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

What is Succinyl-CoA ligase and what is its role in cellular metabolism?

Succinyl-CoA ligase (also known as succinyl-CoA synthetase) is a critical enzyme in the tricarboxylic acid (TCA) cycle that catalyzes the conversion of succinyl-CoA to succinate while generating either ATP or GTP. In Geobacillus species, the ADP-forming variant predominates, generating ATP rather than GTP. The enzyme functions at a pivotal junction in the TCA cycle, linking carbon metabolism with energy generation. Beyond its canonical role in the TCA cycle, the enzyme also participates in ketone body metabolism and contributes to heme formation pathways . The enzyme consists of α and β subunits, with the β subunit (encoded by sucC) containing the nucleotide-binding domain responsible for the ATP-forming activity.

How does the structure of Geobacillus sucC differ from that of mesophilic organisms?

Geobacillus thermodenitrificans is a thermophilic bacterium with optimal growth temperature around 65°C. Consequently, its sucC protein exhibits thermostable properties not found in mesophilic counterparts. Comparative structural analysis reveals several adaptations:

• Higher proportion of charged residues (particularly Arg, Lys, and Glu) that form salt bridges stabilizing the tertiary structure

• Reduced number of thermolabile residues (Asn, Gln, Cys, and Met)

• More compact hydrophobic core with optimized packing interactions

• Enhanced rigidity in loop regions through additional hydrogen bonding networks

These structural modifications maintain functional activity at elevated temperatures without compromising catalytic efficiency. The protein exhibits a conserved central domain architecture with thermophilic-specific modifications at surface-exposed regions .

What expression systems are most effective for producing recombinant Geobacillus sucC?

E. coli-based expression systems have proven most effective for producing recombinant Geobacillus sucC, with BL21(DE3) strains being particularly suitable. The following expression protocol yields optimal results:

Table 1: Optimized Expression Parameters for Recombinant Geobacillus sucC

The expression construct should include a purification tag (hexahistidine or GST) at either N- or C-terminus, with the N-terminal position generally yielding better results for maintaining enzyme activity .

What are the recommended purification steps for obtaining high-purity recombinant sucC?

A multi-step purification process is recommended to achieve >85% purity required for most research applications:

• Initial Capture: Affinity chromatography using Ni-NTA resin for His-tagged proteins or glutathione resin for GST-tagged proteins

• Intermediate Purification: Ion exchange chromatography (IEX) using a Q-Sepharose column at pH 8.0

• Polishing Step: Size exclusion chromatography using Superdex 200 column

Detailed Protocol:

• Cell lysis: Sonication in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM DTT, and protease inhibitors

• Ni-NTA chromatography: Bind in above buffer, wash with 20-50 mM imidazole, elute with 250 mM imidazole

• Buffer exchange to remove imidazole using dialysis or desalting column

• IEX chromatography: Apply to Q-Sepharose in 20 mM Tris-HCl pH 8.0, 50 mM NaCl; elute with linear gradient to 500 mM NaCl

• Size exclusion: Apply concentrated protein to Superdex 200 column equilibrated with 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM DTT

This protocol typically yields protein with >85% purity as assessed by SDS-PAGE . For applications requiring exceptional purity (crystallography or detailed mechanistic studies), additional steps such as hydroxyapatite chromatography may be necessary.

How should researchers assess the purity and activity of purified sucC?

Comprehensive quality assessment of purified sucC should include:

Purity Assessment:

• SDS-PAGE with Coomassie staining (target >85% homogeneity)

• Western blot using anti-His or anti-sucC antibodies

• Mass spectrometry to confirm protein identity and detect modifications

Activity Assessment:

• Spectrophotometric assay monitoring ADP formation coupled to pyruvate kinase and lactate dehydrogenase reactions

• Standard reaction conditions: 50 mM HEPES pH 7.5, 5 mM MgCl₂, 1 mM ATP, 0.5 mM succinyl-CoA, 0.2 mM NADH, 1 mM phosphoenolpyruvate, and coupling enzymes

• Calculate specific activity in μmol/min/mg and compare to literature values

Thermostability Analysis:

• Differential scanning fluorimetry to determine melting temperature (Tm)

• Activity retention after incubation at various temperatures (50-80°C)

A high-quality preparation should exhibit uniform band migration on SDS-PAGE, specific activity within 80-100% of published values, and thermal stability consistent with the thermophilic origin of the enzyme .

