Recombinant Nicotiana attenuata 5-epi-aristolochene synthase 2, partial

What is 5-epi-aristolochene synthase and what is its role in Nicotiana attenuata?

5-epi-aristolochene synthase (EAS) is a sesquiterpene cyclase that catalyzes the conversion of farnesyl diphosphate (FPP) to 5-epi-aristolochene, which serves as the precursor for the antimicrobial compound capsidiol. In Nicotiana attenuata (coyote tobacco), multiple copies of the EAS gene have been identified, including EAS1 (NaEAS34) and EAS2 (NaEAS37), with EAS2 being classified as EC 4.2.3.61 . These enzymes play a crucial role in the plant's defense mechanisms against pathogens and herbivores. EAS is expressed constitutively in roots of N. attenuata, while its expression can be induced in shoots when the plant is under stress, such as herbivory from the tobacco hornworm (Manduca sexta) .

What is the difference between constitutive and inducible expression of EAS in N. attenuata?

N. attenuata exhibits differential patterns of EAS gene expression between plant tissues. In roots, EAS is expressed constitutively, meaning it is continuously produced regardless of external stimuli, resulting in constant capsidiol formation for baseline protection. This constitutive expression corresponds with continuous capsidiol formation in roots of both N. attenuata and N. sylvestris .

How does the biosynthetic pathway involving EAS2 function in plant defense?

EAS2 functions in a multi-step biosynthetic pathway that contributes to both constitutive and inducible plant defense mechanisms. The pathway begins with farnesyl diphosphate (FPP), which EAS2 converts to 5-epi-aristolochene. This compound is further modified to produce capsidiol, an antimicrobial sesquiterpenoid phytoalexin with activity against pathogens .

In N. attenuata, this pathway operates differently in various plant tissues. The constitutive pathway in roots provides continuous protection against soil-borne pathogens, while the inducible pathway in shoots activates only upon herbivore or pathogen attack, conserving metabolic resources . The induction can occur rapidly, with studies showing that simulated herbivory using Manduca sexta oral secretions (OS) applied to wounded leaves (W+OS treatment) recapitulates most changes in the N. attenuata transcriptome and metabolome that occur during actual insect feeding .

What expression systems are optimal for recombinant production of N. attenuata EAS2?

E. coli has proven to be the most effective and commonly used expression system for recombinant N. attenuata EAS2 production. The protein can be expressed with a purity level greater than 85% as determined by SDS-PAGE . While other systems including yeast, baculovirus, and mammalian cells are possible alternatives, E. coli offers advantages in terms of simplicity, cost-effectiveness, and yield.

For optimal expression in E. coli, the following methodology has proven effective:

• The EAS2 gene can be cloned into vectors such as pET series (e.g., pET28a or pET32)

• Expression is typically induced using isopropyl thio-β-d-thiogalactoside (IPTG)

• His-tagged protein can be generated to facilitate purification

• Induction conditions typically involve growth at 30-37°C with IPTG concentrations in the 0.1-1.0 mM range

What analytical methods can be used to assess enzymatic activity of recombinant EAS2?

Multiple complementary analytical methods can be employed to assess EAS2 activity:

• Gas Chromatography-Mass Spectrometry (GC-MS):

  • Both non-polar (5% diphenyl/95% dimethylsiloxane) and chiral (20% β-cyclodextrin) stationary phases are complementary for separating reaction products

  • The identity of 5-epi-aristolochene can be confirmed by comparing its mass spectrum with authentic standards

  • This technique allows identification and quantification of both the main product and alternative products

• Radiolabeled Assays:

  • Using [1-3H]-FPP as substrate, followed by organic extraction and scintillation counting

  • This assay can be used for kinetic studies and inhibition analyses

• Enzyme Kinetics Analysis:

  • Determination of kinetic parameters (Km, kcat) through concentration-dependent studies

  • Km values for FPP typically range from 3-7 μM for EAS enzymes

What purification strategies yield highest activity and purity for recombinant EAS2?

