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

What is Succinyl-CoA ligase [ADP-forming] subunit beta (sucC) and what is its role in Brevibacillus brevis metabolism?

Succinyl-CoA ligase (also known as succinyl-CoA synthetase) is a critical enzyme in the TCA cycle that catalyzes the reversible reaction: succinyl-CoA + ADP + Pi ⇌ succinate + ATP + CoA. The β-subunit (encoded by sucC) contains the catalytic site for the phosphorylation reaction. In Brevibacillus brevis, this enzyme plays a central role in energy metabolism, participating in both catabolic and anabolic processes. The sucC gene in B. brevis encodes the ADP-forming β-subunit, which determines the nucleotide specificity of the reaction .

Within the bacterial genome, sucC is often organized in an operon with sucD (encoding the α-subunit). Together, these subunits form a functional heterodimer that catalyzes the conversion of succinyl-CoA to succinate, a key step in the TCA cycle. This reaction is particularly important in B. brevis metabolism as it represents one of the few steps in central metabolism where substrate-level phosphorylation occurs, generating ATP directly without requiring the electron transport chain .

What expression systems are currently used for producing recombinant Brevibacillus brevis sucC protein?

Several expression systems have been successfully employed for producing recombinant B. brevis sucC protein, each with distinct advantages:

• Brevibacillus choshinensis expression system: This system has demonstrated particular success for B. brevis proteins. The system utilizes an efficient promoter and the secretion signal of its surface layer protein, allowing for direct secretion of the recombinant protein into the culture medium . This approach simplifies downstream purification and maintains protein functionality.

• Escherichia coli expression systems: E. coli remains a popular choice, particularly with amino-terminal His-tag modifications (His-sucC) that facilitate purification using Ni-NTA affinity chromatography. When expressed in E. coli cytoplasm, recombinant proteins can be purified to homogeneity using column chromatography techniques .

• Cell-free protein synthesis: For proteins that might be toxic to host cells, cell-free systems based on bacterial lysates provide an alternative production method, though yields may be lower than in vivo systems.

The choice of expression system should be guided by specific research requirements, including protein yield, purity needs, post-translational modifications, and intended downstream applications.

How can researchers verify the enzymatic activity of recombinant Brevibacillus brevis sucC protein?

Verification of enzymatic activity for recombinant B. brevis sucC protein involves several complementary approaches:

• Thiol-disulfide oxidoreductase activity assay: Similar to other thioredoxin superfamily proteins, activity can be measured using standard thiol-disulfide exchange reactions . This assay monitors the ability of the enzyme to facilitate electron transfer between substrate molecules.

• Coupled enzyme assays: The ADP-forming activity can be measured by coupling succinyl-CoA conversion to succinate with a secondary enzyme system that monitors either ADP consumption or ATP production. Common coupling systems include pyruvate kinase/lactate dehydrogenase, which link ATP production to NADH oxidation (measurable at 340 nm).

• Isotopic exchange assays: Using radiolabeled substrates (such as 14C-succinate or 32P-ATP) to monitor the forward and reverse reactions catalyzed by the enzyme.

The specific assay conditions should be optimized for B. brevis sucC, including appropriate pH (typically 7.2-7.8), temperature (30-37°C), and buffer composition (often containing divalent cations like Mg2+ that are essential for activity) .

What are the optimal experimental design principles for studying recombinant Brevibacillus brevis sucC function?

When designing experiments to study recombinant B. brevis sucC function, researchers should adhere to four fundamental pillars of experimental design:

• Replication: Ensure adequate biological and technical replicates to establish statistical validity. For enzyme kinetic studies, a minimum of three independent protein preparations should be tested, with each assay performed in triplicate .

• Randomization: Implement proper randomization of experimental units to minimize systematic errors or biases. This is particularly important when testing multiple conditions or comparing different protein variants .

• Blocking: Use appropriate blocking strategies to control for confounding variables. For instance, when comparing wild-type and mutant versions of sucC, ensure that all proteins are expressed and purified under identical conditions .

