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

What is Succinyl-CoA ligase [ADP-forming] subunit beta (sucC) and what role does it play in E. coli metabolism?

Succinyl-CoA ligase [ADP-forming] subunit beta (sucC) is a critical enzyme component in E. coli's tricarboxylic acid (TCA) cycle, catalyzing the reversible conversion of succinyl-CoA to succinate while generating ADP from AMP and inorganic phosphate. This reaction represents a crucial substrate-level phosphorylation step in the TCA cycle. The beta subunit (sucC) provides nucleotide specificity to the enzyme complex and contains binding sites for succinate, while the alpha subunit (sucD) contains binding sites for coenzyme A and phosphate . This enzyme plays a pivotal role in energy metabolism, connecting the TCA cycle with oxidative phosphorylation and serving as a critical junction for carbon flux distribution.

Unlike human SUCLA2 which functions primarily in the mitochondrial matrix, E. coli sucC operates in the cytoplasmic environment. The enzyme's activity is particularly important under anaerobic conditions when the TCA cycle operates in a branched mode rather than as a complete cycle, contributing to redox balance maintenance and metabolic flexibility.

How does the structure of sucC relate to its function in metabolic pathways?

The sucC protein forms a heterodimer with sucD (the alpha subunit) to create the functional Succinyl-CoA ligase complex. This structural arrangement is essential for proper enzymatic function. The beta subunit contains the nucleotide-binding domain that determines whether the enzyme utilizes ADP or GDP for the reaction mechanism. In E. coli, the ADP-forming variant predominates, differentiating it from some other organisms that utilize GDP-forming variants.

The protein's three-dimensional structure features characteristic nucleotide-binding motifs, including the Walker A and B motifs typical of ATP/ADP-binding proteins. These structural elements position the nucleotide correctly for phosphoryl transfer during catalysis. The interface between the alpha and beta subunits creates a substrate channel and active site pocket that accommodates succinyl-CoA and facilitates the catalytic reaction.

What are the optimal conditions for expressing functional recombinant sucC in E. coli expression systems?

For optimal expression of functional recombinant sucC in E. coli, researchers should consider several key parameters:

The E. coli expression system has been successfully used to produce recombinant Succinyl-CoA ligase proteins with high purity levels (>90% as determined by SDS-PAGE) . When designing an expression strategy:

• Expression vector selection: pTrc99a has shown good results for metabolic engineering applications involving TCA cycle enzymes .

• Induction conditions: IPTG induction at concentrations of 0.1-1.0 mM when the culture reaches OD600 of 0.6-0.8 typically yields good expression levels.

• Growth temperature: Post-induction growth at lower temperatures (16-25°C) often enhances protein solubility compared to standard 37°C incubation.

• Co-expression considerations: For complete enzymatic activity, co-expression with the alpha subunit (sucD) may be necessary, as the functional enzyme exists as an α2β2 heterotetramer.

• Affinity tags: Both N-terminal and C-terminal tagging approaches have been successful, with His-tags (6x-10x) being commonly used for efficient purification .

What purification strategies are most effective for obtaining high-quality recombinant sucC protein?

Purification of recombinant sucC typically follows these methodological steps:

• Initial clarification: After cell lysis (typically using sonication or high-pressure homogenization), centrifugation at 10,000-15,000 × g for 30-45 minutes removes cellular debris.

• Affinity chromatography: For His-tagged constructs, immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resins offers excellent initial purification. Typical washing buffers contain 20-50 mM imidazole, while elution is performed with 250-500 mM imidazole .

• Secondary purification: Size exclusion chromatography (SEC) effectively separates monomeric, dimeric, and aggregated forms of the protein.

• Buffer optimization: The final purified protein is typically stored in Tris or phosphate-based buffers (pH 7.5-8.0) with 5-50% glycerol to maintain stability .

• Storage recommendations: For long-term storage, aliquoting and storage at -80°C is recommended. Repeated freeze-thaw cycles should be avoided to maintain enzymatic activity .

Researchers should aim for purity levels of at least 90% as determined by SDS-PAGE for functional and structural studies .

How can manipulation of sucC expression contribute to succinate production in engineered E. coli strains?

Modifying sucC expression levels represents one approach in a comprehensive metabolic engineering strategy for enhancing succinate production in E. coli. The evidence suggests several key considerations:

• Pathway context is critical: While sucC overexpression alone may have limited impact, its combination with other genetic modifications has shown significant improvements in succinate yield. Key complementary modifications include:

  • Deletion of succinate dehydrogenase genes (sdhAB) to prevent succinate oxidation

  • Inactivation of the isocitrate lyase regulator (iclR) to enhance glyoxylate shunt activity

  • Deletion of malic enzyme gene (maeB) to reduce carbon flux toward gluconeogenesis

• Carbon source considerations: When using acetate as a carbon source, proper expression of acetyl-CoA synthetase (acs) alongside TCA cycle enzymes including sucC can significantly enhance succinate production .

