Recombinant Artemisia spiciformis Monoterpene synthase FDS-5, chloroplastic (FDS-5)

What is the role of monoterpene synthase FDS-5 in Artemisia spiciformis?

FDS-5 in Artemisia spiciformis likely catalyzes the formation of specific monoterpenes from precursor molecules. Based on research on related enzymes in Artemisia species, monoterpene synthases typically catalyze the conversion of geranyl diphosphate (GPP) or dimethylallyl diphosphate (DMAPP) to various monoterpene products. In the case of FDS-5, which appears similar to farnesyl diphosphate synthase (FDS), it may catalyze reactions involving the sequential condensation of isopentenyl diphosphate (IPP) with DMAPP to form intermediates like GPP and potentially further products. Monoterpene synthases in Artemisia species are known to produce a wealth of terpenoid compounds that play important roles in plant defense, signaling, and ecological interactions .

How does FDS-5 compare structurally to other monoterpene synthases in Artemisia species?

FDS-5 likely shares significant structural similarities with other characterized monoterpene synthases from Artemisia species. Research on Artemisia tridentata has shown that chrysanthemyl diphosphate synthase (CDS) shares 75% identity and 96% similarity with farnesyl diphosphate synthase (FDS) from the same species . This high degree of homology suggests conservation of key structural elements. Specifically, FDS-5 would be expected to contain conserved aspartate-rich motifs (such as the DDXXD motif) that are crucial for substrate binding and catalysis in terpene synthases. Additionally, studies of monoterpene synthases in Artemisia annua have identified specific N-terminal targeting peptides that direct these enzymes to plastids where monoterpene biosynthesis occurs . As FDS-5 is described as chloroplastic, it likely possesses similar plastid-targeting sequences.

What is known about the expression patterns of FDS-5 in different tissues of Artemisia spiciformis?

While specific data on FDS-5 expression patterns in Artemisia spiciformis tissues is not directly presented in the available research, patterns can be inferred from studies of related monoterpene synthases. In Artemisia annua, monoterpene synthases like AaTPS2, AaTPS5, and AaTPS6 are expressed in both aerial tissues and roots, with higher expression typically observed in glandular trichomes where many specialized metabolites accumulate . Expression analysis using techniques such as qRT-PCR would reveal tissue-specific expression patterns of FDS-5. Researchers should examine expression in leaves, stems, roots, flowers, and specialized structures like glandular trichomes. This information would provide insights into the potential ecological roles of FDS-5-derived products in different plant tissues and developmental stages.

What are the optimal conditions for expressing recombinant FDS-5 in heterologous systems?

For optimal heterologous expression of recombinant FDS-5, researchers should consider several critical parameters based on successful approaches with related enzymes. For bacterial expression, E. coli BL21(AI) strain has proven effective for expressing Artemisia monoterpene synthases . The expression construct should contain the enzyme-coding sequence without the N-terminal plastid-targeting peptide, as this can interfere with proper folding in bacterial systems. Expression should be induced at lower temperatures (approximately 18°C) for 16-20 hours with appropriate inducers (0.02% L-arabinose has worked well for related enzymes) .

For protein purification, a buffer system containing 50 mM Tris-HCl (pH 7.5), 300 mM NaCl, and 1.4 mM β-mercaptoethanol should be used, followed by sonication (6 × 10 seconds) and centrifugation at 13,000 × g . Including a histidine tag facilitates purification via nickel affinity chromatography. Alternative expression systems worth considering include yeast (Saccharomyces cerevisiae or Pichia pastoris) for enzymes that prove difficult to express in bacteria. Codon optimization for the expression host organism may significantly improve yield and functional activity.

What mechanisms regulate FDS-5 activity in response to environmental stresses?

The regulation of FDS-5 in response to environmental stresses likely follows similar patterns to those observed in related monoterpene synthases. Research on monoterpene synthases in Artemisia annua demonstrated that mechanical wounding induces the expression of monoterpene synthase genes, suggesting their role in plant defense responses . Additionally, treatments with phytohormones including methyl jasmonate, salicylic acid, and gibberellin have been shown to elevate transcript levels of certain monoterpene synthases (AaTPS5 and AaTPS6) .

