Recombinant Nocardia farcinica Bifunctional protein FolD (folD)

What is Nocardia farcinica and why is its FolD protein significant for research?

Nocardia farcinica is a filamentous-growing Gram-positive bacterium belonging to the Actinomycetales family, which includes other clinically and industrially important genera such as Mycobacterium, Streptomyces, and Corynebacterium . It is an opportunistic pathogen that causes nocardiosis in humans and animals, affecting the lungs, central nervous system, brain, and cutaneous tissues . The complete genome of N. farcinica IFM 10152 has been sequenced, revealing 5,674 putative protein-coding sequences that contribute to its pathogenicity, multidrug resistance, and ability to produce bioactive molecules .

The bifunctional protein FolD in N. farcinica is significant because it likely plays a crucial role in folate metabolism through its 5,10-methylene-tetrahydrofolate dehydrogenase and 5,10-methenyltetrahydrofolate cyclohydrolase activities. These activities are essential for one-carbon metabolism, which supports nucleotide synthesis and amino acid metabolism. Given N. farcinica's pathogenic nature and multidrug resistance, studying FolD could provide insights into bacterial survival mechanisms and potential drug targets.

What expression systems are most effective for producing recombinant N. farcinica FolD?

When expressing recombinant N. farcinica FolD, researchers should consider the high G+C content of the organism's genome (70.8%) , which can present challenges in heterologous expression systems. The following expression systems have proven effective for proteins from high G+C content organisms:

E. coli Expression System:

• Recommended strains: BL21(DE3), Rosetta(DE3), or Arctic Express for proteins that may fold poorly at higher temperatures

• Vector options: pET series vectors with T7 promoter for high expression

• Expression conditions: Induction at lower temperatures (16-20°C) often improves solubility

• Codon optimization: Consider optimizing the gene sequence to account for E. coli codon bias

Methodological approach:

• Clone the folD gene with appropriate restriction sites into a vector containing a His-tag or other purification tag

• Transform into an E. coli expression strain

• Grow cultures to OD600 of 0.6-0.8 before induction

• Induce with IPTG (0.1-0.5 mM) and express at 16-20°C overnight

• Harvest cells and lyse using sonication or pressure-based methods

• Purify using affinity chromatography followed by size exclusion chromatography

What purification strategies yield the highest purity and activity for recombinant N. farcinica FolD?

A multi-step purification approach is recommended for obtaining high-purity, active N. farcinica FolD:

Primary Purification (Affinity Chromatography):

• For His-tagged constructs: Ni-NTA or TALON resin with imidazole gradient elution

• For GST-tagged constructs: Glutathione Sepharose with reduced glutathione elution

• Buffer recommendation: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT

Secondary Purification:

• Ion Exchange Chromatography: Based on the predicted pI of N. farcinica FolD

• Size Exclusion Chromatography: To remove aggregates and ensure monodispersity

Activity Preservation Measures:

• Addition of folate or folate derivatives (0.1-0.5 mM) in purification buffers

• Inclusion of reducing agents (1-5 mM DTT or 1-2 mM β-mercaptoethanol)

• Storage in 20-30% glycerol at -80°C to maintain long-term activity

How does the genomic context of the folD gene in N. farcinica inform its function?

The genomic context of folD in N. farcinica provides valuable insights into its metabolic role and regulation. The genome of N. farcinica IFM 10152 consists of a single circular chromosome of 6,021,225 bp with a high G+C content of 70.8%, plus two plasmids .

While the search results don't specifically mention the genomic context of folD, we can make informed inferences based on typical arrangements in related bacteria:

• FolD is typically part of one-carbon metabolism pathways, which integrate with:

  • Purine biosynthesis pathways

  • Methionine biosynthesis genes

  • Glycine cleavage system components

• In pathogenic bacteria, folate metabolism genes often show coordinated expression with:

  • Stress response elements

  • Virulence factors

  • Drug resistance mechanisms

N. farcinica has evolved gene duplications that enable it to survive in both soil environments and animal tissues , suggesting that folate metabolism genes may have specialized regulatory mechanisms to respond to different environmental conditions.

What structural features distinguish N. farcinica FolD from homologs in other bacterial species?

N. farcinica FolD likely possesses distinct structural features compared to homologs in other bacteria, particularly in substrate binding pockets and allosteric regulatory sites. Although specific structural data for N. farcinica FolD is not available in the search results, the following comparative analysis is based on known FolD structures from related species:

Expected Structural Features:

The high G+C content of N. farcinica (70.8%) likely influences its codon usage and potentially protein folding, which may result in unique structural adaptations compared to homologs from lower G+C content organisms.

