Recombinant Brucella suis Succinyl-CoA ligase [ADP-forming] subunit beta (sucC)

What is Succinyl-CoA ligase [ADP-forming] subunit beta (sucC) in Brucella suis?

Succinyl-CoA ligase [ADP-forming] subunit beta (sucC) in Brucella suis is a key enzyme in the tricarboxylic acid (TCA) cycle that catalyzes the reversible conversion of succinyl-CoA to succinate, coupled with the synthesis of ADP from AMP and inorganic phosphate. The protein structure is likely similar to that of Brucella abortus sucC, which consists of 398 amino acids as shown in available sequence data. The enzyme plays a fundamental role in energy metabolism within the bacteria, contributing to its survival and pathogenicity. Structurally, the protein contains multiple functional domains that facilitate substrate binding and catalytic activity essential for TCA cycle function .

How is the protein structure of Brucella suis sucC organized?

Based on homology with the closely related Brucella abortus sucC protein, the Brucella suis sucC protein contains several key structural domains. The amino acid sequence begins with an N-terminal region involved in protein-protein interactions, followed by nucleotide-binding domains that facilitate interaction with ADP/ATP. The central region contains the catalytic domain responsible for the enzymatic conversion of succinyl-CoA to succinate. The sequence from B. abortus sucC (which shares high similarity with B. suis) reveals conserved motifs critical for substrate binding and catalysis, including the characteristic GGRGKGKFK motif involved in nucleotide binding .

What expression systems are typically used to produce recombinant B. suis sucC?

Recombinant B. suis sucC is most commonly expressed in E. coli expression systems, similar to other Brucella proteins. The process typically involves cloning the sucC gene from the Brucella suis genome, inserting it into an appropriate expression vector, and transforming it into a suitable E. coli strain. Expression is usually induced under optimized conditions, followed by protein purification through techniques such as affinity chromatography. The purification protocol may include a tag system (such as His-tag) to facilitate isolation of the protein. The final product typically achieves >85% purity as determined by SDS-PAGE, similar to other recombinant Brucella proteins .

What are the optimal storage conditions for recombinant B. suis sucC?

For optimal preservation of recombinant B. suis sucC, storage recommendations based on similar recombinant proteins include maintaining the protein at -20°C for regular use and -80°C for extended storage. Addition of glycerol (typically 5-50% final concentration) is recommended to prevent freeze-thaw damage. It is advisable to avoid repeated freeze-thaw cycles, as these can significantly reduce protein activity. The shelf life in liquid form is generally around 6 months at -20°C/-80°C, while lyophilized forms may remain stable for up to 12 months at these temperatures. For working aliquots, storage at 4°C is suitable for up to one week .

What purification strategies yield the highest activity for recombinant B. suis sucC?

Based on established protocols for similar proteins, the optimal purification strategy for recombinant B. suis sucC typically involves a multi-step approach:

• Initial capture: Affinity chromatography using tags such as His-tag is the primary purification method

• Intermediate purification: Ion-exchange chromatography to remove contaminants with different charge properties

• Polishing: Size-exclusion chromatography to achieve final purity and remove aggregates

Buffer optimization is crucial, with most protocols using a neutral pH buffer (pH 7.0-8.0) containing 150-300 mM NaCl. Addition of reducing agents like DTT or β-mercaptoethanol may be necessary to maintain enzymatic activity by preventing oxidation of cysteine residues. The final product should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL for optimal stability and activity .

What assays can be used to measure enzymatic activity of recombinant B. suis sucC?

The choice of assay depends on available equipment, required sensitivity, and the specific aspect of enzyme kinetics being studied .

How does B. suis sucC compare to homologous proteins in other species?

These comparative differences highlight potential species-specific features that could be exploited for targeted drug development .

What role might sucC play in Brucella suis virulence and host-pathogen interactions?

SucC likely contributes to Brucella suis virulence through multiple mechanisms. As an essential component of the TCA cycle, it generates energy required for invasion of host cells and resistance to host defense mechanisms. During infection, Brucella must adapt to nutrient-limited environments within host cells, and the TCA cycle plays a crucial role in this metabolic adaptation. Drawing parallels from research on SUCLA2 in eukaryotic cells, sucC might also have non-canonical functions related to stress resistance, potentially contributing to survival under oxidative stress conditions encountered within macrophages .

Research approaches to investigate these roles include:

How do mutations in the sucC gene affect Brucella suis metabolic flux?

Mutations in the sucC gene would likely cause significant perturbations in Brucella suis metabolism. Based on the enzyme's role in the TCA cycle, potential effects include:

• Altered TCA cycle flux: Accumulation of succinyl-CoA and upstream metabolites, with decreased levels of succinate and downstream metabolites

• Energy production deficits: Disruption of substrate-level phosphorylation would reduce ADP generation

• Metabolic rewiring: Alternative pathways might be upregulated to compensate, such as the glyoxylate shunt

• Conditional essentiality: The importance of sucC likely varies depending on available carbon sources

A comprehensive research approach would involve creating defined sucC mutants, measuring growth under various conditions, conducting metabolomic analysis to quantify TCA cycle intermediates, and using isotope labeling to track carbon flux through central metabolism .

What is the relationship between sucC and oxidative stress response in Brucella?

