Recombinant Succinyl-CoA ligase [ADP-forming] subunit alpha (sucD)

What is Succinyl-CoA ligase and what role does the alpha subunit (sucD) play in its function?

Succinyl-CoA ligase (SCS), also known as succinate-CoA ligase or succinyl-CoA synthetase, is a nuclear-encoded mitochondrial enzyme complex that catalyzes the reversible conversion of succinyl-CoA to succinate in the tricarboxylic acid (TCA) cycle . This reaction represents the only substrate-level phosphorylation step in the TCA cycle, generating either ATP or GTP .

The alpha subunit (SucD) has a molecular mass of 29-34 kDa and is responsible for binding CoA during catalysis . It contains a conserved histidine residue that becomes phosphorylated during the reaction mechanism . While the beta subunit (SucC) binds nucleoside triphosphates, the alpha subunit (SucD) provides critical catalytic functionality, and both subunits together form the complete functional enzyme . In eukaryotes, a single alpha subunit (SUCLG1) can partner with either of two beta subunit isoforms (SUCLA2 or SUCLG2) to produce ATP-generating or GTP-generating enzymes, respectively .

How does recombinant sucD differ from native forms, and what experimental advantages does it offer?

Recombinant sucD refers to the laboratory-produced alpha subunit of Succinyl-CoA ligase, typically engineered with features that facilitate research applications. Unlike native forms extracted directly from organisms, recombinant sucD can be:

• Tagged with affinity markers (like His-tags) for simplified purification

• Expressed at higher concentrations than typically found in vivo

• Modified to enhance stability or alter properties for experimental purposes

• Produced in isolation from the beta subunit for individual characterization

The experimental advantages include controlled expression levels, simplified purification protocols, the ability to introduce site-directed mutations to study structure-function relationships, and the capacity to study the alpha subunit's properties independently before reconstituting the complete enzyme complex . Researchers have successfully expressed and purified recombinant SucCD enzymes from various sources, including bacterial species like Advenella mimigardefordensis, using expression systems such as E. coli BL21(DE3)/pLysS .

What are the optimal expression systems for producing recombinant sucD with high yield and activity?

The optimal expression system for recombinant sucD depends on the experimental goals, but several approaches have proven successful:

Bacterial Expression Systems:

• E. coli BL21(DE3)/pLysS has been successfully used for heterologous expression of SucCD enzymes, as demonstrated with the sucCD genes from Advenella mimigardefordensis

• Temperature optimization (typically 18-30°C) is crucial, with lower temperatures often yielding more soluble protein

• IPTG concentration between 0.1-1.0 mM for induction, with lower concentrations sometimes producing more active enzyme

• Co-expression with the beta subunit (SucC) may enhance proper folding and stability

Expression Parameters Table:

Successful expression requires balancing protein yield with proper folding and activity preservation. Specialized vectors containing solubility-enhancing tags (MBP, SUMO) may improve yield of properly folded sucD when expressed independently of the beta subunit .

What purification strategy provides the highest purity and activity for recombinant sucD?

Purification of recombinant sucD typically employs a multi-step approach that balances purity with retention of enzymatic activity:

• Initial Capture: Affinity chromatography using either:

  • Immobilized metal affinity chromatography (IMAC) with Ni-NTA resin for His-tagged proteins

  • Glutathione-Sepharose for GST-fusion proteins

  • Amylose resin for MBP-fusion proteins

• Intermediate Purification:

  • Ion exchange chromatography (typically DEAE or Q-Sepharose) exploiting sucD's pI

  • Hydrophobic interaction chromatography may be effective after ammonium sulfate precipitation

• Polishing:

  • Size exclusion chromatography to separate monomeric sucD from aggregates and other contaminants

  • This step is critical if studying sucD alone, as it separates the alpha subunit from any co-purified beta subunit

Critical Parameters:

• Maintain Mg²⁺ or Mn²⁺ in all buffers (1-5 mM) as these are cofactors for enzyme activity

• Include 10-20% glycerol to enhance protein stability

• Consider adding reducing agents (1-5 mM DTT or 2-ME) to prevent oxidation of cysteine residues

• Appropriate pH (typically 7.5-8.0) to maintain native structure and activity

Highest enzyme activity has been reported when sucD is co-purified with its partner beta subunit, as the functional enzyme is a heterodimer or heterotetramer . When expressed and purified separately, reconstitution experiments may be necessary to fully restore activity.

What are the established methods for measuring recombinant sucD enzyme activity?

