Recombinant Tuber borchii Enolase (eno-1)

What is the function of enolase in Tuber borchii metabolism?

Enolase (EC 4.2.1.11) in Tuber borchii functions as a key glycolytic enzyme that catalyzes the conversion of 2-phosphoglycerate (2-PG) to phosphoenolpyruvate (PEP) in the penultimate step of glycolysis. In T. borchii, enolase appears to play a significant role in the fungus's adaptive metabolism, particularly in conditions where fermentative rather than respiratory metabolism is dominant. Transcriptome analyses of T. borchii SP1 strain suggest that even under aerobic conditions, this truffle species relies heavily on fermentative metabolism . The enzyme is likely involved in the fungus's ability to adapt to varying nutrient conditions, similar to other highly regulated proteins in this organism such as TbSP1 .

What expression systems are most effective for producing recombinant T. borchii enolase?

For expression of recombinant T. borchii enolase, E. coli-based systems have proven effective, particularly those utilizing N-terminal His-tag fusions for subsequent purification. The methodology mirrors successful approaches used for other T. borchii proteins such as TbSP1, where the coding sequence (minus the native signal peptide) was cloned into an expression vector, expressed in E. coli, and purified via metal affinity chromatography . For optimal expression, BL21(DE3) or Rosetta strains with pET-based vectors are recommended, with expression typically induced using IPTG (0.5-1 mM) at temperatures between 18-25°C to maximize soluble protein yield.

What purification strategy yields the highest purity recombinant T. borchii enolase?

A multi-step purification strategy is recommended for obtaining high-purity recombinant T. borchii enolase:

• Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin as the initial capture step for His-tagged protein

• Anion exchange chromatography (typically Q-Sepharose) at pH 8.0 to separate the target from E. coli proteins

• Size exclusion chromatography as a polishing step to remove aggregates and achieve >95% purity

This strategy has been successfully utilized for other T. borchii recombinant proteins, yielding preparations suitable for enzymatic assays and structural studies . The purification process should be monitored by SDS-PAGE, with the expected molecular weight for recombinant T. borchii enolase being approximately 45-48 kDa.

What assay methods are most reliable for measuring T. borchii enolase activity?

The most reliable methods for measuring T. borchii enolase activity include:

Spectrophotometric assay (forward reaction):
This measures the conversion of 2-phosphoglycerate to phosphoenolpyruvate by monitoring the increase in absorbance at 240 nm due to the formation of the enol structure. The standard reaction mixture typically contains:

Coupled enzyme assay (reverse reaction):
This measures the conversion of phosphoenolpyruvate to 2-phosphoglycerate coupled with the pyruvate kinase and lactate dehydrogenase reactions, monitoring the oxidation of NADH at 340 nm.

For both assays, optimal activity for fungal enolases typically occurs at pH 6.5-7.5 and temperatures of 25-30°C, though these parameters should be experimentally determined specifically for the T. borchii enzyme.

How does the kinetic profile of recombinant T. borchii enolase compare to enolases from other fungi?

While specific kinetic data for T. borchii enolase is not provided in the search results, fungal enolases typically exhibit the following kinetic parameters, which can serve as a comparative framework:

The predicted values for T. borchii enolase are based on the organism's growth preferences, as T. borchii displays optimal growth at 22°C in laboratory conditions . Like other truffle species, T. borchii likely has metabolic adaptations to its ecological niche, which may be reflected in its enolase properties.

What structural features distinguish T. borchii enolase from other fungal enolases?

T. borchii enolase is expected to share the typical homodimeric structure of fungal enolases, with each monomer consisting of a smaller N-terminal domain and a larger C-terminal domain containing the active site. Key distinguishing features may include:

• Metal binding sites: Like other enolases, the T. borchii enzyme requires divalent metal ions (typically Mg²⁺) for catalytic activity

• Surface-exposed residues: These may reflect adaptations to the hypogeous (underground) lifestyle of truffles

• Post-translational modifications: Glycosylation patterns specific to T. borchii

Transcriptome analysis of T. borchii has revealed that this species has adapted its central metabolism to function effectively under the environmental conditions found in its ecological niche . These adaptations may be reflected in subtle structural modifications to enzymes like enolase that are involved in primary metabolism.

How can recombinant T. borchii enolase be used as a marker for truffle identification and authentication?

Recombinant T. borchii enolase can serve as an important reference standard for developing species-specific immunological or mass spectrometry-based detection methods for truffle authentication. The methodology involves:

• Using purified recombinant T. borchii enolase to develop monoclonal or polyclonal antibodies specific to species-unique epitopes

• Developing ELISA or Western blot protocols for truffle extract analysis

• Creating mass spectrometry reference libraries based on tryptic digest patterns of the recombinant protein

These approaches can help distinguish T. borchii from other white truffle species, particularly T. magnatum, which commands significantly higher market prices. Recent research has shown that chemical profiling can effectively differentiate between truffle species . By incorporating protein-based markers like species-specific enolase peptides, researchers can develop multi-parameter authentication systems with greater accuracy than volatile compound analysis alone.

