Recombinant Succinyl-CoA ligase [ADP-forming] subunit beta (sucC)

What is the biochemical function of Succinyl-CoA ligase in cellular metabolism?

Succinyl-CoA ligase (SUCL), also known as succinyl coenzyme A synthetase or succinate thiokinase, catalyzes the reversible conversion of succinyl-CoA and ADP (or GDP) to CoASH, succinate, and ATP (or GTP) in the mitochondrial matrix . This reaction represents the only substrate-level phosphorylation step in the TCA cycle .

The enzyme exists as a heterodimer composed of an invariant α-subunit (SUCLG1) and a substrate-specific β-subunit that can be either SUCLA2 (for ADP-forming reactions) or SUCLG2 (for GDP-forming reactions) . The beta subunit provides nucleotide specificity and binds succinate, while the alpha subunit contains binding sites for coenzyme A and phosphate .

Beyond its canonical role in the TCA cycle, SUCL serves as:

• An entry point for amino acid and fatty acid catabolism through the propionyl-CoA pathway

• A critical component in ketone body metabolism

• An intermediate in heme synthesis

• A factor in mtDNA maintenance through association with nucleotide diphosphate kinase

How can researchers accurately measure Succinyl-CoA ligase activity in experimental settings?

Succinyl-CoA ligase activity can be measured spectrophotometrically using the following methodology:

• Reaction direction: Activity is commonly measured in the direction of succinate to succinyl-CoA (reverse physiological direction) due to easier assay setup .

• Standard assay mixture composition:

  • 50 mM potassium phosphate buffer (pH 7.2)

  • 10 mM MgCl₂

  • 0.2 mM succinyl-CoA

  • 2 mM ADP (for A-SCS activity) or 1 mM GDP (for G-SCS activity)

  • 0.2 mM DTNB (5,5'-dithiobis(2-nitrobenzoic acid))

• Reaction initiation: The reaction is initiated by adding succinyl-CoA and DTNB in quick succession to the assay mixture containing cell lysates or purified enzyme .

• Detection method: The formation of thionitrobenzoate (TNB) is monitored at 412 nm. The molar extinction coefficient for TNB is 13,600 M⁻¹cm⁻¹ .

• Controls and corrections: Rates should be corrected by subtracting the rate observed in the absence of ADP or GDP. Control reactions should demonstrate that the observed activities are dependent on magnesium, succinyl-CoA, and nucleoside diphosphate .

• Quantification: Activities are typically calculated as nmoles/min/mg protein and expressed as a percentage of control activity .

What expression systems are optimal for producing functional recombinant sucC protein?

For producing functional recombinant sucC protein, the following expression systems and conditions have proven effective:

• Expression host: Escherichia coli is the most commonly used expression system, particularly the BD7G strain which incorporates GroELS chaperone co-expression .

• Protein solubility optimization: Co-expression with GroELS chaperone in the BD7G strain has been shown to enhance enzyme solubility to over 90%, achieving yields of 1.155 g of protein per liter .

• Expression vector considerations:

  • T7 promoter systems generally yield higher expression levels compared to T5 promoter systems

  • N-terminal and/or C-terminal tags can be incorporated to facilitate purification

  • Tag selection should consider tag-protein stability interactions

• Purification strategies:

  • Affinity chromatography using the incorporated tags

  • Ion-exchange chromatography

  • Size-exclusion chromatography for final polishing

• Functional validation: Activity assays should be performed on the purified recombinant protein to ensure proper folding and catalytic function .

What are the key structural features of recombinant sucC that influence its activity?

Several key structural features of the sucC subunit are critical for its catalytic function:

• Nucleotide binding domain: The beta subunit contains a nucleotide grasp domain that determines specificity for ADP vs. GDP . This domain includes multiple conserved lysine residues that are essential for substrate binding.

• Conserved lysine residues: Several lysine residues in SUCLA2 (Lys108, Lys116, and Lys143) are located on the edge of the ADP-binding cleft and are critical for substrate binding through interactions with negatively charged residues (Glu193, Asp194, and Glu198) on the opposite side of the binding cleft .

• Succinyl-CoA binding site: Lys66 in the alpha subunit (SUCLG1) is positioned close to the negatively charged phosphate groups of succinyl-CoA and is critical for substrate binding .

• Post-translational modifications: Lysine succinylation of these conserved residues can alter local charge distribution, affecting substrate binding affinity. For example:

  • Succinylation of Lys66 in SUCLG1 changes local charge, potentially decreasing affinity for succinyl-CoA

  • Succinylation of Lys108, Lys116, and Lys143 in SUCLA2 changes local positive charge to negative, potentially decreasing affinity for ADP

These structural insights can guide protein engineering approaches to enhance enzyme activity or substrate specificity.