• Experimental unit size: Determine appropriate sample size through power analysis to ensure sufficient statistical power while minimizing resource waste .

Remember that experimental design should be viewed not as a rigid recipe but as a creative problem-solving process tailored to address specific research questions about sucC function .

How should researchers approach contradictory data when studying Brevibacillus brevis sucC enzyme kinetics?

When faced with data that contradicts hypotheses about B. brevis sucC enzyme kinetics, researchers should follow a systematic approach:

Contradictory data often leads to important discoveries. For example, unexpected findings in succinyl-CoA ligase studies have revealed its involvement in mtDNA maintenance through interaction with nucleoside diphosphate kinase, a connection not initially predicted from its primary metabolic role .

What strategies are effective for optimizing recombinant Brevibacillus brevis sucC expression yield and solubility?

Optimizing recombinant B. brevis sucC expression requires careful consideration of several factors:

• Codon optimization: Analyze the codon usage bias of the expression host and modify the sucC sequence accordingly. For expression in E. coli, codon adaptation index (CAI) values of >0.8 typically yield better results.

• Expression vector selection: Choose vectors with appropriate promoters based on expression goals:

  • For high-level expression, T7 or tac promoters in E. coli systems

  • For controlled expression, arabinose or tetracycline-inducible systems

  • For secreted expression in Brevibacillus, vectors containing the cell wall protein promoter and signal sequence

• Fusion tags selection: Strategic use of fusion partners can enhance solubility:

  • N-terminal His-tags facilitate purification while minimally affecting activity

  • MBP (maltose-binding protein) or SUMO tags can significantly improve solubility

  • Thioredoxin fusion may enhance disulfide bond formation

• Culture conditions optimization: Fine-tune expression conditions:

  • Reduce induction temperature to 16-25°C to improve folding

  • Adjust induction timing to mid-log phase (OD600 ~0.6-0.8)

  • Supplement with specific additives (glycylglycine, sorbitol, arginine) to improve protein folding

• Co-expression strategies: Consider co-expressing molecular chaperones (GroEL/ES, DnaK/J) or the partner α-subunit (sucD) to enhance proper folding and assembly of the functional heterodimer.

A systematic approach testing these variables in combination often yields the best results for challenging proteins like sucC.

How can researchers effectively analyze the interaction between recombinant Brevibacillus brevis sucC and the α-subunit (sucD)?

Analyzing the interaction between sucC (β-subunit) and sucD (α-subunit) requires multiple complementary approaches:

• Co-immunoprecipitation (Co-IP): Using antibodies against either subunit to pull down the complex, followed by western blot analysis to detect the partner protein. This confirms direct interaction in solution.

• Biolayer interferometry (BLI) or surface plasmon resonance (SPR): These techniques provide quantitative binding kinetics (kon, koff) and affinity (KD) measurements between purified sucC and sucD subunits.

• Size-exclusion chromatography with multi-angle light scattering (SEC-MALS): This approach determines the absolute molecular weight of the complex and confirms the expected stoichiometry (α2β2 tetramer or αβ dimer).

• Isothermal titration calorimetry (ITC): Provides thermodynamic parameters (ΔH, ΔS, ΔG) of subunit interaction, offering insights into the driving forces of complex formation.

• Functional complementation: Activity assays comparing individual subunits versus the reconstituted complex can demonstrate the functional significance of the interaction.

When analyzing subunit interactions, it's crucial to consider the role of divalent cations (typically Mg2+) and nucleotides, as these factors often stabilize the complex and are essential for proper assembly .

What are the key structural features of Brevibacillus brevis sucC that determine its catalytic properties?

The catalytic properties of B. brevis sucC (β-subunit) are determined by several critical structural features:

• Nucleotide-binding domain: Contains the characteristic P-loop (phosphate-binding loop) motif that coordinates the phosphate groups of ADP/ATP. This glycine-rich sequence (typically GXXXXGK[T/S]) is essential for nucleotide specificity .

