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

What are the optimal storage conditions for maintaining long-term stability of the recombinant sucC protein?

For optimal stability, store the recombinant Herpetosiphon aurantiacus Succinyl-CoA ligase [ADP-forming] subunit beta at -20°C for routine storage or at -80°C for extended storage periods. The shelf life varies depending on the formulation: liquid preparations typically maintain stability for 6 months at -20°C/-80°C, while lyophilized preparations remain stable for approximately 12 months at the same temperatures .

To minimize protein degradation through multiple freeze-thaw cycles, it is recommended to prepare small working aliquots that can be stored at 4°C for up to one week. Repeated freezing and thawing should be avoided as this can significantly compromise protein integrity and enzymatic activity .

How should researchers properly reconstitute lyophilized recombinant sucC protein for experimental use?

For optimal reconstitution of lyophilized recombinant sucC protein:

• Centrifuge the vial briefly before opening to ensure all material is at the bottom of the tube.

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

• Add glycerol to a final concentration of 5-50% (with 50% being the standard recommendation) for cryoprotection during storage.

• Prepare multiple small-volume aliquots to avoid repeated freeze-thaw cycles.

• Store aliquots at -20°C or -80°C for long-term storage, or at 4°C for up to one week if being actively used .

This methodology ensures maximum retention of enzymatic activity while preventing protein aggregation or degradation that could interfere with experimental results.

What are the key functional assays for measuring Succinyl-CoA ligase activity in recombinant sucC preparations?

The standard methodology for assessing Succinyl-CoA ligase [ADP-forming] activity includes:

• ATP-PPi Exchange Assay: This assay measures the reverse reaction of succinyl-CoA formation by monitoring the incorporation of radioactive pyrophosphate into ATP. Similar to the γ-18O4-ATP pyrophosphate exchange assay referenced for other adenylation domains, this method can be adapted for sucC activity assessment .

• Coupled Spectrophotometric Assay: This assay couples ADP formation to NADH oxidation through pyruvate kinase and lactate dehydrogenase, allowing continuous monitoring at 340 nm.

• Direct Product Quantification: Using HPLC or LC-MS to quantify succinyl-CoA formation from succinate, CoA, and ATP.

The experimental conditions typically include:

• Buffer: 50 mM HEPES or Tris-HCl (pH 7.5-8.0)

• MgCl₂: 5-10 mM

• KCl: 50-100 mM

• ATP: 1-5 mM

• CoA: 0.1-0.5 mM

• Succinate: 1-10 mM

• Protein: 50-200 ng of purified enzyme

How can researchers verify the quaternary structure of recombinant Succinyl-CoA ligase to ensure proper α-β subunit association?

Since Succinyl-CoA ligase functions as a heterodimer consisting of α and β subunits, verifying proper quaternary structure is essential. The methodological approach should include:

• Size Exclusion Chromatography (SEC): Run the recombinant sucC protein through a calibrated SEC column to determine if it exists in monomeric form or forms higher-order structures.

• Native PAGE: Compare the migration pattern of recombinant sucC with and without the α subunit to assess complex formation.

• Dynamic Light Scattering (DLS): Measure the hydrodynamic radius to determine if the protein exists as a monomer or as part of a larger complex.

• Analytical Ultracentrifugation: Determine the sedimentation coefficient to assess the molecular weight and shape of the protein complex.

• Functional Assays: Compare the activity of the β subunit alone versus in combination with the α subunit to confirm that heterodimer formation enhances catalytic activity.

When working with the β subunit (sucC) alone, researchers should be aware that full enzymatic activity typically requires association with the α subunit (sucD), which may need to be co-expressed or added separately to functional assays.

What strategies can be employed to investigate the role of sucC in the broader metabolic network of Herpetosiphon aurantiacus?

Investigating the metabolic context of sucC requires a systems biology approach:

This multi-faceted approach provides a comprehensive understanding of how sucC contributes to central metabolism in H. aurantiacus beyond its catalytic function in the TCA cycle.

What are the comparative advantages of E. coli versus yeast expression systems for producing recombinant Herpetosiphon aurantiacus sucC?

Both E. coli and yeast expression systems are utilized for recombinant H. aurantiacus sucC production, with distinct advantages depending on research objectives:

The choice between these systems should be guided by the intended application. For structural studies requiring large protein quantities, E. coli may be preferable. For functional studies where proper folding and post-translational modifications are critical, the yeast system may produce a more native-like protein .

What purification challenges are specific to recombinant Succinyl-CoA ligase β subunit, and how can they be addressed?

