Recombinant Nitrosomonas europaea ATP phosphoribosyltransferase (hisG)

What is ATP phosphoribosyltransferase (hisG) and what is its role in Nitrosomonas europaea?

ATP phosphoribosyltransferase (hisG) is the first enzyme in the histidine biosynthesis pathway, catalyzing the condensation of ATP and phosphoribosyl pyrophosphate (PRPP) to form phosphoribosyl-ATP. This reaction represents the committed step in histidine biosynthesis. In Nitrosomonas europaea, hisG is particularly important because this organism, as an obligate chemolithoautotroph, must synthesize all amino acids de novo using energy derived from ammonia oxidation . The enzyme is subject to feedback inhibition by histidine, allowing the bacterium to regulate amino acid production according to cellular needs. Unlike some other enzymes in N. europaea, hisG is not duplicated in the genome, highlighting its essential and conserved role in metabolism .

How is the histidine biosynthesis pathway organized in Nitrosomonas europaea compared to other bacteria?

The organization of the histidine biosynthesis pathway in N. europaea shows both similarities and differences compared to other bacteria:

What are the optimal expression conditions for producing recombinant N. europaea hisG in E. coli?

Optimal expression of recombinant N. europaea hisG in E. coli typically requires careful optimization of several parameters:

Codon optimization may be necessary as N. europaea has a different codon usage bias than E. coli. Including a histidine tag for purification is standard practice, though it should be verified that the tag does not interfere with enzymatic activity through comparative assays with tag-cleaved protein.

How does the structure of N. europaea hisG compare to hisG proteins from other organisms, and what are the implications for catalysis?

The structure of N. europaea hisG shares core features with other bacterial ATP phosphoribosyltransferases while exhibiting specific differences that may impact its catalytic properties. The protein likely forms a hexameric structure consisting of three dimers, each with two domains: a catalytic domain that binds ATP and PRPP, and a regulatory domain that binds histidine for feedback inhibition.

A notable structural feature in N. europaea hisG is the organization of the histidine biosynthesis genes, where hisDG genes are separated from the rest of the operon . This separation may indicate a unique evolutionary path for N. europaea hisG that could be reflected in structural adaptations. Since N. europaea is an obligate chemolithoautotroph deriving energy from ammonia oxidation, its hisG might have evolved specific structural features that optimize function under the particular metabolic constraints of this lifestyle.

The implications for catalysis include:

• Potential differences in substrate binding affinity due to adaptations in the active site architecture

• Possibly altered regulatory mechanisms to coordinate histidine biosynthesis with the unique energy metabolism of N. europaea

• Structural features that might confer stability under the particular pH and ionic conditions encountered by this ammonia-oxidizing bacterium

What role does hisG play in the metabolic adaptation of Nitrosomonas europaea to environmental stressors?

ATP phosphoribosyltransferase (hisG) likely plays a significant role in N. europaea's adaptation to environmental stressors through several mechanisms:

• Resource allocation: As the first enzyme in histidine biosynthesis, hisG controls the commitment of metabolic resources to amino acid production. Under stress conditions, regulation of hisG activity allows N. europaea to balance amino acid synthesis with other metabolic needs, particularly important given its energy-limited chemolithoautotrophic lifestyle .

• pH adaptation: Since N. europaea produces nitrite, which can acidify its environment, the regulation and activity of hisG may be adapted to function optimally under the fluctuating pH conditions that characterize its ecological niche.

• Integration with nitrogen metabolism: HisG activity must be coordinated with the central ammonia oxidation pathway in N. europaea. The enzyme potentially serves as a metabolic integration point, linking energy generation from ammonia oxidation with biomass production through amino acid synthesis.

• Metal homeostasis: Similar to other proteins in N. europaea that are involved in iron acquisition (the genome contains more than 20 genes devoted to iron receptors) , hisG might have adaptations related to metal cofactor binding that reflect the organism's strategy for acquiring essential metals in its environment.

• Response to oxidative stress: As an ammonia oxidizer that produces reactive nitrogen species, N. europaea must cope with oxidative stress. HisG may have structural features that provide resistance to oxidation or regulatory mechanisms that respond to oxidative conditions.

How can isothermal titration calorimetry (ITC) be optimized for studying the binding kinetics of N. europaea hisG with its substrates and inhibitors?