Methodological approach for structural comparison:

• Generate homology models using related bacterial FolD structures as templates

• Perform molecular dynamics simulations under conditions mimicking both soil and human host environments

• Analyze binding pocket differences using computational docking of substrates and inhibitors

• Validate structural predictions through site-directed mutagenesis of predicted key residues

How might FolD contribute to N. farcinica pathogenicity or antibiotic resistance?

FolD's potential contributions to N. farcinica pathogenicity and antibiotic resistance are multifaceted, particularly considering that N. farcinica possesses multiple drug resistance mechanisms and virulence factors .

Pathogenicity Contributions:

• One-carbon metabolism supported by FolD is essential for nucleotide synthesis and methylation reactions, both critical for bacterial growth within host tissues

• Folate-dependent pathways may contribute to bacterial survival under oxidative stress conditions within phagocytes

• N. farcinica possesses multiple catalases, superoxide dismutases, and an alkylhydroperoxidase that protect against reactive oxygen species produced by phagocytes , and folate metabolism may support these defense mechanisms

Antibiotic Resistance Connections:

• N. farcinica is known to be resistant to many front-line antibiotics

• The bacterium has a rifampicin monooxygenase that catalyzes hydroxylation of rifampicin as the first step in its degradation pathway

• Folate metabolism inhibitors (sulfonamides and trimethoprim) are commonly used antibiotics, and modifications in folate pathway enzymes could contribute to resistance

Methodological approach to investigate these connections:

• Generate folD knockout or knockdown strains and assess changes in virulence using cell culture infection models

• Compare gene expression levels of folD under different antibiotic stresses

• Perform metabolomic analysis comparing wild-type and folD-mutant strains to identify metabolic adaptations

• Test synergistic effects between FolD inhibitors and other antibiotics against clinical isolates

What techniques are recommended for crystallization and structural determination of N. farcinica FolD?

Successfully determining the crystal structure of N. farcinica FolD requires careful optimization of protein preparation, crystallization conditions, and diffraction data collection:

Protein Sample Preparation:

• Express with removable affinity tags to ensure native protein structure

• Ensure >95% purity by SDS-PAGE and monodispersity by dynamic light scattering

• Concentrate to 10-15 mg/mL in a buffer containing 20 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol, and 1 mM DTT

• Co-purify or add ligands (NADP+, folate derivatives) to stabilize the protein

Crystallization Strategy:

• Initial screening: Employ sparse matrix screens at multiple temperatures (4°C, 18°C, and 25°C)

• Optimization techniques:

  • Microseeding from initial crystals

  • Additive screening with divalent cations (Mg2+, Mn2+)

  • Counter-diffusion methods for slower crystal growth

Data Collection and Structure Determination:

• Cryoprotection: Test various cryoprotectants (glycerol, ethylene glycol, PEG 400) at 15-25% concentration

• Collection strategy: High redundancy data collection with fine oscillation angles

• Phasing options:

  • Molecular replacement using homologous structures from related bacteria

  • SAD/MAD phasing using selenomethionine-labeled protein

  • Heavy atom derivatives if molecular replacement fails

Co-crystallization with Ligands:

• For enzyme mechanism studies, co-crystallize with:

  • Substrate analogs at 2-5 mM concentration

  • Inhibitors at concentrations near their Ki values

  • Cofactors (NADP+/NADPH) at 2-5 mM

How can enzyme kinetics studies of N. farcinica FolD inform drug development efforts?

Comprehensive enzyme kinetics studies of N. farcinica FolD can provide critical insights for structure-based drug design and inhibitor development:

Steady-State Kinetics Analysis:

Pre-Steady-State Kinetics:

• Stopped-flow spectroscopy to determine rate-limiting steps

• Analysis of intermediates using rapid quench techniques

• Isothermal titration calorimetry for thermodynamic binding parameters

Environmental Effects:

• Assessing activity under conditions mimicking host environments:

  • Reduced oxygen conditions, as N. farcinica can survive under low-oxygen conditions

  • Various iron concentrations, as N. farcinica produces siderophores for iron acquisition

  • Acidic pH to simulate phagolysosomal environments

Methodological approach for drug development applications:

• Identify differences in kinetic parameters between human and N. farcinica FolD

• Develop a high-throughput screening assay based on the most distinctive activity

• Perform fragment-based screening followed by structure-guided optimization

• Validate leads using whole-cell assays against N. farcinica clinical isolates

What genetic manipulation strategies are most effective for studying FolD function in N. farcinica?

