Recombinant Nocardia farcinica Aspartate carbamoyltransferase (pyrB)

What is the biological role of aspartate carbamoyltransferase in Nocardia farcinica?

Aspartate carbamoyltransferase (ATCase), encoded by the pyrB gene, catalyzes the first committed step in de novo pyrimidine biosynthesis, converting L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate and inorganic phosphate. In Nocardia farcinica, this enzyme is critical for nucleotide biosynthesis and thus cellular replication and survival. The enzyme likely plays a significant role in the organism's ability to adapt to different environments and contribute to its pathogenicity. Similar to other bacterial species, N. farcinica ATCase activity is presumably essential during active growth phases when pyrimidine demand is high, particularly during infection processes when rapid replication is required .

How does the structure of N. farcinica aspartate carbamoyltransferase compare to that of other bacterial species?

While the specific structure of N. farcinica ATCase has not been fully characterized in the provided literature, comparative analysis with other bacterial ATCases suggests likely structural similarities with potential unique features. Based on homology modeling approaches similar to those used for Methanococcus jannaschii ATCase, the N. farcinica enzyme likely preserves key catalytic residues that are evolutionarily conserved . The catalytic subunit (encoded by pyrB) likely forms a trimer, as observed in M. jannaschii where the PyrB gene product was determined to be trimeric . Unlike the well-characterized Escherichia coli ATCase that forms a dodecameric structure consisting of six catalytic and six regulatory subunits, the quaternary structure of N. farcinica ATCase may exhibit differences that reflect its adaptation to the specific metabolic requirements and environmental conditions of this pathogen .

What genomic context surrounds the pyrB gene in N. farcinica, and how might this influence its regulation?

The genomic organization of the pyrB gene and its surrounding regions in N. farcinica likely provides insights into its regulation and coordination with other metabolic pathways. While specific details about N. farcinica are not directly available in the research results, comparative genomic analysis with related species would be valuable. In many bacteria, pyrB is part of an operon structure that can include other pyrimidine biosynthesis genes. Research into the genomic context would reveal potential co-transcribed genes and regulatory elements such as attenuator regions or operator sites that respond to pyrimidine levels. Understanding this context is crucial for interpreting how N. farcinica modulates pyrimidine synthesis in response to environmental conditions and metabolic demands, which could be particularly relevant during infection processes .

What are the optimal expression systems for recombinant N. farcinica aspartate carbamoyltransferase?

Based on comparable research with other bacterial ATCases, E. coli expression systems represent the most practical approach for recombinant production of N. farcinica ATCase. The literature indicates successful heterologous expression of M. jannaschii pyrB and pyrI genes in E. coli at high levels, suggesting a similar approach would be viable for N. farcinica pyrB . The following expression strategy is recommended:

This approach would likely yield sufficient quantities of recombinant enzyme for subsequent purification and characterization studies, similar to what was achieved with M. jannaschii ATCase components .

What purification protocol is most effective for isolating recombinant N. farcinica pyrB with high purity and activity?

A multi-step purification strategy based on protocols developed for similar ATCases would be most effective for N. farcinica pyrB. Drawing from the purification methods described for M. jannaschii PyrB, the following protocol is recommended :

• Initial clarification: Harvest cells by centrifugation, resuspend in an appropriate buffer (typically 50 mM Tris-HCl, pH 8.0, containing 1 mM EDTA and 1 mM DTT), and disrupt by sonication or French press. Remove cell debris by centrifugation at >20,000×g.

• Ammonium sulfate fractionation: Perform sequential precipitation to remove contaminating proteins, collecting the fraction containing ATCase activity.

• Ion-exchange chromatography: Apply the resuspended ammonium sulfate fraction to a Q-Sepharose column and elute with a gradient of NaCl (0-500 mM).

• Affinity chromatography: If a His-tagged construct is used, Ni-NTA chromatography would provide high selectivity.