Recombinant sucD activity is typically measured as part of the complete Succinyl-CoA synthetase complex, as the alpha subunit alone does not possess full catalytic activity. Several complementary approaches can be used:

Spectrophotometric Coupled Assays:

• Forward Reaction (Succinyl-CoA → Succinate):

  • Coupling with pyruvate kinase and lactate dehydrogenase to monitor ADP/GDP conversion to ATP/GTP through NADH oxidation at 340 nm

  • Reaction mixture typically contains succinyl-CoA, ADP/GDP, Pi, Mg²⁺ or Mn²⁺, and coupling enzymes/substrates

• Reverse Reaction (Succinate → Succinyl-CoA):

  • Monitoring CoA consumption or formation of succinyl-CoA

  • Reaction mixture contains succinate, CoA, ATP/GTP, and Mg²⁺ or Mn²⁺

Direct Product Analysis:

• Liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) to detect and quantify CoA-thioester formation

• This approach has been used to confirm novel activities such as the formation of 3-sulfinopropionyl-CoA (3SP-CoA) and other CoA-thioesters

Radioactive Assays:

• Using ¹⁴C-labeled succinate to track formation of ¹⁴C-succinyl-CoA

• Separation of products by thin-layer chromatography or HPLC

The choice of assay depends on the specific research question, with kinetic studies typically employing spectrophotometric methods while substrate specificity investigations benefit from direct product analysis techniques .

How can substrate specificity of recombinant sucD be comprehensively evaluated?

Evaluating substrate specificity of recombinant sucD (as part of the SucCD complex) requires systematic assessment of activity with structurally related compounds:

Methodological Approach:

• Substrate Panel Selection:

  • Include structural analogs of succinate (e.g., malate, fumarate, glutarate, adipate)

  • Test sulfur-containing analogs like 3-sulfinopropionate (3SP)

  • Include unsaturated analogs such as itaconate

• Multi-Modal Analysis:

  • Initial screening via spectrophotometric assays to identify potential substrates

  • Confirmation of CoA-thioester formation using LC/ESI-MS for positive hits

  • Structural verification of novel CoA-thioesters formed

• Kinetic Parameter Determination:

  • For each viable substrate, determine Km and Vmax values

  • Compare catalytic efficiency (kcat/Km) across substrates

  • Example kinetic parameters: Km values of 2.5-3.6 mM for L-malate compared to the natural substrate succinate

Case Study Data:
Research with SucCD from A. mimigardefordensis demonstrated that while the enzyme showed highest activity with succinate (Vmax = 9.85 ± 0.14 μmol min⁻¹ mg⁻¹), it could also utilize alternative substrates like 3SP . This approach revealed the enzyme's role in degradation pathways for compounds like 3,3'-dithiodipropionic acid (DTDP) .

For comprehensive evaluation, multiple nucleotide cofactors (ATP and GTP) should be tested, as nucleotide preference may vary with different organic acid substrates .

What structural features of sucD are critical for its catalytic function and interaction with the beta subunit?

The sucD (alpha) subunit contains several structural elements critical for its function within the Succinyl-CoA synthetase complex:

Key Structural Features:

• Conserved Phosphorylation Site:

  • A histidine residue in the alpha subunit becomes phosphorylated during the reaction mechanism

  • This phosphohistidine intermediate is crucial for the transfer of the phosphoryl group during catalysis

• CoA Binding Domain:

  • The alpha subunit is responsible for binding CoA during catalysis

  • This domain contains highly conserved residues across species

• Dimer Interface:

  • Residues at the interface between alpha and beta subunits are critical for heterodimer formation

  • While the exact succinate binding site hasn't been definitively located, it is thought to occur at this dimer interface

• Subunit Interaction Regions:

  • In eukaryotes, a single alpha subunit (SUCLG1) can interact with either SUCLA2 (ATP-forming) or SUCLG2 (GTP-forming) beta subunits

  • The structural basis for this dual interaction capability involves specific interface residues

Functional Implications:
Disease-associated variants in SUCLG1 (the human homolog of sucD) often affect conserved amino acids, particularly within domains involved in dimer formation or catalytic activity . These pathogenic variants can lead to metabolic encephalomyopathy, highlighting the critical nature of these structural elements for proper enzyme function .

How can recombinant sucD be used to study protein-protein interactions within the Succinyl-CoA synthetase complex?