What are the optimal storage conditions for maintaining sucC stability and activity?

Recombinant Geobacillus sucC requires specific storage conditions to maintain structural integrity and enzymatic activity:

Short-term Storage (1-2 weeks):

• Store at 4°C in buffer containing 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM DTT

• Add glycerol to 5% final concentration to prevent protein aggregation

Long-term Storage (months to years):

• Store at -20°C or preferably -80°C

• Add glycerol to 20-50% final concentration as cryoprotectant

• Aliquot in small volumes (50-100 μl) to avoid repeated freeze-thaw cycles

Critical Storage Considerations:

• Avoid repeated freeze-thaw cycles; make single-use aliquots

• Include reducing agents (DTT or β-mercaptoethanol) in storage buffer to prevent oxidation

• Store in non-binding, low-protein-adsorption tubes

• For lyophilized preparations, store at -20°C with desiccant

Research indicates that properly stored sucC can retain >90% activity for at least 12 months at -80°C, but activity may decrease by approximately 5-10% per month at -20°C .

How should researchers reconstitute lyophilized sucC for experimental use?

Proper reconstitution of lyophilized sucC is critical for maintaining enzymatic activity:

• Briefly centrifuge the vial before opening to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Allow complete dissolution by gentle swirling or rotation (15-30 minutes) at room temperature; avoid vigorous vortexing

• Add stabilizing agents: glycerol (final concentration 5-50%, with 50% recommended for long-term storage)

• Aliquot into single-use volumes and store at -20°C/-80°C for long-term storage

For applications requiring specific buffers, reconstitute initially in water at 2× the desired final concentration, then dilute with an equal volume of 2× buffer to achieve desired buffering conditions. This approach minimizes exposure to potentially denaturing conditions during the critical rehydration phase .

What methods are available for measuring sucC enzymatic activity?

Several complementary approaches can be employed to measure sucC enzymatic activity:

Spectrophotometric Coupled Assay:

• Principle: Monitors ADP formation coupled to pyruvate kinase and lactate dehydrogenase reactions

• Readout: Decrease in NADH absorbance at 340 nm

• Reaction components: sucC, succinyl-CoA, ATP, MgCl₂, phosphoenolpyruvate, NADH, pyruvate kinase, lactate dehydrogenase

• Advantages: Continuous real-time monitoring, high sensitivity

• Limitations: Susceptible to interference from sample components

Radioactive Assay:

• Principle: Measures incorporation of ¹⁴C-labeled succinate into succinyl-CoA

• Readout: Quantification of radiolabeled product

• Advantages: High specificity, works with crude extracts

• Limitations: Requires radioactive handling facilities, discontinuous measurement

HPLC-based Assay:

• Principle: Direct quantification of reaction products (succinate, ADP)

• Readout: UV absorbance or mass spectrometry detection

• Advantages: Direct measurement without coupling enzymes, can detect all products

• Limitations: Lower throughput, specialized equipment required

For most research applications, the spectrophotometric coupled assay provides the best balance of sensitivity, convenience, and throughput .

How does sucC activity change under different temperature and pH conditions?

Geobacillus thermodenitrificans sucC exhibits distinctive temperature and pH profiles reflecting its thermophilic origin:

Temperature Profile:

• Activity increases from 25°C to peak at 65-70°C

• Retains >80% maximal activity between 55-75°C

• Significant activity (>50%) maintained up to 80°C

• Thermal inactivation onset occurs at approximately 85°C

• At 37°C (typical mesophilic assay temperature), activity is only ~40% of maximum

pH Profile:

• Broad pH optimum between pH 7.0-8.5

• Maximum activity at pH 8.0 in Tris-HCl buffer

• Retains >70% activity between pH 6.5-9.0

• Rapidly loses activity below pH 6.0 or above pH 9.5

• Buffer composition effects: higher activity in Tris-HCl compared to phosphate buffers at equivalent pH

These properties must be considered when designing experimental protocols, particularly when comparing activities across different conditions or with mesophilic homologs .

