the evolutionary history of hisE2. Key approaches include:

• Similarity plot analysis to visualize sequence relationships

• Phylogenetic incongruence tests to detect conflicting evolutionary signals

• Statistical tests for recombination detection (e.g., GARD, RDP4)

Gene linkage assessment:
Determine whether hisE is genetically linked to other genes in the histidine biosynthesis pathway, which would affect how it recombines:

• Analyze the genomic context of hisE in N. farcinica and related species

• Assess whether hisE undergoes independent assortment or linked inheritance2

• Consider how linked genes might co-evolve or be transferred together during horizontal gene transfer events

Evolutionary implications:
Interpret recombination data in the context of:

• Selection pressures acting on the histidine biosynthesis pathway

• Potential acquisition of adaptive traits through recombination

• Relationship between recombination patterns and ecological niches of different Nocardia species

The recombination analysis of hisE should be viewed within the broader context of genome evolution in the Actinomycetales family, which exhibits significant genomic plasticity through gene duplication and horizontal gene transfer . Understanding these patterns can provide insights into how essential metabolic pathways like histidine biosynthesis evolve while maintaining their core functionality.

What structural insights can be gained from studying recombinant N. farcinica hisE compared to homologs from other organisms?

Structural analysis of recombinant N. farcinica hisE compared to homologs from other organisms can reveal important insights into enzyme function, evolution, and potential drug targeting. A comprehensive structural investigation would involve:

Protein structure determination:

• X-ray crystallography to determine three-dimensional structure at high resolution (comparable to the 1.25 Å resolution achieved for other phosphoribosyl-ATP pyrophosphohydrolases )

• NMR spectroscopy for solution structure and dynamics analysis

• Cryo-EM for analyzing potential protein complexes with other histidine biosynthesis enzymes

Comparative structural analysis:

• Superimposition of N. farcinica hisE structure with homologs from other bacteria, particularly comparing:

  • Active site architecture and catalytic residues

  • Substrate binding pocket configuration

  • Secondary structural elements and domain organization

• Identification of structural features unique to N. farcinica hisE that might explain:

  • Substrate specificity differences

  • Catalytic efficiency variations

  • Stability under different environmental conditions

Structure-function relationships:

• Site-directed mutagenesis of conserved residues to validate their roles

• Enzyme kinetics correlated with structural features

• Molecular dynamics simulations to understand conformational changes during catalysis

Applied structural insights:

• Rational design of inhibitors targeting unique structural features of N. farcinica hisE

• Structure-guided protein engineering to enhance enzyme properties for biotechnological applications

• Evolutionary analysis based on structural conservation patterns

The high G+C content of N. farcinica's genome (70.8%) might influence codon usage and protein folding in ways that distinguish its enzymes from homologs in organisms with different genomic compositions. These distinctive features could be exploited for selective targeting of N. farcinica enzymes in therapeutic development.

What are the main challenges in purifying active recombinant hisE from N. farcinica?

Purification of active recombinant hisE from N. farcinica presents several technical challenges that researchers must address through methodical approaches:

Expression challenges:

• High G+C content (70.8%) of N. farcinica genome may lead to:

  • Secondary structure formation in mRNA

  • Codon usage bias affecting translation efficiency

  • Premature transcription termination

Solution approaches:

• Codon optimization for expression host

• Use of specialized strains designed for GC-rich genes

• Fusion with solubility-enhancing partners (MBP, SUMO, etc.)

Solubility issues:

• Potential for inclusion body formation

• Aggregation during concentration steps

• Limited stability in standard buffer systems

Solution approaches:

• Systematic screening of expression conditions (temperature, induction, media)

• Development of refolding protocols if inclusion bodies are unavoidable

• Buffer optimization using factorial design experiments

Activity preservation:

• Loss of catalytic activity during purification steps

• Interference from co-purifying proteins or contaminants

• Dependence on specific cofactors or metal ions

Solution approaches:

• Activity assays at each purification stage

• Addition of stabilizing agents (glycerol, reducing agents)

• Inclusion of necessary cofactors in purification buffers

Purification strategy optimization:

• Selection of appropriate chromatography techniques:

  • IMAC for His-tagged constructs

  • Ion exchange chromatography exploiting pI characteristics

  • Size exclusion for final polishing and buffer exchange

• Development of a purification table tracking protein quantity, purity, and specific activity:

Note: Values in this table are representative examples based on typical protein purification workflows and would need to be experimentally determined for N. farcinica hisE.

How can researchers overcome the challenge of potential contamination with endogenous E. coli proteins during recombinant expression?

