Recombinant Solanum lycopersicum Succinyl-CoA ligase [ADP-forming] subunit alpha-2, mitochondrial

What is the structure and function of Succinyl-CoA ligase in Solanum lycopersicum?

Succinyl-CoA ligase in Solanum lycopersicum (tomato) is a heterodimer enzyme composed of an α-subunit and a β-subunit, with the α-subunit generally considered catalytic and the β-subunit regulatory . The enzyme is located in the mitochondrial matrix and catalyzes the reversible conversion of succinyl-CoA and ADP to CoASH, succinate, and ATP within the TCA cycle . This reaction has a ΔG of approximately 0.07 kJ/mol, making it highly reversible . The enzyme functions at the intersection of multiple metabolic pathways, including the citric acid cycle, heme metabolism, ketone body metabolism, and amino acid catabolism .

How does Succinyl-CoA ligase contribute to metabolic networks beyond the TCA cycle?

Succinyl-CoA ligase participates in numerous metabolic pathways beyond its canonical role in the TCA cycle:

• Amino acid and fatty acid catabolism: It serves as an entry point for metabolites derived from odd-chain fatty acids and the catabolism of isoleucine, threonine, methionine, and valine .

• Protein post-translational modifications: Succinyl-CoA serves as the coenzyme A donor for lysine succinylation, a protein modification linked to widespread metabolic and epigenetic effects .

• Mitochondrial DNA maintenance: SCS plays a critical role in maintaining mtDNA content through the provision of phosphorylated deoxyribonucleotides .

• GABA shunt: In specialized cells of the brain and in plants, succinate is the entry point to the citric acid cycle of the 'GABA shunt' .

• Heme synthesis: Succinyl-CoA is a critical intermediate in the heme biosynthetic pathway .

These diverse roles highlight the central importance of this enzyme in cellular metabolism and explain why alterations in its activity can have pleiotropic effects on multiple pathways.

Why does severe reduction in Succinyl-CoA ligase activity produce only mild phenotypes in tomato plants?

Despite dramatic reductions in Succinyl-CoA ligase activity (down to 5% of wild-type levels) in transgenic tomato plants, researchers observed surprisingly mild phenotypic effects . This apparent paradox can be explained by several mechanisms:

• Metabolic plasticity: Plants can upregulate alternative pathways for succinate production, particularly the γ-aminobutyric acid (GABA) shunt, which compensates for reduced SCoAL activity .

• Metabolic remodeling: Analysis of metabolite profiles in SCoAL-reduced plants showed increases in TCA cycle intermediates upstream of succinate (isocitrate, citrate, and 2-oxoglutarate) and elevated levels of GABA, indicating metabolic adjustments that help maintain homeostasis .

• Threshold effect: The remaining low levels of enzyme activity may be sufficient to maintain essential functions, particularly if the enzyme normally operates well below its maximal capacity.

• Reversibility of the reaction: The highly reversible nature of the SCoAL reaction (ΔG ≈ 0.07 kJ/mol) may allow for metabolic adaptations that reduce the impact of decreased enzyme activity .

This metabolic flexibility demonstrates the remarkable adaptability of plant metabolism and suggests that SCoAL may not be rate-limiting for mitochondrial respiration in tomato plants under normal conditions.

What are the established methods for measuring Succinyl-CoA ligase activity in plant tissues?

Accurate measurement of Succinyl-CoA ligase activity in plant tissues requires careful consideration of extraction methods and assay conditions. The following approaches have proven effective:

For the most reliable results, researchers should:

• Carefully extract intact mitochondria to preserve enzyme activity

• Include appropriate controls to account for background activity

• Validate findings using complementary approaches

• Consider tissue-specific and developmental variations in activity

The cycling assay has been successfully employed to measure dramatically reduced SCoAL activity (down to 5% of wild-type levels) in transgenic tomato plants .

How can researchers generate and validate transgenic plants with altered Succinyl-CoA ligase expression?

