Recombinant Nitrosomonas europaea Pantothenate synthetase (panC)

What is Nitrosomonas europaea and why is it significant for biochemical research?

Nitrosomonas europaea (ATCC 19718) is a gram-negative obligate chemolithoautotroph that derives all its energy and reductant for growth from the oxidation of ammonia to nitrite. It plays a crucial role in the biogeochemical nitrogen cycle through the process of nitrification. Its genome consists of a single circular chromosome of 2,812,094 bp with approximately 2,460 protein-encoding genes averaging 1,011 bp in length . The organism's specialized metabolism makes it an ideal model for studying unique biochemical pathways and enzymes, including pantothenate synthetase.

What is pantothenate synthetase (panC) and what role does it play in bacterial metabolism?

Pantothenate synthetase (PS), encoded by the panC gene, catalyzes the final step in pantothenate (vitamin B5) biosynthesis. This ATP-dependent reaction involves the condensation of pantoate and β-alanine to form pantothenate . The pantothenate biosynthetic pathway consists of four steps catalyzed by enzymes encoded by panB, panC, panD, and panE genes. Pantothenate is essential as a precursor for coenzyme A (CoA), which plays a central role in numerous metabolic processes including the citric acid cycle, fatty acid metabolism, and various biosynthetic pathways.

Why focus on recombinant expression of Nitrosomonas europaea panC for research?

Recombinant expression of N. europaea panC allows researchers to study this enzyme's unique properties compared to better-characterized pantothenate synthetases from other organisms. As N. europaea has a specialized metabolism dependent on ammonia oxidation, its pantothenate synthetase may possess distinctive catalytic properties, substrate preferences, or regulatory mechanisms. Recombinant expression facilitates protein purification, structural analysis, and functional characterization that would be difficult to achieve with native enzyme production due to the challenging growth requirements of N. europaea.

How does the pantothenate biosynthesis pathway in Nitrosomonas europaea compare to other bacteria?

While the core pantothenate biosynthesis pathway involving panB, panC, panD, and panE is conserved across many bacterial species, N. europaea's specialized metabolism may influence how this pathway is regulated and integrated with other metabolic networks. The genome analysis of N. europaea reveals that genes encoding transporters for organic molecules are scarce compared to those for inorganic ions , suggesting the organism's preference for autotrophic metabolism. This metabolic specialization may affect how pantothenate biosynthesis is regulated in N. europaea compared to heterotrophic bacteria like E. coli.

What expression systems are most suitable for producing recombinant N. europaea panC?

For expressing recombinant N. europaea panC, E. coli expression systems typically provide good yields and are experimentally tractable. The choice of expression vector should include an appropriate promoter (such as T7 or tac), affinity tag (such as His6 or GST) for purification, and optimal codon usage for E. coli. Temperature modulation (typically 16-25°C post-induction) often improves soluble protein yield. Alternative expression hosts such as Bacillus subtilis or Pseudomonas species might be considered if E. coli-based expression results in inclusion bodies or inactive protein.

What purification strategy should be employed for recombinant N. europaea panC?

A typical purification strategy would include:

• Affinity chromatography (e.g., Ni-NTA for His-tagged protein)

• Ion exchange chromatography (based on the protein's theoretical pI)

• Size exclusion chromatography for final polishing

Purification buffers should contain components that maintain enzyme stability, potentially including:

• pH buffer in the range of 7.0-8.0 (e.g., Tris-HCl or phosphate)

• Salt (typically 100-300 mM NaCl)

• Reducing agent (e.g., 1-5 mM DTT or β-mercaptoethanol)

• Glycerol (10-20%) for long-term storage

Optimization of these conditions should be determined empirically based on enzyme activity and stability assessments.

How do the kinetic parameters of N. europaea panC compare with those of other bacterial pantothenate synthetases?

While specific kinetic data for N. europaea panC is not directly provided in the search results, comparative analysis with other bacterial pantothenate synthetases would typically include parameters such as Km, kcat, and substrate specificity. Based on studies of pantothenate synthetase from other organisms, researchers should:

• Determine Km values for ATP, pantoate, and β-alanine

• Calculate kcat and catalytic efficiency (kcat/Km)

• Assess substrate specificity using pantoate/β-alanine analogs

• Evaluate pH and temperature optima

• Investigate metal ion dependencies

Comparison of these parameters with those from E. coli, M. tuberculosis, and other bacterial species would provide insights into potential adaptations of N. europaea panC to its specialized metabolism.

What structural features distinguish N. europaea panC from other characterized pantothenate synthetases?