• Expression level optimization: The data from engineered strains indicates that balancing sucC expression relative to other TCA cycle enzymes is more important than simply maximizing its expression. For instance, in strain WCY-7, coordinated expression of acs, gltA (citrate synthase), and acnB (aconitate hydratase) led to significantly improved succinate titers of 11.23 mM compared to strains lacking this balanced approach .

What genetic modifications have been most effective when combined with sucC manipulation for optimizing succinate production?

Research demonstrates that a multi-target approach to metabolic engineering yields superior results compared to single-gene modifications. The most effective genetic modifications that synergize with sucC manipulation include:

• TCA cycle redirection:

  • Deletion of sdhAB (encoding succinate dehydrogenase): This prevents succinate oxidation to fumarate, creating a metabolic block that allows succinate accumulation. In WCY-1, this modification alone increased succinate production to 1.68 mM from non-detectable levels in wild-type .

• Glyoxylate shunt enhancement:

  • Deletion of iclR (encoding the isocitrate lyase regulator): This derepresses the aceBAK operon, increasing carbon flux through the glyoxylate bypass. When combined with sdhAB deletion (strain WCY-2), succinate production increased to 2.04 mM .

• Blocking competing pathways:

  • Deletion of maeB (encoding malic enzyme): This prevents malate conversion to pyruvate, further directing carbon flux toward succinate production. The triple deletion strain WCY-3 (ΔiclRΔsdhABΔmaeB) achieved 4.62 mM succinate .

• Coordinated enzyme overexpression:

  • Overexpression of acs (acetyl-CoA synthetase): Essential for efficient acetate utilization

  • Overexpression of gltA (citrate synthase): Enhances carbon entry into the TCA cycle

  • Overexpression of acnB (aconitate hydratase): Ensures efficient conversion through the early steps of the TCA cycle

The combination of these three enzyme overexpressions in the triple deletion background (strain WCY-7) achieved the highest reported succinate titer of 11.23 mM, representing a 2.4-fold improvement over the triple deletion strain alone .

What assays can be used to accurately measure sucC activity in recombinant protein preparations?

Several robust assays are available for measuring sucC enzymatic activity:

• Coupled spectrophotometric assay: This approach monitors the formation of ADP or ATP coupled to pyruvate kinase and lactate dehydrogenase reactions, tracking NADH oxidation at 340 nm. The reaction mixture typically contains:

  • 50 mM HEPES buffer (pH 7.5)

  • 10 mM MgCl₂

  • 0.1 mM CoA

  • 0.2 mM succinyl-CoA

  • 0.5 mM ADP or ATP (depending on direction)

  • 0.2 mM NADH

  • 1 mM phosphoenolpyruvate

  • Pyruvate kinase and lactate dehydrogenase (2-5 U each)

• Radioactive assay: Using ¹⁴C-labeled succinate or succinyl-CoA to directly measure the forward or reverse reaction.

• Direct HPLC quantification: Measuring the consumption of succinyl-CoA or production of succinate using HPLC with appropriate standards.

When conducting these assays, researchers should:

• Include appropriate negative controls (heat-inactivated enzyme)

• Establish linear range of the assay with respect to enzyme concentration

• Determine Km and Vmax values under various conditions to characterize enzymatic properties

How do different tags affect the activity and stability of recombinant sucC protein?

The choice of affinity tag can significantly impact both the activity and stability of recombinant sucC:

• His-tags (6x-10x): Generally well-tolerated at either N- or C-terminus, with minimal impact on enzyme activity. The data shows that His-tagged recombinant proteins can maintain >90% purity and functionality .

• Myc-tag: Commonly used for immunodetection, the Myc-tag has been successfully combined with His-tags in dual-tagging approaches (His&Myc) for recombinant Succinyl-CoA ligase proteins .

• GST-tag: While useful for improving solubility, the large size (~26 kDa) may interfere with dimerization or activity, often necessitating tag removal for functional studies.

• SUMO-tag: Can enhance solubility while allowing for precise tag removal via SUMO protease, preserving the native N-terminus.

For optimal results when studying enzymatic activity:

• Compare tagged and untagged (tag-cleaved) versions of the protein

• Position the tag at the terminus predicted to have minimal interference with active site or dimerization interface

• Consider dual expression systems with differentially tagged alpha and beta subunits to study subunit interactions

How can isotope labeling be used to track carbon flux through sucC-catalyzed reactions in metabolic engineering studies?

