Recombinant Arabidopsis thaliana 2-succinylbenzoate--CoA ligase, chloroplastic/peroxisomal (AAE14)

What is AAE14 and what is its function in Arabidopsis thaliana?

AAE14 (At1g30520) encodes the o-succinylbenzoyl-coenzyme A (OSB-CoA) ligase in Arabidopsis thaliana, which catalyzes a crucial step in phylloquinone (vitamin K1) biosynthesis. Phylloquinone serves as an essential one-electron carrier at the A1 site of photosystem I, making it indispensable for photosynthesis . The enzyme facilitates the conversion of o-succinylbenzoate (OSB) to OSB-CoA, representing a critical junction in the phylloquinone synthesis pathway. This reaction is analogous to what the bacterial menE gene product catalyzes in menaquinone (vitamin K2) biosynthesis in prokaryotes .

Where is AAE14 localized within plant cells?

AAE14 exhibits a complex subcellular localization pattern. Studies using AAE14:GFP reporter constructs have revealed that the protein accumulates primarily in discrete foci within chloroplasts . This punctate distribution pattern suggests that AAE14 might be part of a metabolic enzyme complex in the chloroplast stroma, potentially facilitating the efficient channeling of intermediates through the phylloquinone biosynthesis pathway. Despite its annotation as "chloroplastic/peroxisomal," the predominant functional localization appears to be within chloroplasts, which aligns with the role of phylloquinone in photosynthesis .

How can recombinant AAE14 be effectively expressed and purified?

Recombinant AAE14 can be expressed in E. coli using a His-tag fusion system. The full-length mature protein (amino acids 16-560) can be cloned into an expression vector with an N-terminal His-tag. For optimal expression:

• Transform the construct into an E. coli strain optimized for protein expression (e.g., BL21(DE3)).

• Culture cells at 37°C until reaching OD₆₀₀ of 0.5-0.7.

• Induce protein expression with IPTG (typically 0.1-1.0 mM) at a reduced temperature of 16-22°C overnight to enhance protein solubility.

• Harvest cells by centrifugation and lyse using appropriate buffer systems.

• Purify using Ni-NTA affinity chromatography.

The purified protein should be stored in Tris/PBS-based buffer with 6% trehalose at pH 8.0 to maintain stability . For long-term storage, addition of 5-50% glycerol (final concentration) followed by aliquoting and storage at -20°C/-80°C is recommended . Avoid repeated freeze-thaw cycles as they may compromise enzyme activity.

What assays can be used to measure AAE14 enzymatic activity?

AAE14 activity can be assessed through several complementary approaches:

1. Direct enzymatic assay:

• Measure the formation of OSB-CoA by monitoring the release of pyrophosphate (PPi) during the reaction.

• Utilize HPLC or LC-MS to detect and quantify OSB-CoA production.

• Assess the consumption of ATP, which is required for the reaction.

2. Complementation assay:

• Express AAE14 in E. coli mutants disrupted in the menE gene.

• Evaluate restoration of menaquinone biosynthesis, which indicates functional equivalence.

• This approach has been validated in previous research demonstrating that AAE14 can functionally substitute for the bacterial OSB-CoA ligase .

3. Indirect measurement via phylloquinone quantification:

• In planta activity can be assessed by measuring phylloquinone levels using HPLC with fluorescence detection.

• Wild-type Arabidopsis typically contains approximately 10 pmol mg⁻¹ fresh weight of phylloquinone in leaves, while aae14 mutants contain undetectable levels (<0.1 pmol mg⁻¹) .

How can AAE14 mutants be generated and characterized?

Generation and characterization of AAE14 mutants involve several methodological steps:

1. Mutant generation approaches:

• T-DNA insertion lines: Utilize publicly available T-DNA insertion collections in Arabidopsis.

• CRISPR/Cas9 genome editing: Design guide RNAs targeting specific regions of the AAE14 gene.

• EMS mutagenesis: Screen for mutations in AAE14 using TILLING.

2. Phenotypic characterization:

• Growth analysis: AAE14 mutants typically exhibit seedling lethality, even when grown on sucrose-supplemented media, indicating their inability to become autotrophic .

• Chlorophyll fluorescence: Measure photosystem I activity to assess the impact of phylloquinone deficiency.

• Metabolite analysis: Quantify phylloquinone levels and upstream intermediates like OSB.

3. Genetic complementation:

• Transform mutants with functional AAE14 under various promoters (e.g., CaMV 35S or inducible XVE system).

• Partial complementation can be achieved with weak expression, resulting in chlorotic, slow-growing plants with intermediate phylloquinone levels (approximately 4.7 pmol mg⁻¹ fresh weight) .

• Full complementation restores wild-type phenotype and phylloquinone content.