Drawing parallels from research on SUCLA2 in mammalian cells, there may be important connections between sucC and oxidative stress response in Brucella. The TCA cycle, including the reaction catalyzed by sucC, influences redox balance through NADH production and consumption. Research has shown that in eukaryotic cells, SUCLA2 can relocate from mitochondria to cytosol under stress conditions and promote the formation of stress granules that facilitate the translation of antioxidant enzymes like catalase .

Similar mechanisms might exist in Brucella, where sucC could potentially have functions beyond its metabolic role. TCA cycle intermediates can act as signaling molecules, and changes in succinyl-CoA or succinate levels due to sucC activity might influence regulatory pathways involved in stress response. This connection is particularly relevant for intracellular pathogens like Brucella that must contend with oxidative stress during infection .

What techniques can be used to study protein-protein interactions of B. suis sucC?

Multiple complementary techniques can be employed to comprehensively study sucC protein-protein interactions:

• Co-immunoprecipitation (Co-IP) using antibodies against sucC, followed by mass spectrometry

• Bacterial two-hybrid systems adapted for prokaryotic protein interactions

• Chemical cross-linking coupled with mass spectrometry (XL-MS) to capture transient interactions

• Proximity labeling techniques using BioID or APEX2 fusions to label proximal proteins

• Surface plasmon resonance (SPR) or microscale thermophoresis (MST) for in vitro validation

• Fluorescence techniques such as FRET or BiFC for visualizing interactions in living cells

These methods could reveal whether sucC forms complexes beyond its known interaction with the alpha subunit and whether these interactions change under different environmental conditions, potentially uncovering non-canonical functions similar to those observed with mammalian SUCLA2 .

How can researchers differentiate between metabolic and potential non-metabolic functions of sucC?

Distinguishing between the metabolic and potential non-metabolic functions of sucC requires sophisticated experimental approaches:

• Domain mapping and mutational analysis: Creating mutants that retain structure but lack catalytic activity to determine whether non-metabolic functions persist

• Localization studies: Tracking sucC localization under different conditions, similar to how SUCLA2 was found to relocate from mitochondria to cytosol under stress

• Separation-of-function mutants: Identifying residues responsible for specific functions

• Metabolic bypass experiments: Providing intermediates to complement metabolic defects while observing whether other phenotypes persist

• Temporal analysis: Examining the timing of different phenotypes following sucC perturbation

• Heterologous complementation: Testing whether homologs from other species can complement specific functions

These approaches can help build a comprehensive understanding of sucC's complete functional repertoire in Brucella .

What are potential applications of recombinant B. suis sucC in vaccine development?

Recombinant B. suis sucC could be explored for vaccine development through several approaches:

• Subunit vaccine component: As part of a multi-antigen formulation with appropriate adjuvants

• Diagnostic tool development: Using recombinant sucC in serological assays to detect antibodies

• Target for attenuated live vaccines: Creating sucC mutants with reduced virulence but retained immunogenicity

• Epitope mapping: Identifying immunodominant regions that elicit protective immune responses

• Carrier protein: Using sucC as a carrier for Brucella polysaccharide antigens

Any vaccine development would require extensive testing for safety and efficacy. It's important to note that research-grade recombinant proteins "can only be used for research purposes" and "CANNOT be used directly on humans or animals" without proper development and regulatory approval .

How could structural information about sucC guide drug discovery against Brucella?

Structural insights into sucC could facilitate antimicrobial drug discovery through:

• Structure-based drug design: Using crystal structures or homology models to identify binding pockets for virtual screening

• Species-specific targeting: Identifying structural differences between B. suis sucC and mammalian SUCLA2 to design selective inhibitors

• Allosteric modulation: Finding regulatory sites that could lock the enzyme in an inactive conformation

• Protein-protein interaction disruptors: Developing compounds that interfere with essential protein complexes

• Covalent inhibitor development: Designing compounds that form irreversible bonds with reactive residues

The amino acid sequence of Brucella abortus sucC provides valuable information for homology modeling, with conserved motifs like "GGRGKGKFK" representing potential targets for inhibitor development. The sequence "MNIHEYQAKRLLHTYGAPIANG..." reveals the N-terminal region that could be exploited for species-specific targeting .

What are the major knowledge gaps in understanding B. suis sucC function?

Despite advances in characterizing sucC, significant knowledge gaps remain. These include: 1) The complete three-dimensional structure of B. suis sucC and how it differs from homologs; 2) Potential non-canonical functions beyond its established role in the TCA cycle; 3) The regulatory mechanisms controlling sucC expression during infection; 4) Whether sucC undergoes post-translational modifications that affect its function; and 5) The full interactome of sucC in different environmental conditions. Addressing these gaps would provide comprehensive understanding of this enzyme's role in Brucella physiology and pathogenesis .

What emerging technologies could advance research on B. suis sucC?

Several cutting-edge technologies could significantly advance our understanding of sucC:

• Cryo-electron microscopy for high-resolution structural determination

• Single-cell metabolomics to understand sucC's role in metabolic heterogeneity within bacterial populations

• CRISPR interference for precise temporal control of sucC expression

• Protein engineering approaches to create biosensors for monitoring sucC activity in vivo

• Advanced computational methods combining molecular dynamics simulations with machine learning for drug discovery

• Synthetic biology approaches to create Brucella strains with engineered sucC variants