Creating transgenic plants with altered Succinyl-CoA ligase expression requires a systematic approach:

• Genetic modification strategies:

  • Antisense orientation: Express a fragment of the target gene in reverse orientation to reduce endogenous expression

  • RNA interference (RNAi): Use double-stranded RNA to trigger targeted mRNA degradation, which has achieved up to 95% reduction in SCoAL activity

  • CRISPR/Cas9: Enable precise gene editing for knockout, knockdown, or specific mutations

• Comprehensive validation protocol:

  • Molecular confirmation: PCR and sequencing to verify transgene integration

  • Expression analysis: RT-qPCR to quantify transcript levels

  • Protein detection: Western blotting with specific antibodies

  • Enzyme activity: Cycling assays to measure functional impact

  • Metabolite profiling: GC-MS to assess TCA cycle intermediates and related metabolites

• Phenotypic characterization:

  • Growth parameters and developmental timing

  • Photosynthetic capacity and respiratory rates

  • Response to environmental stresses

  • Tissue-specific effects

Researchers should generate multiple independent transformation lines to account for positional effects and variability in transgene expression. The use of appropriate controls, including wild-type and empty vector controls, is essential for accurate interpretation of results.

What bioinformatic approaches can help predict structure-function relationships in Succinyl-CoA ligase?

Bioinformatic approaches offer valuable insights into structure-function relationships of Succinyl-CoA ligase without requiring extensive experimental work:

A case study of ATP synthase (another mitochondrial enzyme) demonstrates how structural analysis can predict functional effects of mutations: a single de novo mutation (Arg207His) in the α-subunit creates a negatively-charged side chain that interferes with the stability of the α-β interface, causing severe enzyme dysfunction that surprisingly resolves during development .

How does the regulation of Succinyl-CoA ligase subunits differ between plant and animal systems?

The regulation of Succinyl-CoA ligase subunits reveals both conserved mechanisms and divergent strategies between plant and animal systems:

In mice, partial reduction in Sucla2 (ATP-forming) elicits rebound increases in Suclg2 (GTP-forming) expression, a compensation mechanism so dominant it can overcome even a concomitant deletion of one Suclg2 allele . In contrast, plants appear to rely more on alternative metabolic pathways rather than isoform compensation, highlighting the evolved differences in metabolic regulation between kingdoms.

What is the relationship between Succinyl-CoA ligase activity and protein succinylation in plant systems?

Protein succinylation is an emerging post-translational modification with significant regulatory implications. The relationship between Succinyl-CoA ligase and protein succinylation in plants involves several key aspects:

Understanding this relationship offers potential for metabolic engineering strategies targeting not only direct pathway flux but also the broader regulatory landscape affected by protein modifications.

How do mtDNA maintenance mechanisms relate to Succinyl-CoA ligase function across different species?

Mitochondrial DNA (mtDNA) maintenance is critically linked to Succinyl-CoA ligase function, though with species-specific variations:

• Mechanistic connection: SUCL associates with nucleotide diphosphate kinase, which is important for providing phosphorylated deoxyribonucleotides required for mtDNA synthesis and maintenance . This physical and functional interaction creates a direct link between metabolism and mtDNA homeostasis.

• Clinical evidence: In humans, deficiency of SUCL leads to mitochondrial DNA depletion syndrome, often associated with encephalomyopathy . This underscores the essential role of SUCL in maintaining mtDNA integrity in mammals.

• Animal model insights: Studies in mice show that Sucla2 heterozygote animals exhibit moderate decreases in mtDNA content, supporting the connection between SUCL activity and mtDNA maintenance .

• Plant systems: The relationship between SCoAL and mtDNA in plants is less characterized, though the conservation of basic mitochondrial functions suggests similar mechanisms may exist.

• Evolutionary conservation: The association between SUCL and mtDNA maintenance appears to be conserved across diverse eukaryotic lineages, suggesting an ancient and fundamental connection between central metabolism and mitochondrial genome stability.

The dual role of SUCL in both metabolism and mtDNA maintenance exemplifies the integrated nature of mitochondrial functions and highlights how defects in metabolic enzymes can have far-reaching consequences beyond their primary pathways.

What metabolic flux analysis techniques are most appropriate for studying Succinyl-CoA ligase function in tomato?

