Recombinant Nitrosomonas europaea GTP cyclohydrolase folE2 (folE2)

What is GTP cyclohydrolase folE2 and what is its function in Nitrosomonas europaea?

GTP cyclohydrolase folE2 is an enzyme involved in the tetrahydrobiopterin biosynthetic pathway, catalyzing the conversion of GTP to dihydroneopterin triphosphate. In Nitrosomonas europaea, this enzyme plays a critical role in folate metabolism and potentially in nitrogen metabolism pathways. As a member of the GTP cyclohydrolase family (EC 3.5.4.16), folE2 is structurally distinct from folE1 but performs related catalytic functions in pteridine biosynthesis .

What is the molecular structure and key properties of Nitrosomonas europaea folE2?

Nitrosomonas europaea folE2 is a full-length protein consisting of 268 amino acids. The recombinant form expressed for research purposes maintains the complete amino acid sequence from position 1-268. The protein has the following key properties:

• Molecular weight: Approximately 20.2 kDa

• Amino acid sequence: MNKQIDLPIADVQGSLDTRHIAIDRVGIKAIVAIRHPVVVADKGGGSQHTVAQFNMYVNLPHNFKGTHMSRFVEILNSHEREISVESFEEILRSMVSRLESDSGHIEMAFPYFINKSAPVSGVKSLLDYEVTFIGEIKHGNQYSFTMKVIVPVTSLCPCSKISDYGAHNQRSHVTISVRTNSFIWIEDIIRIAEEQASCELYGLLKRPDEKYVOIRTRAYLNNPKFVEDIVRDVIAEVLNHDDRIDAYIVESENESIHNHSAYALIERODKRIR

How is folE2 evolutionarily conserved across bacterial species?

Nitrosomonas europaea folE2 shares structural and functional homology with similar enzymes found across diverse bacterial genera. Comparative analysis shows approximately 56.7% sequence identity with human GTP cyclohydrolase 1. This degree of conservation suggests essential metabolic functions. When examining folE2 from Pseudomonas aeruginosa, it belongs to a large ortholog group (POG003716) with 533 members, indicating wide distribution across bacterial species . The presence of folE2 across 471 genera further emphasizes its evolutionary significance and common occurrence in both pathogenic and non-pathogenic bacterial strains .

What expression systems are most effective for producing functional recombinant Nitrosomonas europaea folE2?

Multiple expression systems have been successfully employed for the production of recombinant Nitrosomonas europaea folE2, each with distinct advantages:

The E. coli expression system represents the most commonly used approach due to its efficiency and cost-effectiveness for basic research applications .

What purification protocols yield the highest enzyme activity for recombinant folE2?

For optimal purification of functional recombinant folE2, a multi-step protocol is recommended:

• Initial clarification: Centrifugation of lysed cells (10,000g, 30 minutes, 4°C)

• Capture step: Immobilized metal affinity chromatography (IMAC) using Ni-NTA for His-tagged folE2

• Intermediate purification: Ion exchange chromatography (recommended buffer: 20 mM Tris-HCl, pH 8.0)

• Polishing step: Size exclusion chromatography

• Quality control: SDS-PAGE analysis to confirm >85% purity

For biotinylated variants (e.g., CSB-EP774299NHH-B), the E. coli biotin ligase (BirA) catalyzes the covalent attachment of biotin to the AviTag peptide with high specificity, enabling additional purification options via avidin-based affinity chromatography .

How should recombinant folE2 be reconstituted after lyophilization to maintain optimal activity?

Proper reconstitution is critical for maintaining enzymatic activity. The recommended protocol includes:

• Brief centrifugation of the vial prior to opening to ensure all lyophilized material is at the bottom

• Reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Addition of glycerol to a final concentration of 5-50% to enhance stability

• Gentle mixing by slow inversion rather than vortexing to prevent protein denaturation

• Storage at -20°C for medium-term use or -80°C for long-term storage

• Avoidance of repeated freeze-thaw cycles

What assays are most reliable for measuring GTP cyclohydrolase activity of recombinant folE2?

Several analytical methods can quantify the enzymatic activity of recombinant folE2:

• Spectrophotometric assay: Monitoring the conversion of GTP to dihydroneopterin triphosphate at 330 nm

• HPLC analysis: Quantifying reaction products using reverse-phase HPLC with UV detection

• Coupled enzyme assay: Using alkaline phosphatase to remove phosphates from the product, followed by iodine oxidation and fluorescence detection

• Mass spectrometry: LC-MS/MS analysis for precise quantification of reaction products

The selection of the appropriate assay depends on available instrumentation and required sensitivity. For high-throughput screening applications, the fluorescence-based coupled assay offers the best combination of sensitivity and throughput .