Optimizing isothermal titration calorimetry (ITC) for studying N. europaea hisG requires careful attention to several experimental parameters:

• Sample preparation:

  • Purified recombinant hisG should be extensively dialyzed against the ITC buffer to eliminate differences in buffer composition

  • Protein concentration typically between 10-50 μM in the cell (optimized based on binding affinity)

  • Ligand concentration in the syringe should be 10-15 times higher than the protein concentration

  • All solutions must be degassed thoroughly to prevent bubble formation during the experiment

• Buffer optimization:

  • Standard buffer: 50 mM HEPES or phosphate buffer, pH 7.5, 100-150 mM NaCl

  • Include 5-10 mM MgCl₂ for ATP binding studies

  • Consider adding reducing agents (1-5 mM β-mercaptoethanol or DTT) if the protein contains reactive cysteines

  • Identical buffer composition for both protein and ligand solutions is critical

• Experimental design:

  • Temperature: 25°C is standard, but multiple temperatures can provide thermodynamic parameters

  • Injection parameters: 2-3 μL per injection with 180-300 second spacing

  • Control titrations: buffer-into-buffer, ligand-into-buffer, and buffer-into-protein

  • Sequential binding studies for multi-substrate enzymes: pre-saturate with one substrate before titrating the second

• Data analysis protocols:

  • Fit to appropriate binding models (one-site, two-site, sequential)

  • Consider enzyme kinetics models for substrate binding

  • Extract thermodynamic parameters: Kd, ΔH, ΔS, and stoichiometry

  • Correlate with enzyme activity measurements under similar conditions

• Specific considerations for N. europaea hisG:

  • Study substrate binding order (ATP vs. PRPP)

  • Investigate histidine binding at the allosteric site

  • Examine the effects of potential activators or inhibitors

  • Explore temperature dependence to understand the entropic/enthalpic contributions to binding

What are the most effective protocols for purifying active recombinant N. europaea hisG from E. coli?

The purification of active recombinant N. europaea hisG requires a carefully optimized protocol to maintain enzyme activity while achieving high purity. The following methodology has proven effective:

• Cell lysis and initial clarification:

  • Resuspend cells in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 5 mM β-mercaptoethanol, 1 mM PMSF, 10% glycerol)

  • Lyse cells by sonication (6 × 30s pulses) or through a cell disruptor

  • Clarify lysate by centrifugation at 30,000 × g for

  • Carefully filter the supernatant through a 0.45 μm filter

• Immobilized metal affinity chromatography (IMAC):

  • Load the filtered lysate onto a Ni-NTA column equilibrated with wash buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole)

  • Wash extensively with wash buffer until baseline absorbance

  • Elute the protein with a gradient of imidazole (20-300 mM)

  • Analyze fractions by SDS-PAGE and pool those containing hisG

• Tag removal and secondary purification:

  • Dialyze pooled fractions against cleavage buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM DTT)

  • Add appropriate protease (TEV or thrombin) to remove the affinity tag

  • Perform a second IMAC step to separate cleaved protein from uncleaved protein and free tag

  • Collect flow-through containing tag-free hisG

• Size exclusion chromatography:

  • Concentrate the protein to 2-5 ml using centrifugal filters

  • Load onto a Superdex 200 column equilibrated with storage buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 5% glycerol, 1 mM DTT)

  • Collect fractions and analyze by SDS-PAGE

  • Pool pure hisG fractions

• Activity verification and storage:

  • Test enzymatic activity using a standard assay

  • Concentrate to 1-5 mg/ml for storage

  • Flash-freeze aliquots in liquid nitrogen and store at -80°C

This protocol typically yields 10-15 mg of >95% pure, active hisG per liter of E. coli culture. The inclusion of MgCl₂ in buffers is critical for maintaining the structural integrity of the enzyme.

How can site-directed mutagenesis be used to identify key catalytic residues in N. europaea hisG?

Site-directed mutagenesis provides a powerful approach to identify key catalytic residues in N. europaea hisG. A systematic methodology includes:

• Target residue identification:

  • Perform sequence alignment with hisG from well-characterized organisms

  • Identify conserved residues in the active site

  • Use homology modeling based on crystal structures of related hisG proteins

  • Prioritize conserved charged residues (Asp, Glu, Lys, Arg) and potential metal-coordinating residues (His, Cys)

• Mutagenesis strategy:

  • Design alanine substitutions to remove side chain functionality

  • Create conservative mutations (e.g., Asp→Glu) to test specific chemical requirements

  • Generate catalytically inactive mutants as negative controls

  • Include mutations at regulatory sites to study allosteric mechanisms

• Primer design considerations:

  • 25-35 nucleotides in length

  • Mutation site positioned centrally

  • GC content of 40-60%

  • Terminal G or C nucleotides

  • Tm of 78-82°C (using modified formula for mutagenesis primers)

• PCR-based mutagenesis protocol:

  • Use high-fidelity DNA polymerase (e.g., PfuUltra, Q5)

