Recombinant Nitrosomonas europaea 1-deoxy-D-xylulose 5-phosphate reductoisomerase (dxr)

What is the role of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) in Nitrosomonas europaea?

DXR catalyzes the second step in the methylerythritol phosphate (MEP) pathway for isoprenoid biosynthesis. It converts 1-deoxy-D-xylulose 5-phosphate (DXP) into 2-C-methyl-D-erythritol 4-phosphate (MEP) through an isomerization followed by a NADPH-dependent reduction . In Nitrosomonas europaea, this pathway is critical for the production of essential isoprenoid compounds needed for cell membrane integrity and various cellular processes, enabling this chemolithoautotrophic ammonia-oxidizing bacterium to maintain its unique metabolic functions.

How does DXR activity relate to the core metabolic processes of Nitrosomonas europaea?

While the specific relationship between DXR and nitrification processes in N. europaea isn't directly described in available literature, the MEP pathway in which DXR functions represents a crucial metabolic route that likely supports the specialized energy metabolism of this organism. N. europaea generates energy primarily through the oxidation of ammonia to nitrite , and the isoprenoids produced via the MEP pathway likely contribute to maintaining cellular structures necessary for this chemolithoautotrophic lifestyle. The regulation of DXR may be integrated with other metabolic pathways that respond to environmental conditions such as oxygen availability and nitrite concentration, which are known to affect gene expression patterns in N. europaea .

What genetic characteristics of the dxr gene in Nitrosomonas europaea are important to understand?

Similar to other functional genes in N. europaea, the dxr gene likely has specific regulatory elements that respond to the organism's unique environmental adaptations. N. europaea is known to regulate gene expression in response to environmental factors such as dissolved oxygen levels and nitrite concentrations . When working with the dxr gene, researchers should consider potential regulatory interactions with other metabolic pathways, especially considering that N. europaea has evolved complex gene clusters such as those observed with nirK and associated genes (ncgABC) that work together functionally .

What are the optimal expression systems for producing recombinant Nitrosomonas europaea DXR?

When expressing recombinant N. europaea DXR, researchers should consider the following methodological approaches:

• Expression system selection: E. coli BL21(DE3) or similar strains are typically suitable for expressing bacterial enzymes like DXR.

• Codon optimization: N. europaea has distinct codon usage patterns that may require optimization for efficient expression in heterologous hosts.

• Expression conditions: Based on knowledge of other N. europaea proteins, optimal expression may require:

  • Induction at lower temperatures (16-25°C)

  • Extended expression periods (18-24 hours)

  • IPTG concentrations of 0.1-0.5 mM

• Vector design: Incorporating affinity tags (His6, GST) while ensuring they don't interfere with the active site is crucial for downstream purification.

Researchers should monitor expression levels through SDS-PAGE and Western blot analysis, similar to methods used for monitoring NirK expression in N. europaea mutants .

What purification challenges are specific to recombinant Nitrosomonas europaea DXR?

Purification of recombinant N. europaea DXR presents several challenges requiring strategic approaches:

• Maintaining enzyme stability: Buffer optimization is critical, typically requiring:

  • pH maintenance between 7.0-8.0

  • Inclusion of glycerol (10-20%)

  • Addition of reducing agents (DTT or β-mercaptoethanol)

  • Potential inclusion of divalent cations (Mg²⁺ or Mn²⁺)

• Solubility issues: N. europaea proteins may have unique solubility properties requiring:

  • Solubility screening with different detergents if membrane association occurs

  • Optimization of salt concentrations (typically 100-300 mM NaCl)

• Purification strategy:

  • Initial capture: affinity chromatography (IMAC for His-tagged constructs)

  • Intermediate purification: ion exchange chromatography

  • Polishing: size exclusion chromatography to obtain homogenous enzyme preparations

Protein quality should be assessed through activity assays specific to DXR function, ensuring the conversion of DXP to MEP in the presence of NADPH .

How can isotope labeling be effectively incorporated when expressing recombinant Nitrosomonas europaea DXR for structural studies?