Crystal structures of pantothenate synthetase from M. tuberculosis have revealed important insights about the enzyme's structure-function relationship . When studying N. europaea panC, researchers should focus on:

• Sequence alignment with characterized pantothenate synthetases to identify conserved and divergent regions

• Homology modeling based on available crystal structures (if N. europaea panC structure is undetermined)

• Analysis of active site architecture and substrate binding pockets

• Assessment of oligomeric state (dimer vs tetramer) as E. coli PS was found to be a dimer in crystal structures but potentially tetrameric in solution

• Identification of unique structural elements that might relate to N. europaea's specialized metabolism

How does the quaternary structure of N. europaea panC influence its catalytic activity?

Based on studies of E. coli pantothenate synthetase, which exists as a dimer in crystal structures but potentially as a tetramer in solution , researchers should investigate:

• The oligomeric state of N. europaea panC using methods such as:

  • Size exclusion chromatography

  • Analytical ultracentrifugation

  • Native PAGE

  • Cross-linking studies

• The functional significance of oligomerization:

  • Compare activity of monomeric vs oligomeric forms

  • Identify interface residues through structural analysis

  • Generate interface mutants to disrupt oligomerization and assess effects on activity

What are the key considerations for designing experiments to characterize the catalytic mechanism of N. europaea panC?

When investigating the catalytic mechanism of N. europaea panC, researchers should consider:

• Reaction order analysis:

  • Determine if the mechanism follows a sequential or ping-pong mechanism

  • Use initial velocity studies with varying concentrations of substrates

• Transition state analysis:

  • Employ kinetic isotope effects with labeled substrates

  • Use transition state analogs as inhibitors

• Catalytic residue identification:

  • Site-directed mutagenesis of predicted catalytic residues

  • pH-rate profiles to identify essential ionizable groups

• Intermediate capture:

  • Use rapid quench techniques to trap reaction intermediates

  • Employ product analogs that can trap the enzyme in intermediate conformations

The experimental approach should parallel methodologies used for M. tuberculosis pantothenate synthetase, where crystal structures with various ligands (AMPCPP, pantoate, and reaction intermediates) provided insights into the catalytic mechanism .

What spectroscopic techniques are most informative for studying N. europaea panC structure and function?

Several spectroscopic approaches can provide valuable information:

• Circular Dichroism (CD):

  • Secondary structure composition

  • Thermal stability

  • Conformational changes upon substrate binding

• Fluorescence Spectroscopy:

  • Intrinsic tryptophan fluorescence for tertiary structure assessment

  • Ligand binding studies using fluorescence quenching

  • FRET-based approaches for monitoring conformational changes

• Nuclear Magnetic Resonance (NMR):

  • Backbone dynamics

  • Substrate binding interactions

  • Conformational changes during catalysis

• X-ray Crystallography:

  • High-resolution structure determination

  • Substrate and inhibitor complex structures

  • Mechanistic insights through intermediate-bound structures

The approach used for M. tuberculosis PS, which involved crystallographic studies with various ligands including AMPCPP, pantoate, and reaction intermediates , provides a valuable template for similar studies with N. europaea panC.

What experimental design would best elucidate the role of metal ions in N. europaea panC catalysis?

To investigate metal ion requirements for N. europaea panC:

• Metal depletion studies:

  • Dialysis against chelating agents (EDTA, EGTA)

  • Verification of metal removal by atomic absorption spectroscopy

• Metal reconstitution experiments:

  • Systematic testing of various divalent cations (Mg²⁺, Mn²⁺, Ca²⁺, Zn²⁺)

  • Determination of metal-dependent activity restoration

• Spectroscopic analysis:

  • Electron paramagnetic resonance (EPR) for paramagnetic metals

  • Isothermal titration calorimetry (ITC) for metal binding affinity

• Structural studies:

  • Crystallography with and without metal cofactors

  • Identification of metal coordination sites

• Mutagenesis of potential metal-coordinating residues:

  • Target conserved acidic residues (Asp, Glu) in the active site

  • Assess activity of mutants with various metal ions

This experimental design would provide comprehensive insights into the metal ion dependency of N. europaea panC and potential differences from other bacterial pantothenate synthetases.

How should researchers analyze kinetic data for N. europaea panC to distinguish between different mechanistic models?

Proper kinetic data analysis involves:

• Initial velocity studies:

  • Plot initial velocity data using Lineweaver-Burk, Eadie-Hofstee, or Hanes-Woolf transformations

  • Determine kinetic parameters (Km, Vmax) for each substrate

  • Analyze patterns of intersecting lines to distinguish sequential from ping-pong mechanisms

• Product inhibition studies:

  • Determine inhibition patterns (competitive, noncompetitive, uncompetitive)

  • Use inhibition patterns to confirm reaction order and mechanism

• Global data fitting:

  • Employ software like DynaFit or KinTek Explorer for simultaneous fitting to multiple models

  • Use statistical criteria (AIC, BIC) to select the best-fitting model

• Isotope effects:

  • Analyze primary and secondary kinetic isotope effects

  • Interpret results in terms of rate-limiting steps and transition state structure

Table 1: Comparative Kinetic Parameters for Bacterial Pantothenate Synthetases

Note: This table provides representative values based on literature for E. coli and M. tuberculosis PS. N. europaea PS parameters would need to be experimentally determined.

