Recombinant Nitrosomonas europaea Phosphoglycerate kinase (pgk)

What is the role of phosphoglycerate kinase in Nitrosomonas europaea metabolism?

Phosphoglycerate kinase in Nitrosomonas europaea functions as a key enzyme in central carbon metabolism, specifically within the gluconeogenesis pathway and the Calvin-Benson-Bassham (CBB) cycle used for carbon fixation. Unlike heterotrophic organisms that primarily use PGK in glycolysis, N. europaea, being a chemolithoautotroph, utilizes PGK predominantly in the direction of 3-phosphoglycerate to 1,3-bisphosphoglycerate conversion, consuming rather than generating ATP.

N. europaea obtains energy by oxidizing ammonia in the presence of oxygen and fixes CO2 via the Calvin-Benson cycle . Within this metabolic context, PGK works alongside other enzymes like phosphoglycerate mutase (PGM) to support the organism's autotrophic lifestyle. While N. europaea lacks carboxysomes present in some other autotrophs, it regulates carbon fixation primarily at the level of RuBisCO enzyme concentration .

How is the pgk gene typically cloned and expressed as a recombinant protein?

The pgk gene from Nitrosomonas europaea can be amplified from genomic DNA using PCR with high-fidelity DNA polymerase systems such as the Expand High Fidelity PCR System. Based on protocols used for other N. europaea enzymes, the following approach is recommended:

• Design primers to amplify the full-length pgk gene with appropriate restriction sites for directional cloning into an expression vector like pET-21a, which allows for C-terminal His6-tag fusion .

• Clone the amplified gene into the expression vector and verify the construct by DNA sequencing.

• Transform the verified construct into an E. coli expression host such as T7 Express strain.

• Induce protein expression with IPTG, typically at concentrations around 0.3 mM for 3-4 hours at 37°C .

• Extract and purify the His-tagged protein using nickel affinity chromatography under native conditions.

This approach has been successfully employed for other N. europaea enzymes like ADP-glucose pyrophosphorylase and glycogen synthase .

What are the typical cofactor requirements for N. europaea PGK activity?

As with most PGK enzymes, N. europaea PGK likely requires magnesium ions (Mg²⁺) as a cofactor for optimal activity. Based on studies of other bacterial enzymes from N. europaea, the following cofactor considerations are important:

• Divalent metal ions: While Mg²⁺ is the primary cofactor, N. europaea enzymes often exhibit broader metal ion specificity. For instance, the ADP-glucose pyrophosphorylase from N. europaea showed activity with different divalent metal ions as cofactors .

• pH optimum: Typically, bacterial PGK enzymes function optimally in the pH range of 7.0-7.5, which is consistent with the intracellular pH of N. europaea.

• Temperature stability: N. europaea enzymes often display broad thermal stability, which may reflect the organism's adaptability to various environmental conditions .

To determine specific cofactor requirements, a systematic analysis varying metal ion concentrations, pH, and temperature would be necessary for the purified recombinant enzyme.

How does oxygen limitation affect expression of glycolytic enzymes like PGK in N. europaea?

Oxygen limitation has profound effects on N. europaea metabolism, which likely extends to glycolytic/gluconeogenic enzymes like PGK. Transcriptomic studies of N. europaea under oxygen-limited conditions have revealed:

• While genes for central metabolic pathways show relatively stable expression, specific adjustments occur to adapt to oxygen stress.

• Under oxygen limitation, N. europaea maintains expression of core metabolic genes while significantly altering nitrogen metabolism genes. Transcriptome analysis detected 98.5% of protein-coding genes expressed under both ammonia- and oxygen-limited conditions .

• Most genes in central metabolic pathways (glycolysis/gluconeogenesis, TCA cycle) showed no significant differential expression between ammonia-limited and oxygen-limited conditions .

• Energy storage mechanisms are altered - polyphosphate metabolism genes show differential expression, with polyphosphate kinase (ppk) transcripts increased 2.1-fold under oxygen-limited conditions .