4. Bypass experiments:

• Provide downstream intermediates like 1,4-dihydroxy-2-naphthoate to verify the specific enzymatic step disrupted in the mutants .

How does AAE14 contribute to the phylloquinone biosynthetic pathway?

AAE14 catalyzes a critical step in phylloquinone biosynthesis, functioning at the interface between primary and secondary metabolism. The pathway proceeds as follows:

• Chorismate (from shikimate pathway) → Isochorismate

• Isochorismate → 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate

• 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate → o-succinylbenzoate (OSB)

• OSB → OSB-CoA (catalyzed by AAE14)

• OSB-CoA → 1,4-dihydroxy-2-naphthoyl-CoA

• 1,4-dihydroxy-2-naphthoyl-CoA → 1,4-dihydroxy-2-naphthoate

• 1,4-dihydroxy-2-naphthoate → Phylloquinone (after phytyl addition and methylation)

Research indicates that enzymes involved in phylloquinone synthesis from isochorismate may form a metabolic complex in the chloroplast stroma, facilitating efficient channeling of intermediates . The punctate localization pattern of AAE14 supports this hypothesis, suggesting that the enzyme participates in a multi-enzyme complex rather than functioning as a solitary catalyst.

What is the relationship between AAE14 function and photosynthetic efficiency?

AAE14 function directly impacts photosynthetic efficiency through its role in phylloquinone biosynthesis. The relationship can be characterized as follows:

1. Electron transport impact:

• Phylloquinone serves as the one-electron carrier at the A₁ site of photosystem I (PSI).

• In AAE14-deficient plants, the absence of phylloquinone disrupts electron transport through PSI.

• This disruption prevents effective light harvesting and energy conversion.

2. Photosystem I stability:

• Phylloquinone is integral to PSI structure and function.

• Without phylloquinone, PSI assembly or stability may be compromised.

3. Oxidative stress management:

• Disruption of electron transport in PSI can lead to increased production of reactive oxygen species.

• This may contribute to the seedling lethality observed in aae14 mutants.

4. Quantitative relationship:

• Research suggests that even partial restoration of phylloquinone synthesis (to approximately 47% of wild-type levels) can support photoautotrophic growth, albeit with chlorosis and reduced growth rate .

• This indicates a threshold effect rather than a strictly linear relationship between phylloquinone content and photosynthetic capability.

How does AAE14 compare to analogous enzymes in other species?

AAE14 belongs to the acyl-activating enzyme family and shows functional homology to enzymes across diverse organisms:

Despite the functional equivalence, AAE14 and its homologs display several key differences:

• Substrate specificity patterns may vary, particularly for secondary substrates

• Regulatory mechanisms differ between prokaryotic and eukaryotic systems

• Arabidopsis AAE14 contains targeting sequences for dual organellar localization

• Kinetic parameters may vary across species, reflecting evolutionary adaptation

How can issues with protein solubility and stability be addressed when working with recombinant AAE14?

Recombinant AAE14 may present solubility and stability challenges common to many plant enzymes expressed in heterologous systems. These issues can be addressed through various approaches:

1. Solubility enhancement strategies:

• Optimize induction conditions: Lower temperatures (16-22°C) and reduced IPTG concentrations often improve solubility.

• Use solubility-enhancing fusion tags: Beyond His-tags, consider MBP (maltose-binding protein) or SUMO tags.

• Modify buffer compositions: Include osmolytes like glycerol, sucrose, or trehalose.

• Try co-expression with chaperones: GroEL/GroES or trigger factor can assist proper folding.

2. Stability considerations:

• Store purified protein in Tris/PBS-based buffer with 6% trehalose at pH 8.0 .

• Add glycerol (5-50% final concentration) for long-term storage at -20°C/-80°C .

• Aliquot the protein to avoid repeated freeze-thaw cycles.

• Consider lyophilization as an alternative storage approach.

3. Protein reconstitution protocol:

• Centrifuge the vial briefly before opening to bring contents to the bottom.

• Reconstitute in deionized sterile water to 0.1-1.0 mg/mL.

• For extended storage, add glycerol to a final concentration of 50% .

4. Alternative expression systems:

• If E. coli expression yields poor results, consider plant-based expression systems.

• Cell-free protein synthesis may be suitable for difficult-to-express variants.

What are the common pitfalls in AAE14 mutant analysis and how can they be overcome?

Analysis of AAE14 mutants presents several challenges that require careful experimental design:

1. Seedling lethality complications:

• Challenge: AAE14 knockout mutants are seedling lethal, limiting developmental studies.

• Solution: Utilize inducible expression systems (e.g., XVE) to create partial complementation lines that are viable but retain phenotypic differences .

• Alternative: Develop tissue-specific or conditional knockout strategies.

2. Pleiotropic effects:

• Challenge: Distinguishing direct effects of AAE14 deficiency from secondary consequences.