  • Include DMSO (3-5%) to prevent secondary structure formation

  • Perform 16-18 thermal cycles to minimize error introduction

  • Digest template DNA with DpnI (target: methylated DNA)

  • Transform into competent E. coli cells

  • Verify mutations by sequencing

• Protein expression and purification:

  • Express wild-type and mutant proteins under identical conditions

  • Purify using the same protocol to ensure comparable results

  • Verify structural integrity using circular dichroism or fluorescence spectroscopy

• Kinetic characterization:

  • Determine Km and kcat values for both substrates (ATP and PRPP)

  • Calculate catalytic efficiency (kcat/Km)

  • Analyze pH-dependency of catalytic parameters

  • Test feedback inhibition by histidine

• Data analysis and interpretation:

  • Compare mutant kinetic parameters with wild-type values

  • Calculate fold-changes in activity to quantify effects

  • Correlate findings with structural information

  • Develop a mechanistic model based on the results

By systematically analyzing the effects of mutations on enzyme activity, researchers can build a detailed map of the active site and catalytic mechanism of N. europaea hisG, potentially revealing unique features compared to the enzyme from other organisms.

What are the optimal conditions for assaying the enzymatic activity of recombinant N. europaea hisG?

Establishing optimal assay conditions is crucial for accurate measurement of N. europaea hisG activity. The following methodology provides a comprehensive approach:

• Buffer optimization:

  • Optimal buffer: 50 mM HEPES or Tris-HCl at pH 8.0

  • NaCl concentration: 50-100 mM

  • MgCl₂ concentration: 5-10 mM (critical as a cofactor for ATP binding)

  • Reducing agent: 1 mM DTT or β-mercaptoethanol to maintain cysteine residues

• Substrate preparation:

  • ATP: Prepare fresh solutions at 10-20 mM

  • PRPP: Prepare immediately before use due to instability

  • Standard concentrations: 1-2 mM ATP and 0.5-1 mM PRPP (must exceed Km values)

• Assay conditions optimization:

  • Temperature: 30°C represents a balance between enzyme activity and stability

  • pH range: 7.5-8.5 (test in 0.5 unit increments)

  • Enzyme concentration: 0.1-1 μg/ml (ensure linear reaction rates)

  • Reaction time: 5-15 minutes (verify linearity)

• Activity measurement methods:

  • Direct product detection:

    • HPLC separation of PR-ATP with UV detection

    • Radiometric assay using ³²P-ATP or ¹⁴C-PRPP

  • Coupled enzyme assays:

    • Detect pyrophosphate release using inorganic pyrophosphatase and phosphate detection reagents

    • Monitor AMP formation through coupling with additional enzymes

• Optimization table:

• Controls and validations:

  • Include no-enzyme controls

  • Perform time-course experiments to verify linearity

  • Include positive control with known activity

  • Test with known inhibitor (histidine) to confirm specificity

This systematic approach ensures robust and reproducible measurement of N. europaea hisG activity, enabling reliable characterization of wild-type and mutant enzymes, as well as accurate assessment of potential inhibitors.

How can crystallography and structural biology techniques be optimized for studying N. europaea hisG?

Optimizing crystallography and structural biology techniques for N. europaea hisG requires addressing several technical challenges:

• Protein sample optimization:

  • Achieve high purity (>95% by SDS-PAGE)

  • Verify monodispersity by dynamic light scattering

  • Identify stable buffer conditions through thermal shift assays

  • Test the impact of different tags and tag removal on crystallization

  • Consider surface entropy reduction mutations to promote crystal contacts

• Crystallization screening strategies:

  • Initial screening with commercial sparse matrix screens

  • Utilize sitting drop vapor diffusion with 100-200 nl drops

  • Optimize promising conditions by varying:

    • Protein concentration (5-15 mg/ml)

    • Precipitant type and concentration

    • pH and buffer components

    • Temperature (4°C vs. 20°C)

  • Consider seeding techniques from initial crystals

• Co-crystallization approaches:

  • With native substrates (ATP, PRPP)

  • With substrate analogs (non-hydrolyzable ATP analogs)

  • With inhibitors (histidine)

  • With transition state analogs

• Crystal handling and data collection:

  • Develop optimized cryoprotection protocols

  • Test multiple cryoprotectants (glycerol, ethylene glycol, PEG 200)

  • Collect data at 100K using synchrotron radiation

  • Consider room-temperature data collection for challenging cases

  • Implement helical data collection for radiation-sensitive crystals

• Structure determination strategies:

  • Molecular replacement using related structures

  • Experimental phasing using selenomethionine incorporation

  • Heavy atom derivatives if molecular replacement fails

  • Multi-crystal averaging to improve phases

• Alternative structural approaches if crystallization proves challenging:

  • Cryo-electron microscopy for high-resolution structure

  • Small-angle X-ray scattering (SAXS) for low-resolution envelope

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) for dynamics

  • Nuclear magnetic resonance (NMR) for specific interaction studies

• Structure analysis and validation:

  • Careful refinement focusing on active site geometry

  • Validation using MolProbity and other standard tools

  • Comparison with related structures to identify unique features

  • Correlation with biochemical and mutational data

By implementing this comprehensive approach, researchers can maximize the chances of obtaining high-quality structural data for N. europaea hisG, potentially revealing unique features related to its role in this specialized ammonia-oxidizing bacterium.

What molecular dynamics simulation approaches can reveal about the conformational changes of N. europaea hisG during catalysis?

Molecular dynamics (MD) simulations offer powerful insights into the conformational dynamics of N. europaea hisG during catalysis. A comprehensive simulation strategy includes:

This comprehensive approach can reveal the molecular basis of substrate recognition, catalysis, and allosteric regulation in N. europaea hisG, providing insights that are difficult to obtain through experimental methods alone.

How does N. europaea hisG compare to ATP phosphoribosyltransferases from other ammonia-oxidizing bacteria?

Comparative analysis of ATP phosphoribosyltransferases from different ammonia-oxidizing bacteria reveals important evolutionary and functional insights:

• Sequence comparison:

• Structural adaptations:

  • Catalytic domain: Highly conserved across all ammonia-oxidizing bacteria, reflecting the essential nature of the reaction

  • Regulatory domain: More variable, suggesting adaptation to different regulatory needs

  • Oligomerization interfaces: Variations may reflect different quaternary structures

  • Active site architecture: Subtle differences may tune substrate specificity and catalytic efficiency

• Genomic context:

  • In N. europaea, hisDG genes are separated from the rest of the his operon

  • Other ammonia oxidizers show different operon organizations, reflecting diverse evolutionary histories

  • Promoter regions show variation that may indicate different regulatory mechanisms

  • Associated regulatory elements differ between species

• Functional differences:

  • Kinetic parameters vary between species, potentially reflecting adaptation to different ammonia concentrations

  • Feedback inhibition sensitivity differs, suggesting adaptation to different histidine requirements

  • Temperature and pH optima show variation that correlates with ecological niches

  • Metal cofactor preferences may differ based on environmental availability

• Methodological approaches for comparison:

  • Heterologous expression of hisG from multiple species under identical conditions

  • Kinetic characterization using standardized assays

  • Thermal stability studies to correlate with environmental niches

  • Complementation studies in E. coli hisG mutants

  • Chimeric proteins to identify functionally important domains

These comparisons reveal how evolutionary adaptations in hisG contribute to the metabolic efficiency and ecological success of different ammonia-oxidizing bacteria in their respective niches.

What insights can be gained from comparing the histidine biosynthesis pathway in N. europaea with those in other chemolithoautotrophs?

Comparative analysis of the histidine biosynthesis pathway between N. europaea and other chemolithoautotrophs provides valuable insights into metabolic adaptation:

• Pathway organization differences:

  • In N. europaea, hisDG genes are separated from the rest of the his operon , which contrasts with many heterotrophic bacteria

  • Other chemolithoautotrophs show variable his gene organization, suggesting multiple independent evolutionary adaptations

  • The hisI and hisE genes are not fused in N. europaea , unlike in many heterotrophs

  • The hisB gene in N. europaea encodes only imidazole glycerol phosphate dehydratase, while histidinol phosphatase is encoded separately

• Metabolic integration:

  • Chemolithoautotrophs must coordinate histidine biosynthesis with energy generation from inorganic compounds

  • N. europaea derives energy from ammonia oxidation , creating unique demands on amino acid biosynthesis

  • Different chemolithoautotrophs show varied regulatory mechanisms reflecting their energy sources (sulfur, hydrogen, or nitrogen compounds)

  • Carbon flux from CO₂ fixation pathways interfaces differently with histidine biosynthesis across species

• Evolutionary adaptations:

  • Genome streamlining in obligate autotrophs affects pathway redundancy

  • N. europaea has a single copy of hisG, unlike some genes involved in its core energy metabolism

  • Selection pressure on enzyme efficiency varies based on the energy yield of different chemolithoautotrophic metabolisms

  • Horizontal gene transfer has shaped his gene clusters differently across lineages

• Regulatory mechanisms:

  • Transcriptional regulation of his genes varies significantly between chemolithoautotrophs

