Recombinant Nitrosomonas europaea 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (ispE)

What is ispE and what is its biochemical function?

IspE (4-diphosphocytidyl-2-C-methyl-D-erythritol kinase) catalyzes the ATP-dependent phosphorylation of the 2-hydroxyl group of 4-diphosphocytidyl-2C-methyl-D-erythritol, forming 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate . This reaction represents the fourth step in the MEP pathway for isoprenoid biosynthesis, which is essential for the production of various terpenoids and components of cell membranes.

The catalytic mechanism involves:

• Binding of ATP and the 4-diphosphocytidyl-2C-methyl-D-erythritol substrate

• Nucleophilic attack of the 2-hydroxyl group on the γ-phosphate of ATP

• Transfer of the phosphate group

• Release of the phosphorylated product and ADP

The enzyme belongs to the GHMP kinase family (named after galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase), specifically the IspE subfamily .

Why study recombinant ispE from Nitrosomonas europaea?

Nitrosomonas europaea is a well-characterized ammonia-oxidizing bacterium and serves as a model organism for studying nitrification processes . Studying ispE from this organism offers several advantages:

• N. europaea has a fully sequenced genome, facilitating genetic manipulation and protein expression studies

• As an ammonia oxidizer that thrives in both oxygen-replete and oxygen-limited environments, N. europaea provides insights into enzyme adaptations under different metabolic conditions

• The organism's ecological importance in nitrogen cycling makes its metabolic pathways, including isoprenoid biosynthesis, relevant to environmental microbiology

• Comparison of N. europaea ispE with homologs from other organisms can reveal evolutionary adaptations in enzyme function

What are the structural characteristics of N. europaea ispE?

N. europaea ispE consists of 278 amino acids with a partial sequence beginning with MDIFPAPAKLNLFLHVIGRREDGYHLLQTVFRFIDHSDRLHFDITHDGVIRHENLIPGLTETDDLCVRAAKLL . Though the complete crystal structure of N. europaea ispE has not been reported in the provided search results, based on other characterized ispE enzymes, it likely features:

• A typical GHMP kinase fold with distinct N-terminal and C-terminal domains

• A central ATP-binding site with conserved motifs for phosphate binding

• A substrate-binding pocket that accommodates 4-diphosphocytidyl-2C-methyl-D-erythritol

• Critical residues for catalysis, including those involved in metal coordination (usually Mg²⁺)

The protein likely adopts a similar structure to other bacterial ispE enzymes, with specific variations that may relate to the adaptation of N. europaea to its ecological niche.

What expression systems are recommended for recombinant N. europaea ispE?

For successful expression of recombinant N. europaea ispE, researchers should consider the following methodological approaches:

• E. coli expression systems: BL21(DE3) or similar strains are recommended due to their reduced protease activity and controllable expression via T7 promoter systems.

• Expression vectors: pET series vectors containing N- or C-terminal affinity tags (His6, GST, or MBP) facilitate purification while potentially enhancing solubility.

• Expression conditions:

  • Induce at OD₆₀₀ of 0.6-0.8 with 0.1-0.5 mM IPTG

  • Lower the temperature to 16-20°C after induction

  • Extend expression time to 16-20 hours

  • Supplement media with 5-10 mM MgCl₂ to stabilize the enzyme

• Codon optimization: Consider codon optimization for E. coli if expression levels are low, as N. europaea and E. coli have different codon usage preferences.

Expression testing should include analysis of both soluble and insoluble fractions to determine optimal conditions for producing active enzyme.

How should researchers purify recombinant N. europaea ispE?

A comprehensive purification strategy for recombinant N. europaea ispE should include:

• Cell lysis buffer optimization:

  • 50 mM Tris-HCl or HEPES, pH 7.5-8.0

  • 100-300 mM NaCl

  • 5-10% glycerol for stability

  • 1-5 mM MgCl₂ (cofactor)

  • 1-5 mM β-mercaptoethanol or DTT

  • Protease inhibitor cocktail

• Affinity chromatography (primary purification):

  • For His-tagged constructs: Ni-NTA or TALON resin

  • Wash with increasing imidazole concentrations (20-40 mM)

  • Elute with 250-300 mM imidazole

• Secondary purification:

  • Ion-exchange chromatography (IEX) using Q or SP columns depending on the isoelectric point

  • Size-exclusion chromatography (SEC) for highest purity and removal of aggregates

• Quality control assessments:

  • SDS-PAGE for purity analysis (>95% for structural studies)

  • Western blot for identity confirmation

  • Dynamic light scattering (DLS) for monodispersity

  • Mass spectrometry for accurate mass determination

  • Circular dichroism (CD) for secondary structure confirmation

Enzyme activity should be assessed at each purification step to monitor retention of catalytic function.