The following purification strategy has been demonstrated to yield high purity and maintained activity for recombinant EAS2:

• Initial Extraction:

  • Cell lysis via sonication followed by centrifugation to separate soluble fraction

• Affinity Chromatography:

  • Immobilized Metal Affinity Chromatography (IMAC) using nickel or cobalt resins is the method of choice

  • His-tagged EAS2 binds with high affinity, allowing effective separation from bacterial proteins

  • This method yields approximately 95% purity based on Coomassie Blue staining of SDS-PAGE gels

• Storage Conditions:

  • The purified protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Addition of 5-50% glycerol (with 50% being the default final concentration)

  • Storage at -20°C/-80°C for long-term stability

  • The shelf life is approximately 6 months for liquid form and 12 months for lyophilized form at -20°C/-80°C

What are the key residues involved in catalytic activity and product specificity of EAS2?

Several critical residues have been identified that control both the catalytic activity and product specificity of EAS enzymes:

These residues represent central waypoints in the biochemical pathway of EAS enzymes and are part of the active site involved in the conversion of farnesyl diphosphate to 5-epi-aristolochene . Mutations in these residues can significantly alter the product profile of the enzyme, potentially redirecting catalysis toward alternative sesquiterpene products.

What is the product specificity profile of EAS enzymes and how does it compare across Nicotiana species?

While EAS enzymes primarily catalyze the formation of 5-epi-aristolochene, they also produce several alternative products. The product profile of tobacco 5-epi-aristolochene synthase (TEAS), which serves as a model for understanding N. attenuata EAS2, has been characterized in detail:

This spectrum of products demonstrates that even highly specific EAS enzymes produce a range of related sesquiterpenes . Natural variation in EAS-like enzymes across Nicotiana species results in differences in product profiles, with some variants showing distinct product distributions compared to the well-characterized N. tabacum TEAS .

How do mutations affect the catalytic properties and product profiles of EAS2?

Mutations in specific residues can dramatically alter both the catalytic efficiency and product specificity of EAS enzymes. Research has identified several types of mutations that affect EAS function:

• Active Site Mutations:

  • Alterations in residues that interact directly with the substrate can change binding affinity and orientation, affecting Km values

  • Mutations in residues that stabilize carbocation intermediates can redirect the cyclization pathway toward alternative products

• Second-Tier Residues:

  • Mutations in residues that don't directly contact the substrate but influence the active site architecture can have substantial effects on product specificity

  • These mutations may alter the conformational dynamics of the enzyme during catalysis

• Product Specificity Switch Residues:

  • Certain key positions act as "switches" that determine product outcome

  • Chapter 4 of a comprehensive dissertation on EAS enzymes specifically addresses "Identification of a product specificity switch residue in the TEAS family of enzymes"

These findings have important implications for protein engineering efforts aimed at creating EAS variants with novel catalytic properties or altered product profiles.

How can recombinant EAS2 be used to study plant-herbivore interactions?

Recombinant EAS2 provides valuable tools for investigating plant-herbivore interactions through several experimental approaches:

• Comparative Expression Studies:

  • Using recombinant EAS2 as a standard, researchers can quantify native EAS expression levels in plant tissues under various herbivory conditions

  • For example, measuring EAS induction in N. attenuata shoots in response to Manduca sexta feeding

• Standardized Herbivory Simulation:

  • Wounding plant leaves and applying insect oral secretions (W+OS treatment) can standardize herbivore elicitation for reproducible studies

  • This approach "recapitulates most changes in the N. attenuata transcriptome and metabolome, which are repeatedly activated during continuous insect feeding"

• Temporal Defense Response Analysis:

  • Time-course studies sampling plants every 4 hours can capture both early and late activity phases of herbivore-induced responses

  • Correlating EAS expression with metabolite production helps elucidate defense response kinetics

• Functional Validation:

  • Recombinant EAS2 can be used to validate the activity of enzyme variants identified in different plant populations adapted to specific herbivore pressures

What insights can transcriptome-metabolome integration provide regarding EAS2 function?