Metabolic flux analysis (MFA) provides crucial insights into the dynamic operation of metabolic networks. For studying Succinyl-CoA ligase function in tomato, several complementary approaches are recommended:

• Steady-state 13C-MFA:

  • Feed plants with 13C-labeled substrates (glucose, pyruvate, or glutamate)

  • Analyze isotopic enrichment patterns in TCA cycle intermediates

  • Use computational models to calculate flux distributions

  • Compare flux maps between wild-type and SCoAL-modified plants

• Non-stationary 13C-MFA:

  • Track isotope incorporation over time to capture dynamic flux changes

  • Particularly valuable for plant systems with multiple metabolic pools

  • Provides higher resolution of fluxes in cyclic pathways like the TCA cycle

• Multi-omics integration:

  • Combine flux data with:

    • Transcriptomics (gene expression changes)

    • Proteomics (enzyme abundance)

    • Metabolomics (metabolite concentrations)

  • Build comprehensive models of metabolic adaptation

• Tissue-specific analysis:

  • Separate analysis of different plant tissues (leaves, fruits, roots)

  • Microdissection techniques for analyzing specific cell types

  • Comparison of developmental stages

• Environmental perturbation:

  • Flux analysis under different growth conditions (light/dark, stress)

  • Reveals condition-dependent roles of SCoAL

This multi-faceted approach can reveal how metabolic networks reorganize in response to altered SCoAL activity, identifying potential compensatory pathways such as the GABA shunt that was shown to be upregulated in tomato plants with reduced SCoAL activity .

How should researchers design experiments to investigate the role of Succinyl-CoA ligase in stress responses?

Investigating the role of Succinyl-CoA ligase in plant stress responses requires a systematic experimental design:

• Experimental setup:

• Multi-level analysis approach:

  • Physiological parameters:

    • Photosynthetic efficiency

    • Respiratory rates

    • Growth metrics

    • Visible stress symptoms

  • Biochemical measurements:

    • SCoAL enzyme activity under stress conditions

    • TCA cycle flux using isotope labeling

    • ROS production and antioxidant capacity

    • Energy status (ATP/ADP ratio)

  • Molecular analyses:

    • Transcript levels of SCoAL and related genes

    • Protein abundance and post-translational modifications

    • Metabolite profiling focusing on TCA cycle intermediates

    • Protein succinylation patterns

• Mechanistic validation:

  • Complementation experiments to confirm specificity

  • Pharmacological interventions targeting specific pathways

  • Cell-type specific analyses to identify primary responders

This comprehensive approach will reveal whether SCoAL serves as a metabolic sensor during stress conditions and how its activity influences stress adaptation mechanisms in plants.

What approaches can identify proteins regulated by succinylation in relation to Succinyl-CoA ligase activity?

Identifying the protein succinylation landscape in relation to Succinyl-CoA ligase activity requires sophisticated proteomic approaches:

• Global succinylome analysis:

  • Enrich succinylated peptides using anti-succinyl-lysine antibodies

  • Perform liquid chromatography-tandem mass spectrometry (LC-MS/MS)

  • Compare succinylation patterns between wild-type and SCoAL-modified plants

  • Quantify using SILAC, TMT, or label-free quantification methods

• Site-specific functional validation:

  • Identify critical succinylation sites on key metabolic enzymes

  • Generate site-directed mutants (K→R to prevent succinylation; K→Q to mimic succinylation)

  • Assess effects on enzyme activity, protein-protein interactions, and stability

  • Correlate with metabolic phenotypes

• Dynamic succinylation analysis:

  • Monitor temporal changes in succinylation in response to:

    • Metabolic perturbations

    • Environmental stresses

    • Developmental transitions

  • Identify regulatory sites with high turnover rates

• Succinylation enzyme identification:

  • Investigate potential succinyltransferases in plants

  • Characterize desuccinylases (primarily sirtuins)

  • Determine how these enzymes respond to changes in SCoAL activity

The discovery that disruption of succinyl-CoA ligase in yeast leads to a 3-fold increase in protein succinylation suggests that similar regulatory mechanisms may exist in plants, potentially affecting numerous metabolic pathways beyond the TCA cycle.

How can researchers distinguish between direct and indirect effects of altered Succinyl-CoA ligase activity?

