Recombinant Nitrosomonas europaea Dephospho-CoA kinase (coaE)

What is the role of dephospho-CoA kinase (coaE) in Nitrosomonas europaea metabolism?

Dephospho-CoA kinase (coaE) catalyzes the final step in coenzyme A biosynthesis in Nitrosomonas europaea, specifically phosphorylating the 3′-hydroxy group of the ribose sugar moiety in dephospho-CoA to form CoA. This reaction is critical for the organism's metabolism since CoA is an essential cofactor in numerous biochemical pathways. Based on studies in other organisms, approximately 4% of all enzymes use CoA or thioesters of CoA as substrates, highlighting the central importance of this cofactor in cellular metabolism . In N. europaea, which is a gram-negative obligate chemolithoautotroph that derives all its energy and reductant from ammonia oxidation, CoA plays crucial roles in energy generation and carbon fixation pathways . The functional coaE enzyme ensures adequate CoA supplies to maintain these essential metabolic processes.

How is the coaE gene organized in the Nitrosomonas europaea genome?

The coaE gene in Nitrosomonas europaea is located within its single circular chromosome of 2,812,094 bp . Like other bacteria, N. europaea's coaE was likely previously designated as a hypothetical kinase gene (similar to yacE in E. coli) before its function was characterized . Interestingly, genomic analysis of N. europaea reveals that genes are distributed relatively evenly around the genome, with approximately 47% transcribed from one strand and 53% from the complementary strand . Unlike some metabolic pathways that show gene clustering, coaE in bacterial genomes is typically not clustered with other genes related to CoA biosynthesis or metabolism, suggesting independent regulation . The average protein-encoding gene in N. europaea is approximately 1,011 bp in length, with intergenic regions averaging 117 bp , providing context for the expected size parameters of the coaE gene in this organism.

What are the predicted structural features of N. europaea coaE based on homology studies?

Based on homology with characterized dephospho-CoA kinases from other bacteria, N. europaea coaE likely contains several conserved structural features. The enzyme would be expected to contain the Walker kinase motif (residues 9-17 in the E. coli enzyme), which is critical for ATP binding and phosphoryl transfer activity . The predicted molecular mass would be approximately 22-25 kDa, similar to the 22.6-kDa monomeric recombinant dephospho-CoA kinase from E. coli . Homologues of dephospho-CoA kinase have been identified across a diverse range of organisms including bacteria, fungi, and animals, with high sequence conservation in critical functional regions . The enzyme would likely function as a monomer in N. europaea, unlike in some mammalian systems where dephospho-CoA kinase activity is part of a bifunctional enzyme complex that also includes phosphopantetheine adenylyltransferase activity .

What are the optimal conditions for heterologous expression of recombinant N. europaea coaE?

For optimal heterologous expression of recombinant N. europaea coaE, an E. coli expression system using BL21(DE3) cells with a pET-based vector (such as pEt28b) is recommended, based on successful expression strategies for homologous enzymes . The expression protocol should include the following key parameters:

• Culture medium: Luria-Bertani medium supplemented with appropriate antibiotic (e.g., kanamycin at 50 μg/ml for pET28b vectors)

• Growth temperature: 37°C until optical density at 600 nm reaches 0.6

• Induction: IPTG at a final concentration of 100 μM

• Post-induction growth: 6 hours at 37°C

• Cell harvest: Centrifugation followed by disruption with a French press

This approach has been effective for the expression of E. coli coaE, yielding significant amounts of soluble, active enzyme . If inclusion body formation occurs, optimization may include reducing the growth temperature to 25-30°C after induction or decreasing IPTG concentration to 50 μM.

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

A multistep chromatographic purification strategy is recommended for recombinant N. europaea coaE, based on successful purification methods used for homologous enzymes:

This purification protocol is adapted from methods used for dephospho-CoA kinase from C. ammoniagenes and recombinant E. coli dephospho-CoA kinase . Purification can be monitored by SDS-PAGE analysis and enzyme activity assays at each step. Expected purity should be >95% after the Q Sepharose step, with potential for further purification using ATP-affinity chromatography if needed for crystallographic studies.

How can enzyme activity be reliably measured for N. europaea coaE?