• Size-exclusion chromatography: As a final polishing step, apply the partially purified enzyme to a Superdex 200 column to separate according to molecular size.

Throughout purification, enzyme activity should be monitored using a colorimetric assay for ATCase activity, and protein purity assessed by SDS-PAGE. This approach typically yields enzyme with >95% purity suitable for kinetic and structural studies .

How can researchers overcome solubility challenges when expressing recombinant N. farcinica pyrB?

Recombinant expression of N. farcinica pyrB may face solubility challenges similar to those encountered with other bacterial proteins. To address these issues, researchers can implement several strategies:

These approaches should be tested systematically, assessing their impact on both yield and activity of the recombinant enzyme. Additionally, if the native N. farcinica pyrB forms complexes with regulatory subunits, co-expression with these partners might enhance solubility and stability .

What techniques are most appropriate for elucidating the quaternary structure of N. farcinica aspartate carbamoyltransferase?

To determine the quaternary structure of N. farcinica ATCase, a combination of biophysical techniques should be employed, similar to those used for other bacterial ATCases. Based on the approach used for M. jannaschii ATCase, the following methods are recommended :

• Size-exclusion chromatography: Using calibrated columns (Superdex 200 or similar) to estimate the native molecular weight of the enzyme complex.

• Native PAGE: Non-denaturing gel electrophoresis to analyze the intact enzyme complex and compare with known standards.

• Analytical ultracentrifugation: Sedimentation velocity and equilibrium experiments to determine precise molecular weight and assess homogeneity.

• Dynamic light scattering: To measure the hydrodynamic radius and estimate molecular weight.

• Mass spectrometry: Native mass spectrometry can provide accurate mass determination of intact complexes.

• Chemical cross-linking followed by mass spectrometry: To identify interacting subunits and their orientation within the complex.

• Cryo-electron microscopy: For direct visualization of the quaternary structure, particularly valuable if the complex is large.

These techniques would collectively provide comprehensive information about the oligomeric state of N. farcinica ATCase, whether it forms a trimer like the M. jannaschii catalytic subunit or assembles into a larger complex with potential regulatory subunits .

How does temperature affect the catalytic activity and stability of recombinant N. farcinica aspartate carbamoyltransferase?

Understanding temperature effects on N. farcinica ATCase is crucial given the potential adaptation of this pathogen to host environments. Although specific data for N. farcinica is not available from the search results, a systematic characterization approach similar to that used for M. jannaschii ATCase would include:

• Thermal stability profile: Determine enzyme activity retention after pre-incubation at temperatures ranging from 25-70°C, measuring residual activity at standard conditions.

• Temperature-activity relationship: Measure enzyme activity directly at different temperatures to determine the temperature optimum.

• Arrhenius plot analysis: Calculate activation energy by measuring reaction rates at different temperatures and constructing an Arrhenius plot. For M. jannaschii catalytic trimer, the activation energy was found to be similar to that of E. coli catalytic trimer .

• Differential scanning calorimetry (DSC): Determine the melting temperature (Tm) as a measure of thermal stability.

• Circular dichroism thermal melt: Monitor secondary structure changes in response to temperature.

What are the kinetic parameters of N. farcinica aspartate carbamoyltransferase, and how do they compare to those of other bacterial species?

A comprehensive kinetic characterization of N. farcinica ATCase would involve determining its fundamental catalytic parameters and comparing them to well-characterized bacterial ATCases. Based on approaches used for similar enzymes, the following kinetic analysis is recommended :

The kinetic profile would likely reveal whether N. farcinica ATCase exhibits hyperbolic kinetics similar to the M. jannaschii catalytic trimer or displays cooperativity like the E. coli holoenzyme . Additionally, testing the effects of nucleotide effectors (ATP, CTP, UTP) would determine whether the enzyme is subject to allosteric regulation. This characterization is essential for understanding the enzyme's role in the metabolic network of N. farcinica and may provide insights into potential differences that could be exploited for targeted therapies .

