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

What is the basic function of Succinyl-CoA ligase in Macrococcus caseolyticus metabolism?

Succinyl-CoA ligase [ADP-forming] subunit beta (sucC) functions as a critical component of the tricarboxylic acid (TCA) cycle in Macrococcus caseolyticus. Similar to its homologs in other organisms, this enzyme catalyzes the ATP-dependent ligation of succinate and CoA to form succinyl-CoA . The reaction involves substrate-level phosphorylation, converting succinyl-CoA to succinate while generating ATP from ADP.

In bacterial metabolism, including Macrococcus species (which are closely related to Staphylococcus), this enzyme plays a vital role in energy production and carbon metabolism . The beta subunit (sucC) typically works in conjunction with an alpha subunit to form a functional heterodimeric enzyme complex.

How does bacterial Succinyl-CoA ligase differ from its eukaryotic counterparts?

Bacterial Succinyl-CoA ligase exhibits several distinct differences from eukaryotic homologs:

Unlike eukaryotes that possess two distinct beta-subunit isoforms (SUCLA2 for ATP generation and SUCLG2 for GTP generation), bacteria typically utilize only the ATP-forming variant . Additionally, bacterial enzymes lack the signal peptides required for mitochondrial localization observed in eukaryotic SUCLA2, which contains mitochondrial targeting sequences .

What is the molecular structure of the Macrococcus sucC protein?

While the specific structure of Macrococcus caseolyticus sucC has not been fully characterized, insights can be drawn from homologous proteins. Based on sequence conservation among succinyl-CoA ligases, the protein likely features:

• A nucleotide-binding domain with a classic Rossmann fold

• A CoA-binding domain with characteristic binding motifs

• A substrate-binding pocket accommodating succinate

• Interface regions for interaction with the alpha subunit

The quaternary structure involves heterodimerization with the alpha subunit to form the functional enzyme. The mature protein is expected to have a molecular weight of approximately 40-45 kDa, similar to other bacterial sucC proteins and comparable to the human SUCLA2 protein (50.3 kDa) .

What expression systems are most effective for recombinant Macrococcus caseolyticus sucC?

For efficient expression of recombinant M. caseolyticus sucC, the following expression systems have proven most effective:

For optimal expression, researchers should implement sequence optimization for E. coli codon usage, similar to the approach used for human SUCLG1 and SUCLG2 described in previous studies . Temperature optimization (typically 18-25°C) and IPTG concentration adjustment (0.1-0.5 mM) are necessary to maximize soluble protein yield.

What purification strategy yields the highest activity for recombinant sucC?

A multi-step purification approach yields the highest enzymatic activity:

• Initial capture: Affinity chromatography using His6-tag or GST-tag fusion proteins

• Intermediate purification: Ion exchange chromatography (typically DEAE or Q-Sepharose)

• Final polishing: Size exclusion chromatography to confirm oligomeric state

Critical factors affecting enzyme activity during purification include:

• Buffer composition: 50 mM Tris-HCl or phosphate buffer (pH 7.5-8.0)

• Salt concentration: 150-300 mM NaCl to maintain stability

• Reducing agents: 1-5 mM DTT or β-mercaptoethanol to prevent oxidation

• Stabilizing additives: 10-20% glycerol and 1-5 mM MgCl₂

• Temperature management: All purification steps at 4°C

For optimal enzymatic activity, co-expression or co-purification with the alpha subunit (sucD) is essential, as the functional enzyme exists as a heterodimer.

How can researchers optimize solubility when expressing recombinant sucC?

To enhance solubility of recombinant M. caseolyticus sucC, researchers should implement the following strategies:

• Temperature optimization:

  • Lower induction temperature (16-25°C)

  • Extended expression time (12-24 hours)

• Expression construct modifications:

  • Addition of solubility-enhancing tags (MBP, SUMO, Thioredoxin)

  • Co-expression with the alpha subunit (sucD)

  • Strategic removal of hydrophobic regions if they don't affect function

• Media and induction optimization:

  • Use of enriched media (Terrific Broth, Super Broth)

  • Reduced IPTG concentration (0.1-0.3 mM)

  • Addition of osmolytes (sorbitol, betaine) to culture media

• Lysis buffer optimization:

  • Inclusion of mild detergents (0.1% Triton X-100)

  • Higher salt concentration (300-500 mM NaCl)

  • Addition of stabilizing cofactors (1-5 mM ATP, 5-10 mM MgCl₂)

Co-expression with bacterial chaperones (GroEL/GroES, DnaK/DnaJ/GrpE) has also shown significant improvements in solubility for challenging recombinant proteins and may be beneficial for sucC expression.

What enzymatic assays can accurately measure Macrococcus caseolyticus sucC activity?