• CoA-binding pocket: A hydrophobic cavity with specific residues that form hydrogen bonds with the pantetheine moiety of CoA. This region is critical for substrate recognition and positioning.

• Catalytic histidine: A conserved histidine residue serves as the catalytic base, facilitating the formation of the phosphorylated enzyme intermediate during the reaction cycle.

• Interface with α-subunit: The β-subunit contains specific residues that form the interface with the α-subunit, creating the composite active site necessary for full catalytic function.

• Metal-binding sites: Coordination sites for divalent cations (typically Mg2+) that are essential for both structural stability and catalytic activity.

How can site-directed mutagenesis be used to investigate the functional domains of recombinant Brevibacillus brevis sucC?

Site-directed mutagenesis provides a powerful approach to dissect the structure-function relationships in recombinant B. brevis sucC:

• Target selection strategy:

  • Conserved residues identified through multiple sequence alignment

  • Residues predicted to interact with substrates or cofactors

  • Interface residues involved in α/β subunit interaction

  • Residues implicated in conformational changes during catalysis

• Mutation design principles:

  • Conservative substitutions (e.g., Asp→Glu) to probe specific chemical properties

  • Non-conservative substitutions (e.g., Asp→Ala) to eliminate side chain functions

  • Introduction of cysteine residues for site-specific labeling studies

  • Creation of chimeric proteins by swapping domains with homologous enzymes

• Functional analysis workflow:

• Interpretation framework:

  • Compare multiple mutations affecting the same functional element

  • Correlate functional changes with structural predictions

  • Use molecular dynamics simulations to interpret experimental results

  • Develop comprehensive models of enzyme mechanism based on mutagenesis data

Recent studies on related succinyl-CoA ligases have demonstrated that mutations in the nucleotide-binding domain can alter specificity between ATP and GTP, while mutations in the CoA-binding pocket can affect the balance between forward and reverse reactions .

How can genomic analysis inform our understanding of Brevibacillus brevis sucC evolution and function?

Genomic analysis provides critical insights into B. brevis sucC evolution and function through several approaches:

• Comparative genomics: Analysis of sucC across Brevibacillus species reveals conservation patterns and evolutionary relationships. The B. brevis genome contains 6107 protein-coding genes, with sucC showing high conservation across the genus, suggesting its fundamental metabolic importance .

• Operon structure analysis: Examination of the genomic context surrounding sucC can reveal co-regulated genes and functional associations. In many bacteria, sucC and sucD form an operon, sometimes with additional metabolic genes that participate in related pathways.

• Phylogenetic profiling: Comparing the presence/absence patterns of sucC across diverse bacteria identifies co-occurring genes that may function in the same pathways or protein complexes.

• Horizontal gene transfer (HGT) assessment: Analysis of GC content (B. brevis has a GC content of approximately 54.3%) and codon usage bias can identify potential HGT events that might have shaped sucC evolution .

• Regulatory element identification: Analyzing promoter regions and transcription factor binding sites upstream of sucC provides insights into its regulation under different metabolic conditions.

Recent genomic studies have revealed unexpected connections between succinyl-CoA ligase and nucleoside diphosphate kinase function, highlighting how genomic context analysis can reveal non-obvious functional relationships .

What approaches are effective for investigating the potential role of Brevibacillus brevis sucC in mitochondrial DNA maintenance?

Investigating B. brevis sucC's potential role in mitochondrial DNA maintenance requires specialized approaches that connect bacterial model systems to eukaryotic mitochondrial function:

• Heterologous expression systems: Express B. brevis sucC in eukaryotic cells with mitochondrial targeting sequences to assess complementation of deficient succinyl-CoA ligase activity and rescue of mtDNA depletion phenotypes.

• Protein-protein interaction studies: Identify potential interactions between sucC and nucleoside diphosphate kinase (NDPK) proteins, as this interaction has been implicated in mtDNA maintenance mechanisms .