Genetic manipulation of N. farcinica presents challenges due to its thick cell wall and high G+C content, but several approaches can be effective:

Gene Knockout/Knockdown Strategies:

• Homologous recombination with suicide vectors containing antibiotic resistance markers

• CRISPR-Cas9 system optimized for high G+C genomes

• Antisense RNA or CRISPRi for conditional knockdown if folD is essential

Expression Systems for Complementation:

• Integrative vectors based on mycobacteriophage integration systems

• Replicative plasmids derived from the native plasmids pNF1 (184,027 bp) and pNF2 (87,093 bp)

• Inducible promoters responsive to tetracycline or acetamide

Site-Directed Mutagenesis Protocol:

• Select residues based on structural models or alignments

• Generate point mutations using overlap extension PCR

• Complement knockout strains with mutant variants

• Assess enzyme activity, protein stability, and bacterial phenotypes

Phenotypic Analysis:

• Growth curves under various nutritional conditions

• Virulence assays in cell culture models

• Metabolomic profiling to identify changes in one-carbon metabolism

• Antibiotic susceptibility testing, particularly to folate pathway inhibitors

Methodological approach for gene replacement:

• Construct a suicide vector containing:

  • ~1 kb homology arms flanking the folD gene

  • A selectable marker (e.g., kanamycin resistance)

  • A counter-selectable marker (e.g., sacB)

• Transform N. farcinica via electroporation with specialized parameters:

  • High voltage (2.5 kV)

  • Cell wall weakening pretreatment with glycine or cell wall hydrolases

• Select for single crossover events on antibiotic-containing media

• Counter-select for double crossover events

• Confirm gene replacement by PCR and sequencing

How can isotope labeling of N. farcinica FolD be optimized for NMR studies?

NMR studies of N. farcinica FolD require efficient isotope labeling strategies that account for the protein's size and the high G+C content of the organism:

Uniform 15N and 13C Labeling Protocol:

• Express in minimal M9 media containing:

  • 15NH4Cl (1 g/L) as the sole nitrogen source

  • 13C-glucose (2-4 g/L) as the sole carbon source

  • Supplemented with trace elements and vitamins

• For E. coli expression systems:

  • Use auto-induction media formulated with labeled components

  • Grow at lower temperatures (18-20°C) for 24-36 hours

• Purification considerations:

  • Minimize number of purification steps to preserve sample

  • Use deuterated buffers for final NMR samples

Selective Labeling Strategies:

• Amino acid-specific labeling:

  • Supplement minimal media with specifically labeled amino acids

  • Use auxotrophic E. coli strains to prevent scrambling of labels

• Segmental labeling using protein trans-splicing:

  • Split FolD into domains

  • Label each domain separately

  • Reassemble using split inteins

Deuteration Protocols:

• Grow in D2O-based minimal media with deuterated glucose

• Implement step-wise adaptation to D2O (50%, 70%, 90%, 100%)

• Back-exchange amide protons after purification

NMR Experiment Selection:

• For backbone assignment: TROSY-based triple-resonance experiments

• For dynamics studies: 15N relaxation experiments and hydrogen-deuterium exchange

• For ligand binding: Chemical shift perturbation experiments using 15N-HSQC

What are the recommended protocols for assessing both enzymatic activities of N. farcinica FolD?

The bifunctional nature of FolD requires specialized assays to measure both 5,10-methylene-tetrahydrofolate dehydrogenase and 5,10-methenyltetrahydrofolate cyclohydrolase activities:

Dehydrogenase Activity Assay:

• Reaction components:

  • 50 mM HEPES buffer, pH 7.5

  • 0.1 mM 5,10-methylene-tetrahydrofolate

  • 0.1 mM NADP+

  • 1-10 μg purified FolD enzyme

• Monitoring method:

  • Spectrophotometric measurement of NADPH formation at 340 nm (ε = 6,220 M-1cm-1)

  • Continuous assay at 25°C for 5-10 minutes

• Data analysis:

  • Calculate initial rates from linear portion of progress curves

  • Determine kcat and Km using Michaelis-Menten kinetics

Cyclohydrolase Activity Assay:

• Reaction components:

  • 50 mM MOPS buffer, pH 7.0

  • 0.1 mM 5,10-methenyltetrahydrofolate

  • 1-10 μg purified FolD enzyme

• Monitoring method:

  • Spectrophotometric measurement of 5,10-methenyltetrahydrofolate disappearance at 350 nm (ε = 24,900 M-1cm-1)

  • Continuous assay at 25°C for 2-5 minutes

• Data analysis:

  • Calculate initial rates from linear portion of progress curves

  • Determine kcat and Km using Michaelis-Menten kinetics

Coupled Assay for Both Activities:

• Reaction components:

  • 50 mM HEPES buffer, pH 7.5

  • 0.1 mM tetrahydrofolate

  • 0.1 mM NADP+

  • 10 mM formaldehyde

  • 1-10 μg purified FolD enzyme

• Monitoring method:

  • Measure NADPH formation at 340 nm

  • This measures the combined action of both activities plus the non-enzymatic condensation of tetrahydrofolate with formaldehyde

Assay Controls and Validations:

• Substrate stability controls in the absence of enzyme

• Inhibition studies using known folate pathway inhibitors

• pH and temperature optimum determination

• Effects of divalent cations (Mg2+, Mn2+, Zn2+) on activity

How can the substrate specificity of N. farcinica FolD be comprehensively analyzed?

Understanding the substrate specificity of N. farcinica FolD provides insights into its metabolic role and can inform inhibitor design:

Natural Substrate Panel Testing:

• Test the following folate derivatives as substrates:

  • 5,10-methylene-tetrahydrofolate (natural substrate)

  • 5,10-methylene-tetrahydrodihydrofolate

  • 5,10-methenyltetrahydrofolate

  • Various polyglutamated forms of these substrates

• Compare kinetic parameters (kcat, Km, kcat/Km) for each substrate

Cofactor Specificity Analysis:

• Test alternative cofactors:

  • NADP+ vs. NAD+ for dehydrogenase activity

  • Various metal ions for potential cyclohydrolase activity enhancement

• Determine relative efficiency with each cofactor

Structural Analog Testing:

• Synthetic folate analogs with modifications at:

  • Pteridine ring

  • p-aminobenzoic acid moiety

  • Glutamate chain

• Assess as substrates and/or inhibitors

Methodological approaches:

• High-throughput screening using a spectrophotometric plate reader

• LC-MS analysis to detect product formation with non-chromogenic substrates

• Isothermal titration calorimetry for binding studies independent of catalytic activity

• In silico docking studies to predict binding modes of various substrates

Data Analysis Framework:

How can structural information about N. farcinica FolD be applied to antimicrobial drug development?

Structural insights into N. farcinica FolD can drive rational drug design efforts for novel antimicrobials:

Structure-Based Drug Design Strategy:

• Identify unique structural features in N. farcinica FolD compared to human homologs

• Focus on differences in:

  • Active site architecture

  • Substrate binding pockets

  • Allosteric regulation sites

  • Domain interfaces

Virtual Screening Workflow:

• Generate a pharmacophore model based on:

  • Known FolD inhibitors

  • Natural substrates

  • Transition state structures

• Screen compound libraries using:

  • Shape-based methods

  • Pharmacophore filtering

  • Molecular docking

• Score compounds based on:

  • Predicted binding affinity

  • Selectivity over human enzymes

  • Drug-like properties

Lead Optimization Cycle:

• Synthesize or acquire hit compounds

• Test inhibitory activity in enzyme assays

• Determine co-crystal structures with FolD

• Optimize based on structure-activity relationships

• Evaluate antimicrobial activity against N. farcinica

Target Product Profile:

• Selective inhibition of bacterial over human FolD (>100-fold)

• Active against multidrug-resistant N. farcinica strains

• Low potential for resistance development

• Favorable pharmacokinetic properties for treating disseminated nocardiosis

What role might FolD play in the adaptation of N. farcinica to different environmental conditions?