The dephospho-CoA kinase activity of recombinant N. europaea coaE can be measured using several complementary assay methods:

• Coupled enzyme assay:

  • Reaction mixture containing ATP, dephospho-CoA, and recombinant enzyme

  • Coupling to ADP formation through pyruvate kinase and lactate dehydrogenase

  • Monitoring NADH oxidation spectrophotometrically at 340 nm

• Direct product analysis by HPLC:

  • Reaction mixture containing ATP, dephospho-CoA, and enzyme

  • Incubation at optimal temperature (typically 37°C)

  • Separation of reaction products by reversed-phase HPLC

  • Monitoring CoA formation by UV absorbance at 260 nm

• NMR analysis for product verification:

  • Larger-scale reaction for product accumulation

  • Purification of product

  • 31P-NMR analysis to confirm 3′-phosphorylation

For kinetic characterization, the Km for dephospho-CoA is expected to be in the range of 0.1-0.8 mM, based on values from C. ammoniagenes (0.12 mM) and E. coli (0.74 mM) . The enzyme typically shows maximum activity at high pH values, similar to other characterized dephospho-CoA kinases .

What is the substrate specificity profile of N. europaea coaE?

While dephospho-CoA is the natural substrate for coaE, assessment of substrate specificity is important for understanding enzyme function and evolution. Based on studies with recombinant E. coli dephospho-CoA kinase, the N. europaea enzyme would be expected to show activity with several alternative substrates:

This substrate specificity profile indicates that while the enzyme can utilize alternative adenosine-containing molecules, its activity is highly specific for dephospho-CoA . The significantly lower activities with adenosine, AMP, and APS (4-8% of activity compared to dephospho-CoA) reflect the importance of the pantetheine moiety for optimal substrate recognition and catalytic efficiency . The ability to phosphorylate the 3′-hydroxyl group is a relatively rare enzymatic activity, shared primarily by dephospho-CoA kinase and APS kinase .

How do pH and temperature affect the activity of recombinant N. europaea coaE?

The pH and temperature dependence of recombinant N. europaea coaE activity would be expected to follow patterns similar to those observed for homologous enzymes. Based on studies of dephospho-CoA kinases from other sources:

• pH dependence:

  • Maximum activity typically observed at high pH values

  • Optimal pH range likely between 8.0-9.0

  • Activity decreases significantly below pH 7.0

  • This pH profile is consistent with the catalytic mechanism involving deprotonation of the 3′-hydroxyl group

• Temperature dependence:

  • Optimal temperature likely between 30-40°C

  • Activity increases with temperature up to the optimal point

  • Thermostability may be moderate, with significant loss of activity above 45-50°C

  • The temperature profile reflects both the catalytic rate increase with temperature and protein stability constraints

These parameters would need to be experimentally determined for the N. europaea enzyme, as they may differ somewhat from the well-characterized E. coli and C. ammoniagenes enzymes due to adaptations to N. europaea's ecological niche .

What are the kinetic parameters for ATP utilization by N. europaea coaE?

The kinetic parameters for ATP utilization by N. europaea coaE would be crucial for understanding its catalytic mechanism and efficiency. Based on studies of dephospho-CoA kinases from related organisms, the following parameters would be expected:

The enzyme would be expected to follow Michaelis-Menten kinetics with respect to ATP, with potential substrate inhibition at high ATP concentrations. The catalytic efficiency (kcat/Km) would reflect the enzyme's evolutionary adaptation to cellular ATP concentrations in N. europaea . Given that N. europaea is an ammonia-oxidizing bacterium with a unique energy metabolism compared to heterotrophic bacteria, its coaE might exhibit kinetic parameters optimized for its specialized metabolic niche .

What approaches can be used to determine the crystal structure of N. europaea coaE?

Determining the crystal structure of N. europaea coaE would require a systematic approach:

• High-purity protein preparation:

  • Multiple chromatography steps as described previously

  • Size-exclusion chromatography as a final polishing step

  • Concentration to 10-20 mg/ml in a stabilizing buffer

  • Verification of homogeneity by dynamic light scattering

• Crystallization screening:

  • Initial sparse-matrix screening at various temperatures (4°C, 16°C, 20°C)

  • Testing both apo-enzyme and enzyme-substrate complexes

  • Inclusion of ATP analogs (AMP-PNP, ATPγS) to stabilize active site

  • Optimization of promising conditions by varying pH, precipitant concentration, and additives

• Structural determination:

  • X-ray diffraction data collection at synchrotron radiation facility

  • Phase determination by molecular replacement using E. coli coaE as a model

  • Refinement and validation of the structure

  • Analysis of active site architecture and substrate binding pocket

The anticipated resolution would be 1.5-2.5 Å, sufficient to identify key catalytic residues and the structural basis for substrate specificity . The Walker kinase motif (residues 9-17 in E. coli) would be a key structural feature to examine in detail .