Distinguishing between direct and indirect effects of altered Succinyl-CoA ligase activity presents a significant challenge in metabolic research. A multi-faceted approach is necessary:

• Temporal analysis:

  • Monitor metabolic changes at multiple time points after SCoAL activity reduction

  • Early changes (hours) likely represent direct effects

  • Later changes (days) may reflect compensatory or secondary responses

  • Time-course experiments can establish causality chains

• Concentration-dependent responses:

  • Create an allelic series with varying degrees of SCoAL activity reduction

  • Plot metabolic parameters against enzyme activity levels

  • Direct effects typically show linear relationships with enzyme activity

  • Indirect effects may exhibit threshold responses

• Metabolic network analysis:

  • Map observed changes onto metabolic pathway maps

  • Changes in direct substrates and products (succinyl-CoA, succinate, ATP/GTP) are likely direct effects

  • Changes in distant metabolites require intermediate steps and are likely indirect

  • Network modeling can predict expected propagation of metabolic perturbations

• Complementary approaches:

  • In vitro enzyme assays to confirm direct biochemical effects

  • Isotope labeling to track metabolic flux changes

  • Pharmacological interventions to block specific pathways

  • Genetic suppressor screens to identify compensatory mechanisms

In tomato plants with reduced SCoAL activity, researchers observed increases in TCA cycle intermediates upstream of succinate (isocitrate, citrate, and 2-oxoglutarate) as direct effects, while increases in GABA likely represented an indirect effect through activation of the GABA shunt as a compensatory mechanism .

What control experiments are essential when analyzing metabolite changes in plants with altered Succinyl-CoA ligase?

Robust control experiments are crucial for reliable interpretation of metabolite changes in plants with altered Succinyl-CoA ligase:

• Essential control groups:

• Methodological controls:

  • Internal standards for metabolite quantification

  • Technical replicates to assess measurement variability

  • Sample processing controls to account for degradation or modification

  • Recovery standards to measure extraction efficiency

  • Randomized sample processing order to minimize batch effects

• Environmental controls:

  • Strictly controlled growth conditions (light, temperature, humidity)

  • Time of day standardization (circadian rhythm effects)

  • Consistent tissue sampling protocols

  • Minimized stress during sampling

• Validation experiments:

  • Orthogonal analytical methods for key metabolites

  • Enzyme activity assays to confirm functional changes

  • Transcript analysis of related pathway genes

  • In vitro experiments to confirm direct biochemical effects

When analyzing metabolite changes in SCoAL-modified tomato plants, researchers incorporated appropriate controls that allowed them to identify specific metabolic signatures, including increases in TCA cycle intermediates upstream of succinate and activation of the GABA shunt .

How should researchers interpret conflicting results between transcript, protein, and metabolite levels in Succinyl-CoA ligase studies?

Conflicting results between transcript, protein, and metabolite levels are common in metabolic studies and require careful interpretation:

• Multi-level regulatory mechanisms:

  • Transcriptional regulation: Changes in gene expression

  • Post-transcriptional regulation: mRNA stability, translation efficiency

  • Post-translational regulation: Protein stability, modifications, allosteric control

  • Metabolic regulation: Substrate availability, product inhibition, metabolite signaling

• Temporal considerations:

  • Different response times at each level:

    • Transcriptional changes: Hours

    • Protein abundance changes: Hours to days

    • Metabolic adjustments: Minutes to hours

  • Time-course sampling can reveal sequential regulatory events

• Tissue and subcellular heterogeneity:

  • Bulk tissue measurements may mask cell-type specific responses

  • Changes in subcellular localization without total abundance changes

  • Redistribution between different metabolic pools

• Technical limitations:

  • Different detection sensitivities across platforms

  • Linear range limitations in different analytical methods

  • Specific isoform detection challenges

• Biological interpretation strategies:

  • Integrate data using pathway-based approaches

  • Consider enzyme kinetics and metabolic control analysis principles

  • Identify rate-limiting steps that explain non-intuitive relationships

  • Develop mathematical models that incorporate multiple regulatory layers

In studies of tomato plants with reduced SCoAL β-subunit expression, researchers observed that dramatic reductions in enzymatic activity (down to 5% of wild-type levels) resulted in surprisingly mild phenotypic effects, highlighting the importance of integrating multiple data types to understand complex metabolic adaptations .

What emerging technologies could revolutionize our understanding of Succinyl-CoA ligase in plant metabolism?