Researchers investigating FDS-5 regulation should employ a multi-level approach to characterize its response to stresses: 1) Transcript level analysis using qRT-PCR after exposure to various stresses and phytohormones; 2) Protein abundance measurement using western blotting with specific antibodies; 3) Enzyme activity assays under different stress conditions; and 4) Metabolite profiling to correlate enzyme activity with accumulation of end products. Additionally, promoter analysis could identify stress-responsive elements that control FDS-5 expression. This comprehensive approach would elucidate the precise mechanisms through which environmental cues modulate FDS-5 function in stress adaptation.

How do metal cofactors influence the catalytic activity and product spectrum of FDS-5?

Metal cofactors likely play crucial roles in FDS-5 catalytic activity and product formation, as demonstrated in related monoterpene synthases. Studies with Artemisia annua monoterpene synthases revealed that both Mg²⁺ and Mn²⁺ support catalytic activities, but Mn²⁺ can significantly alter the product spectrum for certain enzymes (AaTPS2 and AaTPS5) . This metal-dependent product switching is an important consideration when characterizing terpene synthases.

What are the most effective methods for assaying FDS-5 enzymatic activity?

For comprehensive characterization of FDS-5 enzymatic activity, researchers should employ a combination of approaches. The recommended in vitro assay system should contain the following components: 15 mM MOPSO buffer (pH 7.0), 2 mM dithioerythreitol, 12.5% (v/v) glycerol, 1-5 mM MgCl₂, 1 mM ascorbic acid, 0.1% (v/v) Tween 20, and varying concentrations of potential substrates (DMAPP, IPP, GPP) . After adding purified enzyme (30-50 μg), the reaction mixture should be overlaid with 100 μl of pentane and incubated at 30°C for an appropriate time period (20-96 hours depending on expected activity level) .

Products should be extracted with pentane or hexane and analyzed by GC-MS using an HP5 MS column with the following parameters: injection port temperature 250°C, interface temperature 290°C, MS source temperature 180°C, and an oven program starting at 45°C for 1 min with a ramp of 15°C min⁻¹ to 280°C . For diphosphate products, the aqueous phase should be treated with alkaline phosphatase to release alcohols before extraction and analysis. Activity should be quantified using authentic standards or by comparing mass spectra with library data. This comprehensive approach enables identification of all potential products and determination of kinetic parameters.

How can site-directed mutagenesis be effectively applied to study FDS-5 structure-function relationships?

Site-directed mutagenesis represents a powerful approach for investigating structure-function relationships in FDS-5. Based on research with related enzymes, key residues likely involved in catalysis include those in the conserved DDXXD and N/DXXD motifs that coordinate divalent metal ions and substrate binding . To perform effective site-directed mutagenesis studies:

• Identify conserved residues through multiple sequence alignment of FDS-5 with related terpene synthases, particularly focusing on catalytic domains.

• Design mutagenesis primers that introduce specific substitutions at these conserved positions. For example, primers similar to those used for CDS mutagenesis: TcCDS-N283D_F, 5′-GGTATGTATTATCAAATTCAGGATGATTATCTCGACAC-3′ and TcCDS-N283D_R, 5′-GTGTCGAGATAATCATCCTGAATTTGATAATACATACC-3′ .

• Employ QuikChange or similar commercial kits for efficient mutagenesis.

• Verify mutations by DNA sequencing before protein expression.

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

• Compare kinetic parameters, product profiles, and metal ion requirements between wild-type and mutant enzymes.

Particularly informative mutations would include conversions of aspartate to alanine in the DDXXD motif, which should reduce or eliminate catalytic activity, and asparagine to aspartate conversions in NDXXD motifs, which might alter product specificity as observed in related enzymes .

What are the most reliable methods for quantifying FDS-5 gene expression in different tissues?

For reliable quantification of FDS-5 gene expression across different tissues, researchers should employ a multi-technique approach. Quantitative real-time PCR (qRT-PCR) represents the most accessible and reliable technique. The recommended protocol includes:

• RNA extraction from different tissues (leaves, stems, roots, trichomes) using RNeasy Plant Mini Kit or TRIzol reagent with modifications to address terpenoid and polysaccharide content.