• Solution: Perform time-course analyses to establish temporal sequences of physiological changes.

• Alternative: Supply downstream intermediates (e.g., 1,4-dihydroxy-2-naphthoate) to bypass the blocked step and confirm pathway specificity .

3. Metabolite quantification:

• Challenge: Accurate measurement of phylloquinone and pathway intermediates.

• Solution: Develop sensitive HPLC or LC-MS methods with appropriate internal standards.

• Reference: Wild-type Arabidopsis leaves typically contain approximately 10 pmol mg⁻¹ fresh weight of phylloquinone .

4. Genetic background effects:

• Challenge: Variability in phenotype severity due to genetic background differences.

• Solution: Generate multiple independent mutant alleles and perform complementation tests.

• Alternative: Create mutations in different Arabidopsis ecotypes to assess genetic background influence.

How can Single-Subject Experimental Design (SSED) be applied to AAE14 research?

Single-Subject Experimental Design (SSED) approaches can be adapted for AAE14 research to improve experimental rigor and reproducibility:

1. SSED implementation in AAE14 studies:

• Establish stable baseline measurements before applying interventions.

• Use repeated measurements to track changes over time within the same experimental units.

• Implement interventions sequentially to isolate effects.

• Return to baseline conditions to verify intervention-specific effects.

2. SSED advantages for AAE14 research:

• Accounts for individual variation in plant responses to genetic manipulation or treatment.

• Reduces required sample sizes while maintaining statistical power.

• Facilitates identification of temporal patterns in physiological responses.

• Improves detection of subtle phenotypic changes.

3. Quality standards for SSED in plant research:

• Experiments should meet design standards with or without reservations .

• Visual analysis of results should support experimental effects.

• A minimum of five supporting SSED studies meeting evidence standards is required if studies are to be combined .

• Studies should be conducted by at least three different research teams at three different geographical locations with a combined minimum of 20 participants or cases .

4. Application to AAE14 inducible complementation studies:

• Monitor phenotypic parameters before, during, and after induction of AAE14 expression.

• Quantify phylloquinone synthesis rates at multiple timepoints following induction.

• Track photosynthetic parameters in response to changing AAE14 expression levels.

How can QTL mapping approaches be used to study natural variation in AAE14 function?

Quantitative Trait Locus (QTL) mapping offers powerful approaches to investigate natural variation in AAE14 function across Arabidopsis accessions:

1. Advanced Intercross-Recombinant Inbred Line (AI-RIL) methodology:

• Utilize AI-RIL populations such as EstC and KendC, which provide high genetic resolution .

• These populations capture increased recombination events compared to traditional RILs.

• Average intermarker distance of 600 kb in these populations enables precise QTL localization .

• Genetic map expansion in AI-RILs corresponds to approximately 50 kb/cM resolution .

2. Phenotyping strategies:

• Quantify phylloquinone content across accessions and mapping populations.

• Measure photosystem I efficiency parameters.

• Assess growth and development under varying light conditions.

• Quantify AAE14 expression levels to identify expression QTLs (eQTLs).

3. QTL analysis framework:

• Map QTLs for phylloquinone content and related traits.

• Identify naturally occurring AAE14 variants contributing to phenotypic differences.

• Examine epistatic interactions between AAE14 and other loci.

• Perform comparative QTL mapping using multiple AI-RIL populations genotyped with common markers .

4. Follow-up validation:

• Confirm QTL effects through near-isogenic lines.

• Perform complementation tests with different AAE14 alleles.

• Utilize genome editing to introduce specific AAE14 variants.

What approaches can be used to study protein-protein interactions involving AAE14?

Understanding AAE14's interactions with other proteins is crucial for elucidating its regulation and metabolic context. Several complementary approaches can be employed:

1. In vivo approaches:

• Bimolecular Fluorescence Complementation (BiFC) to visualize interactions in plant cells.

• Förster Resonance Energy Transfer (FRET) to detect proximity between AAE14 and potential interactors.

• Co-immunoprecipitation followed by mass spectrometry to identify interaction partners.

• Yeast two-hybrid screening to identify binary interactions.

2. In vitro approaches:

• Pull-down assays using recombinant His-tagged AAE14 .

• Surface Plasmon Resonance (SPR) to measure binding kinetics.

• Isothermal Titration Calorimetry (ITC) to determine thermodynamic parameters of interactions.

• Native gel electrophoresis to detect stable complex formation.

3. Computational predictions:

• Protein-protein interaction prediction algorithms.

• Structural modeling to identify potential interaction interfaces.

• Co-expression network analysis to identify functionally related genes.

4. Multi-enzyme complex investigation:

• Size-exclusion chromatography to isolate native complexes.

• Cross-linking mass spectrometry to capture transient interactions.

• Investigate associations with other phylloquinone biosynthesis enzymes, particularly those involved in adjacent pathway steps.