How can site-directed mutagenesis be used to investigate catalytic mechanisms of N. europaea coaE?

Site-directed mutagenesis offers powerful tools for investigating the catalytic mechanism of N. europaea coaE. Based on comparative sequence analysis with homologous enzymes, the following experimental approach is recommended:

• Target residue selection:

  • Conserved residues in the Walker A motif (likely involved in ATP binding)

  • Conserved basic residues near the active site (potential role in transition state stabilization)

  • Residues unique to N. europaea coaE (potential adaptation to its metabolic niche)

• Mutagenesis strategy:

• Functional characterization:

  • Purification of mutant proteins using the same protocol as wild-type

  • Detailed kinetic analysis comparing wild-type and mutant enzymes

  • Stability assessment to distinguish catalytic defects from structural disruption

  • Crystallization of informative mutants to correlate structure with function

This approach would provide insights into the catalytic mechanism, identifying residues essential for substrate binding, transition state stabilization, and phosphoryl transfer .

What computational approaches can predict substrate binding and catalytic mechanisms for N. europaea coaE?

Computational approaches can provide valuable insights into substrate binding and catalytic mechanisms of N. europaea coaE:

• Homology modeling:

  • Generation of 3D model using E. coli coaE crystal structure as template

  • Refinement with molecular dynamics simulations

  • Validation through Ramachandran plot analysis and energy minimization

  • Comparison with other known dephospho-CoA kinase structures

• Molecular docking studies:

  • Docking of dephospho-CoA and ATP into the active site

  • Analysis of binding modes and key interactions

  • Comparison of docking scores for natural substrate versus alternative substrates

  • Identification of residues controlling substrate specificity

• Molecular dynamics simulations:

  • Simulations of enzyme-substrate complexes in explicit solvent

  • Analysis of conformational changes during substrate binding

  • Investigation of water molecules in the active site

  • Calculation of binding free energies

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

  • Modeling of transition state during phosphoryl transfer

  • Calculation of activation energy barriers

  • Identification of catalytic residues and their roles

  • Proposal of detailed reaction mechanism

These computational approaches would complement experimental studies and provide atomic-level insights into the catalytic mechanism of N. europaea coaE that might be difficult to obtain experimentally .

How does N. europaea coaE compare to homologous enzymes from other organisms?

Comparative analysis of N. europaea coaE with homologous enzymes from diverse organisms provides evolutionary context and functional insights:

This comparative analysis highlights that while the core function of dephospho-CoA kinase is conserved across diverse organisms, significant structural and organizational variations have evolved . The monofunctional nature of bacterial enzymes contrasts with the bifunctional or complexed forms in higher organisms, suggesting different evolutionary pressures on CoA biosynthesis regulation . Sequence analysis across these homologues would reveal conservation patterns indicating essential catalytic residues versus those that have diverged for specialized functions.

What genomic context surrounds the coaE gene in N. europaea compared to other bacteria?

The genomic context of coaE in N. europaea provides insights into its regulation and potential functional relationships:

• Lack of pathway clustering:

  • Unlike some metabolic pathways, coaE is typically not clustered with other CoA biosynthesis genes in bacterial genomes

  • This suggests independent regulation rather than operon-based coordination

• Potential neighboring genes:

  • In some bacteria, coaE shows chromosomal clustering with genes involved in NAD de novo biosynthesis

  • This potential association might reflect coordination between CoA and NAD metabolism, both essential cofactors

• Comparison with nitrification genes:

  • N. europaea has key ammonia oxidation genes (amoCAB) and hydroxylamine oxidoreductase genes (hao) with specific genomic organization

  • The relative position of coaE to these central metabolism genes in N. europaea would indicate potential regulatory relationships

• Insertion sequences and genome plasticity:

  • N. europaea contains 85 predicted insertion sequence elements in eight different families

  • The proximity of these elements to coaE could affect its expression and evolutionary stability

This genomic context analysis would reveal whether coaE in N. europaea shows unique organizational features compared to other bacteria, potentially reflecting adaptations to its specialized metabolism .

How has the structure and function of coaE evolved across different bacterial phyla?