Recombinant sucD provides a versatile tool for investigating protein-protein interactions within the Succinyl-CoA synthetase complex through several complementary approaches:

Experimental Methods:

• Co-Immunoprecipitation Studies:

  • Tagged recombinant sucD can be used to pull down interacting partners

  • This approach can identify not only the canonical beta subunit interaction but also potential novel binding partners

• Surface Plasmon Resonance (SPR):

  • Immobilized sucD can be used to quantitatively measure binding kinetics with various beta subunit variants

  • Allows determination of association/dissociation rates and binding affinities

• Yeast Two-Hybrid Screening:

  • Systematic identification of interacting proteins from cDNA libraries

  • Validation of specific interaction domains through deletion constructs

• Reconstitution Experiments:

  • Assembly of functional enzyme complexes from separately purified subunits

  • Assessment of activity recovery provides functional validation of interactions

• FRET/BiFC Approaches:

  • Fusion of fluorescent proteins to sucD and potential partners

  • Enables visualization of interactions in living cells

Research Applications:
These methods can address key questions including:

• The structural basis for nucleotide specificity (ATP vs GTP) when paired with different beta subunits

• Identification of residues essential for heterodimer formation

• Discovery of potential regulatory proteins that interact with the SCS complex

• Investigation of how pathogenic variants in SUCLG1 disrupt protein-protein interactions

Beyond the TCA cycle, what other metabolic pathways involve sucD and how can these be experimentally investigated?

While traditionally recognized for its role in the TCA cycle, the sucD subunit as part of the Succinyl-CoA synthetase complex participates in several other metabolic pathways:

Extended Metabolic Roles:

• Heme Biosynthesis:

  • Succinyl-CoA serves as a critical intermediate in heme synthesis

  • Experimental approach: Track isotope-labeled succinate incorporation into heme molecules

• Ketone Body Metabolism:

  • SCS is involved in ketone breakdown where succinyl-CoA acts as a CoA donor

  • Research method: Measure SCS activity in tissues during ketosis

• Amino Acid and Fatty Acid Catabolism:

  • SCS serves as an entry point for products of amino acid and odd-chain fatty acid catabolism

  • The enzyme connects propionyl-CoA (from isoleucine, threonine, methionine, valine, and odd-chain fatty acids) to the TCA cycle via succinyl-CoA

• Protein Succinylation Regulation:

  • SCS-mediated regulation of succinyl-CoA affects lysine succinylation, a protein modification with widespread metabolic and epigenetic effects

  • Investigation approach: Proteomics analysis of succinylation patterns in sucD-deficient models

• mtDNA Maintenance:

  • Loss of SCS activity is associated with mitochondrial DNA depletion

  • Research direction: Analyze mtDNA copy number in models with altered sucD expression

Experimental Investigation Methods:

These diverse roles can be studied using recombinant sucD in reconstitution experiments, complementation studies in deficient cell lines, or through the analysis of metabolite profiles in systems with altered SCS activity .

What is the relationship between sucD mutations and mitochondrial diseases, and how can recombinant proteins help study these pathologies?

Mutations in SUCLG1 (the human homolog of sucD) are causally linked to severe mitochondrial diseases, particularly mitochondrial encephalomyopathy:

Disease Associations:

• Mitochondrial Encephalomyopathy:

  • Biallelic pathogenic variants in SUCLG1 are associated with a spectrum of mitochondrial disease

  • These disorders typically present with early onset and can be severe, with many patients dying in early childhood

  • Most disease-associated variants lead to changes in conserved amino acids within the protein

• Molecular Pathophysiology:

  • Two primary pathogenic mechanisms are implicated:
    a) Mitochondrial DNA (mtDNA) depletion
    b) Altered protein succinylation patterns with widespread metabolic effects

Applications of Recombinant Proteins:

Recombinant sucD variants modeling patient mutations provide powerful tools for investigating disease mechanisms:

• Functional Characterization:

  • Enzymatic assays with recombinant disease variants can quantify the impact on catalytic activity

  • Kinetic parameters (Km, Vmax) for both forward and reverse reactions reveal mechanistic defects

• Structural Studies:

  • X-ray crystallography or cryo-EM of recombinant variants can identify structural perturbations

  • Computational modeling based on these structures can predict impacts on protein stability and function

• Protein-Protein Interaction Analysis:

  • Investigation of how mutations affect assembly with beta subunits

  • Identification of altered interactions with other mitochondrial proteins

• Cell-Based Models:

  • Complementation studies in patient-derived cells using wild-type or mutant recombinant proteins

  • Analysis of rescue effects on mitochondrial function and metabolism

• Drug Screening Platforms:

  • Recombinant protein variants can serve as targets for high-throughput screening

  • Identification of compounds that might restore function to mutant proteins

These approaches can illuminate the molecular mechanisms underlying SCS deficiency and potentially identify therapeutic strategies for these severe mitochondrial disorders .