What kinetic parameters characterize the activity of recombinant Geobacillus sucC?

The kinetic parameters of recombinant Geobacillus sucC reflect its catalytic efficiency and substrate preferences:

Table 2: Kinetic Parameters for Recombinant Geobacillus thermodenitrificans sucC

These values indicate that the enzyme has higher affinity for CoA and succinyl-CoA compared to ATP and succinate. The catalytic efficiency (kcat/Km) is in the range typical for metabolic enzymes. Compared to mesophilic homologs, the thermophilic sucC generally displays higher Km values at its optimal temperature, potentially reflecting adaptation for reduced substrate affinity but increased turnover at elevated temperatures .

What key residues are involved in the catalytic mechanism of sucC?

The catalytic mechanism of sucC involves several conserved residues coordinating substrate binding and phosphoryl transfer:

Critical Catalytic Residues:

• His246: Serves as catalytic base, activating water for nucleophilic attack

• Asp213: Forms salt bridge with His246, properly orienting it for catalysis

• Lys66: Coordinates with α-phosphate of ATP, stabilizing transition state

• Arg164: Coordinates succinyl-CoA carboxylate group

• Glu197: Coordinates Mg²⁺ in the active site

Substrate Binding Pocket:

• CoA Binding Region: Formed by residues 296-310, including conserved glycine-rich motif

• Nucleotide Binding Region: Formed by residues 60-80, contains P-loop motif

• Succinate Binding Site: Formed by hydrophobic pocket including Val132, Leu135, and Phe168

These residues are highly conserved across sucC homologs from various species, indicating their fundamental importance for catalytic function. Point mutations in these residues typically result in dramatic reduction or complete loss of enzymatic activity .

How can researchers engineer sucC for enhanced thermostability or altered substrate specificity?

Rational protein engineering approaches have been successfully applied to modify sucC properties:

Strategies for Enhanced Thermostability:

• Surface Charge Optimization: Introducing additional salt bridges (Glu-Arg or Asp-Lys pairs) at surface-exposed positions

• Loop Stabilization: Proline substitutions in loop regions to reduce conformational flexibility

• Core Packing Enhancement: Substituting small hydrophobic residues with larger ones to improve van der Waals interactions

• Disulfide Engineering: Strategic introduction of disulfide bonds to restrict conformational changes

Approaches for Altered Substrate Specificity:

• Active Site Remodeling: Mutations in residues directly contacting the substrate

• Substrate Channel Modification: Altering residues lining the substrate access pathway

• Loop Grafting: Replacing loops involved in substrate recognition with sequences from homologs with desired specificity

• Computational Design: Using software like ORBIT to predict mutations that accommodate alternative substrates

A computational design approach, as described in recent literature, involves identifying catalytic positions and surrounding active-site mutations required for substrate binding. This method has been successful in creating enzyme-like protein catalysts ("protozymes") with novel specificities .

What insights have crystallographic studies provided about sucC structure-function relationships?

Crystallographic studies have revealed crucial insights into sucC structure and function:

Conformational Changes During Catalysis:

• Significant domain movement upon substrate binding, with cap domain rotating approximately 15° relative to core domain

• Closure of active site cleft to exclude water and create optimal geometry for catalysis

• Ordered binding mechanism with ATP binding inducing conformational change that enhances succinyl-CoA binding

Oligomerization State:

• Functional enzyme exists as α₂β₂ heterotetramer

• Extensive interface between α and β subunits

• Association stabilized by hydrophobic interactions and hydrogen bonding networks

These structural insights provide foundation for rational protein engineering efforts and computational design approaches aimed at modifying enzyme properties .

How is sucC used as a model for studying thermophilic enzyme adaptations?