What role does enolase play in the stress response and nutrient adaptation of T. borchii?

Enolase likely plays a pivotal role in T. borchii's response to nutrient stress, similar to other highly regulated proteins in this organism. Research on T. borchii has demonstrated that nutrient deprivation causes strong and reversible up-regulation of certain proteins, such as the phospholipase TbSP1 . While the search results don't specifically address enolase regulation, the transcriptome analysis of T. borchii SP1 indicated that:

• The fungus adopts predominantly fermentative metabolism even under aerobic conditions

• The expression profile of central metabolism genes varies subtly under different culture conditions

• Approximately 20% of genes account for 80% of the transcription effort

Given these patterns, enolase (as a central metabolic enzyme) may show altered expression under different nutrient conditions. This adaptation would be particularly important during the transition between free-living mycelium and symbiotic mycorrhizal states, where nutrient acquisition strategies change dramatically.

How can protein-protein interaction studies with T. borchii enolase reveal insights into moonlighting functions?

Fungal enolases are known to have moonlighting functions beyond their canonical role in glycolysis. To investigate potential non-glycolytic functions of T. borchii enolase, the following methodological approaches are recommended:

• Pull-down assays: Using recombinant His-tagged T. borchii enolase as bait to identify interacting proteins from truffle extracts

• Yeast two-hybrid screening: Creating a T. borchii cDNA library to screen for potential protein partners

• Surface plasmon resonance: Measuring binding kinetics between purified recombinant enolase and candidate interacting proteins

• Crosslinking coupled with mass spectrometry: Identifying transient protein interactions in vivo

Potential moonlighting functions worth investigating include roles in:

• Cell wall association (similar to TbSP1, which shows dual localization as both secreted and cell wall-associated )

• Adhesion to host plants during mycorrhizal formation

• RNA binding and potential involvement in stress granules

How is enolase expression modulated during the establishment of mycorrhizal symbiosis between T. borchii and host plants?

Enolase expression in T. borchii likely undergoes significant modulation during the establishment of mycorrhizal symbiosis, similar to other proteins involved in nutrient acquisition and metabolism. While the search results don't provide specific data on enolase regulation during symbiosis, research on other T. borchii proteins offers valuable insights. For instance, TbSP1 shows nutrient-dependent regulation and is present in both free-living hyphae and symbiotic structures .

A methodological approach to studying enolase expression during mycorrhizal formation would include:

• Establishing in vitro mycorrhizal synthesis systems (e.g., with Arbutus unedo or other host plants)

• Collecting samples at different stages of symbiosis development

• Using RT-qPCR and Western blotting to quantify enolase mRNA and protein levels

• Performing immunolocalization studies to track changes in enolase distribution

Expected findings might include increased enolase expression during the early stages of contact with host roots, followed by stabilization once the mycorrhizal network is established.

Can recombinant T. borchii enolase be used to study host immune responses during mycorrhizal colonization?

Recombinant T. borchii enolase offers a valuable tool for studying potential host immune responses during mycorrhizal colonization. The methodology would involve:

• Treating host plant cells or seedlings with purified recombinant T. borchii enolase at physiologically relevant concentrations

• Monitoring immune-related gene expression using RT-qPCR or RNA-seq

• Assessing immune signaling pathway activation (e.g., MAPK cascades, calcium signaling)

• Comparing responses to enolase with those elicited by other T. borchii proteins

This approach could reveal whether enolase acts as a microbe-associated molecular pattern (MAMP) that triggers pattern-triggered immunity (PTI) responses in the host plant. If so, successful mycorrhization would require mechanisms to suppress or evade these immune responses.

The study design should include appropriate controls such as heat-denatured enolase, enolases from non-mycorrhizal fungi, and known MAMPs like chitin fragments.

What computational approaches can identify regulatory elements controlling T. borchii enolase expression?

To identify regulatory elements controlling T. borchii enolase expression, researchers should employ the following computational approaches:

• Promoter analysis: Extract the 1-2 kb region upstream of the enolase coding sequence from the T. borchii genome (available from the JGI Mycocosm database as mentioned in the search results ). Use tools like MEME, JASPAR, and TRANSFAC to identify potential transcription factor binding sites.

• Comparative genomics: Compare the promoter region of T. borchii enolase with those of related species to identify conserved regulatory elements, which often indicate functional importance.

• Expression correlation networks: Analyze transcriptome data to identify genes with expression patterns that correlate with enolase, suggesting co-regulation.