• DNase treatment to eliminate genomic DNA contamination.

• cDNA synthesis using SuperScript IV or similar reverse transcriptase.

• qRT-PCR with SYBR Green or TaqMan chemistry using gene-specific primers designed to amplify 100-200 bp fragments.

• Multiple reference genes (such as actin, ubiquitin, and GAPDH) should be evaluated with tools like geNorm or NormFinder to identify the most stable references for normalization.

• Data analysis using the 2^(-ΔΔCt) method with proper statistical evaluation.

For more comprehensive analysis, RNA-Seq can provide genome-wide expression context, while in situ hybridization would reveal cell-type specific expression patterns within tissues. Northern blotting can confirm transcript size and alternative splicing. Western blotting with specific antibodies would correlate transcript levels with protein abundance. This multi-technique approach ensures reliable tissue-specific expression profiles of FDS-5.

How should kinetic parameters for FDS-5 be determined and interpreted?

Accurate determination and interpretation of kinetic parameters for FDS-5 requires careful experimental design and data analysis. Based on methodologies used for related enzymes, researchers should:

• Perform enzyme assays with varying substrate concentrations (8-10 concentrations ranging from 0.1× to 10× the expected Km value).

• Ensure reactions remain in the linear range by conducting time-course experiments.

• Use appropriate enzyme concentrations where less than 10% of substrate is consumed.

• Include proper controls (heat-inactivated enzyme, no substrate, no enzyme).

• Perform all assays in triplicate to ensure statistical reliability.

The kinetic data should be fitted to appropriate equations using software like KaleidaGraph , GraphPad Prism, or R to determine parameters:

• For standard Michaelis-Menten kinetics: Km, Vmax, kcat, and kcat/Km

• For substrate inhibition: Ki values in addition to standard parameters

• For multiple substrate reactions: perform initial velocity studies varying one substrate while keeping others constant

When interpreting these values, consider that the Km for related enzymes with DMAPP is relatively high (600 μM for CDS) compared to estimated plastidial concentrations (approximately 30 μM) , suggesting potential regulatory mechanisms. Additionally, compare parameters with those of related enzymes across Artemisia species to identify evolutionary patterns in catalytic efficiency.

What are the best approaches for analyzing the complex product mixtures generated by FDS-5?

Analyzing complex product mixtures generated by FDS-5 requires sophisticated analytical techniques and data processing. Based on approaches used for related monoterpene synthases, researchers should employ:

• Gas Chromatography-Mass Spectrometry (GC-MS) as the primary analytical method, using an HP5 MS column (30 m × 0.25 mm inner diameter, 0.25 μm film thickness) with optimized temperature programs starting at 45°C and ramping to 280°C .

• Multiple identification approaches:

  • Comparison with authentic standards

  • Matching mass spectra with commercial libraries (NIST, Wiley)

  • Calculation of Kovats retention indices

  • NMR analysis for structural confirmation of major products

• Quantification strategies:

  • Integration of total ion chromatograms or selected ion monitoring

  • Use of internal standards like camphor or menthol

  • Standard curves with authentic compounds when available

• Statistical approaches for complex mixture analysis:

  • Principal Component Analysis (PCA) to identify patterns in product distribution

  • Hierarchical Clustering to group similar product profiles

  • ANOVA with post-hoc tests to identify significant differences between conditions

For comprehensive characterization, combine GC-MS with other techniques like liquid chromatography (for less volatile products) and NMR spectroscopy (for structural confirmation). Additionally, chiral GC columns should be employed to determine enantiomeric composition of products, as stereochemistry is crucial for biological activity.

How do different analytical techniques compare for studying FDS-5 products and their biosynthetic pathways?

Different analytical techniques offer complementary information about FDS-5 products and biosynthetic pathways. Researchers should consider the following comparison to select appropriate methods:

For comprehensive characterization of FDS-5 function, these techniques should be used in combination. Initial screening with GC-MS identifies products, followed by purification and NMR analysis of major components. Isotope labeling experiments with ¹³C or ²H-labeled precursors can confirm biosynthetic relationships, while enzyme assays with potential intermediates can validate proposed pathways. This multi-technique approach provides robust evidence for enzyme function and pathway elucidation.