Several complementary assays can be employed to precisely measure sucC enzymatic activity:

For kinetic studies, the coupled spectrophotometric assay offers the best combination of convenience and accuracy. Typical reaction conditions include: 50 mM HEPES (pH 7.5), 10 mM MgCl₂, 0.2 mM NADH, 1 mM phosphoenolpyruvate, 5 units pyruvate kinase, 5 units lactate dehydrogenase, varying concentrations of succinate, CoA, and ATP.

How do substrate concentrations and environmental conditions affect sucC activity?

The kinetic properties of Macrococcus caseolyticus sucC are significantly influenced by multiple factors:

• Substrate concentration effects:

  • Succinate: Typical Km = 0.2-0.5 mM

  • CoA: Typical Km = 0.01-0.05 mM

  • ATP: Typical Km = 0.1-0.3 mM

  • Substrate inhibition may occur at high concentrations (>5 mM for succinate)

• Environmental conditions:

  • pH optimum: Typically 7.4-8.0

  • Temperature optimum: 30-37°C (species-dependent)

  • Ionic strength effect: Activity decreases above 300 mM salt

  • Divalent cation requirement: Mg²⁺ (optimal at 5-10 mM)

• Allosteric regulation:

  • Inhibited by: GTP, high energy charge (ATP:ADP ratio)

  • Activated by: ADP, inorganic phosphate

These parameters should be systematically investigated when characterizing recombinant sucC to establish optimal assay conditions that reflect physiologically relevant activity.

What structural elements are critical for sucC catalytic activity?

Based on homology with characterized succinyl-CoA ligases, several structural elements are critical for catalytic function:

• Nucleotide binding site: Contains the signature P-loop motif (Gly-X-X-Gly-X-Gly-Lys-Thr) essential for ATP binding and hydrolysis

• CoA binding domain: Features a characteristic CoA-binding motif with conserved basic residues that interact with the phosphate groups of CoA

• Succinate binding pocket: Lined with hydrophilic residues that coordinate the carboxyl groups of succinate

• Catalytic histidine residue: Forms the succinyl-phosphate intermediate during the reaction mechanism

• Subunit interface: Contains complementary charged and hydrophobic residues that facilitate heterodimerization with the alpha subunit

Site-directed mutagenesis studies targeting these regions would provide valuable insights into the specific catalytic mechanism of M. caseolyticus sucC and could identify unique features compared to homologs from other species.

How is the sucC gene organized in the Macrococcus caseolyticus genome?

In Macrococcus caseolyticus, the sucC gene organization follows patterns typical of bacteria in the Staphylococcaceae family:

• Operon structure: The sucC gene is typically co-transcribed with sucD (encoding the alpha subunit) in a bicistronic operon

• Genomic context: The suc operon is frequently positioned near other TCA cycle genes

• Regulatory elements:

  • Promoter contains recognition sites for carbon metabolism regulators

  • CcpA (catabolite control protein A) binding sites regulate expression based on carbon source availability

  • Potential anaerobic response elements control expression under oxygen limitation

• Conserved sequence elements: The coding sequence contains conserved motifs for substrate binding and catalysis that show high similarity to those in related genera like Staphylococcus

This genomic organization ensures coordinated expression of both subunits required for the functional enzyme complex, allowing precise regulation in response to metabolic demands.

How does sucC from Macrococcus caseolyticus compare evolutionarily to homologs in other bacteria?

Evolutionary analysis of sucC reveals important relationships across bacterial taxa:

Phylogenetic analysis suggests that sucC in Macrococcus evolved from a common ancestor shared with Staphylococcus, with subsequent divergence reflecting adaptation to different ecological niches . Conservation is highest in catalytic domains, while regulatory regions show greater variability, suggesting differential metabolic regulation across bacterial taxa.

The relatively high conservation of sucC across diverse bacterial species highlights the essential nature of this enzyme in central metabolism, despite the considerable ecological diversity of these organisms.

What can studying Macrococcus caseolyticus sucC reveal about bacterial metabolic evolution?

Investigating M. caseolyticus sucC provides several important insights into bacterial metabolic evolution:

• Metabolic pathway conservation: The high conservation of sucC across diverse bacteria demonstrates the fundamental nature of the TCA cycle in bacterial metabolism

• Specialization vs. conservation: Comparing variations in sucC sequence and regulation between pathogenic Staphylococcus and non-pathogenic Macrococcus can reveal adaptations related to different lifestyles

• Horizontal gene transfer assessment: Analysis of GC content, codon usage, and phylogenetic incongruence can identify potential horizontal gene transfer events affecting TCA cycle genes

• Metabolic adaptation: Differences in kinetic parameters and regulatory mechanisms can reflect adaptation to different environmental niches and nutrient availability

• Enzyme evolution: Comparison with eukaryotic homologs (like human SUCLA2 and SUCLG2) can trace the evolutionary divergence of substrate specificity (ATP vs. GTP utilizing enzymes)

These evolutionary insights contribute to our broader understanding of metabolic pathway evolution and adaptation in bacteria.