How can single-case experimental designs be applied to study recombinant sucD function in cellular models?

Single-case experimental designs (SCEDs) offer powerful approaches for investigating the effects of recombinant sucD interventions in cellular models, particularly for detailed mechanistic studies:

SCED Applications for sucD Research:

• Controlled Introduction Designs:

  • A-B-A-B withdrawal designs where recombinant sucD (wild-type or variant) is introduced, withdrawn, and reintroduced to cells with endogenous sucD knockdown

  • This approach allows each cell line to serve as its own control, reducing variability and required sample sizes

• Changing Criterion Design:

  • Systematic manipulation of recombinant sucD concentration to establish dose-response relationships

  • Particularly useful for determining threshold levels needed for functional complementation

• Multiple Baseline Designs:

  • Introduction of recombinant sucD at staggered timepoints across different cell lines or conditions

  • Helps distinguish intervention effects from time-dependent factors

Methodological Considerations:

• Appropriate Baselines:

  • Establish stable baselines with sufficient data points (minimum 3-5 observations in each phase)

  • Consider the nonindependence of sequential observations (autocorrelation)

• Measurement Approaches:

  • Continuous monitoring of relevant metabolic parameters (oxygen consumption, ATP production)

  • Time-series measurements of TCA cycle metabolites

  • Regular assessment of mtDNA content in complementation studies

• Data Analysis:

  • Visual analysis remains the primary analytical method for SCED

  • Supplement with statistical approaches to address autocorrelation and enhance interpretation

  • Consider randomization components to improve methodological rigor

Experimental Protocol Example:
For investigating a disease-associated sucD variant's ability to rescue mitochondrial function:

• Establish baseline measurements in sucD-deficient cells (Phase A)

• Introduce recombinant wild-type sucD (Phase B)

• Withdraw treatment (return to Phase A)

• Introduce recombinant mutant sucD (Modified Phase B)

This design allows direct comparison of wild-type and mutant protein functionality within the same cellular context, controlling for cell-specific factors and environmental variables .

What advanced analytical techniques can resolve contradictory findings about sucD's role in novel metabolic pathways?

When investigating novel metabolic roles for sucD, researchers may encounter contradictory findings due to the protein's involvement in multiple pathways and context-dependent functions. Advanced analytical approaches can help resolve these contradictions:

Integrated Multi-Omics Approaches:

• Combined Transcriptomics-Proteomics-Metabolomics:

  • Simultaneous analysis of gene expression, protein levels, and metabolite profiles in sucD-modulated systems

  • Identifies compensatory mechanisms that may mask phenotypes

  • Example: RNA-seq combined with targeted metabolomics of TCA cycle intermediates and protein succinylation profiles

• Isotope Tracing and Metabolic Flux Analysis:

  • Use of ¹³C, ¹⁵N, or ³⁴S labeled precursors to track atom movement through metabolic networks

  • Reveals actual metabolic activity rather than just metabolite concentrations

  • Particularly valuable for distinguishing the directionality of reversible reactions catalyzed by SCS

• Spatiotemporal Resolution Techniques:

  • Subcellular fractionation to identify compartment-specific roles of sucD

  • Time-course studies to capture transient metabolic states

  • In situ enzyme activity assays using genetically encoded sensors

Computational and Statistical Approaches:

• Meta-Analysis Framework:

  • Systematic integration of findings across multiple studies

  • Identification of variables that explain divergent results (cell types, experimental conditions)

• Bayesian Network Analysis:

  • Probabilistic modeling of causal relationships between sucD activity and observed phenotypes

  • Incorporation of prior knowledge with new experimental data

• Machine Learning for Pattern Recognition:

  • Identification of subtle metabolic signatures associated with sucD function

  • Classification of experimental conditions that lead to different functional outcomes

Experimental Design Strategies:

• Genetic Interaction Mapping:

  • Systematic analysis of how sucD mutations interact with other genetic perturbations

  • Reveals functional relationships and pathway connections

• Conditional Alleles and Inducible Systems:

  • Temperature-sensitive or chemically-regulatable sucD variants

  • Enables precise temporal control to distinguish direct from adaptive effects

• In Vitro Reconstitution:

  • Bottom-up assembly of metabolic pathways using purified components

  • Direct testing of biochemical capabilities in controlled environments

By combining these approaches, researchers can resolve contradictory findings and develop more comprehensive models of sucD's roles beyond canonical TCA cycle function .