How does FDS-5 compare functionally to similar enzymes in other Artemisia species?

FDS-5 from Artemisia spiciformis likely shares functional similarities with monoterpene synthases from other Artemisia species, but with distinct catalytic properties and product profiles. Comparative analysis should consider:

The CDS from Artemisia tridentata ssp. spiciformis shares 75% identity and 96% similarity with FDS from the same species . This high sequence similarity suggests recent evolutionary divergence, yet they catalyze different reactions: FDS forms farnesyl diphosphate via c1′-4 condensation reactions, while CDS performs c1′-2-3 cyclopropanation reactions .

Researchers should conduct comparative enzyme assays under identical conditions to directly compare substrate preferences, product distributions, and catalytic efficiencies. Additionally, homology modeling and structural analyses could identify key active site residues responsible for functional differences. This comparative approach would provide insights into the molecular evolution of specialized metabolism in Artemisia species and the functional diversification of terpene synthases.

What evolutionary patterns explain the diversification of monoterpene synthases in Artemisia species?

The diversification of monoterpene synthases in Artemisia species likely reflects evolutionary processes driven by ecological adaptation. Several patterns are evident:

Monoterpene synthases like FDS-5 appear to have evolved through gene duplication and neofunctionalization from ancestral terpene synthases involved in primary metabolism. For example, CDS evolved from FDS, with high sequence similarity (75% identity) but distinct enzymatic functions . This represents a classic case of enzyme evolution through relatively minor sequence changes leading to significant functional divergence.

Comparative genomic studies across Artemisia species would likely reveal expansion of monoterpene synthase gene families through duplication events, followed by sequence divergence. This is consistent with the observation that multiple related monoterpene synthases exist within a single species (e.g., AaTPS2, AaTPS5, and AaTPS6 in A. annua) .

The ecological drivers for this diversification likely include selection pressures from herbivores, pathogens, and environmental stresses. The observation that monoterpene synthase expression is induced by wounding and phytohormones associated with defense responses supports this hypothesis . Researchers should employ phylogenetic analyses combined with functional characterization and ecological studies to fully understand the evolutionary trajectories of these enzymes and their roles in plant adaptation.

How can computational approaches aid in predicting structure-function relationships in FDS-5?

Computational approaches offer powerful tools for predicting structure-function relationships in FDS-5 without extensive experimental work. Researchers should consider the following methodology:

The computational predictions should be validated experimentally through site-directed mutagenesis of predicted key residues. This integrated computational-experimental approach can accelerate understanding of structure-function relationships and guide rational enzyme engineering for production of specific monoterpenes of interest.

How can FDS-5 be engineered for altered product specificity?

Engineering FDS-5 for altered product specificity requires strategic modifications based on structure-function understanding. Researchers should consider the following approaches:

• Site-directed mutagenesis of active site residues: Target residues within 5Å of the substrate binding site, particularly those lining the enzyme cavity that may influence substrate folding. For related terpene synthases, mutations in the DDXXD and N/DXXD motifs have been shown to alter product profiles . Researchers should create small libraries of variants (10-20) focusing on these conserved regions.

• Domain swapping: Exchange functional domains between FDS-5 and related monoterpene synthases with different product profiles. Chimeric enzymes may produce novel product mixtures or shift specificity toward desired products.

• Semi-rational approaches: Combine computational predictions with directed evolution. Create focused mutant libraries based on computational hot spots, then screen for desired activities.

• Metal cofactor engineering: Exploit the observation that different metal ions (Mg²⁺ vs. Mn²⁺) can alter product profiles in related enzymes . Engineer metal binding sites to favor specific conformational states that promote formation of targeted products.

Success should be evaluated using high-throughput GC-MS screening approaches, comparing product profiles of variants against wild-type enzyme. Promising variants should undergo full kinetic characterization and structural analysis to understand the molecular basis of altered specificity. This engineering approach could yield enzyme variants producing valuable monoterpenes for pharmaceutical or fragrance applications.