The evolution of coaE structure and function across bacterial phyla reveals adaptation patterns and functional constraints:

• Sequence conservation patterns:

  • The Walker kinase motif (residues 9-17 in E. coli) is highly conserved across all phyla

  • ATP-binding residues show stronger conservation than substrate-binding regions

  • Catalytic residues show near-complete conservation across diverse bacteria

• Structural adaptations:

  • Core fold remains conserved across bacterial phyla

  • Surface residues show higher variability, reflecting different cellular environments

  • Substrate-binding pocket shows subtle variations potentially related to differences in cellular dephospho-CoA concentration

• Functional adaptations:

  • Kinetic parameters (Km, kcat) vary across phyla

  • Thermophilic bacteria show adaptations for protein stability at high temperatures

  • Psychrophilic bacteria show opposite adaptations for activity at low temperatures

  • Chemolithoautotrophs like N. europaea may show adaptations to their unique energy metabolism

• Regulatory adaptations:

  • Different mechanisms for regulation of expression and activity

  • Variable allosteric regulation by metabolites

  • Different patterns of post-translational modification

This evolutionary analysis would position N. europaea coaE within the broader context of bacterial dephospho-CoA kinases, highlighting both conserved features essential to function and specialized adaptations to its ecological niche .

How can isotope labeling be used to track CoA synthesis and utilization in N. europaea?

Isotope labeling provides powerful tools for tracking CoA synthesis and utilization pathways in N. europaea:

• 13C-labeled precursor incorporation:

  • Feeding N. europaea cultures with 13C-labeled pantothenate

  • Analysis of labeled intermediates by LC-MS/MS

  • Quantification of flux through the CoA biosynthetic pathway

  • Identification of branch points and regulatory steps

• 15N-labeling of adenine moiety:

  • Incorporation of 15N-labeled adenine into ATP pool

  • Tracking incorporation into newly synthesized CoA

  • Measuring turnover rates of CoA under different growth conditions

  • Distinguishing between de novo synthesis and salvage pathways

• 32P or 33P pulse-chase experiments:

  • Brief exposure to radioactive phosphate

  • Chase with unlabeled phosphate

  • Isolation of CoA and intermediates at different time points

  • Determination of phosphorylation kinetics in vivo

• 2H-labeling to track metabolic utilization:

  • Growth in 2H2O (heavy water) medium

  • Isolation of CoA-linked metabolites

  • Mass spectrometric analysis of isotope incorporation patterns

  • Determination of metabolic flux through CoA-dependent pathways

These isotope labeling approaches would provide insights into how N. europaea's unique metabolic lifestyle as an ammonia-oxidizing chemolithoautotroph affects CoA metabolism and utilization patterns .

What strategies can identify potential inhibitors of N. europaea coaE for metabolic studies?

Identifying specific inhibitors of N. europaea coaE would provide valuable tools for metabolic studies:

• Structure-based inhibitor design:

  • Virtual screening of compound libraries against coaE homology model

  • Focus on compounds targeting the ATP-binding pocket or substrate-binding site

  • Molecular dynamics simulations to refine binding predictions

  • Synthesis or acquisition of top virtual hits for experimental testing

• High-throughput screening approach:

  • Development of a fluorescence-based assay suitable for plate format

  • Screening of diverse compound libraries (natural products, synthetic libraries)

  • Hit validation using secondary assays and dose-response curves

  • Structure-activity relationship studies of promising scaffolds

• Targeted analog approach:

  • Design of dephospho-CoA analogs with modifications at the 3′-hydroxyl position

  • Synthesis of non-hydrolyzable ATP analogs as competitive inhibitors

  • Testing of transition state mimics as potential inhibitors

  • Evaluation of selectivity against mammalian bifunctional enzymes

• Inhibitor characterization workflow:

These approaches would yield chemical probes for studying coaE function in N. europaea metabolism and potentially provide insights into specialized adaptations of the enzyme in this ammonia-oxidizing bacterium .

How can systems biology approaches integrate coaE function into N. europaea metabolic networks?