These findings suggest that while PGK expression may remain relatively stable under oxygen limitation, its activity might be affected by broader metabolic adjustments, particularly in ATP availability and carbon flux through central pathways.

What purification strategies are most effective for recombinant N. europaea PGK?

Based on successful purification of other recombinant enzymes from N. europaea, the following purification strategy is recommended:

• Affinity chromatography: Nickel affinity chromatography for His-tagged recombinant PGK provides an efficient first purification step .

• Buffer optimization: For N. europaea enzymes, purification buffers typically contain:

  • 30 mM Tris-HCl, pH 7.0-7.5

  • 5 mM MgSO₄ or MgCl₂

  • 0.1-1.0 mM DTT (to protect cysteine residues)

  • 100-300 mM NaCl (to maintain protein solubility)

• Additional purification steps if needed:

  • Size exclusion chromatography to remove aggregates and achieve higher purity

  • Ion exchange chromatography as a polishing step

• Storage conditions: Purified N. europaea enzymes are typically stored in buffer containing 20% glycerol at -80°C to maintain activity.

This approach has yielded functionally active recombinant enzymes from N. europaea with sufficient purity for biochemical characterization and crystallization studies .

How can enzyme activity assays be optimized for recombinant N. europaea PGK?

PGK activity can be measured using coupled enzyme assays that monitor either the forward or reverse reaction. For N. europaea PGK, the following considerations are important for assay optimization:

• Standard coupled assay components:

  • 30 mM Tris-HCl, pH 7.0

  • 5 mM MgSO₄

  • 0.2 mM NADH (for spectrophotometric detection)

  • Coupling enzymes (such as glyceraldehyde-3-phosphate dehydrogenase for the reverse reaction)

  • Appropriate substrates (3-phosphoglycerate, ATP or 1,3-bisphosphoglycerate, ADP)

• Metal ion optimization: Testing different divalent cations (Mg²⁺, Mn²⁺, Co²⁺) at various concentrations can identify optimal cofactor requirements, as N. europaea enzymes may show activity with different metal ions .

• pH optimization: Testing activity across pH ranges (6.5-8.0) can identify the optimal pH for the recombinant enzyme.

• Temperature effects: Assessing activity at different temperatures (25-45°C) can determine thermal optima and stability profiles.

Activity measurements should include appropriate controls and be conducted under conditions where coupling enzyme activities are not rate-limiting.

How might the structure of N. europaea PGK relate to its function in this chemolithoautotroph?

While specific structural information for N. europaea PGK is not currently available in the literature, several insights can be drawn from comparative analysis:

• Domain organization: Like other PGKs, N. europaea PGK likely possesses two domains with a deep cleft containing the active site, allowing for the large conformational change associated with catalysis.

• Adaptations to autotrophic metabolism: N. europaea PGK may feature structural adaptations that favor the gluconeogenic direction of the reaction, supporting carbon fixation via the Calvin-Benson cycle.

• Potential regulatory interfaces: As a key metabolic enzyme in a chemolithoautotroph that must balance energy generation (ammonia oxidation) with carbon fixation, N. europaea PGK might possess unique regulatory features or protein-protein interaction sites.

• Thermal and pH stability features: Given N. europaea's environmental adaptability, its PGK may contain structural elements that confer broader stability across environmental conditions, similar to the ADP-glucose pyrophosphorylase that showed broad thermal and pH stability .

Structural studies through X-ray crystallography or cryo-EM would be needed to confirm these hypotheses and identify unique features of N. europaea PGK.

What are the challenges in expressing active N. europaea PGK in heterologous systems?

Expressing active N. europaea PGK in heterologous systems may face several challenges:

• Codon usage bias: N. europaea has different codon preferences than common expression hosts like E. coli, which may necessitate codon optimization for efficient expression.