How does SUCLA2 contribute to cancer metastasis independent of its conventional metabolic role?

Recent research has uncovered a novel, non-canonical function of SUCLA2 in promoting cancer metastasis:

• Subcellular relocalization: Upon cancer cell detachment, SUCLA2 (but not the alpha subunit of the enzyme complex) relocates from mitochondria to the cytosol .

• Stress granule formation: In the cytosol, SUCLA2 binds to and promotes the formation of stress granules .

• Antioxidant enzyme translation: SUCLA2-mediated stress granules facilitate the protein translation of antioxidant enzymes, particularly catalase .

• Oxidative stress mitigation: The increased catalase expression mitigates oxidative stress that typically occurs during cell detachment .

• Anoikis resistance: By reducing oxidative stress, SUCLA2 renders cancer cells resistant to anoikis (detachment-induced cell death), which is a critical step in cancer metastasis .

• Clinical correlation: SUCLA2 expression correlates with catalase levels and metastatic potential in lung and breast cancer patients .

This metastasis-promoting function of SUCLA2 is independent of its role in the TCA cycle and represents a unique mechanism that cancer cells co-opt to facilitate metastasis, suggesting SUCLA2 as a potential therapeutic target for anti-metastatic strategies.

What mechanisms underlie mitochondrial DNA depletion in SUCL deficiency disorders?

Mitochondrial DNA (mtDNA) depletion is a hallmark feature of SUCL deficiency disorders, and several interconnected mechanisms have been proposed:

• Nucleotide metabolism disruption: SUCL forms a complex with mitochondrial nucleoside diphosphate kinase (NDPK), which is crucial for maintaining balanced deoxyribonucleotide pools required for mtDNA replication .

• Tissue-specific effects: The severity of mtDNA depletion varies by tissue and correlates with the expression patterns of SUCLA2 (predominantly in brain, heart, and muscle) and SUCLG2 (primarily in liver and kidney) .

• Compensatory mechanisms: In heterozygous mouse models, deletion of one Sucla2 allele leads to rebound increases in Suclg2 expression, which can partially compensate for the deficiency .

• Progressive nature: mtDNA depletion becomes more severe over time, contributing to the progressive nature of clinical symptoms .

• Differential outcomes:

  • In Sucla2 heterozygote mice, mtDNA content is moderately decreased (typically to 50-65% of normal)

  • In double heterozygote mice (with deletion of one Sucla2 and one Suclg2 allele), more pronounced changes in mtDNA content are observed

Understanding these mechanisms is crucial for developing potential therapeutic approaches for SUCL deficiency disorders.

How does protein succinylation resulting from SUCLA2 deficiency affect cellular functions?

SUCLA2 deficiency leads to accumulation of succinyl-CoA, resulting in increased global protein succinylation with significant functional consequences:

• Mechanism of increased succinylation:

  • Disruption of the succinyl-CoA ligase reaction increases the cellular pool of succinyl-CoA

  • Elevated succinyl-CoA serves as a donor for both enzymatic and non-enzymatic protein succinylation reactions

• Global protein succinylation profile:

  • Mass spectrometry analysis reveals widespread succinylation of proteins in various cellular compartments

  • TCA cycle proteins are particularly susceptible to succinylation, with most carrying several succinylation sites

• Autoregulatory feedback loop:

  • SUCL itself becomes succinylated at multiple conserved lysine residues

  • Succinylation of SUCL lysines is predicted to decrease substrate binding affinity, potentially functioning as a negative feedback mechanism

• Regulation by desuccinylase SIRT5:

  • SIRT5 is the primary enzyme responsible for removing succinyl modifications from proteins

  • In SIRT5-deficient models, succinylation of TCA cycle proteins increases dramatically

  • Increased SIRT5 activity can reverse pathological protein succinylation in disease models

• Functional consequences:

  • Disruption of enzyme activities in various metabolic pathways

  • Altered protein-protein interactions

  • Changes in protein stability and subcellular localization

  • Potential epigenetic effects through histone succinylation

These findings highlight protein succinylation as a crucial mechanism linking SUCLA2 deficiency to cellular dysfunction.

What strategies can researchers employ for designing selective inhibitors of Succinyl-CoA ligase as potential therapeutic targets?