Isotope labeling provides powerful insights into metabolic flux distribution through sucC-catalyzed reactions:

• ¹³C-labeled substrate experiments: Using ¹³C-labeled acetate or glucose allows tracking of carbon atoms through the TCA cycle and specifically through the sucC-catalyzed reaction. This approach helps determine:

  • Relative flux through competing pathways

  • Contribution of different carbon entry points to succinate production

  • Effectiveness of genetic modifications in redirecting carbon flux

• Metabolic flux analysis methodology:

  • Feed cells with ¹³C-labeled substrate (e.g., 1-¹³C acetate or U-¹³C acetate)

  • Extract intracellular metabolites at steady state

  • Analyze isotopomer distribution using GC-MS or LC-MS/MS

  • Apply computational models to determine relative flux through different pathways

• Dynamic labeling studies: Time-course measurements following introduction of labeled substrate can reveal:

  • Rate-limiting steps in the pathway

  • Metabolic bottlenecks that could be targeted for further engineering

  • Temporal changes in flux distribution following genetic modifications

This approach is particularly valuable when evaluating the impact of sucC expression levels relative to other TCA cycle enzymes in engineered strains like WCY-7, where coordinated expression of multiple enzymes led to significantly improved succinate production .

How does the structure-function relationship in sucC compare between E. coli and human SUCLA2?

Despite their evolutionary distance, E. coli sucC and human SUCLA2 share several key structural and functional features while maintaining important differences:

• Sequence and structural similarities:

  • Both function as beta subunits of Succinyl-CoA ligase in their respective organisms

  • Both provide nucleotide specificity to the enzyme complex

  • Both contain binding sites for the substrate succinate

• Functional differences:

  • Human SUCLA2 is ATP-specific, functioning in the mitochondrial TCA cycle

  • E. coli sucC is primarily ADP-forming in its native context

  • Human SUCLA2 functions exclusively in the mitochondrion, while E. coli sucC operates in the cytoplasm

• Expression and regulation:

  • SUCLA2 shows tissue-specific expression patterns, being widely expressed in human tissues but notably absent from liver and lung

  • E. coli sucC expression responds to environmental conditions, particularly oxygen availability and carbon source

• Clinical relevance:

  • Mutations in human SUCLA2 are associated with mitochondrial DNA depletion syndrome 5 (MTDPS5), encephalomyopathy, and methylmalonic aciduria

  • These conditions illustrate the critical role of proper Succinyl-CoA ligase function in human metabolism

Understanding these similarities and differences provides valuable insights for researchers studying metabolic pathways across species and can inform biomedical research on mitochondrial disorders associated with SUCLA2 mutations.

What are common challenges in recombinant sucC expression and how can they be addressed?

Researchers commonly encounter several challenges when working with recombinant sucC:

• Solubility issues:

  • Problem: Formation of inclusion bodies

  • Solution: Lower induction temperature (16-25°C), reduce IPTG concentration, or co-express with chaperones (GroEL/GroES system)

• Incomplete activity without alpha subunit:

  • Problem: The beta subunit alone may show limited or no activity

  • Solution: Co-express with the alpha subunit (sucD) using dual-expression vectors or co-transformation strategies

• Stability concerns:

  • Problem: Loss of activity during storage

  • Solution: Add 5-50% glycerol to storage buffer, avoid repeated freeze-thaw cycles, and store working aliquots at 4°C for up to one week

• Purification challenges:

  • Problem: Co-purification of contaminants with similar properties

  • Solution: Implement multi-step purification strategy including affinity chromatography followed by size exclusion or ion exchange chromatography

• Activity verification:

  • Problem: Difficulty distinguishing sucC activity from background enzymatic activities

  • Solution: Include appropriate controls (heat-inactivated enzyme, assays lacking key substrates) and optimize assay conditions for maximal signal-to-noise ratio

How can researchers optimize expression constructs for maximum yield and activity of recombinant sucC?

Optimization of expression constructs involves several strategic considerations:

• Codon optimization:

  • Analyze the codon usage bias in the expression host

  • Optimize codons for E. coli if expressing in an E. coli system

  • Consider using specialized strains like Rosetta for genes with rare codons

• Vector selection:

  • For high expression: pTrc99a with strong promoter has demonstrated success for TCA cycle enzymes

  • For controlled expression: pCL1920 provides more moderate expression levels which may improve solubility

• Promoter and induction system:

  • T7 promoter systems offer high expression but may lead to inclusion body formation

  • Arabinose-inducible promoters (pBAD) allow finer control over expression levels

  • Auto-induction media can simplify the process while providing good yields

• Fusion partners:

  • SUMO or MBP tags can significantly enhance solubility

  • Thioredoxin fusion may assist proper disulfide bond formation if present

• Expression testing strategy:

  • Conduct small-scale expression trials varying temperature, inducer concentration, and induction time

  • Evaluate both soluble fraction yield and enzymatic activity

  • Scale up using conditions that optimize the yield of active enzyme rather than total protein

Experimental data indicates that optimizing expression constructs can significantly impact end-product yields, as demonstrated in the succinate production strains where different expression systems for the same genes resulted in varying titers (compare WCY-6 and WCY-7, showing different expression levels of the same genes led to succinate production of 9.87 mM versus 11.23 mM) .