• Nucleotide pool analysis: Measure the impact of altered sucC activity on cellular dNTP pools, which are critical for mtDNA replication fidelity. Imbalances in nucleotide triphosphates have been proposed as the mechanism linking succinyl-CoA ligase dysfunction to mtDNA depletion .

• Mitochondrial DNA quantification: Assess mtDNA copy number in cells expressing wild-type versus mutant forms of sucC to establish direct connections between enzyme activity and DNA maintenance.

• Mouse models: Develop transgenic mouse models expressing B. brevis sucC variants to study in vivo effects on mitochondrial function and mtDNA stability across different tissues.

Research on human patients with SUCLG1 mutations has shown that even modest reductions in mtDNA (to 65-50% of normal levels) can significantly impact mitochondrial function, suggesting a critical threshold effect that should be considered when designing experiments .

How can structural biology techniques be applied to understand the catalytic mechanism of Brevibacillus brevis sucC?

Advanced structural biology techniques provide deep insights into the catalytic mechanism of B. brevis sucC:

• X-ray crystallography: Obtaining high-resolution crystal structures of sucC in various states:

  • Apo-enzyme (without ligands)

  • Binary complexes with individual substrates/products

  • Ternary complexes with transition state analogs

  • Structures with site-directed mutations

• Cryo-electron microscopy (cryo-EM): Particularly valuable for visualizing the complete α2β2 tetramer structure and capturing different conformational states during the catalytic cycle.

• Nuclear magnetic resonance (NMR) spectroscopy: Provides dynamic information about protein motions during catalysis:

  • Chemical shift perturbation experiments to map ligand binding sites

  • Relaxation dispersion experiments to detect conformational exchange

  • Hydrogen-deuterium exchange to identify flexible regions

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Maps regions of protein flexibility and solvent accessibility changes upon ligand binding or subunit association.

• Molecular dynamics simulations: Complements experimental data by simulating:

  • Substrate approach and binding

  • Conformational changes during catalysis

  • Water molecule roles in active site chemistry

  • Effects of mutations on protein dynamics

Correlation of structural insights with biochemical data is essential for developing a complete mechanistic model. For example, structures of related succinyl-CoA ligases have revealed substantial conformational changes during the catalytic cycle, particularly in the positioning of the histidine residue that forms the phosphorylated enzyme intermediate .

What strategies can address challenges in purifying recombinant Brevibacillus brevis sucC to homogeneity?

Purifying recombinant B. brevis sucC to homogeneity presents several challenges that can be addressed with specialized strategies:

• Affinity tag selection and placement:

  • N-terminal His-tags have proven successful for purifying related proteins to homogeneity using Ni-NTA chromatography

  • C-terminal tags may be preferable if the N-terminus is involved in folding or activity

  • Cleavable tags with precision protease sites allow tag removal after purification

• Multi-step purification protocols:

• Stabilization strategies during purification:

  • Addition of glycerol (10-20%) to prevent aggregation

  • Including reducing agents (DTT, β-mercaptoethanol) to prevent disulfide formation

  • Maintaining physiological concentrations of Mg2+ (1-5 mM) for structural stability

  • Adding ADP or ATP analogs to stabilize the native conformation

• Addressing proteolytic degradation:

  • Use of protease inhibitor cocktails throughout purification

  • Performing all steps at 4°C to minimize proteolysis

  • Expression in protease-deficient strains

  • Engineering out potential protease recognition sites

• Secretion-based purification:

  • Utilizing Brevibacillus choshinensis expression systems that secrete the protein directly into the culture medium, simplifying initial purification steps

Successful purification typically requires optimization of multiple parameters simultaneously, with protein activity assays at each step to monitor recovery of functional enzyme.

How can researchers effectively design and analyze site-directed mutagenesis experiments for Brevibacillus brevis sucC?