How can PCR-based methods be adapted to specifically identify the N. farcinica pyrB gene in clinical or environmental samples?

PCR-based detection of N. farcinica pyrB could be developed following principles similar to those used for species identification. Drawing from the N. farcinica-specific PCR method described in the literature, a targeted approach for the pyrB gene would involve :

• Sequence analysis: Perform comparative genomic analysis of the pyrB gene across Nocardia species to identify unique regions specific to N. farcinica.

• Primer design: Design primers targeting conserved regions within pyrB that contain species-specific polymorphisms, aiming for amplicons of 300-400 bp.

• PCR optimization: Determine optimal annealing temperatures, MgCl₂ concentrations, and cycle parameters to ensure specificity and sensitivity.

• Validation: Test the primers against a panel of Nocardia species and related actinomycetes to confirm specificity, similar to the approach that verified no amplification products from heterologous nocardial species (n = 59) or other related bacterial genera (n = 41) .

• Restriction analysis confirmation: Identify distinctive restriction enzyme sites within the amplicon that could provide additional verification through RFLP analysis, as was done with CfoI for the 314-bp N. farcinica specific fragment .

This approach would provide a rapid molecular method for identifying N. farcinica pyrB, complementing existing diagnostic methods and potentially offering insights into strain variations that might correlate with virulence or metabolic capabilities .

What is the potential of metagenomic next-generation sequencing (mNGS) for identifying N. farcinica and characterizing its pyrB gene in complex clinical samples?

Metagenomic next-generation sequencing (mNGS) offers powerful capabilities for detecting N. farcinica in complex clinical samples where traditional culture methods might fail. Based on the successful application of mNGS in clinical diagnosis of N. farcinica infection, the following approach would be effective for pyrB characterization :

• Sample processing: Extract total DNA from clinical specimens (blood, BALF, tissue) using methods that efficiently lyse Nocardia cell walls.

• Library preparation: Prepare shotgun metagenomic libraries using standard protocols, ensuring sufficient depth to detect low-abundance microbial sequences.

• Sequencing: Perform high-throughput sequencing on platforms such as Illumina NextSeq (as used in the case report) .

• Bioinformatic analysis:

  • Filter human reads to enrich for microbial sequences

  • Align remaining reads to reference databases containing Nocardia genomes

  • Specifically analyze coverage and sequence variants of the pyrB gene

  • Perform phylogenetic analysis to place the identified pyrB sequence in evolutionary context

• Validation: Confirm findings with targeted PCR or diagnostic tests specific for N. farcinica.

This approach enables not only identification of N. farcinica but also characterization of its pyrB gene sequence without prior isolation and culture, providing valuable epidemiological and research insights. In clinical contexts, mNGS can lead to rapid diagnosis and appropriate treatment, as demonstrated in the case report where appropriate antibiotic therapy was initiated promptly after mNGS identification .

How can structural information about N. farcinica aspartate carbamoyltransferase contribute to the development of novel diagnostic methods?

Structural characterization of N. farcinica ATCase can significantly advance diagnostic method development through several approaches:

• Epitope identification: Structural analysis can reveal surface-exposed regions unique to N. farcinica ATCase that could serve as targets for specific antibodies. These epitopes, identified through homology modeling and comparison with structures of homologous enzymes from other species, could form the basis of immunodiagnostic assays .

• Aptamer development: Knowledge of the three-dimensional structure facilitates the design of aptamers (DNA or RNA molecules) that specifically bind to N. farcinica ATCase with high affinity. These aptamers could be incorporated into biosensor platforms for rapid detection.

• Activity-based probes: Understanding the catalytic mechanism and active site architecture enables the design of mechanism-based inhibitors or substrate analogs that could be developed into activity-based probes specific for N. farcinica ATCase.

• Structure-guided PCR design: Structural insights can inform the selection of PCR primer binding sites that target regions with unique structural constraints, increasing specificity beyond sequence-based design alone .