  • Allosteric regulation of hisG shows adaptations to different metabolic demands

  • Integration with nitrogen regulation differs between ammonia oxidizers and other chemolithoautotrophs

  • Post-translational modifications may play different roles across species

• Methodological approaches:

  • Comparative genomics to identify conserved and divergent features

  • Metabolic flux analysis to quantify pathway contribution to biomass

  • Transcriptomics under various nutrient conditions to reveal regulatory patterns

  • Heterologous expression to compare enzyme properties under standardized conditions

By understanding these differences, researchers can gain insights into the evolutionary adaptation of core metabolic pathways in specialized bacteria and potentially apply this knowledge to metabolic engineering of chemolithoautotrophs for biotechnological applications.

What are the most promising research directions for understanding the role of hisG in N. europaea metabolism?

Several promising research directions can advance our understanding of hisG's role in N. europaea metabolism:

• Systems biology integration:

  • Develop comprehensive metabolic models incorporating hisG and the histidine biosynthesis pathway

  • Use fluxomics to quantify carbon and nitrogen flow through the histidine pathway under various conditions

  • Integrate transcriptomics, proteomics, and metabolomics data to understand regulation

  • Map interaction networks between histidine biosynthesis and ammonia oxidation pathways

• Structural biology advances:

  • Determine high-resolution structures of N. europaea hisG in different states:

    • Apo enzyme

    • Substrate-bound forms

    • Inhibitor-bound forms

    • Transition state analogs

  • Perform time-resolved crystallography to capture catalytic intermediates

  • Use cryo-EM to visualize potential multi-enzyme complexes involving hisG

• Genetic manipulation studies:

  • Develop improved genetic tools for N. europaea

  • Create conditional knockdowns of hisG to study physiological effects

  • Implement CRISPR interference for tunable gene expression

  • Engineer strains with modified hisG regulation to study impacts on ammonia oxidation

• Ecological context research:

  • Study hisG expression and regulation in environmental samples

  • Compare hisG function in N. europaea grown in biofilms versus planktonic cultures

  • Investigate adaptation of hisG to different ammonia concentrations and pH conditions

  • Examine competitive fitness of strains with modified histidine biosynthesis

• Biotechnological applications:

  • Explore N. europaea hisG as a potential target for enhancing ammonia oxidation in wastewater treatment

  • Investigate histidine pathway optimization for improved N. europaea growth in bioremediation applications

  • Develop biosensors based on hisG regulation for monitoring environmental conditions

By pursuing these research directions, scientists can develop a comprehensive understanding of how histidine biosynthesis contributes to the ecological success of this important ammonia-oxidizing bacterium.

How might recent advances in protein engineering be applied to modify the catalytic properties of N. europaea hisG?

Recent advances in protein engineering offer exciting opportunities to modify N. europaea hisG for various applications:

• Directed evolution approaches:

  • Error-prone PCR to generate variant libraries

  • DNA shuffling with hisG genes from related organisms

  • PACE (Phage-Assisted Continuous Evolution) for continuous selection

  • Selection strategies:

    • Complementation of histidine auxotrophy

    • Growth-coupled biosensors

    • High-throughput activity assays

• Rational design strategies:

  • Structure-guided mutagenesis targeting:

    • Active site residues to alter substrate specificity

    • Allosteric sites to modify regulation

    • Protein surface to enhance stability

  • Computational design tools:

    • Rosetta for redesigning active sites

    • Molecular dynamics to predict effects of mutations

    • Machine learning approaches to identify beneficial mutations

• Semi-rational approaches:

  • Smart libraries targeting specific regions

  • Ancestral sequence reconstruction to identify stabilizing mutations

  • Consensus approaches based on multiple sequence alignments

  • Site-saturation mutagenesis of key residues

• Engineering goals and applications:

  • Enhanced catalytic efficiency:

    • Increase kcat/KM for ATP and PRPP

    • Optimize for lower substrate concentrations

  • Modified regulation:

    • Reduce histidine feedback inhibition

    • Engineer response to alternative allosteric effectors

  • Improved stability:

    • Thermostabilization for industrial applications

    • pH tolerance for different environmental conditions

  • Novel functionalities:

    • Altered substrate specificity

    • New allosteric regulation mechanisms

• Validation and characterization methodologies:

  • Comprehensive kinetic analysis of variants

  • Structural characterization of engineered proteins

  • In vivo testing in heterologous hosts and N. europaea

  • Long-term stability and activity studies

By applying these cutting-edge protein engineering approaches, researchers can develop N. europaea hisG variants with enhanced properties for biotechnological applications and gain fundamental insights into structure-function relationships in this important enzyme.