What are the most effective strategies for expressing FDS-5 in plant systems for metabolic engineering?

For effective expression of FDS-5 in plant systems for metabolic engineering, researchers should consider the following comprehensive strategies:

• Promoter selection: For constitutive expression, the CaMV 35S promoter provides high expression levels, while inducible promoters (e.g., dexamethasone-inducible or ethanol-inducible) offer temporal control. Tissue-specific promoters like trichome-specific promoters can target expression to specialized metabolite production sites.

• Subcellular targeting: Since FDS-5 is naturally chloroplastic, researchers should retain its native transit peptide or replace it with well-characterized plastid-targeting sequences. Alternative targeting to mitochondria or cytosol could be explored to access different substrate pools.

• Expression optimization:

  • Codon optimization for the target plant species

  • Inclusion of appropriate 5' and 3' UTRs for mRNA stability

  • Use of introns to enhance expression levels

  • Consideration of gene silencing mechanisms

• Vector systems:

  • For stable transformation: Binary vectors compatible with Agrobacterium-mediated transformation

  • For transient expression: Viral vectors or agroinfiltration systems

  • For chloroplast transformation: Vectors with homologous recombination regions

• Host selection: Consider both model plants (Arabidopsis, tobacco) for proof-of-concept studies and target species (medicinal plants, crop plants) for application development.

To confirm successful expression, researchers should quantify transcript levels via qRT-PCR, protein abundance via western blotting, and ultimately measure monoterpene product formation using headspace analysis or tissue extraction followed by GC-MS. For optimal pathway engineering, FDS-5 expression should be coordinated with upstream precursor pathway enhancement and potential downstream modifications.

How can isotope labeling experiments be designed to elucidate the reaction mechanism of FDS-5?

Isotope labeling experiments provide crucial insights into FDS-5 reaction mechanisms and can resolve mechanistic ambiguities. Researchers should design experiments following these guidelines:

• Selection of labeled precursors:

  • ¹³C-labeled DMAPP at specific positions (particularly C1, C2, and C3)

  • ²H-labeled DMAPP to track hydrogen migrations

  • ¹⁸O-labeled diphosphate groups to track oxygen fate

  • Combinations of differently labeled precursors for complex analyses

• Experimental design:

  • In vitro assays with purified recombinant FDS-5 and labeled substrates

  • Incubation times optimized to capture potential intermediates

  • Quenching methods that preserve unstable intermediates

  • Multiple extraction protocols to capture diverse product types

• Analytical approaches:

  • GC-MS with careful analysis of mass fragmentation patterns

  • NMR spectroscopy (¹³C-NMR, HSQC, HMBC) for positional assignment of labels

  • Time-course experiments to capture reaction progression

• Data interpretation:

  • Comparison with theoretical fragmentation patterns predicted for different mechanistic scenarios

  • Mathematical modeling of label incorporation percentages

  • Integration with computational predictions of reaction pathways

These experiments could distinguish between concerted and stepwise mechanisms, identify carbocation intermediates, and clarify the stereochemical course of the reaction. Particularly informative would be experiments examining whether FDS-5 follows similar bifunctional mechanisms as observed with CDS, which catalyzes both the formation of CPP from DMAPP and the conversion of CPP to chrysanthemol . Determination of rate-limiting steps and identification of trapped intermediates would provide a comprehensive mechanistic picture of FDS-5 catalysis.

What are common challenges in expressing recombinant FDS-5 and how can they be addressed?

Researchers frequently encounter several challenges when expressing recombinant monoterpene synthases like FDS-5. Based on experiences with related enzymes, these issues and their solutions include:

• Low solubility and inclusion body formation:

  • Lower induction temperature (16-18°C instead of 37°C)

  • Reduce inducer concentration (0.01-0.02% L-arabinose instead of 0.2%)

  • Use solubility-enhancing fusion tags (MBP, SUMO, or thioredoxin)

  • Add solubility enhancers to growth media (sorbitol, glycine betaine)

• Poor enzyme activity:

  • Ensure proper metal cofactor concentration (1-5 mM Mg²⁺ or Mn²⁺)