Purification of recombinant sucC presents several challenges that require specific methodological approaches:

• Solubility Issues: The β subunit may form inclusion bodies in heterologous expression systems. This can be addressed by:

  • Optimizing growth temperature (typically lowering to 16-20°C)

  • Co-expressing with molecular chaperones

  • Using solubility-enhancing fusion partners like SUMO or MBP

• Maintaining Enzymatic Activity: The β subunit alone may show reduced activity without its partner α subunit. Consider:

  • Co-purifying with the α subunit

  • Including stabilizing agents like glycerol (5-10%) in purification buffers

  • Maintaining reducing conditions with DTT or β-mercaptoethanol

• Purification Strategy: A typical optimized protocol would include:

  • Initial capture using affinity chromatography (His-tag based purification shows >85% purity by SDS-PAGE)

  • Secondary purification by ion exchange chromatography

  • Final polishing by size exclusion chromatography

  • Buffer optimization to include stabilizing agents

• Quality Control: Verify purity and activity by:

  • SDS-PAGE (ensuring >85% purity as reported)

  • Western blotting

  • Activity assays comparing to known standards

This methodological framework provides a systematic approach to obtaining pure, active recombinant sucC suitable for downstream applications.

How can researchers determine the substrate specificity of H. aurantiacus Succinyl-CoA ligase compared to orthologous enzymes from other species?

To systematically characterize the substrate specificity of H. aurantiacus Succinyl-CoA ligase:

• Kinetic Analysis with Various Substrates: Test the enzyme activity with:

  • Different dicarboxylic acids (succinate, glutarate, adipate)

  • Alternative nucleotides (ATP, GTP, ITP)

  • CoA analogs or derivatives

• Structure-Based Approaches:

  • Homology modeling based on the known crystal structures of Succinyl-CoA ligases from other species

  • Molecular docking simulations to predict substrate binding modes

  • Site-directed mutagenesis of predicted substrate-binding residues

• Comparative Analysis: Create a substrate specificity profile comparing H. aurantiacus Succinyl-CoA ligase with orthologous enzymes from model organisms. This should include:

  • Km values for each substrate

  • kcat values

  • Catalytic efficiency (kcat/Km) ratios

  • Inhibition profiles

• Isothermal Titration Calorimetry (ITC): Directly measure binding affinities for different substrates to complement kinetic data.

This comprehensive approach will reveal unique features of the H. aurantiacus enzyme that may reflect its adaptation to the organism's specific metabolic requirements or ecological niche.

What computational approaches can be used to predict the three-dimensional structure and catalytic mechanism of H. aurantiacus sucC?

Contemporary computational approaches for structural and mechanistic analysis of sucC include:

• Homology Modeling:

  • Identify suitable templates from solved structures of Succinyl-CoA ligases

  • Generate multiple models using different algorithms (SWISS-MODEL, I-TASSER, AlphaFold2)

  • Validate models using tools like PROCHECK, VERIFY3D, and ProSA

• Molecular Dynamics Simulations:

  • Run long-timescale simulations (>100 ns) to sample conformational space

  • Analyze protein flexibility, especially around the active site

  • Identify water networks and ion-binding sites critical for catalysis

• Quantum Mechanics/Molecular Mechanics (QM/MM):

  • Model the reaction mechanism at the electronic level

  • Calculate energy barriers for different proposed catalytic pathways

  • Predict the roles of specific residues in catalysis

• Evolutionary Coupling Analysis:

  • Identify co-evolving residues that may be functionally linked

  • Predict residue networks important for allosteric regulation

  • Guide mutagenesis experiments to test predicted functional interactions

These computational approaches should be integrated with experimental data from mutagenesis, kinetic studies, and structural biology to develop a comprehensive understanding of sucC function.

How does H. aurantiacus Succinyl-CoA ligase compare to homologous enzymes in terms of sequence conservation and evolutionary history?

Evolutionary analysis of H. aurantiacus Succinyl-CoA ligase [ADP-forming] subunit beta reveals important insights into its conservation and adaptation:

The protein sequence from H. aurantiacus (UniProt: A9B0I2) shows several conserved domains characteristic of this enzyme family, including nucleotide-binding motifs and substrate recognition regions . Comparative analysis with homologs from other bacterial phyla reveals:

• Core Catalytic Domain: The ATP-binding region and CoA-binding pocket show high conservation across bacterial species, indicating the fundamental importance of these regions for catalytic function.

• Lineage-Specific Adaptations: Regions involved in subunit interaction (α-β interface) show greater variability, suggesting adaptation to specific partner proteins in different bacterial lineages.

• Horizontal Gene Transfer: Similar to observations for other metabolic genes in Asgard archaea, sucC homologs may have been subject to horizontal gene transfer events during bacterial evolution . This is consistent with the broader patterns of recombination observed in bacterial population genetics .

• Functional Conservation: Despite sequence divergence, the enzymatic function of Succinyl-CoA ligase is highly conserved across prokaryotes, reflecting its central role in the TCA cycle and energy metabolism.

The evolutionary trajectory of sucC genes provides valuable context for understanding metabolic adaptation in different bacterial lineages, including the specialized metabolism observed in H. aurantiacus.