How does folE2 activity correlate with ammonia oxidation in Nitrosomonas europaea?

Nitrosomonas europaea is an obligate ammonia-oxidizing bacterium, and the relationship between folE2 activity and ammonia oxidation represents an important area of investigation. Current research suggests:

• Folate metabolism, influenced by folE2 activity, may impact cellular energy production required for ammonia oxidation

• Changes in environmental ammonia concentrations (e.g., 50 mM ammonium media) can affect metabolic pathways involving folE2

• The production of nitrite, a primary indicator of ammonia oxidation activity, may correlate with folE2 expression levels

How does folE2 function differ under varying environmental conditions in Nitrosomonas europaea?

The function of folE2 in Nitrosomonas europaea can be significantly influenced by environmental conditions:

Research methodologies should incorporate careful control of these variables when studying folE2 function, particularly when extrapolating laboratory findings to environmental contexts .

What structural features of folE2 contribute to its catalytic mechanism?

The catalytic mechanism of folE2 depends on several key structural features:

• Active site residues: Critical amino acids involved in substrate binding and catalysis

• Metal ion coordination: Potential requirement for zinc or other divalent cations

• Protein folding dynamics: Conformational changes during catalysis

• Oligomerization state: Functional unit may be monomeric, dimeric, or higher-order structures

X-ray crystallographic studies of related folE2 proteins have revealed insights into inhibitor binding. For example, research on folE2 from Burkholderia thailandensis demonstrated that dehydrocostus lactone (DHL) functions as a mechanism-based inhibitor, with crystal structures capturing the covalently inhibited enzyme .

How does folE2 from Nitrosomonas europaea compare with folE2 from pathogenic bacteria for antimicrobial development?

Comparative analysis between Nitrosomonas europaea folE2 and folE2 from pathogenic bacteria reveals important considerations for antimicrobial research:

• FolE2 has been identified as conditionally essential in Burkholderia thailandensis and Burkholderia pseudomallei in the presence of subinhibitory doses of trimethoprim

• Chemical synthetic lethality approaches have identified inhibitors like dehydrocostus lactone (DHL), parthenolide, and β-lapachone that form lethal combinations with low-dose trimethoprim

• The concept of targeting folE2 may represent a promising strategy for developing new antimicrobial combinations against specific pathogens

• Structural similarities between folE2 proteins across bacterial species suggest potential for broad-spectrum inhibitor development

Researchers studying Nitrosomonas europaea folE2 can leverage these insights for comparative structural analysis and inhibitor design, potentially contributing to antimicrobial discovery efforts .

What controls are essential when studying folE2 enzymatic activity in vitro?

Robust experimental design for folE2 enzymatic studies requires multiple controls:

• Negative enzyme control: Reaction mixture without folE2 to establish baseline

• Denatured enzyme control: Heat-inactivated folE2 to confirm activity is enzyme-dependent

• Substrate saturation control: Varying GTP concentrations to ensure non-limiting conditions

• Buffer composition control: Testing activity in different buffer systems to optimize conditions

• Metal ion dependency control: Addition/chelation of potential cofactors

• Thermal stability control: Activity measurements at different temperatures

These controls help ensure experimental reproducibility and valid interpretation of results when characterizing folE2 enzymatic properties .

How can researchers design experiments to investigate potential inhibitors of folE2?

When screening for folE2 inhibitors, consider the following experimental design principles:

• Primary screening: High-throughput fluorescence-based assays to identify initial hits

• Secondary validation: Orthogonal assays using different detection methods to confirm true positives

• Mechanism studies: Kinetic analysis to determine inhibition type (competitive, non-competitive, uncompetitive)

• Structure-activity relationship analysis: Testing structural analogs to identify key pharmacophores

• X-ray crystallography: Obtaining structures of enzyme-inhibitor complexes for rational design

• Cellular assays: Testing inhibitor effects in appropriate bacterial models

Recent research exemplifies this approach, where researchers identified dehydrocostus lactone as a mechanism-based inhibitor of folE2, confirmed through X-ray crystallography of the covalently inhibited enzyme .

What methodologies are most appropriate for studying folE2 gene expression regulation?