Designing and analyzing site-directed mutagenesis experiments for B. brevis sucC requires careful planning and robust analytical approaches:

• Strategic mutation selection:

  • Use sequence alignments with homologous proteins to identify conserved residues

  • Analyze available structures of related succinyl-CoA ligases to identify potential catalytic residues

  • Design mutation series (e.g., Asp → Glu → Asn → Ala) to systematically alter chemical properties

  • Consider double mutant cycles to test functional coupling between residues

• Mutagenesis method selection:

  • QuikChange PCR for single amino acid substitutions

  • Gibson Assembly for multiple mutations or larger modifications

  • Golden Gate Assembly for creating libraries of variants at specific positions

• Control inclusion:

  • Wild-type protein expressed and purified in parallel

  • Neutral mutations (e.g., conservative surface residue changes) as negative controls

  • Known catalytic mutations from homologous enzymes as positive controls

• Comprehensive characterization workflow:

• Data interpretation framework:

  • Calculate mutation effects as ΔΔG (change in activation energy)

  • Construct free energy profiles for the reaction with different mutations

  • Map mutations to specific steps in the catalytic mechanism

  • Integrate results with computational modeling (e.g., QM/MM)

Successful mutagenesis studies on related enzymes have revealed that subtle changes in the active site can dramatically alter the rate-limiting step or cause unexpected effects on substrate specificity, highlighting the importance of comprehensive characterization .

What are the future research directions for Brevibacillus brevis sucC that hold the most promise?

Several promising future research directions for B. brevis sucC warrant investigation:

• Structural biology advancements: Obtaining high-resolution structures of the complete B. brevis succinyl-CoA ligase heterotetramer in multiple conformational states would provide unprecedented insights into its catalytic mechanism and allosteric regulation.

• Synthetic biology applications: Engineering B. brevis sucC variants with altered nucleotide specificity or enhanced catalytic efficiency could create valuable biocatalysts for synthesizing CoA derivatives or generating high-energy compounds.

• Systems biology integration: Investigating how sucC activity is coordinated with other TCA cycle enzymes and peripheral metabolic pathways in B. brevis would enhance our understanding of bacterial metabolic regulation and adaptation.

• Comparative analysis with mitochondrial enzymes: Detailed functional comparisons between bacterial sucC and human mitochondrial SUCLG1/SUCLG2 could identify both conserved catalytic features and species-specific regulatory mechanisms, potentially informing therapeutic approaches for mitochondrial disorders .

• Development of activity-based probes: Creating chemical probes specific for active sucC would enable monitoring of enzyme activity in complex biological samples and facilitate drug discovery for targeting bacterial metabolism.

The intersection of these research directions with emerging technologies in structural proteomics, synthetic biology, and systems biology promises to deliver comprehensive insights into this evolutionarily conserved and metabolically central enzyme.

How can researchers address the challenges of data reproducibility when studying recombinant Brevibacillus brevis sucC?

Addressing reproducibility challenges in recombinant B. brevis sucC research requires systematic approaches:

• Standardized expression and purification protocols:

  • Detailed documentation of expression conditions (temperature, induction time, media composition)

  • Specific purification parameters (column types, buffer compositions, flow rates)

  • Consistent quality control criteria (purity thresholds, activity benchmarks)

• Rigorous experimental design principles:

  • Sufficient replication (biological and technical)

  • Appropriate randomization and blinding procedures

  • Inclusion of proper positive and negative controls

  • Power analysis to determine adequate sample sizes

• Comprehensive reporting standards:

  • Complete methods documentation including all variables that could affect outcomes

  • Raw data preservation and sharing through repositories

  • Detailed statistical analysis methods

  • Transparent reporting of both successful and failed experiments

• Cross-validation approaches:

  • Verification of key findings using alternative experimental methods

  • Collaborative validation across different laboratories

  • Use of orthogonal analytical techniques for critical measurements

• Managing biological variability:

  • Consistent source materials (plasmid verification, host strain authentication)

  • Controlled growth conditions for expression hosts

  • Standardized enzyme storage conditions

  • Regular activity benchmarking with reference substrates

• Dealing with unexpected data:

  • Systematic approach to investigating contradictory results

  • Consideration of alternative hypotheses

  • Refinement of methods based on new information