What approaches should be used to analyze potential allosteric regulation of N. europaea panC?

To investigate allosteric regulation:

• Substrate saturation curves:

  • Test for sigmoidal kinetics indicating cooperativity

  • Calculate Hill coefficients to quantify cooperativity

  • Perform these studies at various concentrations of potential allosteric effectors

• Equilibrium binding studies:

  • Use isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR)

  • Compare binding parameters in the presence and absence of potential allosteric modulators

• Structural analysis:

  • Identify potential allosteric sites through structural comparison with related enzymes

  • Perform crystallography or HDX-MS in the presence of potential allosteric modulators

• Mutagenesis of predicted allosteric sites:

  • Generate mutations at residues in predicted allosteric sites

  • Assess changes in cooperativity and response to allosteric modulators

• Thermal shift assays:

  • Measure protein stability changes in response to ligand binding

  • Compare thermostability profiles with and without potential allosteric modulators

How can contradictory experimental results in N. europaea panC research be reconciled and interpreted?

When faced with contradictory experimental results:

• Methodological reconciliation:

  • Carefully compare experimental conditions (pH, temperature, buffer components)

  • Assess protein quality and integrity across different preparations

  • Consider differences in protein constructs (tags, truncations, fusion partners)

• Statistical analysis:

  • Apply appropriate statistical tests to determine significance of differences

  • Consider sample sizes and experimental replication

  • Perform power analysis to ensure adequate statistical power

• Integrative analysis:

  • Combine multiple experimental approaches (kinetic, structural, biophysical)

  • Look for consistent patterns across different experimental modalities

  • Use computational modeling to reconcile apparently contradictory data

• Experimental design considerations:

  • Apply experimental and quasi-experimental designs as outlined by Shadish, Cook, and Campbell

  • Consider counterfactual reasoning and control for confounding variables

  • Implement qualitative research questions when appropriate

What are promising applications of structure-based drug design targeting N. europaea panC?

While N. europaea itself is not typically a pathogen, research on its pantothenate synthetase could inform drug development against pathogenic bacteria:

• Comparative structural analysis:

  • Identify conserved features between N. europaea panC and pathogenic bacterial homologs

  • Leverage unique features of N. europaea panC to understand evolutionary adaptation

• Inhibitor development workflow:

  • Virtual screening against N. europaea panC structure

  • Fragment-based drug design targeting active site or allosteric sites

  • Structure-activity relationship studies of identified inhibitors

• Resistance mechanisms:

  • Investigate natural variations in panC sequences that confer resistance

  • Engineer resistance mutations and characterize their effects on enzyme function and inhibitor binding

The approach used for M. tuberculosis PS, which involved crystal structure determination with various ligands and complexes , provides valuable methodology for similar drug discovery efforts.

How might systems biology approaches enhance our understanding of N. europaea panC in the context of the organism's metabolism?

Integration of panC function into systems-level understanding requires:

• Metabolic flux analysis:

  • Isotope labeling experiments to track carbon flow through pantothenate synthesis pathway

  • Integration of pantothenate synthesis with ammonia oxidation and carbon fixation pathways

• Transcriptomic and proteomic profiling:

  • RNA-Seq under various growth conditions to assess panC regulation

  • Proteomic analysis to quantify PanC protein levels and post-translational modifications

• Computational modeling:

  • Incorporate pantothenate synthesis into genome-scale metabolic models of N. europaea

  • Perform flux balance analysis to predict metabolic responses to environmental changes

• Interactome analysis:

  • Identify protein-protein interactions involving PanC

  • Characterize potential multienzyme complexes involving pantothenate biosynthesis enzymes

This systems approach would provide context for understanding how N. europaea's specialized metabolism influences pantothenate biosynthesis regulation and integration with other metabolic pathways.

What novel experimental techniques could advance research on N. europaea panC beyond current methodologies?

Emerging techniques with potential applications include:

• Single-molecule enzymology:

  • FRET-based approaches to monitor conformational changes during catalysis

  • Optical tweezers or magnetic tweezers to study mechanical properties

• Cryo-electron microscopy:

  • High-resolution structural determination without crystallization

  • Time-resolved structures capturing catalytic intermediates

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Mapping conformational dynamics and ligand-induced changes

  • Identifying allosteric networks within the protein structure

• Microfluidics and droplet-based assays:

  • Ultra-high-throughput screening of enzyme variants

  • Single-cell analysis of enzyme function in vivo

• Computational advances:

  • Molecular dynamics simulations at extended timescales

  • Machine learning approaches for predicting enzyme properties and designing variants

These advanced methodologies could provide unprecedented insights into N. europaea panC structure, dynamics, and function that are not accessible through conventional approaches.