Designing selective inhibitors for Succinyl-CoA ligase presents unique challenges and opportunities:

• Structural considerations:

  • Target the unique nucleotide binding pocket of the beta subunit

  • Exploit differences between SUCLA2 and SUCLG2 isoforms for selective targeting

  • Focus on key catalytic residues identified through structural analyses (e.g., Lys108, Lys116, and Lys143 in SUCLA2)

• Rational inhibitor design approaches:

  • Structure-based design using crystal structures of sucC

  • Fragment-based screening to identify initial binding scaffolds

  • Computer-aided drug design leveraging the known structural information

  • Modification of known substrate analogs or transition state mimics

• Potential inhibition mechanisms:

  • Competitive inhibition at the ADP/GDP binding site

  • Allosteric inhibition affecting subunit interactions or conformational changes

  • Covalent modification of critical residues

  • Disruption of protein-protein interactions (e.g., between SUCLA2 and stress granule components)

• Context-specific targeting:

  • For cancer applications, target the cytosolic functions of SUCLA2 rather than its mitochondrial role to minimize metabolic side effects

  • In other contexts, consider tissue-specific distribution of SUCLA2 vs. SUCLG2 to minimize off-target effects

• Validation methodologies:

  • Enzymatic assays to measure SCS activity inhibition

  • Cell-based assays focused on specific pathways (e.g., stress granule formation, catalase expression)

  • Animal models of SUCL-associated diseases

This multifaceted approach can guide the development of selective SUCL inhibitors for potential therapeutic applications in cancer metastasis or other contexts.

How do enzymatic properties differ between recombinant sucC from different bacterial sources?

The enzymatic properties of succinyl-CoA ligase can vary significantly between different bacterial sources, with implications for research applications:

• Substrate specificity variations:

  • While succinate is the primary substrate, SucCD enzymes from different bacteria show varying abilities to form CoA-thioesters with alternative substrates

  • Liquid chromatography/electrospray ionization-mass spectrometry analyses have confirmed the ability of various SucCD enzymes to form CoA-thioesters of adipate, glutarate, and fumarate

  • Some bacterial SucCD enzymes can activate 3-sulfinopropionate (3SP) to 3SP-CoA, though this is not universal across all species

• Kinetic parameters for alternative substrates:

  • Km values for L-malate range from 2.5 to 3.6 mM across different bacterial SucCD enzymes

  • Km values for D-malate range from 3.6 to 4.2 mM

  • Activities with these alternative substrates are typically much lower than with succinate

• Evolutionary relationships:

  • Structural comparisons suggest a strong resemblance between SucCD and L-malate-CoA ligase

  • This similarity suggests that malate-CoA ligases and succinate-CoA ligases may share the same evolutionary origin

• Temperature and pH optima:

  • Optimal temperature for activity varies between species, with most showing maximum activity between 30°C and 37°C

  • pH optima typically range from 7.0 to 7.5, though some bacterial enzymes show activity across a broader pH range

These differences highlight the importance of source selection when using recombinant sucC for specific research applications, particularly when alternative substrate utilization is relevant.

What are the transgenic mouse models available for studying SUCL subunit functions, and what phenotypes do they exhibit?

Several transgenic mouse models have been developed to study SUCL subunit functions:

• Available transgenic models:

  • Sucla2 heterozygote mice (with deletion of one Sucla2 allele)

  • Suclg2 heterozygote mice (with deletion of one Suclg2 allele)

  • Double heterozygote mice (with deletion of one Sucla2 and one Suclg2 allele)

• Phenotype of Sucla2 heterozygote mice:

  • Tissue- and age-dependent decreases in Sucla2 expression

  • Decreased ATP-forming activity

  • Rebound increases in cardiac Suclg2 expression and GTP-forming activity

  • Normal bioenergetic parameters, including substrate-level phosphorylation (SLP), unless a submaximal pharmacological inhibition of SUCL is present

  • Moderately decreased mtDNA content

  • Significantly elevated blood carnitine esters

• Phenotype of Suclg2 heterozygote mice:

  • Decreased Suclg2 expression

  • No rebound increases in Sucla2 expression

  • No changes in bioenergetic parameters

• Phenotype of double heterozygote mice:

  • Rebound but protracted increase in Suclg2 expression

  • No alterations in GTP-forming activity or SLP

  • More pronounced changes in mtDNA content and blood carnitine esters

  • Increased succinate dehydrogenase activity

• Compensation mechanisms:

  • Partial reduction in Sucla2 elicits rebound increases in Suclg2 expression

  • This compensatory mechanism is sufficiently dominant to overcome even a concomitant deletion of one Suclg2 allele

  • These compensatory changes pleiotropically affect metabolic pathways associated with SUCL