• Protein folding issues: As with other enzymes from specialized metabolic pathways, N. europaea PGK may require specific chaperones or folding conditions that differ from those available in conventional expression hosts.

• Post-translational modifications: While bacterial PGKs typically undergo limited post-translational modifications, any N. europaea-specific modifications would be absent in heterologous hosts.

• Expression temperature: Lower induction temperatures (15-25°C) may be required to ensure proper folding, as has been observed with other enzymes from non-model organisms.

• Solubility enhancement: Fusion tags beyond the His-tag (such as MBP or SUMO) may improve solubility of recombinant N. europaea PGK.

Empirical optimization of expression conditions, including strain selection, media composition, induction parameters, and inclusion of folding enhancers, would be necessary to overcome these challenges.

How can site-directed mutagenesis be used to investigate the catalytic mechanism of N. europaea PGK?

Site-directed mutagenesis offers a powerful approach to investigate the catalytic mechanism of N. europaea PGK through targeted amino acid substitutions:

• Identification of catalytic residues: Based on sequence alignment with well-characterized PGKs, key residues likely involved in substrate binding and catalysis can be identified and mutated (typically to alanine) to assess their functional importance.

• Experimental design for mutagenesis:

  • Design primers containing the desired mutation following standard overlap extension PCR protocols

  • Confirm mutations by DNA sequencing

  • Express and purify mutant proteins using the same protocols as for wild-type

  • Compare kinetic parameters (Km, kcat, kcat/Km) between wild-type and mutant enzymes

• Key residues to investigate:

  • Basic residues coordinating phosphate groups of substrates

  • Residues involved in domain movement during catalysis

  • Residues potentially providing chemolithoautotroph-specific functions

• Analytical techniques for mutant characterization:

  • Steady-state kinetics

  • Isothermal titration calorimetry for binding studies

  • Thermal shift assays for stability assessment

  • Structural studies of mutants to correlate structural changes with functional effects

This approach has been successfully applied to study other enzymes from N. europaea, such as the functional characterization of glycogen synthase .

What is the role of PGK in integrating carbon and nitrogen metabolism in N. europaea?

The role of PGK in integrating carbon and nitrogen metabolism in N. europaea is complex and multifaceted:

• Metabolic connection points: PGK likely serves as a critical node connecting energy generation (from ammonia oxidation) with carbon fixation (via the Calvin-Benson cycle), as it participates in generating high-energy phosphate bonds.

• Energetic considerations: Under oxygen-limited conditions, N. europaea shows significant metabolic adjustments that affect energy production and consumption pathways . PGK activity may be regulated in response to changing energy availability, as evidenced by the differential regulation of energy storage mechanisms like polyphosphate accumulation .

• Response to environmental conditions: When N. europaea transitions between ammonia-replete and ammonia-limited conditions, or between oxygen-replete and oxygen-limited conditions, the flux through central carbon metabolism changes . PGK likely plays a key role in these adjustments.

• Coordination with nitrogen metabolism: In N. europaea, carbon fixation must be balanced with nitrogen oxidation for optimal growth. PGK, as a central metabolic enzyme, likely responds to regulatory cues that coordinate these two essential processes.

• Potential involvement in stress responses: During environmental stress, such as oxygen limitation, N. europaea shows transcriptional changes in key metabolic pathways . PGK activity may be modulated as part of this adaptive response.

Understanding these integrative roles would require systems biology approaches, including metabolic flux analysis under various growth conditions, to determine how PGK activity is regulated in response to changing nitrogen and carbon availability.

What are the best approaches for analyzing kinetic parameters of recombinant N. europaea PGK?