  • Include reducing agents (2-5 mM DTT or β-mercaptoethanol) to maintain cysteine residues

  • Optimize buffer composition and pH (typically MOPSO buffer at pH 7.0)

  • Add glycerol (10-20%) to stabilize protein structure

• Proteolytic degradation:

  • Include protease inhibitors during purification

  • Reduce processing time and temperature

  • Use protease-deficient expression strains

• Improper folding:

  • Co-express with molecular chaperones (GroEL/ES, DnaK/J)

  • Slow down expression rate with lower temperatures and inducer concentrations

  • Remove plastid-targeting peptide that may interfere with bacterial expression

• Low yield:

  • Optimize codon usage for expression host

  • Use high-copy number expression vectors

  • Implement fed-batch cultivation techniques

A systematic approach addressing these challenges sequentially can significantly improve the yield and activity of recombinant FDS-5, enabling more comprehensive biochemical characterization and biotechnological applications.

How can contradictory data about FDS-5 substrate specificity be reconciled?

When confronted with contradictory data regarding FDS-5 substrate specificity, researchers should implement a systematic approach to reconcile discrepancies:

• Methodological comparison:

  • Examine differences in enzyme preparation methods (bacterial vs. yeast expression, purification protocols)

  • Compare assay conditions (buffer composition, pH, metal cofactors, incubation time)

  • Evaluate analytical techniques used (GC-MS parameters, extraction methods)

• Enzyme state assessment:

  • Verify enzyme purity via SDS-PAGE and mass spectrometry

  • Assess enzyme stability under different storage conditions

  • Check for post-translational modifications that might affect activity

• Substrate quality control:

  • Verify purity and stereochemistry of substrates used

  • Ensure substrates haven't degraded during storage

  • Consider different substrate batches or suppliers

• Concentration effects:

  • Test wide substrate concentration ranges (10 μM to 1 mM)

  • Look for substrate inhibition at higher concentrations as observed with related enzymes

  • Consider substrate sequestration by detergents or vessel surfaces

• Comprehensive product analysis:

  • Use multiple analytical approaches (GC-MS, LC-MS, NMR)

  • Implement chiral separation for stereoisomer detection

  • Search for potential unstable or volatile products that might be missed

• Biological context:

  • Test enzyme in cellular contexts to account for potential protein-protein interactions

  • Consider compartmentalization effects when interpreting in vivo versus in vitro results

By systematically addressing these factors, researchers can often identify the source of contradictory results and develop a more nuanced understanding of FDS-5 substrate specificity under different conditions.

What are the critical quality control steps for ensuring reliable FDS-5 activity data?

Ensuring reliable FDS-5 activity data requires rigorous quality control measures throughout the experimental workflow. Critical steps include:

• Enzyme preparation quality control:

  • Assess purity by SDS-PAGE (>95% homogeneity)

  • Confirm identity by western blotting and/or mass spectrometry

  • Verify protein concentration using multiple methods (Bradford assay, BCA, absorbance at 280 nm)

  • Evaluate batch-to-batch consistency with standard activity assays

• Substrate and reagent verification:

  • Confirm substrate purity by analytical methods (NMR, MS)

  • Prepare fresh buffers and verify pH before each experiment

  • Use high-purity metal salts and verify concentrations

  • Implement standard operating procedures for reagent preparation

• Assay controls:

  • Include negative controls (heat-inactivated enzyme, no substrate)

  • Use positive controls (known active enzyme preparations)

  • Perform time-course experiments to ensure linearity

  • Verify product identity with authentic standards

• Technical replication:

  • Perform all measurements in triplicate at minimum

  • Conduct assays on multiple days with different enzyme preparations

  • Calculate coefficient of variation (<15% is typically acceptable)

• Data analysis rigor:

  • Apply appropriate statistical tests (ANOVA, t-tests)

  • Use proper regression analysis for kinetic parameter determination

  • Report all data with standard deviations or standard errors

  • Avoid cherry-picking data points that fit hypotheses

• Method validation:

  • Calibrate all instruments regularly

  • Validate analytical methods with standard compounds

  • Determine limits of detection and quantification

  • Document all protocols in detail for reproducibility