• Epigenetic modification prediction: Analyze the promoter sequence for potential DNA methylation sites or histone modification regions that might influence expression.

How does the amino acid sequence of T. borchii enolase compare with enolases from other truffle species?

While specific sequence comparisons for T. borchii enolase are not provided in the search results, a typical comparative analysis would examine:

The T. borchii genome has been sequenced and contains 12,346 predicted genes , which provides the foundation for such comparative analyses. A comprehensive comparison would include enolases from:

• Other Tuber species (T. magnatum, T. melanosporum, T. aestivum)

• Related ectomycorrhizal fungi

• Non-mycorrhizal ascomycetes as outgroups

Researchers can use this comparative information to identify regions that might be responsible for adaptation to the specific ecological niche of T. borchii.

What are the common challenges in obtaining active recombinant T. borchii enolase and how can they be overcome?

Researchers working with recombinant T. borchii enolase may encounter several challenges:

Additionally, researchers should consider fungal-specific post-translational modifications that might be required for full activity. If E. coli-expressed protein shows limited activity, expression in yeast systems (e.g., Pichia pastoris) might be necessary to achieve proper folding and modifications.

How can the stability of purified recombinant T. borchii enolase be maximized for long-term studies?

To maximize the stability of purified recombinant T. borchii enolase:

• Buffer optimization:

  • Test various buffers (HEPES, phosphate, Tris) at pH 7.0-8.0

  • Include stabilizing agents: 10-20% glycerol, 1-5 mM DTT or β-mercaptoethanol

  • Add divalent cations (1-2 mM MgCl₂) to stabilize the native conformation

• Storage conditions:

  • Divide into small aliquots (50-100 μL) to minimize freeze-thaw cycles

  • Flash-freeze in liquid nitrogen before storing at -80°C

  • For short-term storage (1-2 weeks), 4°C may be preferable to freezing

• Concentration effects:

  • Determine optimal protein concentration (typically 1-5 mg/mL)

  • Too high: risk of aggregation; too low: surface adsorption and denaturation

  • Consider adding carrier proteins (BSA) for very dilute solutions

• Lyophilization:

  • Add lyoprotectants (sucrose, trehalose) at 1:1 weight ratio

  • Reconstitute in original buffer containing reducing agents

Thermal shift assays (Thermofluor) can be used to systematically screen different buffer conditions and additives to identify those that maximize thermal stability, which often correlates with long-term storage stability.

How can studies of T. borchii enolase contribute to understanding broader fungal adaptation mechanisms?

Research on T. borchii enolase can provide insights into broader fungal adaptation mechanisms through:

• Metabolic adaptation: Understanding how glycolytic enzymes in T. borchii are regulated under different environmental conditions can reveal mechanisms of metabolic plasticity in fungi. Transcriptome studies already suggest that T. borchii employs fermentative metabolism even under aerobic conditions , which may represent a specialized adaptation.

• Evolution of symbiosis: Comparing enolase structure and regulation between mycorrhizal fungi like T. borchii and non-symbiotic fungi can help identify adaptations specific to the mycorrhizal lifestyle.

• Stress response mechanisms: Enolase often plays roles beyond glycolysis during stress conditions. For example, the strong up-regulation of TbSP1 during carbon and nitrogen starvation suggests that T. borchii has sophisticated nutrient-sensing and response mechanisms that might also involve enolase.

• Protein moonlighting: Studying non-canonical functions of T. borchii enolase could reveal novel aspects of protein moonlighting in fungi, particularly in the context of host interaction.

What potential biotechnological applications exist for recombinant T. borchii enolase in research settings?

Recombinant T. borchii enolase has several potential biotechnological applications in research settings:

• Biomarker development:

  • Species-specific peptides from T. borchii enolase can serve as biomarkers for truffle identification and authentication

  • Antibodies raised against these unique epitopes can be used in immunoassays for food quality control

• Enzyme engineering:

  • Understanding the structural and functional properties of T. borchii enolase can inform protein engineering efforts

  • Cold-adapted enzymes from organisms growing at lower temperatures (like T. borchii, which grows optimally at 22°C ) can be valuable for biotechnological applications

• Mycorrhizal research tools:

  • Fluorescently labeled recombinant enolase can be used to study protein uptake during mycorrhizal interactions

  • Enolase-specific antibodies can serve as markers for T. borchii in environmental or agricultural samples

• Structural biology:

  • T. borchii enolase crystal structures could reveal unique features related to the organism's ecological adaptations

  • These insights could guide the design of inhibitors or activators of fungal enolases

By focusing on these research applications rather than commercial applications, scientists can utilize T. borchii enolase as a tool to advance our understanding of fungal biology, symbiosis, and metabolism.