Several cutting-edge technologies promise to transform our understanding of Succinyl-CoA ligase in plant metabolism:

• Single-cell omics technologies:

  • Single-cell transcriptomics to reveal cell-type specific expression patterns

  • Single-cell metabolomics to capture metabolic heterogeneity

  • Spatial transcriptomics/metabolomics to map SCoAL activity across tissues

  • These approaches would reveal how SCoAL function varies across different plant cell types

• Real-time metabolic monitoring:

  • Genetically encoded biosensors for key metabolites (succinyl-CoA, succinate)

  • FRET-based sensors for enzyme activity

  • Live-cell imaging of metabolic dynamics

  • Optogenetic control of enzyme activity

• Advanced structural biology:

  • Cryo-electron microscopy of plant SCoAL at near-atomic resolution

  • Time-resolved structural analysis during catalytic cycle

  • In situ structural studies within mitochondrial context

  • These would reveal precise mechanistic details of SCoAL function

• Genome editing advances:

  • Base editing for precise single-nucleotide modifications

  • Prime editing for targeted sequence replacements

  • Multiplex CRISPR for simultaneous modification of multiple pathways

  • Inducible/reversible gene regulation systems

• Systems biology integration:

  • Multi-omics data integration through machine learning

  • Whole-cell metabolic modeling incorporating enzyme kinetics

  • Digital plant twins for predicting metabolic responses to environmental changes

  • Network analysis tools to map enzyme-enzyme and enzyme-metabolite interactions

These technologies would allow researchers to move beyond static measurements to dynamic, spatially resolved understanding of SCoAL's role in plant metabolism, potentially revealing novel regulatory mechanisms and metabolic interactions.

How might our understanding of Succinyl-CoA ligase inform metabolic engineering strategies for crop improvement?

Insights into Succinyl-CoA ligase function offer numerous opportunities for metabolic engineering strategies in crops:

• Stress resilience enhancement:

  • Fine-tuning SCoAL activity to optimize energy production under stress conditions

  • Engineering regulatory elements to improve dynamic responses to environmental challenges

  • Exploiting the connection between SCoAL and the GABA shunt to enhance stress adaptation

• Yield and growth optimization:

  • Balancing carbon flux between the TCA cycle and biosynthetic pathways

  • Engineering tissue-specific SCoAL activity to match metabolic demands

  • Optimizing energetic efficiency of mitochondrial metabolism

• Nutritional quality improvement:

  • Modifying amino acid metabolism linked to SCoAL activity

  • Enhancing vitamin precursor synthesis (e.g., heme-derived compounds)

  • Reducing anti-nutritional factors through altered metabolic flux

• Engineering considerations:

  • Target precision: Specific mutations rather than complete knockdowns

  • Tissue specificity: Using tissue-specific promoters

  • Developmental regulation: Conditional expression systems

  • Compensatory mechanisms: Simultaneous engineering of connected pathways

• Potential applications table:

The finding that tomato plants can tolerate dramatic reductions in SCoAL activity suggests considerable flexibility for metabolic engineering without compromising essential functions .

What are the most promising research questions regarding the evolutionary conservation of Succinyl-CoA ligase function?

The evolutionary conservation of Succinyl-CoA ligase across diverse organisms raises intriguing research questions:

• Structural evolution of SCoAL:

  • How have the catalytic and regulatory domains evolved across prokaryotes, plants, and animals?

  • What structural features determine substrate specificity (ATP vs. GTP formation)?

  • How has the interaction between α and β subunits evolved?

  • Are there lineage-specific structural adaptations that reflect metabolic demands?

• Functional diversification:

  • When did the dual roles in metabolism and mtDNA maintenance emerge during evolution?

  • How did the connection to protein succinylation evolve as a regulatory mechanism?

  • What explains the different phenotypic consequences of SCoAL deficiency across species?

  • Have different organisms evolved distinct compensatory mechanisms for reduced SCoAL activity?

• Regulatory evolution:

  • How have transcriptional and post-translational regulatory mechanisms of SCoAL evolved?

  • Do different organisms use similar or distinct allosteric regulators of SCoAL activity?

  • Has the role of SCoAL in stress responses evolved differently across lineages?

  • What selective pressures have shaped SCoAL evolution in different environments?

• Metabolic network integration:

  • How has SCoAL's position at the intersection of multiple metabolic pathways been conserved or modified?

  • Has the relationship between SCoAL and the GABA shunt evolved differently in plants versus animals?

  • How do different organisms balance the dual use of succinyl-CoA for energy metabolism and as a cofactor?

The contrast between severe consequences of SCoAL deficiency in mammals versus mild effects in plants suggests fascinating evolutionary divergence in metabolic regulation that warrants further investigation.