What role might Succinyl-CoA ligase play in the unique metabolic capabilities and ecological adaptations of Herpetosiphon aurantiacus?

H. aurantiacus is known for its gliding motility, predatory behavior, and production of secondary metabolites, all of which may be linked to its central metabolic pathways. The role of Succinyl-CoA ligase in these processes can be analyzed through several perspectives:

• Integration with Secondary Metabolism: The genomic analysis of H. aurantiacus has revealed numerous biosynthetic gene clusters, including PKS/NRPS loci that produce specialized metabolites . Succinyl-CoA, generated partly through the action of Succinyl-CoA ligase, serves as a key precursor for many secondary metabolites, potentially linking primary and secondary metabolism.

• Energy Conservation: As a key enzyme in the TCA cycle, Succinyl-CoA ligase catalyzes substrate-level phosphorylation, generating ATP or GTP. This energy conservation mechanism may be particularly important for H. aurantiacus in its predatory lifestyle, which requires energy for motility and secretion systems.

• Metabolic Flexibility: The ability to modulate Succinyl-CoA ligase activity could allow H. aurantiacus to adapt to different carbon sources and energy states, supporting its survival in diverse ecological niches.

• Coordination with Gliding Motility: The energy requirements for gliding motility may be partially supported by the ATP generated through Succinyl-CoA ligase activity, suggesting a potential link between central metabolism and this distinctive behavioral trait.

Research examining the expression patterns of sucC under different ecological conditions would provide valuable insights into how this enzyme contributes to the unique ecological adaptations of H. aurantiacus.

How can site-directed mutagenesis of H. aurantiacus sucC be used to study structure-function relationships and catalytic mechanisms?

A systematic site-directed mutagenesis approach for studying H. aurantiacus sucC should target key functional regions:

• ATP-Binding Site: Mutations in the conserved ATP-binding motifs would be expected to alter nucleotide preference or catalytic efficiency. Suggested residues include:

  • Lysine residues involved in phosphate coordination

  • Glycine-rich loop residues that form the nucleotide-binding pocket

  • Residues that determine ATP vs. GTP specificity

• CoA-Binding Site: Mutations affecting CoA recognition and binding would provide insights into substrate specificity. Target residues would include those that interact with:

  • The adenosine moiety of CoA

  • The pantetheine arm

  • The terminal thiol group

• Catalytic Residues: Mutations of putative catalytic residues would help elucidate the reaction mechanism:

  • Acidic residues potentially involved in metal coordination

  • Basic residues that might act as general bases

  • Hydrophobic residues forming the succinate-binding pocket

• α-Subunit Interface: Mutations at the interface with the α-subunit would reveal the structural basis for subunit cooperation:

  • Charged residues forming salt bridges

  • Hydrophobic residues contributing to the hydrophobic core

  • Residues involved in allosteric communication

For each mutation, a comprehensive characterization should include:

• Steady-state kinetic parameters (Km, kcat, kcat/Km)

• Binding affinity measurements by ITC or fluorescence

• Thermal stability assessment by differential scanning fluorimetry

• Structural characterization by circular dichroism or X-ray crystallography when possible

This systematic approach would generate a detailed map of structure-function relationships in H. aurantiacus sucC.

What biophysical techniques are most effective for studying the conformational dynamics of Succinyl-CoA ligase during its catalytic cycle?

Understanding the conformational changes that occur during the catalytic cycle of Succinyl-CoA ligase requires a multi-technique approach:

• X-ray Crystallography with Different Ligands:

  • Crystallize the enzyme in different states (apo, ATP-bound, CoA-bound, succinate-bound)

  • Solve structures to identify conformational changes associated with each step

  • Use time-resolved crystallography when possible to capture short-lived intermediates

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

  • Map regions of differential solvent accessibility in various ligand-bound states

  • Identify flexible regions that undergo conformational changes during catalysis

  • Monitor the effects of mutations on protein dynamics

• Single-Molecule Förster Resonance Energy Transfer (smFRET):

  • Introduce fluorescent labels at strategic positions

  • Measure distance changes between domains during substrate binding and catalysis

  • Directly observe conformational trajectories of individual enzyme molecules

• Nuclear Magnetic Resonance (NMR) Spectroscopy:

  • Use chemical shift perturbation experiments to map ligand binding sites

  • Employ relaxation dispersion techniques to characterize millisecond dynamics

  • Apply residual dipolar couplings to determine relative domain orientations

• Molecular Dynamics Simulations:

  • Conduct extensive simulations to sample conformational space

  • Use enhanced sampling techniques to capture rare events

  • Integrate experimental restraints from HDX-MS or FRET to guide simulations

By combining these complementary techniques, researchers can construct a dynamic model of how Succinyl-CoA ligase coordinates structural changes to achieve catalysis, particularly focusing on domain movements and allosteric communication between the α and β subunits.