Recombinant Burkholderia vietnamiensis Succinyl-CoA ligase [ADP-forming] subunit beta (sucC)

What is the function of Succinyl-CoA ligase [ADP-forming] subunit beta in Burkholderia vietnamiensis?

Succinyl-CoA ligase [ADP-forming] subunit beta (sucC) in B. vietnamiensis functions as the β subunit of the ADP-forming succinyl-CoA synthetase (SCS-A). This mitochondrial matrix enzyme catalyzes the formation of succinate and ATP from succinyl-CoA and ADP in the tricarboxylic acid (TCA) cycle in a reversible manner. The β subunit specifically determines the nucleotide specificity of the enzyme complex, with sucC favoring ADP as the phosphate donor in the reaction . While SCS-A shares its α subunit with another form of the enzyme (SCS-G) that uses GDP, the β subunit encoded by sucC provides the specificity for ADP utilization in energy metabolism.

How does the sucC gene in Burkholderia vietnamiensis compare to homologous genes in other bacterial species?

The sucC gene in Burkholderia vietnamiensis belongs to a conserved family of genes encoding ADP-forming succinyl-CoA synthetase β subunits found across bacterial species. While specific sequence variations exist, the core functional domains remain conserved. Multilocus sequence typing (MLST) analyses have revealed that sucC can be reliably amplified and sequenced across various Burkholderia species, indicating significant conservation within the genus . The sequence homology allows researchers to use similar primer sets for amplification across different Burkholderia species, although specific nucleotide differences can be used for species identification and phylogenetic analysis. These sequence variations reflect evolutionary adaptations to different metabolic requirements while maintaining the core catalytic function.

What molecular techniques are most effective for isolating the sucC gene from Burkholderia vietnamiensis?

For isolating the sucC gene from B. vietnamiensis, PCR amplification using redesigned primers targeting conserved regions has proven most effective. Based on expanded MLST approaches for Burkholderia species, primers can be designed to reliably amplify this locus not only from B. vietnamiensis but also from other Burkholderia species . The approach typically involves:

• Genomic DNA extraction using commercial kits optimized for Gram-negative bacteria

• PCR amplification using primers designed for the conserved regions flanking the sucC gene

• Verification by gel electrophoresis followed by sequencing

• Cloning into appropriate vectors for expression studies

This approach has successfully amplified previously difficult-to-amplify genes from various Burkholderia species using the redesigned primers and optimized PCR conditions .

What expression systems are optimal for producing recombinant Burkholderia vietnamiensis Succinyl-CoA ligase?

The optimal expression system for producing recombinant B. vietnamiensis Succinyl-CoA ligase depends on research objectives and downstream applications. For high-yield protein production, E. coli-based systems using pET vectors under T7 promoter control have shown good results. When selecting an expression system, consider the following parameters:

When working with recombinant Burkholderia proteins, researchers should ensure compliance with NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules, which specify appropriate containment practices based on risk assessment .

How can researchers effectively measure the enzymatic activity of recombinant sucC protein?

Measuring the enzymatic activity of recombinant sucC protein (β subunit) requires reconstitution with the α subunit to form a functional Succinyl-CoA synthetase complex. The standard methodology involves:

• Spectrophotometric assays measuring the formation of succinyl-CoA from succinate and CoA in the presence of ATP, coupled with auxiliary enzymes

• Direct measurement of ADP formation using HPLC or coupled enzyme assays

• Radiometric assays tracking the incorporation of labeled substrates

The most reliable approach for determining SCS-A activity is in the direction of succinyl-CoA formation, with typical activity in control samples ranging from 0.18-0.25 U/mg of mitochondrial protein . For accurate measurements, researchers should:

• Ensure the presence of both α and β subunits in appropriate stoichiometry

• Maintain optimal buffer conditions (typically pH 7.4-7.8)

• Include appropriate metal cofactors (Mg²⁺)

• Control temperature (usually 30-37°C)

• Verify linearity of the reaction over the measurement period

Control experiments should include measurements with heat-inactivated enzyme and with individual subunits to confirm the specificity of the measured activity.