N. farcinica demonstrates remarkable versatility, surviving in both soil environments and animal tissues . FolD likely plays a key role in this adaptability:

Environmental Adaptation Mechanisms:

Experimental Approaches to Investigate Environmental Adaptation:

• Transcriptomic analysis:

  • Compare folD expression levels under different growth conditions

  • Identify co-regulated genes that may form functional networks

• Metabolomic profiling:

  • Track folate-dependent metabolites under different environmental stresses

  • Identify metabolic bottlenecks where FolD activity becomes limiting

• Fitness assays:

  • Compare growth of wild-type and folD mutant strains under various conditions

  • Competitive growth experiments to assess relative fitness contributions

Methodological design for environmental adaptation studies:

• Culture N. farcinica under conditions mimicking:

  • Soil environments (nutrient-limited, competing microbes)

  • Host environments (macrophage infections, serum exposure)

  • Treatment conditions (subinhibitory antibiotic concentrations)

• Measure folD expression using:

  • RT-qPCR for targeted analysis

  • RNA-seq for genome-wide expression patterns

• Correlate expression with metabolite levels and growth parameters

How does N. farcinica FolD compare with FolD proteins from other pathogenic bacteria in terms of inhibitor sensitivity?

Comparative analysis of FolD inhibitor sensitivity across pathogenic bacteria can reveal common vulnerabilities or unique resistance mechanisms:

Cross-Species Inhibitor Panel Analysis:

Methodological approach for comparative inhibitor testing:

• Express and purify recombinant FolD from multiple bacterial species

• Test a standardized panel of inhibitors against each enzyme

• Determine IC50 and Ki values under identical assay conditions

• Correlate inhibition patterns with structural features

• Test promising inhibitors against whole cells of each species

Structure-Function Correlations:

• Map resistance-conferring mutations onto structural models

• Identify conserved vs. variable regions of the binding pocket

• Develop pharmacophore models that target conserved features

• Design broad-spectrum inhibitors targeting multiple bacterial FolDs

Synergistic Approaches:

• Test FolD inhibitors in combination with:

  • Other folate pathway inhibitors

  • Cell wall targeting antibiotics

  • Efflux pump inhibitors

• Target multiple steps in one-carbon metabolism simultaneously

What emerging technologies could enhance our understanding of N. farcinica FolD function in vivo?

Several cutting-edge technologies hold promise for elucidating the in vivo functions of FolD in N. farcinica:

Advanced Imaging Techniques:

• Cryo-electron tomography:

  • Visualize FolD localization within bacterial cells

  • Map interactions with other metabolic enzymes

• Super-resolution microscopy:

  • Track FolD dynamics during different growth phases

  • Monitor protein-protein interactions using fluorescent tags

Functional Genomics Approaches:

• CRISPRi screens:

  • Identify genetic interactions with folD

  • Map synthetic lethal relationships

• Transposon sequencing (Tn-seq):

  • Determine essential nature of folD under different conditions

  • Identify compensatory pathways

Systems Biology Integration:

• Multi-omics data integration:

  • Correlate transcriptomics, proteomics, and metabolomics data

  • Build predictive models of FolD regulation

• Flux analysis using stable isotopes:

  • Trace carbon flow through one-carbon metabolism

  • Quantify the contribution of FolD to various biosynthetic pathways

In vivo Infection Models:

• Zebrafish embryo infection model:

  • Real-time visualization of bacterial dissemination

  • Test folD mutants for virulence

• Mouse models of nocardiosis:

  • Evaluate the importance of folD during different infection stages

  • Test inhibitors against established infections

How can computational approaches accelerate the discovery of selective inhibitors for N. farcinica FolD?

Computational methods offer powerful strategies to expedite the discovery of selective FolD inhibitors:

Advanced Computational Screening Methods:

• Machine learning approaches:

  • Train models on known FolD inhibitors

  • Develop target-specific scoring functions

  • Predict selectivity profiles

• Molecular dynamics simulations:

  • Identify cryptic binding sites not visible in static structures

  • Assess protein flexibility and its impact on inhibitor binding

  • Calculate binding free energies more accurately

Fragment-Based Design in silico:

• Computational fragment growing:

  • Start with high-efficiency binding fragments

  • Expand to optimize interactions

  • Link fragments occupying different binding pockets

• Pharmacophore-based scaffold hopping:

  • Maintain essential interaction features

  • Explore novel chemical space

  • Improve drug-like properties

Quantum Mechanical Calculations:

• QM/MM studies of catalytic mechanism:

  • Identify transition states for both enzymatic activities

  • Design transition state analogs as potent inhibitors

• Electronic structure analysis:

  • Map electrostatic potential of binding pocket

  • Optimize polar interactions with inhibitors

Methodological workflow for computational drug discovery:

• Generate homology model of N. farcinica FolD

• Perform molecular dynamics simulations to sample conformational states

• Identify unique binding sites compared to human homologs

• Screen virtual libraries using ensemble docking

• Prioritize compounds based on predicted selectivity and ADME properties

• Validate top candidates with biochemical assays