Systems biology approaches can provide a comprehensive understanding of coaE's role within N. europaea's metabolic network:

• Genome-scale metabolic modeling:

  • Construction of a constraint-based metabolic model of N. europaea

  • Integration of coaE reaction parameters

  • Flux balance analysis under different growth conditions

  • Prediction of metabolic phenotypes upon coaE perturbation

  • Identification of synthetic lethal interactions with coaE

• Transcriptomic and proteomic integration:

  • RNA-Seq analysis under different nitrogen availability conditions

  • Correlation of coaE expression with other metabolic genes

  • Identification of potential co-regulated gene clusters

  • Proteomic analysis to confirm translation patterns and post-translational modifications

• Metabolomic analysis:

  • Targeted analysis of CoA and CoA derivatives

  • Untargeted metabolomics to identify broader metabolic shifts

  • Isotope-assisted metabolomics to track flux through CoA-dependent pathways

  • Integration with transcriptomic data to identify bottlenecks in CoA utilization

• Network analysis approaches:

  • Construction of gene regulatory networks centered on coaE

  • Protein-protein interaction network analysis

  • Metabolite-centric network analysis focusing on CoA and derivatives

  • Identification of network motifs and control points

This systems-level integration would position coaE within the broader context of N. europaea metabolism, revealing how CoA biosynthesis is coordinated with the organism's specialized ammonia-oxidizing lifestyle and energy generation pathways .

What are common challenges in expressing active recombinant N. europaea coaE and their solutions?

Researchers may encounter several challenges when expressing recombinant N. europaea coaE:

Each challenge requires systematic troubleshooting and optimization. For example, if inclusion body formation occurs, a refolding strategy can be developed:

• Isolate inclusion bodies with detergent washes

• Solubilize in denaturants (8M urea or 6M guanidine-HCl)

• Refold by gradual dialysis into native buffer

• Screen refolding additives (L-arginine, glycerol, reduced/oxidized glutathione)

How can researchers address inconsistent kinetic data when characterizing N. europaea coaE?

Inconsistent kinetic data can arise from several sources when characterizing N. europaea coaE:

• Enzyme stability issues:

  • Solution: Include stabilizing agents (glycerol, DTT) in all buffers

  • Monitor enzyme activity over time at experimental temperature

  • Prepare fresh enzyme dilutions for each experiment

  • Pre-incubate enzyme with ATP and metals before activity assays

• Assay interference factors:

  • Solution: Verify linear range of coupled assays

  • Control for background rates without enzyme or substrate

  • Test components individually for interference with detection methods

  • Validate results using multiple independent assay methods

• Substrate quality and consistency:

  • Solution: Verify purity of dephospho-CoA using HPLC

  • Prepare fresh ATP solutions for each experiment

  • Standardize substrate preparation methods

  • Use internal standards in LC-MS based assays

• Data analysis challenges:

  • Solution: Apply appropriate kinetic models (consider substrate inhibition, allosteric effects)

  • Use global fitting approaches for complex kinetic mechanisms

  • Perform replicate experiments with different enzyme preparations

  • Use statistical tests to identify and address outliers

These methodological considerations ensure reliable and reproducible kinetic characterization of N. europaea coaE, providing a solid foundation for understanding its role in the organism's specialized metabolism .

What strategies can overcome difficulties in crystallizing N. europaea coaE for structural studies?

Crystallization of N. europaea coaE may present challenges that can be addressed through systematic approaches:

• Protein sample optimization:

  • Screen multiple constructs with different N- and C-terminal boundaries

  • Test surface entropy reduction mutations (replacing surface lysine/glutamate clusters with alanine)

  • Remove flexible regions identified by limited proteolysis

  • Consider fusion partners that promote crystallization (T4 lysozyme, BRIL)

• Crystallization condition expansion:

  • Extend beyond standard sparse matrix screens to specialized conditions

  • Test wide pH range (4.0-9.5) and various precipitants

  • Explore different temperatures (4°C, 16°C, 20°C)

  • Try seeding techniques from microcrystals or related protein crystals

• Co-crystallization strategies:

  • Include substrate analogs or inhibitors to stabilize active site

  • Try ATP analogs (AMP-PNP, ATPγS) to capture different conformational states

  • Test dephospho-CoA or CoA at various concentrations

  • Co-crystallize with binding partners if identified

• Alternative crystallization methods:

  • Lipidic cubic phase for challenging proteins

  • Counter-diffusion crystallization in capillaries

  • Microfluidic approaches for fine screening

  • In situ proteolysis during crystallization setup

• Alternative structural approaches:

  • Cryo-electron microscopy if molecular weight can be increased (complexes, oligomers)

  • Small-angle X-ray scattering for solution structure

  • NMR spectroscopy for dynamics studies

  • Hydrogen-deuterium exchange mass spectrometry for functional regions

These approaches have proven successful for challenging proteins and could help overcome difficulties in obtaining high-quality crystals of N. europaea coaE for structural studies .