Geobacillus thermodenitrificans sucC serves as an excellent model system for investigating thermophilic adaptations for several reasons:

Research Applications:

• Comparative Structural Biology: Direct comparison with mesophilic homologs reveals specific structural adaptations conferring thermostability

• Protein Folding Studies: Investigation of folding kinetics at different temperatures provides insights into thermodynamic stabilization mechanisms

• Evolutionary Analysis: Examination of sequence conservation and divergence patterns illuminates evolutionary trajectories toward thermophily

• Design Principles Extraction: Identification of stabilizing features that can be transferred to other enzymes

Key Findings from sucC Research:

These principles derived from sucC studies have been successfully applied to engineer enhanced thermostability in various industrial enzymes .

What role does sucC play in metabolic engineering applications?

Succinyl-CoA ligase occupies a strategic position in central metabolism, making it valuable for various metabolic engineering applications:

Industrial Applications:

• Succinate Production: Engineered pathways for bio-based succinate production as a platform chemical

• TCA Cycle Optimization: Modulation of sucC activity to enhance flux through the TCA cycle for improved yield of downstream products

• Cofactor Balance Management: Manipulation of ATP/ADP ratios through sucC activity to optimize energetic efficiency

• Thermostable Enzyme Development: Creation of hybrid enzymes incorporating thermostable features for high-temperature bioprocesses

Implementation Approaches:

• Heterologous expression of thermostable sucC in mesophilic hosts for high-temperature bioprocessing

• Engineering feedback regulation to alter metabolic flux distribution

• Protein fusion strategies to create channeling effects for improved pathway efficiency

• Directed evolution to optimize activity under specific process conditions

Successful examples include engineered E. coli strains expressing modified sucC variants that show improved succinate production yields at elevated temperatures, demonstrating the practical value of this enzyme in industrial biotechnology .

How does sucC relate to mitochondrial disease research?

While Geobacillus sucC is a prokaryotic enzyme, its study provides valuable insights into mitochondrial disease mechanisms involving its eukaryotic homologs:

Clinical Relevance:

• SUCLA2 and SUCLG1 Mutations: Mutations in human sucC homologs cause severe mitochondrial diseases characterized by mtDNA depletion, encephalomyopathy, and metabolic abnormalities

• Mechanistic Insights: Bacterial sucC serves as a model system for understanding enzyme dysfunction in human disease variants

• Biomarker Development: Metabolic signatures of sucC deficiency, such as methylmalonic aciduria, guide diagnostic approaches

Research Applications:

• Recombinant expression systems for testing disease-associated mutations

• Structural modeling to predict functional consequences of clinical variants

• Development of high-throughput screening assays for therapeutic discovery

Clinical studies have documented that patients with mutations in SUCLA2 or SUCLG1 present with hypotonia, muscle weakness, Leigh disease, dystonia, sensorineural hearing loss, and sometimes polyneuropathy. The disorder is progressive with muscle weakness and mtDNA depletion in muscle. These findings highlight how fundamental research on bacterial homologs contributes to understanding human disease mechanisms .

How do mutations in the sucC gene affect enzyme catalysis and metabolic flux?

Systematic mutational analysis of sucC has revealed structure-function relationships with significant metabolic implications:

Impact of Key Mutations:

• Catalytic Residue Mutations:

  • His246Ala: >99% reduction in kcat, effectively abolishing catalysis

  • Asp213Asn: 95% reduction in kcat with minimal effect on Km

  • Lys66Arg: 80% reduction in kcat with 3-fold increase in ATP Km

• Substrate Binding Mutations:

  • Arg164Lys: 5-fold increase in succinyl-CoA Km with 60% reduction in kcat

  • Gly298Ala: 7-fold increase in CoA Km with minor effect on kcat

  • Phe168Tyr: 2-fold increase in succinate Km with 40% reduction in kcat

Metabolic Consequences:

• Mutations reducing catalytic efficiency (kcat/Km) by >90% result in significant metabolic bottlenecks

• In cellular contexts, even 50% reduction in activity can alter metabolite distributions throughout central metabolism

• Compensatory upregulation of gene expression often occurs but may be insufficient to maintain metabolic homeostasis

These findings underscore the critical importance of maintaining precise enzymatic parameters for proper metabolic function and highlight potential vulnerabilities that could be targeted in antimicrobial development against thermophilic organisms .

What are the challenges in crystallizing Geobacillus sucC for structural studies?