For structural studies requiring isotope-labeled DXR, researchers should implement the following methodology:

• Minimal media formulation optimized for N. europaea protein expression:

  • M9 minimal media supplemented with trace elements

  • ¹⁵N-ammonium chloride for nitrogen labeling

  • ¹³C-glucose or ¹³C-glycerol for carbon labeling

  • Deuterated water (D₂O) for deuterium labeling if required

• Expression protocol modifications:

  • Extended adaptation periods in isotope-containing media

  • Lower growth temperatures (16-20°C)

  • Longer induction times with reduced IPTG concentrations

  • Potential supplementation with amino acids for selective labeling

• Verification methods:

  • Mass spectrometry to confirm incorporation levels

  • Preliminary NMR tests to assess spectral quality before full structural studies

This approach allows for the production of labeled DXR suitable for NMR spectroscopy or neutron diffraction studies, enabling detailed analysis of structural dynamics and catalytic mechanisms.

What are the established methods for assaying DXR activity in recombinant Nitrosomonas europaea preparations?

DXR activity can be measured using several complementary approaches:

• Spectrophotometric NADPH oxidation assay:

  • Monitors decrease in absorbance at 340 nm

  • Reaction mixture contains:

    • Purified recombinant DXR

    • DXP substrate (typically 0.1-1 mM)

    • NADPH (0.1-0.2 mM)

    • Buffer (typically HEPES or Tris, pH 7.5-8.0)

    • Divalent cation (usually Mg²⁺ or Mn²⁺, 1-5 mM)

  • Activity calculated using NADPH extinction coefficient (ε₃₄₀ = 6,220 M⁻¹cm⁻¹)

• HPLC-based product formation assay:

  • Directly quantifies MEP production

  • Reaction stopped with acid or heat

  • Products separated by reverse-phase HPLC

  • Detection by UV absorbance or coupled to mass spectrometry

• Coupled enzyme assays:

  • Links MEP formation to a secondary enzyme reaction with colorimetric or fluorescent output

  • Useful for high-throughput screening applications

Similar to approaches used in studying NirK activity in N. europaea , controls should include enzyme-free and substrate-free reactions, with validation using known DXR inhibitors such as fosmidomycin .

How do environmental factors affect the kinetic properties of recombinant Nitrosomonas europaea DXR?

Based on N. europaea's environmental adaptations, the following parameters should be systematically evaluated:

• Temperature effects:

  Temperature (°C)	Expected Relative Activity (%)
  4	10-20
  20	40-60
  30	80-100
  40	60-80
  50	20-40

• pH dependency:

  pH	Expected Relative Activity (%)
  6.0	30-50
  7.0	70-90
  7.5	90-100
  8.0	80-100
  9.0	40-60

• Oxygen sensitivity: Given N. europaea's adaptations to varying oxygen levels , monitor:

  • Activity under aerobic vs. microaerobic conditions

  • Potential oxidative inactivation during extended reactions

  • Effects of oxygen scavengers on enzyme stability

• Ionic strength and cation requirements:

  • Test range of Mg²⁺ and Mn²⁺ concentrations (0.5-10 mM)

  • Evaluate effects of monovalent cations (K⁺, Na⁺)

  • Determine optimal ionic strength for maximal activity

These environmental parameters should be systematically tested using the aforementioned activity assays to establish optimal conditions for enzymatic studies.

How can researchers effectively analyze substrate specificity and inhibition patterns of Nitrosomonas europaea DXR?

Comprehensive analysis of substrate specificity and inhibition requires:

• Substrate specificity assessment:

  • Test DXP analogs with modifications at specific positions

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

  • Create a structure-activity relationship table correlating molecular features with catalytic efficiency

• Inhibition studies methodology:

  • Establish IC₅₀ values for known DXR inhibitors (fosmidomycin, FR900098)

  • Determine inhibition mechanisms through:

    • Lineweaver-Burk plots

    • Dixon plots

    • Cornish-Bowden plots

  • Classify inhibitors as competitive, uncompetitive, or mixed

• Data analysis template:

  Inhibitor	IC₅₀ (μM)	Ki (μM)	Inhibition Type	Structure-Activity Relationship Notes
  Fosmidomycin	[value]	[value]	Competitive	Phosphonate group essential
  FR900098	[value]	[value]	Competitive	N-acetyl modification increases potency
  [Custom inhibitor]	[value]	[value]	[type]	[observations]

• Correlation with other N. europaea enzymes: Compare inhibition patterns with other N. europaea enzymes to identify potential metabolic vulnerabilities, similar to approaches used in studying NirK inhibition .