Ensuring recombinant hisE purity from E. coli host proteins requires a strategic approach to expression and purification:

Prevention strategies during expression:

• Use of knockout E. coli strains lacking endogenous histidine pathway enzymes

• Expression systems with tight transcriptional control to maximize target protein ratio

• Compartmentalization approaches (periplasmic expression or inclusion body formation followed by refolding)

Tag selection strategies:

• Dual affinity tags for tandem purification (e.g., His-tag combined with GST or MBP)

• Cleavable tags with high specificity proteases (TEV, PreScission)

• Uniquely positioned tags (N-terminal, C-terminal, or internal) optimized for accessibility

Enhanced purification strategies:

• Orthogonal chromatography techniques:

  • Affinity chromatography targeting fusion tags

  • Ion exchange chromatography exploiting unique pI characteristics

  • Hydrophobic interaction chromatography

  • Size exclusion as final polishing step

• Selective precipitation techniques:

  • Ammonium sulfate fractionation

  • pH-dependent precipitation

  • Heat treatment if hisE exhibits higher thermostability than E. coli proteins

Contamination assessment and removal:

• Mass spectrometry-based proteomic analysis to identify contaminants

• Western blot analysis with antibodies against common E. coli contaminants

• Subtractive approaches using immobilized antibodies against E. coli proteins

Quality control metrics:

• SDS-PAGE with densitometry analysis (≥95% purity standard)

• Activity assays with specific substrates

• Analytical SEC to confirm monodispersity

Implementing these strategies systematically can significantly reduce contamination issues, resulting in highly pure recombinant hisE suitable for structural and functional studies.

What are the optimal methods for determining the kinetic parameters of recombinant N. farcinica hisE?

Determining accurate kinetic parameters for recombinant N. farcinica hisE requires careful experimental design and rigorous analysis methods:

Assay development considerations:

• Direct measurement options:

  • Spectrophotometric monitoring of phosphoribosyl-ATP consumption (λ = 290 nm)

  • Coupled enzyme assays linking product formation to spectrophotometric readouts

  • Radioactive substrate assays for highest sensitivity

• Reaction condition optimization:

  • pH optimization (typically pH 7.0-8.5 for phosphoribosyl-ATP pyrophosphohydrolases)

  • Buffer selection to avoid inhibition or interference

  • Temperature optimization balancing enzyme stability and activity

  • Metal ion requirements determination

Kinetic parameter determination:

• Initial velocity measurements:

  • Substrate concentration range spanning 0.2-5× Km

  • Multiple time points to ensure linearity

  • Sufficient enzyme dilution to prevent substrate depletion

• Data analysis approaches:

  • Michaelis-Menten equation fitting

  • Lineweaver-Burk, Eadie-Hofstee, or Hanes-Woolf plots for visualization

  • Non-linear regression analysis for direct parameter estimation

Advanced kinetic investigations:

• Inhibition studies:

  • Product inhibition analysis

  • Feedback inhibition by histidine or pathway intermediates

  • Competitive inhibitor studies

• pH-rate profiles:

  • Activity measurements across pH range (5.0-9.0)

  • pKa determination of catalytically important residues

• Temperature effects:

  • Arrhenius plot analysis

  • Thermodynamic parameter calculation (ΔH‡, ΔS‡, ΔG‡)

Expected kinetic parameters range:
Based on related phosphoribosyl-ATP pyrophosphohydrolases:

Note: These values represent typical ranges for this class of enzymes and would need to be experimentally determined for N. farcinica hisE.

How can researchers effectively analyze the role of hisE in N. farcinica metabolism through gene deletion or complementation studies?

Investigating the physiological role of hisE in N. farcinica metabolism requires genetic manipulation approaches that must be carefully designed and executed:

Gene deletion strategy:

• Homologous recombination approach:

  • Construction of deletion cassettes with antibiotic resistance markers flanked by homologous regions

  • Optimization of transformation protocols for N. farcinica (electroporation or conjugation)

  • Screening strategies for identifying successful recombinants

• CRISPR-Cas9 systems:

  • Design of guide RNAs targeting hisE

  • Development of Cas9 expression systems functional in N. farcinica

  • Repair template design for marker insertion or scarless deletion

Complementation approaches:

• Plasmid-based complementation:

  • Selection of appropriate vectors (shuttle vectors for N. farcinica)

  • Promoter selection (native vs. constitutive)

  • Copy number considerations and expression level control

• Chromosomal integration:

  • Site-specific integration systems

  • Ectopic integration at neutral sites

  • Restoration of native regulation through homologous recombination

Phenotypic analysis:

• Growth characterization:

  • Minimal media with and without histidine supplementation

  • Growth curve analysis (lag phase, doubling time, maximum OD)

  • Competition assays with wild-type strain

• Metabolomic analysis:

  • Targeted analysis of histidine pathway intermediates

  • Global metabolite profiling to identify broader metabolic impacts

  • Isotope labeling studies to track metabolic flux

• Transcriptomic response:

  • RNA-Seq analysis of gene expression changes in ΔhisE mutants

  • Identification of compensatory pathways or stress responses

  • Analysis of histidine regulon in N. farcinica

Expected phenotypic outcomes:

• Complete hisE deletion:

  • Histidine auxotrophy (inability to grow without histidine supplementation)

  • Accumulation of phosphoribosyl-ATP

  • Potential polar effects on downstream his genes if operon structure is disrupted

• Conditional knockdown:

  • Growth inhibition under limiting histidine conditions

  • Dose-dependent response to histidine availability

  • Altered virulence or stress resistance phenotypes

The analysis should consider the genomic context of hisE in N. farcinica and the potential for compensatory mechanisms, particularly given the metabolic versatility of this organism revealed by its genome sequence .

How does hisE function potentially relate to N. farcinica pathogenicity or drug resistance?

The connection between hisE function and N. farcinica pathogenicity or drug resistance represents an important area for research, given the clinical significance of this organism:

Pathogenicity connections:

• Amino acid biosynthesis and virulence:

  • Histidine availability in host environments (typically limited in mammalian tissues)

  • Requirement for de novo histidine synthesis during infection

  • Potential attenuation of virulence in histidine auxotrophs

• Stress response and persistence:

  • Role of histidine in protein stability under stress conditions

  • Participation of histidine in pH buffering within phagosomes

  • Contribution to long-term persistence in host tissues

Drug resistance considerations:

Therapeutic potential:

• Inhibitor development strategy:

  • Structure-based design targeting unique features of N. farcinica hisE

  • High-throughput screening approaches

  • Repurposing of known compounds affecting histidine metabolism

• Combination therapy opportunities:

  • Synergy with existing antibiotics used against nocardiosis

  • Targeting multiple steps in histidine biosynthesis

  • Dual-targeting approaches affecting multiple amino acid pathways

N. farcinica exhibits sophisticated drug resistance mechanisms beyond simple permeability barriers, as revealed by its genome sequence . Understanding how hisE and histidine metabolism integrate with these resistance mechanisms could provide new avenues for therapeutic intervention against this challenging pathogen.

What bioinformatic approaches are most effective for analyzing hisE conservation across bacterial species, with implications for broad-spectrum drug development?

Effective bioinformatic analysis of hisE conservation requires integrated computational approaches to identify both conserved features and species-specific characteristics:

Sequence-based approaches:

• Comprehensive homology searches:

  • PSI-BLAST for distant homolog identification

  • HMM-based searches for detecting remote relationships

  • Phylogenetic profiling across diverse bacterial taxa

• Multiple sequence alignment strategies:

  • Progressive alignment methods (MUSCLE, MAFFT)

  • Structure-guided alignments when reference structures are available

  • Conservation scoring using entropy-based methods

• Evolutionary analysis:

  • Maximum likelihood or Bayesian phylogenetic tree construction

  • Selection pressure analysis (dN/dS ratios)

  • Ancestral sequence reconstruction

Structure-based approaches:

• Comparative structural analysis:

  • Homology modeling of N. farcinica hisE based on crystal structures (such as the 1.25 Å resolution structure mentioned )

  • Comparison of active site architecture across species

  • Analysis of structural plasticity and conservation

• Binding site identification:

  • Computational pocket detection algorithms

  • Conservation mapping onto structural models

  • Molecular dynamics simulations to identify cryptic binding sites

• Virtual screening implications:

  • Pharmacophore model development based on conserved features

  • Ensemble docking approaches accounting for structural variability

  • Fragment-based approaches targeting highly conserved sub-pockets

Drug development applications:

• Target assessment metrics:

  • Conservation scores across pathogenic species

  • Absence in human proteome

  • Essentiality predictions based on metabolic modeling

• Selectivity analysis:

  • Identification of unique features in N. farcinica hisE

  • Design of selective inhibitors based on species-specific features

  • Assessment of potential for resistance development

Visualization and data integration:

• Conservation mapping:

  • Heat map representation of sequence conservation across species

  • Structure-based visualization of conserved residues

  • Network analysis of co-evolving residues

These bioinformatic approaches should be applied in the context of N. farcinica's position within the Actinomycetales, which includes other clinically relevant genera such as Mycobacterium . The comparative analysis could reveal important insights into evolutionary adaptation of the histidine biosynthesis pathway across different ecological niches and pathogenic lifestyles.