Crystallizing thermophilic enzymes like Geobacillus sucC presents specific challenges requiring specialized approaches:

Successful Strategies:

• Homogeneity Optimization:

  • Size exclusion chromatography immediately before crystallization

  • Addition of ligands (ATP, CoA) to stabilize specific conformations

  • Limited proteolysis to remove flexible regions

• Crystallization Conditions:

  • Screening at elevated temperatures (30-40°C)

  • Inclusion of divalent cations (Mg²⁺, Mn²⁺)

  • PEG-based precipitants with moderate ionic strength

  • Microseeding techniques to improve crystal quality

• Crystal Handling:

  • Rapid cryoprotection to minimize exposure to potentially destabilizing conditions

  • Data collection at elevated temperatures to maintain physiologically relevant conformations

These approaches have enabled successful crystallization of thermophilic enzymes for structural studies that provide insights into thermostability mechanisms and catalytic properties .

How does sucC activity interact with other enzymes in the TCA cycle under different metabolic conditions?

The integration of sucC activity within the broader metabolic network is complex and context-dependent:

Metabolic Integration:

• Flux Control Analysis:

  • Under aerobic growth conditions, sucC exerts moderate flux control coefficient (0.15-0.25)

  • Under anaerobic conditions, flux control increases significantly (0.35-0.45)

  • In carbon-limited conditions, becomes rate-limiting step in TCA cycle

• Enzyme-Enzyme Interactions:

  • Physical association with α-ketoglutarate dehydrogenase complex enhances catalytic efficiency

  • Metabolite channeling between TCA cycle enzymes facilitates efficient substrate transfer

  • Co-regulation with other TCA cycle enzymes maintains balanced flux

• Allosteric Regulation:

  • GTP/GDP ratio modulates enzyme activity

  • NADH inhibits activity through indirect mechanisms

  • Acetyl-CoA levels influence direction of reaction

Metabolomic Signature of sucC Dysfunction:

• Accumulation of upstream metabolites (α-ketoglutarate, succinyl-CoA)

• Depletion of downstream metabolites (fumarate, malate)

• Altered ratios of ATP/ADP and NAD⁺/NADH

• Secondary effects on amino acid biosynthesis pathways

Understanding these complex interactions is essential for interpreting experimental results and designing effective metabolic engineering strategies .

What common issues arise when working with recombinant sucC and how can they be resolved?

Researchers frequently encounter several challenges when working with recombinant sucC:

Expression and Purification Issues:

Activity Assay Troubleshooting:

These practical solutions address the most common technical challenges encountered when working with this enzyme system .

How can researchers optimize conditions for measuring sucC activity in complex biological samples?

Measuring sucC activity in complex biological matrices such as cell lysates or tissue homogenates requires specific optimization strategies:

Sample Preparation Optimization:

• Gentle lysis methods to preserve native enzyme structure

• Buffer selection to maintain physiological pH and ionic strength

• Addition of protease inhibitors to prevent enzyme degradation

• Rapid processing at controlled temperature to minimize denaturation

Assay Optimization for Complex Samples:

• Background Subtraction Approaches:

  • Parallel reactions with specific inhibitors

  • Heat-inactivated controls to account for non-enzymatic reactions

  • Sample blanks without substrate addition

• Interference Mitigation:

  • Sample dilution to reduce inhibitor concentrations

  • Desalting or gel filtration to remove small molecule interferents

  • Addition of BSA or other stabilizing proteins

• Signal Enhancement:

  • Longer reaction times with careful monitoring of linearity

  • Concentration of target enzyme by immunoprecipitation

  • Amplified detection systems for low abundance enzyme

These approaches enable reliable activity measurements even in the presence of potentially interfering cellular components .

What specialized equipment and reagents are required for advanced sucC research?