• Differential inhibitor profiles: If structural differences exist in the active site compared to human or other bacterial ATCases, specific inhibitors could be developed that selectively interact with N. farcinica ATCase, forming the basis for selective growth inhibition assays.

These structure-based approaches could complement existing molecular methods, potentially offering improved specificity and sensitivity for N. farcinica detection in complex clinical or environmental samples .

How does the pyrB gene sequence and protein structure of N. farcinica compare to those of other clinically relevant Nocardia species?

A comprehensive comparative analysis of pyrB across Nocardia species would reveal evolutionary relationships and potential species-specific features. While detailed comparisons are not directly available in the search results, the approach would include:

• Sequence alignment analysis: Comparing pyrB nucleotide and amino acid sequences across Nocardia species to identify:

  • Conserved regions critical for catalysis and structure

  • Variable regions that might reflect species-specific adaptations

  • Potential signatures that could be used for species differentiation

• Phylogenetic analysis: Constructing phylogenetic trees based on pyrB sequences to understand evolutionary relationships among Nocardia species and assess whether pyrB evolution parallels species evolution.

• Structural comparison: Using homology modeling based on known bacterial ATCase structures (as done for M. jannaschii) to predict and compare N. farcinica ATCase structure with those of other Nocardia species .

• Functional motif analysis: Identifying catalytic residues, substrate-binding sites, and potential regulatory domains across species.

Such analysis would potentially reveal:

• Core ATCase functions preserved across all Nocardia species

• Specific adaptations in N. farcinica that might correlate with its particular ecological niche or virulence

• Potential targets for species-specific identification methods

This comparative approach provides context for understanding N. farcinica pyrB within the broader evolutionary landscape of Nocardia species and their metabolic adaptations .

What insights can be gained from comparing the regulatory mechanisms of aspartate carbamoyltransferase between N. farcinica and model organisms like E. coli?

Comparative analysis of regulatory mechanisms governing ATCase activity in N. farcinica versus well-characterized systems like E. coli could provide valuable insights into metabolic adaptation strategies. Based on observed differences between M. jannaschii and E. coli ATCase regulation, the following comparisons would be informative :

The investigation of these regulatory differences would provide insights into how N. farcinica has adapted its pyrimidine biosynthesis pathway to its specific ecological niche and pathogenic lifestyle. The differences in regulation might reflect adaptations to host environments or competition strategies in soil ecosystems where Nocardia species are commonly found .

How do the catalytic residues and active site architecture of N. farcinica aspartate carbamoyltransferase compare to those of other bacterial and archaeal species?

A detailed analysis of catalytic residues and active site architecture would reveal conservation patterns and potentially unique features of N. farcinica ATCase. Drawing from approaches used to analyze M. jannaschii ATCase, the following comparative strategy is recommended :

• Multiple sequence alignment: Align ATCase sequences from diverse species including:

  • Various Nocardia species

  • E. coli (as a well-characterized reference)

  • M. jannaschii and other archaeal species

  • Other actinomycetes related to Nocardia

• Identification of key functional residues:

  • Catalytic residues directly involved in the reaction mechanism

  • Substrate binding residues that interact with aspartate and carbamoyl phosphate

  • Residues involved in quaternary structure formation

  • Potential allosteric sites

• Homology modeling: Generate structural models of N. farcinica ATCase based on crystal structures of homologous enzymes, with particular focus on active site geometry .

• Active site comparison: Analyze potential differences in:

  • Substrate binding pocket dimensions and electrostatics

  • Positioning of catalytic residues

  • Potential for induced-fit conformational changes

  • Access channels for substrates and products

How can site-directed mutagenesis be used to investigate structure-function relationships in N. farcinica aspartate carbamoyltransferase?