What structural features distinguish Nitrosomonas europaea DXR from other bacterial DXR enzymes?

When analyzing the structural features of N. europaea DXR, researchers should focus on:

• Domain organization comparison:

  • N-terminal NADPH binding domain

  • Central catalytic domain containing the active site

  • C-terminal domain involved in dimerization

  • Comparison with known DXR structures from other organisms

• Active site architecture analysis:

  • Identification of catalytic residues through sequence alignment and structural modeling

  • Comparison with the conserved catalytic triad found in most DXR enzymes

  • Potential adaptations specific to N. europaea's ecological niche

• Oligomeric state determination:

  • Size exclusion chromatography to determine native molecular weight

  • Analytical ultracentrifugation to confirm oligomerization state

  • Cross-linking studies to identify intersubunit interactions

• Structural flexibility assessment:

  • Hydrogen-deuterium exchange mass spectrometry to identify regions of conformational flexibility

  • Molecular dynamics simulations to predict domain movements during catalysis

  • Comparison with conformational changes observed in related DXR enzymes

Understanding these structural features within the context of N. europaea's unique physiological adaptations could reveal novel aspects of DXR function in chemolithoautotrophic bacteria.

How can computational approaches enhance our understanding of Nitrosomonas europaea DXR catalytic mechanism?

Computational methods offer valuable insights into DXR function:

• Homology modeling workflow:

  • Template selection based on sequence identity with known DXR structures

  • Model building using multiple templates when available

  • Refinement focusing on active site geometry

  • Validation through energy minimization and Ramachandran plot analysis

  • Comparison with experimental data when available

• Molecular dynamics simulation protocol:

  • System preparation with proper protonation states and solvation

  • Energy minimization and equilibration

  • Production runs (minimum 100 ns) under various conditions

  • Analysis of:

    • Active site flexibility

    • Water-mediated interactions

    • Conformational changes during substrate binding

    • Effects of pH and temperature on structure

• Quantum mechanics/molecular mechanics (QM/MM) approaches:

  • QM treatment of the active site and substrate

  • MM treatment of the protein environment

  • Calculation of reaction energy profiles

  • Identification of transition states and intermediates

• Integration with experimental data:

  • Validation using site-directed mutagenesis results

  • Refinement based on spectroscopic measurements

  • Correlation with inhibition patterns

These computational approaches can provide valuable insights similar to those that helped understand the functional interactions between NirK and other proteins in N. europaea .

What advanced spectroscopic methods are most informative for analyzing recombinant Nitrosomonas europaea DXR structure-function relationships?

Several spectroscopic techniques provide complementary insights:

• Circular dichroism (CD) spectroscopy applications:

  • Secondary structure composition assessment (190-260 nm)

  • Thermal stability determination through melting curves

  • Conformational changes upon substrate/inhibitor binding

  • Effects of pH, ionic strength, and temperature on structure

• Fluorescence spectroscopy approaches:

  • Intrinsic tryptophan fluorescence to monitor conformational changes

  • Binding studies using:

    • Fluorescence quenching

    • Fluorescence anisotropy

    • Förster resonance energy transfer (FRET)

  • Cofactor binding kinetics (NADPH association/dissociation)

• NMR spectroscopy applications:

  • Backbone assignments of labeled protein

  • Chemical shift perturbation experiments to map binding interfaces

  • Relaxation measurements to assess protein dynamics

  • Hydrogen-deuterium exchange to identify protected regions

• EPR spectroscopy for metal interactions:

  • Characterization of metal cofactor binding

  • Spin-labeling of specific residues to monitor conformational changes

  • Detection of potential radical intermediates during catalysis

Each technique should be applied in conjunction with activity assays to correlate structural observations with functional outcomes, similar to approaches linking NirK structure with its physiological role in N. europaea .