How can recombinant sucC be used to investigate TCA cycle regulation in Macrococcus species?

Recombinant sucC serves as a powerful tool for investigating TCA cycle regulation through several experimental approaches:

• In vitro reconstitution studies:

  • Combine purified recombinant sucC/sucD with other TCA cycle enzymes

  • Measure flux control coefficients to determine rate-limiting steps

  • Assess effects of metabolic intermediates on enzymatic activity

• Allosteric regulation analysis:

  • Systematically test potential allosteric effectors (ATP/ADP ratio, GTP, citrate)

  • Develop binding assays to measure effector interactions

  • Use site-directed mutagenesis to identify allosteric binding sites

• Post-translational modification studies:

  • Investigate phosphorylation, acetylation, or other modifications using mass spectrometry

  • Determine how modifications affect enzyme kinetics

  • Identify responsible kinases/acetyltransferases in Macrococcus

• Protein-protein interaction mapping:

  • Use pull-down assays with tagged recombinant sucC to identify interaction partners

  • Assess potential metabolon formation with other TCA cycle enzymes

  • Characterize interactions with regulatory proteins

These approaches can reveal unique aspects of TCA cycle regulation in Macrococcus compared to other bacterial species, providing insights into metabolic adaptation mechanisms.

What structure-function relationship studies can be performed using site-directed mutagenesis of sucC?

Site-directed mutagenesis enables detailed structure-function relationship studies of M. caseolyticus sucC:

Complementary approaches to enhance these studies include:

• Thermal shift assays to assess structural stability of mutants

• Circular dichroism to verify folding integrity

• Isothermal titration calorimetry to quantify binding affinities

• X-ray crystallography of mutant proteins to visualize structural changes

These studies can reveal unique features of bacterial sucC compared to eukaryotic homologs and identify potential targets for enzyme engineering.

How can metabolic flux analysis be used to study the role of sucC in Macrococcus caseolyticus?

Metabolic flux analysis provides comprehensive insights into the physiological role of sucC:

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

Researchers frequently encounter several challenges when working with recombinant sucC:

For optimal results, consider an integrated approach:

• Design constructs with removable solubility tags

• Express both subunits simultaneously using bicistronic vectors

• Include ATP and Mg²⁺ throughout purification

• Verify complex formation by native PAGE or size exclusion chromatography

• Validate enzyme activity immediately after purification

How can researchers differentiate between sucC activity and background enzymatic reactions?

Distinguishing specific sucC activity from background reactions requires several control experiments and methodological considerations:

• Comprehensive controls:

  • Heat-inactivated enzyme controls

  • Reaction mixtures lacking individual substrates

  • Comparison with known sucC inhibitors (e.g., itaconate)

  • Background measurements in crude lysates vs. purified enzyme

• Assay optimization:

  • Determine optimal substrate concentrations to maximize signal-to-noise ratio

  • Select detection methods with minimal interference from sample components

  • Use multiple orthogonal assay approaches to confirm findings

• Specific activity measurements:

  • Calculate and report enzyme-specific activity (μmol/min/mg protein)

  • Determine enzyme purity by SDS-PAGE and densitometry

  • Account for the heterodimeric nature when calculating molar concentrations

• Advanced techniques for complex samples:

  • Immunoprecipitation to isolate sucC before activity measurements

  • Genetic approaches (knockout/knockdown) to confirm specificity

  • Isotope-labeled substrates to track specific transformations

These approaches ensure accurate attribution of measured activity to the recombinant sucC rather than contaminating enzymes or spontaneous reactions.

What experimental design considerations are important when studying sucC in different physiological contexts?

When investigating sucC under various physiological conditions, researchers should consider:

• Growth condition standardization:

  • Define precise media composition and growth parameters

  • Monitor growth curves to sample at comparable physiological states

  • Consider carbon source effects on TCA cycle regulation

• Sample preparation consistency:

  • Standardize cell disruption methods to preserve enzyme activity

  • Process samples rapidly at low temperature to prevent degradation

  • Verify protein integrity before enzymatic measurements

• Comparative experimental design:

  • Include relevant reference strains (e.g., wild-type vs. sucC mutant)

  • Measure multiple metabolic enzymes simultaneously for context

  • Document environmental parameters (pH, temperature, oxygen availability)

• Data interpretation frameworks:

  • Normalize activity to appropriate references (cell number, protein content)

  • Consider enzyme adaptation timeframes (immediate vs. transcriptional responses)

  • Integrate with other metabolic measurements (oxygen consumption, ATP levels)

• Statistical considerations:

  • Perform biological replicates (different bacterial cultures)

  • Include technical replicates for enzymatic measurements

  • Apply appropriate statistical tests based on data distribution

These methodological considerations ensure reproducible and physiologically relevant insights into sucC function across different experimental contexts.