What purification strategies yield the highest purity and activity for recombinant Burkholderia vietnamiensis sucC?

Purification of recombinant B. vietnamiensis sucC protein requires a multi-step approach to ensure both high purity and preserved enzymatic activity. The most effective strategy typically combines:

• Affinity chromatography: Histidine-tagged recombinant sucC can be purified using Ni-NTA columns, with elution performed using an imidazole gradient (50-300 mM) to reduce co-purification of contaminating proteins

• Ion exchange chromatography: Typically using Q-Sepharose at pH 8.0, which separates the protein based on its charge properties

• Size exclusion chromatography: As a final polishing step to separate monomeric from aggregated forms and remove any remaining impurities

Throughout the purification process, maintaining protein stability is critical. Addition of glycerol (10-15%), reducing agents like DTT or β-mercaptoethanol, and working at 4°C significantly improves retention of enzymatic activity. The purified protein should be assessed for both purity (using SDS-PAGE) and activity (using the enzymatic assays described in section 2.2).

How does the association between sucC and nucleoside diphosphate kinase affect experimental approaches?

The tight association between Succinyl-CoA synthetase and nucleoside diphosphate kinase (NDPK) in both prokaryotes and eukaryotes has significant implications for research approaches . This association creates a functional complex that influences nucleotide metabolism, particularly for maintaining the homeostasis of ribonucleotides and deoxyribonucleotides. When designing experiments with recombinant sucC, researchers should:

• Consider co-expression of NDPK with the sucC protein to maintain physiological relevance

• Evaluate whether observed phenotypes result from disruption of the SCS-NDPK complex rather than just sucC function

• Assess the impact of sucC mutations on both TCA cycle function and nucleotide metabolism

• Design assays that can distinguish between direct effects on succinyl-CoA metabolism and indirect effects on nucleotide homeostasis

The functional significance of this association appears to be conservation of energy through substrate channeling, whereby ADP generated by the sucC reaction can be directly utilized by NDPK for nucleotide phosphorylation . This association may explain why defects in sucC can lead to consequences beyond disruption of the TCA cycle, including potential impacts on DNA replication and repair processes that depend on balanced nucleotide pools.

What are the methodological challenges in studying structure-function relationships of sucC in Burkholderia vietnamiensis?

Studying structure-function relationships of sucC in B. vietnamiensis presents several methodological challenges that require sophisticated approaches:

• Heterodimeric nature: The functional enzyme requires both α and β subunits, making it necessary to co-express and co-purify both proteins for meaningful functional studies. Ensuring proper subunit association while maintaining native conformation is technically demanding.

• Conformational dynamics: The enzyme undergoes significant conformational changes during catalysis, requiring techniques that can capture these dynamic states:

  • Time-resolved X-ray crystallography

  • Hydrogen-deuterium exchange mass spectrometry

  • Single-molecule FRET studies

  • Molecular dynamics simulations calibrated with experimental data

• Species-specific variations: While the catalytic mechanism is conserved, subtle species-specific variations can influence kinetic parameters and regulatory properties, necessitating comparative studies.

• Complex formation with NDPK: The physiological association with NDPK adds another layer of complexity, requiring techniques that can preserve and analyze multi-protein complexes, such as cryo-electron microscopy or native mass spectrometry.

Researchers should consider employing site-directed mutagenesis of conserved catalytic residues to establish structure-function correlations, combined with biochemical assays and structural studies to comprehensively characterize the protein.

How can researchers accurately assess the impact of sucC mutations on cellular metabolism in Burkholderia vietnamiensis?

Accurately assessing the impact of sucC mutations on cellular metabolism requires a multi-faceted approach that integrates various omics technologies and functional assays:

• Metabolomic profiling: Using LC-MS/MS or GC-MS to quantify changes in TCA cycle intermediates, especially succinate, succinyl-CoA, and related metabolites. Isotope labeling experiments with 13C-labeled substrates can track carbon flux through altered pathways.