How does DXR activity in Nitrosomonas europaea relate to its unique ammonia-oxidizing metabolism?

• Metabolic integration hypothesis:
  The MEP pathway likely interfaces with N. europaea's core energy metabolism, which relies on ammonia oxidation to nitrite for energy generation . DXR activity may be coordinated with:

  • Ammonia monooxygenase (AMO) activity

  • Hydroxylamine oxidoreductase (HAO) function

  • Respiratory electron transport chain components

• Stress response connections:
  N. europaea is known to regulate gene expression in response to environmental stressors . DXR activity might be modulated under:

  • Oxygen limitation conditions

  • High nitrite concentrations

  • Nutrient limitation

  • pH stress

• Integration with nitrite detoxification mechanisms:
  N. europaea expresses NirK and other proteins for nitrite tolerance . DXR-dependent isoprenoid production may support:

  • Membrane integrity under nitrite stress

  • Production of specialized lipids for stress tolerance

  • Synthesis of molecules involved in signaling pathways

Understanding these relationships requires correlation of DXR activity with transcriptional data for key metabolic genes (amoA, hao, nirK, norB) under various environmental conditions .

What role might DXR play in Nitrosomonas europaea's adaptation to varying oxygen concentrations?

N. europaea shows complex transcriptional responses to varying oxygen levels , suggesting potential impacts on DXR function:

• Oxygen limitation responses:
  When oxygen is limited, N. europaea increases transcription of ammonia oxidation genes (amoA, hao) , which may correlate with:

  • Altered DXR expression to support membrane adaptations

  • Modified isoprenoid production patterns

  • Shifts in metabolic pathway integration

• Potential regulatory mechanisms:

  • Transcriptional regulation similar to other metabolic genes

  • Post-translational modifications affecting enzyme activity

  • Allosteric regulation by metabolic intermediates

  • Protein-protein interactions modulating function

• Experimental approach to investigate oxygen effects:

  • Controlled batch cultures at defined dissolved oxygen concentrations

  • Measurement of dxr transcript levels via RT-qPCR

  • Analysis of DXR protein levels and activity

  • Correlation with isoprenoid production profiles

• Expected patterns based on N. europaea physiology:

  Oxygen Condition	Expected DXR Expression	Potential Metabolic Significance
  Oxygen-rich	Baseline expression	Normal isoprenoid production
  Oxygen-limited	Potentially increased	Support for membrane adaptations
  Anaerobic stress	Regulatory shift	Integration with alternative respiratory pathways

These hypotheses can be tested using approaches similar to those used for studying other N. europaea genes under varying oxygen conditions .

How do nitrite stress conditions affect DXR expression and activity in Nitrosomonas europaea?

N. europaea has evolved specific mechanisms to tolerate the nitrite it produces , which may involve DXR:

• Nitrite stress response patterns:

  • N. europaea expresses NirK and related proteins (NcgABC) for nitrite tolerance

  • Similar regulatory mechanisms might affect DXR expression

  • Potential coordination between nitrite tolerance mechanisms and isoprenoid biosynthesis

• Experimental design to assess nitrite effects on DXR:

  • Cultures exposed to varying nitrite concentrations (0-300 mg nitrite-N/L)

  • Measurement of dxr transcript levels via RT-qPCR

  • DXR protein quantification via Western blotting

  • Enzyme activity assays from cell extracts

  • Correlation with nirK, ncgABC, and norB expression

• Expected relationship with nitric oxide detoxification:

  • N. europaea expresses nitric oxide reductase (NorB) during aerobic growth

  • Potential coordinated regulation with DXR for holistic stress response

  • MEP pathway products might support membrane integrity during nitrite/NO stress

• Mitigation strategies for nitrite inhibition in enzymatic assays:

  • Buffer optimization to minimize nitrite interference

  • Control experiments with known nitrite concentrations

  • Correlation of in vitro vs. in vivo effects of nitrite on DXR function

These investigations would build upon existing knowledge of N. europaea's nitrite tolerance mechanisms .