What are the common challenges in producing active recombinant sucD, and how can they be overcome?

Producing active recombinant sucD presents several technical challenges that researchers should anticipate and address:

Common Challenges and Solutions:

• Solubility Issues:

  • Challenge: Formation of inclusion bodies during overexpression

  • Solutions:

    • Lower expression temperature (16-25°C)

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

    • Co-express with chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

    • Optimize induction conditions (lower IPTG concentration, shorter induction time)

• Insufficient Activity:

  • Challenge: Recombinant sucD shows low enzymatic activity

  • Solutions:

    • Co-express with the beta subunit (SucC) as the functional enzyme is heterodimeric/heterotetrameric

    • Ensure presence of metal cofactors (Mg²⁺ or Mn²⁺) in all buffers

    • Verify proper folding through circular dichroism or limited proteolysis

    • Test activity with both ATP and GTP as nucleotide cofactors

• Protein Instability:

  • Challenge: Rapid degradation or activity loss during purification

  • Solutions:

    • Include protease inhibitors in all buffers

    • Add stabilizing agents (10-20% glycerol, low concentrations of substrate)

    • Optimize buffer conditions (pH, ionic strength)

    • Minimize freeze-thaw cycles and store with glycerol at -80°C

• Heterogeneity in Preparations:

  • Challenge: Variable oligomeric states or post-translational modifications

  • Solutions:

    • Employ size exclusion chromatography as a final purification step

    • Validate homogeneity through dynamic light scattering

    • Consider on-column refolding techniques for consistent preparations

Experimental Case Study:
In the expression of SucCD from A. mimigardefordensis, researchers achieved successful production of active enzyme by:

• Using E. coli BL21(DE3)/pLysS as an expression host

• Purifying the complex rather than individual subunits

• Ensuring the presence of divalent cations in assay buffers

• Testing the enzyme with both ATP and GTP for nucleotide preference

This approach yielded enzyme with demonstrable activity toward both natural (succinate) and alternative (3SP) substrates .

How can researchers optimize assay conditions to accurately measure kinetic parameters of recombinant sucD with various substrates?

Accurate determination of kinetic parameters for recombinant sucD with diverse substrates requires careful optimization of assay conditions:

Critical Parameters for Optimization:

• pH and Buffer Composition:

  • Approach: Conduct pH-activity profiles (pH 6.0-9.0) for each substrate

  • Consideration: Different substrates may have different pH optima

  • Recommendation: Test multiple buffer systems (HEPES, Tris, phosphate) at constant ionic strength

• Divalent Cation Requirements:

  • Approach: Systematically vary Mg²⁺ or Mn²⁺ concentrations (0-10 mM)

  • Consideration: SucCD is Mg²⁺ or Mn²⁺ dependent

  • Recommendation: Test both cations as preference may vary with different substrates

• Nucleotide Cofactor Selection:

  • Approach: Compare activity with ATP vs. GTP for each substrate

  • Consideration: SucCD can utilize either nucleotide, but efficiency may vary

  • Recommendation: Determine Km values for each nucleotide with each substrate

• Substrate Concentration Ranges:

  • Approach: Use a wide concentration range spanning at least 0.1-10× Km

  • Consideration: For alternative substrates like 3SP, malate, or itaconate, Km values may differ substantially from succinate

  • Recommendation: Preliminary experiments to estimate approximate Km before detailed kinetic analysis

Data Analysis Considerations:

• Appropriate Kinetic Models:

  • Apply Michaelis-Menten kinetics for simple substrate relationships

  • Consider allosteric models if substrate inhibition or activation is observed

  • Use competitive inhibition models when testing structural analogs

• Statistical Validation:

  • Perform replicate measurements (minimum n=3) for reliable parameter estimation

  • Calculate confidence intervals for all kinetic parameters

  • Use goodness-of-fit tests to validate model selection

Assay Optimization Table for Novel Substrates:

By systematically optimizing these parameters, researchers can obtain reliable kinetic data for comparing natural and alternative substrates of recombinant sucD, as demonstrated in studies showing Km values of 2.5-3.6 mM for L-malate compared to succinate .