To determine accurate kinetic parameters for recombinant N. europaea PGK, the following methodological approaches are recommended:

• Steady-state kinetic analysis:

  • Measure initial rates across a range of substrate concentrations

  • Determine Km and Vmax values using appropriate curve-fitting (Michaelis-Menten, Lineweaver-Burk)

  • Analyze both forward and reverse reactions to understand directional preferences

• Cofactor dependency studies:

  • Test activity with different divalent metal ions (Mg²⁺, Mn²⁺, Ca²⁺, Co²⁺, Zn²⁺)

  • Determine optimal cofactor concentration for maximum activity

  • Assess metal ion specificity through comparative kinetic analysis

• pH-rate profiles:

  • Measure activity across a pH range (6.0-9.0) using appropriate buffer systems

  • Identify ionizable groups important for catalysis through pH-dependent kinetic parameters

• Temperature effects:

  • Determine temperature optimum and stability profile

  • Calculate activation energy using Arrhenius plots

• Product inhibition studies:

  • Assess inhibition patterns to elucidate reaction mechanism

  • Determine inhibition constants for reaction products

These approaches have been successfully applied to characterize other metabolic enzymes from N. europaea, yielding valuable insights into their biochemical properties and physiological roles .

How can structural studies of N. europaea PGK inform functional optimization?

Structural studies of N. europaea PGK can provide critical insights for functional optimization:

• X-ray crystallography approaches:

  • Crystallize purified recombinant PGK using screening techniques

  • Solve structures in apo form and in complex with substrates/products

  • Identify key structural elements for catalysis and regulation

• Structure-guided engineering:

  • Target specific residues for mutagenesis based on structural information

  • Design variants with enhanced stability or altered substrate specificity

  • Develop enzyme variants optimized for biotechnological applications

• Comparative structural analysis:

  • Compare N. europaea PGK structure with PGKs from heterotrophs

  • Identify adaptations specific to chemolithoautotrophic metabolism

  • Correlate structural features with kinetic properties

• Molecular dynamics simulations:

  • Model enzyme dynamics during catalytic cycle

  • Identify conformational changes important for activity

  • Simulate effects of environmental conditions on enzyme structure

These structural insights can guide rational design approaches to optimize N. europaea PGK for various research and biotechnological applications.

How does N. europaea PGK compare to PGM in terms of metabolic significance?

While both phosphoglycerate kinase (PGK) and phosphoglycerate mutase (PGM) participate in central carbon metabolism, they serve distinct functions with different metabolic implications:

In N. europaea, PGK likely plays a more direct role in energy metabolism through ATP generation/consumption, while PGM facilitates metabolic flux by catalyzing the interconversion of phosphoglycerate isomers without direct energy involvement . Both enzymes are essential for the proper functioning of the Calvin-Benson cycle in this chemolithoautotroph.

What insights can be gained from comparing PGK across different bacterial metabolic types?

Comparing PGK across different bacterial metabolic types reveals important adaptations to diverse energy-harvesting strategies:

• Sequence and structural adaptations:

  • Chemolithoautotrophs like N. europaea may have PGK adaptations favoring the gluconeogenic direction

  • Heterotrophs typically optimize PGK for the glycolytic direction

  • Extremophiles show structural modifications for stability under extreme conditions

• Regulatory differences:

  • Chemolithoautotrophs like N. europaea likely regulate PGK in coordination with carbon fixation pathways

  • Heterotrophs typically regulate PGK in response to carbon source availability

  • Metabolic versatile bacteria may show more complex regulatory patterns

• Kinetic properties:

  • Substrate affinities and catalytic efficiencies often reflect the predominant metabolic direction

  • Cofactor preferences may vary based on intracellular ion concentrations in different bacterial types

  • Allosteric regulation mechanisms may differ based on metabolic needs

• Genomic context:

  • Gene neighborhood analysis shows different organizational patterns across metabolic types

  • Co-evolution with other enzymes in respective metabolic pathways

  • Different paralogs may exist in metabolically versatile bacteria

These comparative insights can help understand how PGK has been adapted throughout bacterial evolution to support diverse metabolic strategies, from chemolithoautotrophy in N. europaea to heterotrophy in E. coli.