What are the common challenges in obtaining active recombinant Nitrosomonas europaea DXR and how can they be addressed?

Researchers frequently encounter several challenges when working with recombinant N. europaea DXR:

• Low expression levels:

  • Solution: Optimize codon usage for expression host

  • Solution: Test multiple fusion tags (His, GST, MBP)

  • Solution: Evaluate different promoter systems

  • Solution: Screen expression conditions (temperature, induction time, media composition)

• Inclusion body formation:

  • Solution: Lower induction temperature (16-20°C)

  • Solution: Reduce inducer concentration

  • Solution: Co-express with molecular chaperones (GroEL/ES, DnaK/J)

  • Solution: Add solubility-enhancing agents to culture medium (sorbitol, arginine)

• Protein instability:

  • Solution: Include protease inhibitors throughout purification

  • Solution: Add stabilizing agents (glycerol, reducing agents)

  • Solution: Maintain cold temperatures during all processing steps

  • Solution: Consider on-column refolding protocols if necessary

• Loss of activity during purification:

  • Solution: Minimize purification steps

  • Solution: Test activity after each purification stage

  • Solution: Include cofactors (NADPH) or substrate analogs in buffers

  • Solution: Optimize buffer compositions (pH, ionic strength, additives)

Each challenge should be systematically addressed with controlled experiments to identify optimal conditions.

How can researchers troubleshoot inconsistent kinetic data when characterizing Nitrosomonas europaea DXR?

When facing inconsistent kinetic measurements:

• Enzyme quality issues:

  • Verification: Check protein homogeneity by SDS-PAGE and size exclusion chromatography

  • Solution: Implement more stringent purification protocols

  • Solution: Prepare fresh enzyme preparations more frequently

  • Solution: Determine optimal storage conditions through stability tests

• Assay interference factors:

  • Verification: Run control reactions with known DXR enzymes from other sources

  • Solution: Test multiple buffer systems to identify interference

  • Solution: Filter or treat reagents to remove potential contaminants

  • Solution: Validate assays with orthogonal methods (spectrophotometric vs. HPLC)

• Data analysis approach:

  • Verification: Apply multiple fitting methods to the same dataset

  • Solution: Use global fitting approaches for complex kinetic models

  • Solution: Implement statistical analyses to identify outliers

  • Solution: Conduct sufficient replicates (minimum n=3) for each measurement

• Environmental variables:

  • Verification: Monitor temperature stability during reactions

  • Solution: Control oxygen exposure throughout experiments

  • Solution: Standardize reagent preparation protocols

  • Solution: Consider microplate reader calibration if using high-throughput formats

Documenting all experimental conditions and variables is crucial for identifying the source of inconsistency.

What approaches can resolve crystallization challenges with recombinant Nitrosomonas europaea DXR?

For researchers pursuing structural studies:

• Protein sample optimization:

  • Strategy: Further purification steps (ion exchange, size exclusion)

  • Strategy: Limited proteolysis to remove flexible regions

  • Strategy: Surface entropy reduction through engineered mutations

  • Strategy: Thermal stability screening to identify stabilizing conditions

• Crystallization condition screening:

  • Strategy: Expanded commercial screen testing (500+ conditions)

  • Strategy: Systematic grid screens around promising conditions

  • Strategy: Inclusion of substrate, cofactor, or inhibitors as stabilizing ligands

  • Strategy: Testing various protein concentrations (5-20 mg/mL)

• Alternative crystallization approaches:

  • Strategy: Lipidic cubic phase for membrane-associated forms

  • Strategy: Microseeding from initial crystal hits

  • Strategy: Batch crystallization versus vapor diffusion

  • Strategy: Counter-diffusion methods in capillaries

• Construct optimization:

  • Strategy: Test multiple N- and C-terminal truncations

  • Strategy: Surface cysteine mutations to prevent non-specific aggregation

  • Strategy: Co-crystallization with antibody fragments

  • Strategy: Domain-swapping approaches with crystallizable homologs

These approaches have proven successful with challenging proteins and could be adapted specifically for N. europaea DXR.