Site-directed mutagenesis represents a powerful approach to elucidate structure-function relationships in N. farcinica ATCase, building on knowledge from homologous enzymes. A systematic mutagenesis strategy would include:

• Identification of target residues: Select residues for mutation based on:

  • Sequence conservation analysis across multiple species

  • Homology modeling to identify catalytically important residues

  • Comparison with well-characterized residues in E. coli or M. jannaschii ATCase

• Categories of mutations to explore:

• Characterization of mutants: Each mutant should undergo comprehensive analysis including:

  • Expression and purification yield assessment

  • Kinetic parameter determination (kcat, Km, Hill coefficient)

  • Thermostability measurement

  • Quaternary structure analysis

  • Response to potential allosteric effectors

• Structure-function correlation: Use the mutational data to map functional effects onto the structural model, developing a comprehensive understanding of how specific structural elements contribute to the enzyme's catalytic mechanism, regulation, and stability.

This approach would not only illuminate the molecular basis of N. farcinica ATCase function but might also identify unique features that could be exploited for selective inhibition or diagnostic purposes .

What potential does N. farcinica aspartate carbamoyltransferase hold as a drug target, and how might inhibitors be designed?

N. farcinica ATCase represents a potential therapeutic target due to its essential role in pyrimidine biosynthesis and the significant clinical impact of N. farcinica infections, which often show resistance to several antimicrobial agents . A structure-based drug design approach would include:

• Target validation: Confirm the essentiality of ATCase for N. farcinica survival and virulence through genetic approaches or selective inhibition studies.

• Structural analysis: Use homology modeling or experimental structure determination to identify unique features of the N. farcinica ATCase active site or regulatory interfaces that differ from human homologs .

• Virtual screening: Perform in silico screening of compound libraries against the modeled active site to identify potential lead compounds.

• Rational design strategies for inhibitor development:

• Screening cascade: Develop a hierarchical screening approach including:

  • Initial enzymatic assays for activity

  • Secondary assays for selectivity over human ATCase

  • Cellular assays for antimicrobial activity and cytotoxicity

  • Animal models of N. farcinica infection

The challenging multi-drug resistance profile of N. farcinica infections, as highlighted in clinical cases, underscores the value of developing novel therapeutic approaches targeting essential metabolic enzymes like ATCase . Success in this area could provide new treatment options for severe N. farcinica infections, which currently pose significant clinical challenges .

How might the pyrB gene be utilized in molecular evolution studies to understand the phylogeny and adaptive radiation of Nocardia species?

The pyrB gene offers significant potential as a phylogenetic marker for understanding Nocardia evolution and adaptation. A comprehensive approach to utilizing pyrB for evolutionary studies would include:

• Multi-locus sequence analysis (MLSA): Incorporate pyrB alongside other housekeeping genes to construct robust phylogenetic trees of Nocardia species, potentially revealing evolutionary relationships not apparent from 16S rRNA analysis alone.

• Evolutionary rate analysis: Compare nucleotide substitution rates in pyrB with other genes to identify:

  • Conserved regions under purifying selection

  • Variable regions potentially under positive selection

  • Molecular clock calibration for dating divergence events

• Comparative genomic context: Analyze the genomic neighborhood of pyrB across Nocardia species to detect:

  • Gene synteny conservation or rearrangements

  • Horizontal gene transfer events

  • Operon structure variations

• Correlation with ecological niches: Map pyrB sequence variations against known ecological adaptations of different Nocardia species, potentially including:

  • Soil-dwelling generalists

  • Specialized pathogens like N. farcinica

  • Species with unique metabolic capabilities

• Positive selection analysis: Identify amino acid positions under positive selection pressure that might represent adaptations to specific environmental challenges.

This approach would contribute to understanding how metabolic pathways like pyrimidine biosynthesis have evolved during Nocardia speciation and adaptation to various ecological niches, including the human host environment. The results could provide insights into pathogen evolution and potentially reveal molecular signatures associated with increased virulence or host adaptation in species like N. farcinica .