What are the most promising directions for future research on recombinant N. farcinica hisE?

The study of recombinant N. farcinica hisE offers several promising research avenues that could significantly advance our understanding of both basic biology and potential applications:

Structural and functional refinement:

• High-resolution structural studies:

  • Crystal structures with substrate analogs or transition state mimics

  • NMR studies of protein dynamics during catalysis

  • Cryo-EM analysis of potential complexes with other histidine pathway enzymes

• Detailed reaction mechanism elucidation:

  • Isotope effect studies to probe transition states

  • Identification of catalytic residues through systematic mutagenesis

  • Computational simulations of the reaction coordinate

Systems biology integration:

• Metabolic modeling approaches:

  • Integration of hisE function into genome-scale metabolic models of N. farcinica

  • Flux balance analysis to predict effects of hisE perturbation

  • Identification of synthetic lethal interactions with other metabolic genes

• Multi-omics investigations:

  • Proteomics to identify interaction partners of hisE

  • Transcriptomics to understand regulation of the histidine pathway

  • Metabolomics to track metabolic consequences of hisE manipulation

Biotechnological applications:

• Enzyme engineering opportunities:

  • Directed evolution for enhanced stability or altered substrate specificity

  • Development as a biocatalyst for synthesis of phosphoribosyl compounds

  • Creation of biosensors for histidine pathway intermediates

• Potential industrial applications:

  • Production of histidine or histidine derivatives

  • Development of enzymatic assays for related compounds

  • Bioremediation applications leveraging N. farcinica's metabolic versatility

Therapeutic development:

• Target validation studies:

  • In vivo essentiality confirmation in infection models

  • Chemical biology approaches using conditional inhibition

  • Resistance development assessment

• Fragment-based drug discovery:

  • Screening libraries against purified recombinant hisE

  • Structure-guided optimization of hit compounds

  • Development of activity-based probes for target engagement studies

These research directions build upon the unique characteristics of N. farcinica, including its metabolic versatility and dual nature as both a soil saprophyte and a human pathogen , offering opportunities for both basic science advances and applied research outcomes.

How might advances in protein expression systems impact future studies of recombinant N. farcinica enzymes like hisE?

Emerging advances in protein expression technologies are likely to have significant impacts on the study of challenging targets like recombinant N. farcinica hisE:

Next-generation expression systems:

• Cell-free protein synthesis:

  • Advantages for toxic or membrane-associated proteins

  • Rapid production for screening multiple constructs

  • Direct incorporation of non-canonical amino acids for mechanistic studies

• Alternative host organisms:

  • Mycobacterial expression systems better adapted to high G+C content genes

  • Streptomyces expression systems leveraging natural secretion pathways

  • Extremophile-based systems for enhanced protein stability

Genetic code expansion approaches:

• Site-specific incorporation of non-canonical amino acids:

  • Photocrosslinking amino acids for interaction studies

  • Bio-orthogonal handles for selective labeling

  • Fluorescent amino acids for conformational studies

• Applications to N. farcinica hisE research:

  • Mechanistic investigations using chemical probes at catalytic sites

  • Dynamics studies using environmentally sensitive amino acids

  • Mapping of interaction surfaces with crosslinking amino acids

Advanced purification technologies:

• Automated systems:

  • High-throughput parallel purification platforms

  • Real-time activity monitoring during purification

  • Machine learning optimization of purification conditions

• Novel tags and matrices:

  • Self-cleaving purification tags

  • Stimulus-responsive smart polymers for purification

  • Nanobody-based affinity systems

Structural biology integration:

• High-throughput crystallization:

  • Microfluidic approaches to crystal optimization

  • In situ diffraction at synchrotron sources

  • Serial crystallography for microcrystals

• Integrated structural platforms:

  • Combined approaches using X-ray, NMR, and cryo-EM

  • Time-resolved structural studies of enzyme function

  • Computational prediction and experimental validation pipelines

The application of these advanced technologies to N. farcinica enzymes could overcome current technical limitations and accelerate research progress. Particularly for enzymes like hisE, which may present expression challenges due to the high G+C content of the N. farcinica genome (70.8%) , these new approaches could significantly improve protein yield, purity, and functional characterization capabilities.