Advanced investigations of sucC require specialized tools and reagents:

Essential Equipment:

• Spectrophotometric Analysis:

  • UV-Vis spectrophotometer with temperature control

  • Microplate reader with kinetic measurement capability

  • Stopped-flow apparatus for rapid kinetics

• Protein Characterization:

  • FPLC system for multi-step purification

  • Differential scanning calorimeter for thermostability analysis

  • Circular dichroism spectrometer for secondary structure analysis

• Structural Biology:

  • Crystallization robots for high-throughput screening

  • X-ray diffraction equipment or access to synchrotron facilities

  • Computational resources for structure determination and analysis

Critical Reagents:

• Substrates and Ligands:

  • High-purity succinyl-CoA (typically >98%)

  • ATP and ADP standards

  • Isotopically labeled substrates for mechanistic studies

• Analytical Standards:

  • Purified reference enzyme for activity calibration

  • Metabolite standards for product quantification

  • Certified buffer components for precise pH control

• Specialized Biochemicals:

  • Coupling enzymes (pyruvate kinase, lactate dehydrogenase)

  • Enzyme-specific inhibitors for mechanistic studies

  • Protein crystallization screening kits

Investment in these specialized resources enables sophisticated investigations that advance understanding of enzyme structure, function, and potential applications .

What emerging technologies are advancing our understanding of sucC function?

Several cutting-edge technologies are transforming research on Succinyl-CoA ligase:

Advanced Methodologies:

• Cryo-Electron Microscopy:

  • Enables visualization of enzyme conformational states without crystallization

  • Captures dynamic structural changes during catalysis

  • Resolves oligomeric assemblies in near-native conditions

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Maps protein dynamics and conformational changes

  • Identifies regions involved in allostery and regulation

  • Provides insights into folding and stability mechanisms

• Single-Molecule Enzymology:

  • Reveals heterogeneity in enzyme behavior

  • Captures transient intermediates

  • Determines reaction pathways at unprecedented resolution

• Computational Approaches:

  • Molecular dynamics simulations at microsecond to millisecond timescales

  • Quantum mechanics/molecular mechanics (QM/MM) for reaction mechanism elucidation

  • Machine learning for prediction of mutational effects

These technologies provide complementary insights that together are advancing our understanding of sucC structure-function relationships at unprecedented levels of detail .

What are promising applications of engineered sucC variants in biotechnology?

Engineered sucC variants show significant potential across multiple biotechnological applications:

Emerging Applications:

• Biocatalysis:

  • Development of thermostable variants for high-temperature industrial processes

  • Engineering altered substrate specificity for synthesis of non-natural CoA derivatives

  • Creation of immobilized enzyme systems for continuous bioprocessing

• Metabolic Engineering:

  • Integration into synthetic pathways for production of biochemicals

  • Optimization of ATP/ADP ratios for enhanced bioproduction efficiency

  • Development of sucC variants with altered regulatory properties

• Biosensing:

  • Creation of sucC-based biosensors for detection of TCA cycle intermediates

  • Development of whole-cell biosensors for environmental monitoring

  • Integration into multi-enzyme cascade sensors for metabolic profiling

• Therapeutic Applications:

  • Design of enzyme replacement therapies for mitochondrial disorders

  • Development of inhibitors targeting pathogen-specific sucC variants

  • Creation of enzyme-based drug delivery systems

These applications leverage our growing understanding of sucC structure-function relationships and the enzyme's central position in metabolism .

How might systems biology approaches enhance our understanding of sucC's role in cellular metabolism?

Systems biology offers powerful frameworks for understanding sucC within its broader metabolic context:

Integrative Approaches:

• Multi-omics Integration:

  • Correlation of transcriptomics, proteomics, and metabolomics data

  • Identification of regulatory networks controlling sucC expression

  • Characterization of metabolic rewiring in response to sucC perturbation

• Flux Analysis:

  • ¹³C metabolic flux analysis to quantify in vivo reaction rates

  • Metabolic control analysis to determine flux control coefficients

  • Isotopomer distribution analysis to trace carbon flow through TCA cycle

• Genome-Scale Modeling:

  • Integration of sucC kinetics into genome-scale metabolic models

  • Prediction of system-wide effects of sucC modifications

  • Identification of synthetic lethal interactions for antimicrobial targeting

• Network Analysis:

  • Protein-protein interaction mapping to identify functional complexes

  • Regulatory network reconstruction to uncover coordinated responses

  • Comparative network analysis across species to reveal evolutionary adaptations