How does oxygen limitation affect ispE expression in N. europaea?

The transcriptomic response of N. europaea to oxygen limitation provides important context for understanding ispE regulation. While the search results don't specifically mention ispE regulation under oxygen limitation, we can extrapolate based on related metabolic pathways in N. europaea:

Under oxygen-limited conditions, N. europaea experiences:

• Metabolic reprogramming: A significant downregulation of carbon fixation genes, including RuBisCO-encoding genes (reduced by 2.3 to 6.3-fold) . This suggests a general reduction in biosynthetic activities that may extend to isoprenoid biosynthesis.

• Energy allocation shifts: Polyphosphate accumulation increases under oxygen limitation (polyphosphate kinase transcription increases 2.1-fold) , indicating a reallocation of ATP resources. This may impact ATP-dependent reactions such as those catalyzed by ispE.

• Respiratory adaptations: Increased expression of high-affinity terminal oxidases under oxygen limitation , suggesting prioritization of energy generation pathways over biosynthetic pathways.

To specifically study ispE regulation under oxygen limitation, researchers should:

• Perform RT-qPCR targeting ispE during steady-state chemostat growth under both ammonia- and oxygen-limited conditions

• Quantify protein levels using targeted proteomics approaches

• Measure enzyme activity in cell extracts from cultures grown under different oxygen tensions

What are the optimal conditions for assaying N. europaea ispE activity?

When designing kinetic assays for ispE activity, researchers should consider:

• Coupled enzyme assays:

  • ADP production can be coupled to NADH oxidation via pyruvate kinase and lactate dehydrogenase

  • Monitor NADH decrease at 340 nm (ε = 6,220 M⁻¹cm⁻¹)

  • Reaction buffer: 50 mM HEPES or Tris-HCl (pH 7.5-8.0), 10 mM MgCl₂, 50 mM KCl

• Direct phosphorylation assays:

  • Use [γ-³²P]ATP or [γ-³³P]ATP and measure radioactive phosphate incorporation

  • Separate products by thin-layer chromatography or HPLC

  • Quantify using phosphorimaging or scintillation counting

• LC-MS/MS based assays:

  • Directly measure substrate depletion and product formation

  • Allows simultaneous monitoring of side reactions or contaminants

  • Higher sensitivity for detailed kinetic analyses

• Optimization parameters:

  • Temperature range: 25-37°C (test at 5°C intervals)

  • pH range: 6.5-9.0 (test at 0.5 pH unit intervals)

  • Metal cofactors: Test Mg²⁺, Mn²⁺, Co²⁺ at 1-10 mM

  • ATP concentration: 0.1-5 mM

  • Substrate concentration: 0.1-2 mM

How can site-directed mutagenesis elucidate ispE's catalytic mechanism?

Site-directed mutagenesis represents a powerful approach for investigating the catalytic mechanism of N. europaea ispE. Based on conserved residues in GHMP kinases, the following experimental approach is recommended:

• Target residue selection:

  • ATP-binding motifs (conserved glycine-rich regions)

  • Predicted catalytic residues (likely aspartate, lysine, or arginine residues)

  • Substrate-coordinating residues (typically polar amino acids)

  • Metal-coordinating residues (often aspartate or glutamate)

• Mutation design strategy:

  • Conservative mutations: D→E, K→R to preserve charge

  • Non-conservative mutations: D→N, K→A to eliminate charge

  • Size variations: F→Y, S→A to test spatial requirements

• Experimental workflow:

  • Use overlap extension PCR or commercial site-directed mutagenesis kits

  • Express and purify mutant proteins using identical conditions as wild-type

  • Compare expression levels, solubility, and thermal stability

  • Determine kinetic parameters (kcat, Km) for each mutant

  • Perform isothermal titration calorimetry to assess binding changes

• Structural validation:

  • Circular dichroism to confirm proper folding

  • X-ray crystallography of key mutants with substrates/analogs

  • Molecular dynamics simulations to assess subtle structural changes

A systematic mutational analysis could generate a detailed model of the active site architecture and reaction mechanism, contributing to structure-based inhibitor design.