• Transcriptomic analysis: RNA-seq to identify compensatory changes in gene expression that may mask the direct effects of sucC mutations.

• Proteomic studies: Quantitative proteomics to assess changes in enzyme levels and post-translational modifications that might compensate for altered sucC function.

• Bioenergetic measurements: Oxygen consumption rates, ATP/ADP ratios, and membrane potential measurements to assess the impact on cellular energy metabolism.

• Growth phenotyping: Detailed growth analyses under different carbon sources and stress conditions to identify conditional phenotypes that might not be apparent under standard laboratory conditions.

What biosafety considerations apply when working with recombinant Burkholderia vietnamiensis sucC?

Working with recombinant B. vietnamiensis sucC requires adherence to established biosafety guidelines due to the pathogenic potential of some Burkholderia species. According to the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules, researchers must:

• Conduct a comprehensive risk assessment based on the specific strain and experimental procedures

• Implement appropriate physical containment measures based on risk group classification

• Obtain Institutional Biosafety Committee (IBC) approval prior to initiating experiments

• Follow specific containment practices for large-scale cultivation if applicable

B. vietnamiensis generally requires Biosafety Level 2 (BL2) practices, which include limited access to laboratories, biohazard warning signs, minimization of aerosols, and decontamination of waste materials before disposal . When designing expression constructs, researchers should consider using attenuated laboratory strains as hosts to minimize risk while maximizing research value.

How should researchers address potential data inconsistencies when characterizing recombinant sucC function?

When researchers encounter data inconsistencies in characterizing recombinant sucC function, a systematic troubleshooting approach should be employed:

• Validate protein integrity: Confirm that the recombinant protein is properly folded and not degraded using circular dichroism spectroscopy, thermal shift assays, or limited proteolysis.

• Assess oligomeric state: Verify the heterodimeric association of α and β subunits using size exclusion chromatography, native PAGE, or analytical ultracentrifugation.

• Examine experimental conditions: Systematically vary buffer components, pH, temperature, and substrate concentrations to identify condition-dependent effects that might explain inconsistencies.

• Control for post-translational modifications: Compare protein produced in different expression systems to identify potential effects of PTMs on activity.

• Check for interfering factors: Test for the presence of inhibitors or activators in experimental samples that might affect enzyme behavior.

When reporting data, researchers should transparently document all experimental conditions and variations, allowing others to reproduce findings under comparable conditions. Statistical analysis should include appropriate tests for significance and variability measures, with outliers identified and justified if excluded from analysis.

What are the key considerations for designing gene-knockout studies targeting sucC in Burkholderia vietnamiensis?

Designing gene-knockout studies targeting sucC in B. vietnamiensis requires careful planning due to the essential nature of this gene in central metabolism. Key considerations include:

• Selection of knockout strategy:

  • Conditional knockout systems using inducible promoters may be necessary if complete deletion is lethal

  • CRISPR-Cas9 systems have been adapted for Burkholderia species, allowing precise genome editing

  • Insertional inactivation using antibiotic resistance cassettes remains a reliable approach

• Verification of mutants:

  • PCR verification of the intended genetic modification

  • Whole genome sequencing to exclude off-target effects or compensatory mutations

  • Transcriptional and proteomic analysis to confirm absence of target gene expression

• Complementation studies:

  • Generation of complemented strains is essential to confirm phenotype causality

  • Use of native promoters rather than constitutive promoters to maintain physiological expression levels

  • Testing complementation with both B. vietnamiensis sucC and homologs from related species

• Phenotypic characterization:

  • Assessment under various growth conditions, not just optimal laboratory media

  • Measurement of competitive fitness in mixed cultures

  • Evaluation of metabolic flexibility using different carbon sources

All recombinant DNA work must comply with NIH Guidelines, which require experiments to be reviewed and approved by the Institutional Biosafety Committee, particularly for work involving pathogenic species .