Several methodologies can effectively investigate folE2 expression regulation:

Combining multiple approaches provides the most comprehensive understanding of folE2 expression regulation under various environmental and experimental conditions .

How does folE2 from Nitrosomonas europaea compare structurally and functionally with folE2 from other bacterial species?

Comparative analysis reveals both similarities and differences among folE2 proteins across bacterial species:

This comparative approach provides valuable insights into conserved features that may be essential for enzymatic function versus species-specific characteristics that could be exploited for selective targeting .

What are the differences between folE1 and folE2 enzyme families?

The folE1 and folE2 enzyme families represent distinct types of GTP cyclohydrolases with important differences:

• Evolutionary origin: folE2 evolved independently from folE1, representing convergent evolution toward similar catalytic functions

• Structural differences: Different protein folds despite catalyzing similar reactions

• Metal ion requirements: Potentially different dependencies on zinc or other divalent cations

• Substrate specificity: May show different affinities for GTP or substrate analogs

• Inhibition profiles: Typically respond differently to known inhibitors

• Distribution across species: Different patterns of presence across bacterial phyla

These differences have significant implications for researchers studying folE2, particularly when extrapolating findings from folE1 studies or when designing selective inhibitors .

What are common challenges in expressing and purifying functional recombinant folE2?

Researchers frequently encounter several technical challenges when working with recombinant folE2:

• Protein solubility issues: Formation of inclusion bodies in E. coli expression systems

  • Solution: Optimize expression temperature (typically lowering to 18-25°C), use solubility-enhancing tags, or employ refolding protocols

• Enzymatic activity loss during purification:

  • Solution: Include stabilizing agents (glycerol, reducing agents), minimize purification steps, and maintain cold temperature throughout

• Protein aggregation during storage:

  • Solution: Add glycerol (5-50%), aliquot to avoid freeze-thaw cycles, and store at -80°C

• Low expression yields:

  • Solution: Optimize codon usage for expression host, test different promoter systems, or switch to alternative expression hosts

• Protein degradation:

  • Solution: Include protease inhibitors during purification, use freshly prepared buffers, and process samples quickly

How can researchers optimize enzymatic assays to accurately measure folE2 activity?

Optimizing enzymatic assays for folE2 requires attention to several key factors:

• Buffer composition: Test multiple buffer systems (Tris, HEPES, phosphate) at various pH values (typically 7.0-8.5)

• Cofactor requirements: Systematically test divalent cations (Mg²⁺, Zn²⁺, Mn²⁺) at different concentrations

• Substrate concentration optimization: Determine Km value and use 2-5 times Km for routine assays

• Temperature optimization: Test activity at different temperatures (typically 25-37°C)

• Detection method sensitivity: For spectrophotometric methods, ensure measurements are within the linear range; for fluorescence-based methods, control for background fluorescence

• Reaction time course: Establish linear range of product formation to ensure measurements are taken during initial velocity phase

What are promising research avenues for studying folE2 in microbial ecology and environmental applications?

Several emerging research directions for folE2 in Nitrosomonas europaea hold particular promise:

• Role in nitrogen cycling: Investigating how folE2 activity influences ammonia oxidation rates in environmental contexts

• Biosensor development: Exploring folE2-based biosensors for environmental monitoring applications

• Metagenomic analysis: Examining folE2 distribution and variants across diverse environmental microbiomes

• Climate change impacts: Studying how altered environmental conditions affect folE2 expression and function

• Bioremediation applications: Investigating potential roles in systems for nitrogen removal from contaminated sites

These research directions connect fundamental enzyme biochemistry to broader ecological and environmental applications, potentially addressing significant challenges in environmental microbiology and biotechnology .

How might comparative genomics approaches advance our understanding of folE2 evolution and function?

Comparative genomics offers powerful approaches to elucidate folE2 evolution and function:

• Phylogenetic analysis: Constructing evolutionary trees based on folE2 sequences across diverse species

• Synteny analysis: Examining conservation of genomic context around folE2 genes

• Selection pressure analysis: Calculating dN/dS ratios to identify regions under positive or purifying selection

• Structural homology modeling: Predicting folE2 structures in understudied organisms based on resolved structures

• Horizontal gene transfer investigation: Identifying potential HGT events in folE2 evolutionary history

The widespread distribution of folE2 across 471 genera and its presence in a large ortholog group (POG003716) with 533 members provides a rich dataset for such comparative approaches .