How does the N. europaea ispE compare to homologs from other organisms?

Comparative analysis of N. europaea ispE with homologs from other organisms can reveal evolutionary adaptations and functional differences. Consider the following methodological approach:

• Sequence analysis:

  • Multiple sequence alignment with ispE from diverse bacteria, particularly comparing ammonia oxidizers with other bacterial groups

  • Phylogenetic tree construction to determine evolutionary relationships

  • Conservation analysis to identify N. europaea-specific variations

• Structural comparative studies:

  • Homology modeling based on existing crystal structures

  • Molecular dynamics simulations under different conditions

  • Active site comparison focusing on substrate-binding residues

• Functional comparison:

  • Express and purify homologs from representative organisms

  • Compare kinetic parameters under identical conditions

  • Test substrate specificity using substrate analogs

  • Evaluate thermal and pH stability profiles

• Comparative data table for selected bacterial ispE enzymes:

Note: The table contains representative values based on general patterns in the literature. Exact values for N. europaea ispE would need to be experimentally determined.

What approaches can be used to study potential inhibitors of N. europaea ispE?

Developing and studying inhibitors of N. europaea ispE requires a systematic approach:

• Initial screening methods:

  • ATP-competitive inhibitor libraries (kinase inhibitor sets)

  • Fragment-based screening using differential scanning fluorimetry

  • In silico docking with homology models or crystal structures

  • Substrate analog design targeting unique features of the binding pocket

• Secondary validation assays:

  • IC₅₀ determination using the optimized enzyme activity assay

  • Mechanism of inhibition studies (competitive, noncompetitive, uncompetitive)

  • Binding affinity measurements via isothermal titration calorimetry

  • Surface plasmon resonance for association/dissociation kinetics

• Structure-activity relationship studies:

  • Systematic modification of lead compounds to improve potency

  • Focus on selectivity against human kinases

  • Address physicochemical properties for potential cellular penetration

• Cellular validation:

  • Growth inhibition assays using N. europaea cultures

  • Metabolomic analysis to confirm on-target effects (accumulation of substrate)

  • Resistance studies to confirm mechanism of action

• Potential inhibitor classes to explore:

  • Nucleotide analogs targeting the ATP binding site

  • Substrate competitive inhibitors mimicking 4-diphosphocytidyl-2C-methyl-D-erythritol

  • Allosteric inhibitors targeting unique regulatory sites

  • Covalent inhibitors targeting accessible cysteine residues

How can transcriptomic data inform ispE function in N. europaea metabolism?

Integrating transcriptomic data can provide crucial insights into the metabolic context of ispE in N. europaea:

• Co-expression network analysis:

  • Using the transcriptomic data from oxygen limitation experiments , construct co-expression networks

  • Identify genes with expression patterns correlated with known MEP pathway enzymes

  • Map these networks to metabolic pathways to identify functional associations

• Differential expression under varied conditions:

  • Beyond oxygen limitation, examine transcriptomic responses to other stressors

  • Compare ammonia-limited versus oxygen-limited growth conditions as shown in the research

  • Identify potential transcriptional regulators of the MEP pathway

• Integration with metabolomic data:

  • Correlate ispE expression levels with concentrations of isoprenoid precursors and products

  • Use isotope labeling to track carbon flux through the MEP pathway under different growth conditions

  • Develop a comprehensive model of isoprenoid biosynthesis regulation

• Experimental validation approaches:

  • Construct reporter gene fusions to study promoter activity

  • Perform ChIP-seq to identify transcription factors binding to the ispE promoter

  • Create conditional knockdowns to assess the metabolic impact of reduced ispE activity

The transcriptomic study of N. europaea under oxygen limitation revealed significant downregulation of carbon fixation pathways , suggesting that isoprenoid biosynthesis might be similarly affected as part of a broader metabolic reprogramming during environmental stress.