How can recombinant protein-filled vesicle technology be applied to AAE14 research?

Recent advances in recombinant protein-filled vesicle technology present innovative opportunities for AAE14 research:

1. Vesicle-based expression system advantages:

• Compartmentalization of AAE14 within membrane-bound vesicles can enhance stability.

• Short peptide tag-based export system enables efficient production in E. coli .

• Vesicles provide a protected environment that may better maintain enzyme activity.

• The system can facilitate co-expression of multiple phylloquinone pathway enzymes.

2. Implementation strategy:

• Engineer AAE14 with appropriate export tags for vesicle targeting.

• Co-express with other pathway enzymes to create multi-enzyme vesicles.

• Purify vesicles through differential centrifugation or affinity-based methods.

• Characterize enzyme activity within the vesicular context.

3. Applications for AAE14 research:

• Study pathway flux through reconstituted phylloquinone synthesis steps.

• Investigate substrate channeling between sequential enzymes.

• Examine effects of membrane association on enzyme activity.

• Develop in vitro systems for high-throughput inhibitor screening.

4. Experimental design considerations:

• Compare enzymatic efficiency in vesicle-encapsulated versus purified soluble forms.

• Optimize vesicle composition to enhance stability and activity.

• Develop methods to deliver substrate molecules across vesicle membranes.

• Quantify protein incorporation efficiency and vesicle heterogeneity.

What are the key unresolved questions regarding AAE14 structure-function relationships?

Despite significant advances in understanding AAE14, several critical questions about its structure-function relationships remain unaddressed:

1. Structural determinants of substrate specificity:

• Which amino acid residues define the binding pocket for o-succinylbenzoate?

• What structural features differentiate AAE14 from other acyl-activating enzymes with different substrate preferences?

• How does ATP binding and hydrolysis coordinate with substrate binding and product release?

2. Dual targeting mechanisms:

• What molecular features direct AAE14 to both chloroplasts and peroxisomes?

• Are there functional differences between chloroplastic and peroxisomal pools of AAE14?

• How is the distribution between these compartments regulated?

3. Protein dynamics:

• What conformational changes occur during the catalytic cycle?

• How do structural elements contribute to catalytic efficiency?

• Are there allosteric regulation sites that modify enzyme activity?

4. Structure-guided engineering:

• Can rational design approaches enhance AAE14 catalytic efficiency?

• Is it possible to engineer AAE14 variants with altered substrate specificity?

• How might structural modifications affect protein stability and expression?

How can systems biology approaches enhance our understanding of AAE14's role in plant metabolism?

Systems biology offers powerful frameworks to contextualize AAE14 function within broader metabolic networks:

1. Multi-omics integration:

• Combine transcriptomics, proteomics, and metabolomics data from AAE14 mutants.

• Map metabolic flux changes resulting from AAE14 manipulation.

• Identify compensatory responses to AAE14 dysfunction.

2. Network analysis:

• Position AAE14 within global metabolic networks.

• Identify metabolic modules connected to phylloquinone synthesis.

• Predict and validate synthetic lethal gene combinations with AAE14.

• Examine co-expression networks to identify functionally related genes.

3. Computational modeling:

• Develop kinetic models of the phylloquinone synthesis pathway.

• Simulate metabolic flux under varying conditions.

• Predict system-level responses to perturbations in AAE14 activity.

4. Evolutionary systems biology:

• Compare AAE14 network context across plant species.

• Identify co-evolved gene modules related to phylloquinone metabolism.

• Examine AAE14 conservation patterns in relation to photosynthetic adaptations.

What potential biotechnological applications might leverage AAE14 function?

AAE14's pivotal role in phylloquinone synthesis suggests several promising biotechnological applications:

1. Enhancing plant photosynthetic efficiency:

• Engineer plants with optimized AAE14 expression to increase phylloquinone production.

• Evaluate whether increased phylloquinone levels can enhance photosystem I performance under stress conditions.

• Develop stress-responsive AAE14 expression systems to dynamically adjust phylloquinone synthesis.

2. Biofortification strategies:

• Enhance vitamin K content in edible plant tissues through AAE14 engineering.

• Develop tissue-specific expression strategies to target phylloquinone accumulation in harvestable organs.

• Explore whether AAE14 manipulation can increase phylloquinone stability during post-harvest storage.

3. Synthetic biology applications:

• Reconstruct the phylloquinone biosynthetic pathway in heterologous systems.

• Engineer microorganisms for sustainable production of vitamin K.

• Design artificial enzyme complexes incorporating AAE14 for enhanced metabolic channeling.

4. Biosensor development:

• Utilize AAE14 substrate binding properties to develop biosensors for relevant metabolites.

• Create reporter systems that respond to changes in photosynthetic efficiency linked to phylloquinone status.