Integrating transcriptome and metabolome data offers powerful insights into the biological role of EAS2 in plant defense:

• Identification of Gene-Metabolite Networks:

  • Time-course transcriptome analysis combined with UHPLC-qTOFMS metabolome measurements can reveal tissue-specific gene-gene and gene-metabolite associations recruited in response to herbivory

  • This approach helps position EAS2 within broader defense response networks

• Temporal Regulation Patterns:

  • Transcriptome-metabolome studies have revealed that plants sample harvested every 4 hours during a 21-hour period capture distinct early and late activity phases of elicited responses

  • These temporal patterns help determine when EAS2 is activated relative to other defense-related genes

• Comparative Analysis Between Tissues:

  • Differential responses between source/sink transition leaves and roots demonstrate tissue-specific regulation of defense metabolism

  • This approach has revealed that EAS is expressed constitutively in roots but inducibly in shoots of N. attenuata

How can EAS2 serve as a model for understanding terpene synthase mechanisms?

EAS2 provides an excellent model system for investigating fundamental mechanisms of terpene synthases:

• Reaction Mechanism Studies:

  • EAS catalyzes a complex reaction involving initial cyclization followed by 1,2-hydride and methyl migrations

  • The major product, 5-epi-aristolochene, is formed through a reaction pathway involving germacrene A intermediate formation, followed by protonation and sequential migrations on the same face of a eudesmyl carbocation intermediate

• Alternative Reaction Pathways:

  • Detailed analysis of EAS reaction products has revealed a "previously undocumented farnesyl trans-cis isomerization pathway"

  • This discovery highlights how EAS2 can be used to uncover novel enzymatic mechanisms

• Inhibition Studies:

  • Using substrate analogs such as anilinogeranyl diphosphate (AGPP) has led to the discovery of "biocatalytic formation of a novel 13-membered macrocyclic paracyclophane alkaloid"

  • Such studies provide "insights into new biosynthetic means for generating novel, functionally diversified, medium-sized terpene alkaloids"

• Structure-Function Analysis:

  • The well-characterized crystal structure of tobacco EAS serves as "an essential framework for understanding stereochemical selectivity displayed by terpene cyclases"

  • This allows researchers to correlate structural features with catalytic outcomes

How has the EAS gene family evolved across the Nicotiana genus?

The evolution of EAS genes in Nicotiana reveals important patterns of gene duplication and functional diversification:

• Gene Duplication Events:

  • Three copies of the EAS gene have been identified in N. attenuata, indicating gene duplication events

  • These include EAS1 (NaEAS34) and EAS2 (NaEAS37), which have been characterized as distinct variants

• Differential Regulation:

  • Different Nicotiana species show distinct patterns of EAS gene expression, particularly between roots and shoots

  • While N. attenuata and N. sylvestris both have constitutive EAS expression in roots, the regulatory patterns in shoots differ between species

• Functional Diversification:

  • Natural variants of EAS-like enzymes across Nicotiana species show differences in three fundamental properties: thermostability, kinetic efficiency, and product identity/diversity

  • These differences likely reflect adaptation to varying ecological niches and pest pressures

• Product Specificity Evolution:

  • Some EAS-like enzyme variants produce distinct product profiles compared to the well-characterized N. tabacum TEAS

  • This suggests evolution of catalytic functionality, potentially in response to different selection pressures

What factors influence EAS2 expression in response to environmental stresses?

Several environmental factors and molecular mechanisms regulate EAS2 expression:

• Herbivore-Specific Induction:

  • Feeding by the tobacco hornworm (Manduca sexta) induces EAS expression in shoots of N. attenuata

  • This induction can be simulated experimentally by applying insect oral secretions to wounded leaves

• Pathogen Response:

  • In pepper plants, EAS genes are massively up-regulated during incompatible interactions with the Obuda pepper virus (ObPV)

  • This indicates that EAS plays a role in both herbivore and pathogen defense responses

• Hormone Signaling Pathways:

  • Several strongly pathogen-inducible genes (including EAS) show marked induction by salicylic acid and ethylene

  • For example, CaEAS in pepper was up-regulated 10.59-fold (log2 scale) by ObPV and 3.66-fold by ethephon (ethylene precursor)

• Tissue-Specific Regulation:

  • Different regulatory mechanisms control EAS expression in roots (constitutive) versus shoots (inducible)

  • This suggests the existence of tissue-specific transcription factors or epigenetic mechanisms

What biotechnological applications exist for engineered EAS2 variants?

Engineered EAS2 variants offer several promising biotechnological applications:

• Novel Bioactive Compound Production:

  • Modified EAS2 variants could produce novel sesquiterpenes with pharmaceutical potential

  • For example, studying EAS inhibition has led to the discovery of a "novel 13-membered macrocyclic paracyclophane alkaloid"

• Enhanced Biosynthetic Efficiency:

  • Fusion proteins combining farnesyl diphosphate synthase (FPPS) and EAS show "more efficient conversion of isopentenyl diphosphate to epi-aristolochene than the corresponding amount of single enzymes"

  • Such fusion enzymes could improve production efficiency in biotechnological applications

• Pathway Engineering for Defense Compounds:

  • Introducing optimized EAS2 variants into crop plants could enhance natural resistance to pathogens and pests

  • This approach could reduce reliance on chemical pesticides in agriculture

• Product-Specific Enzyme Engineering:

  • Structure-function understanding of EAS2 enables rational design of variants with altered product specificity

  • This could allow selective production of specific sesquiterpenes for use in pharmaceuticals, fragrances, or biopesticides

What are common challenges in expression and purification of active recombinant EAS2?

• Solubility Issues:

  • Terpene synthases can form inclusion bodies when overexpressed in E. coli

  • Solution: Optimize induction conditions (lower temperature, reduced IPTG concentration) and consider fusion tags (thioredoxin, SUMO) to enhance solubility

• Activity Loss During Purification:

  • EAS2 may lose activity during purification due to removal of metal cofactors

  • Solution: Include divalent metal ions (Mg²⁺) in purification buffers and avoid metal chelators like EDTA

• Substrate Availability:

  • Farnesyl diphosphate (FPP) substrate is expensive for large-scale experiments

  • Solution: Consider using a coupled enzyme system with FPPS to generate FPP in situ from cheaper precursors

• Product Analysis Complexity:

  • EAS2 generates multiple products that can be challenging to separate and identify

  • Solution: Use complementary separation techniques (both non-polar and chiral stationary phases) for comprehensive product analysis

How can the catalytic efficiency of recombinant EAS2 be optimized?

Optimization strategies include:

• Buffer Composition:

  • EAS2 requires divalent metal ions (typically Mg²⁺) for activity

  • Optimal buffer conditions: 25 mM HEPES (pH 7.2), 10 mM MgCl₂, 5% glycerol

• Enzyme Fusion Approaches:

  • Creating FPPS/EAS2 fusion proteins has been shown to enhance catalytic efficiency

  • "The bifunctional enzymes showed a more efficient conversion of isopentenyl diphosphate to epi-aristolochene than the corresponding amount of single enzymes"

• Temperature and pH Optimization:

  • Systematic testing of different temperature and pH conditions to identify optimal catalytic parameters

  • Natural variation in thermostability exists among EAS variants from different Nicotiana species

• Directed Evolution:

  • Random mutagenesis followed by screening for variants with enhanced catalytic properties

  • This approach can identify beneficial mutations that might not be predicted through rational design

What analytical methods are most suitable for characterizing novel products from EAS2 variants?

For comprehensive product characterization, a combination of techniques is recommended:

• Gas Chromatography-Mass Spectrometry (GC-MS):

  • Use both non-polar and chiral stationary phases for complete separation of sesquiterpene products

  • Co-injection with authentic standards for product confirmation

  • "The combined use of chiral and non-polar stationary phases for gas chromatography separations proved critical for resolving the numerous sesquiterpene products"

• Nuclear Magnetic Resonance (NMR) Spectroscopy:

  • For complete structural elucidation of novel products

  • Both 1D (¹H, ¹³C) and 2D (COSY, HSQC, HMBC) techniques may be necessary

• High-Resolution Mass Spectrometry:

  • For accurate molecular formula determination of novel products

  • "High-resolution GC-MS and NMR analysis" was used to confirm the structure of a novel macrocyclic compound generated from an EAS variant

• Time-Course Analysis:

  • Monitoring product formation over time can provide insights into reaction mechanisms

  • May reveal intermediate products that are further converted in longer reactions