Recombinant Shewanella baltica Succinyl-CoA ligase [ADP-forming] subunit beta (sucC)

What is Succinyl-CoA ligase and what is its function in Shewanella baltica?

Succinyl-CoA ligase (also known as succinyl-CoA synthetase) catalyzes the reversible conversion of succinyl-CoA and ADP to succinate and ATP. In Shewanella baltica, as in most bacteria, this enzyme is part of the tricarboxylic acid (TCA) cycle, playing a crucial role in energy metabolism. The enzyme consists of two subunits - alpha and beta, with the beta subunit (encoded by sucC) determining the substrate specificity. The ADP-forming enzyme is specifically involved in the conversion of succinyl-CoA and ADP to succinate and ATP, which is essential for energy production in the bacterial cell .

How is Recombinant Shewanella baltica Succinyl-CoA ligase typically expressed and purified?

The recombinant Shewanella baltica Succinyl-CoA ligase [ADP-forming] subunit beta (sucC) is typically expressed in E. coli expression systems. The full-length protein (amino acids 1-388) is expressed with a tag (determined during manufacturing) to facilitate purification. After expression, the protein is purified to >85% purity as confirmed by SDS-PAGE. The purified protein can be stored at -20°C, with extended storage recommended at -20°C or -80°C. For working with the protein, reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL is recommended, with the addition of 5-50% glycerol for long-term storage .

What are the structural characteristics of Shewanella baltica sucC?

The Succinyl-CoA ligase [ADP-forming] subunit beta (sucC) from Shewanella baltica (strain OS195) is a 388-amino acid protein with a UniProt accession number of A9L532. The protein sequence contains domains consistent with its function in catalyzing the conversion of succinyl-CoA to succinate. While specific crystal structure information for the S. baltica enzyme is not provided in the search results, the protein likely shares structural similarities with other bacterial Succinyl-CoA ligases, containing nucleotide-binding domains and substrate-binding regions that facilitate its enzymatic activity .

How does the metabolic efficiency of Shewanella baltica relate to the function of Succinyl-CoA ligase when grown on different carbon sources?

S. baltica exhibits variable metabolic efficiencies depending on the carbon source utilized. Studies on S. baltica KB30 demonstrate its ability to consume multiple substrates including sodium acetate, glucose, tween 80, and peptone. The kinetic behavior follows different mathematical models: Monod model for sodium acetate and tween 80, and Contois model for glucose and peptone. Notably, proteolytic metabolism appears to be favored over lipidic and glucidic metabolism in S. baltica .

What are the implications of horizontal gene transfer on the evolution of sucC in Shewanella baltica strains?

Shewanella baltica demonstrates extraordinarily high levels of horizontal gene transfer (HGT) among spatially co-occurring strains. Research on S. baltica genomes has revealed that genetic exchange through homologous recombination may constitute an important mechanism for population cohesion among spatially co-occurring prokaryotes, similar to sexual reproduction in eukaryotes .

While specific information about HGT of the sucC gene is not directly provided in the search results, the extensive recombination observed in S. baltica genomes suggests that metabolic genes like sucC could be subject to such exchange. This genetic exchange could lead to functional diversification of the Succinyl-CoA ligase enzyme among different S. baltica strains, potentially contributing to their metabolic adaptability in various ecological niches. The rapid genetic exchange in response to environmental settings observed in S. baltica could include adaptations in central metabolic pathways involving sucC, although this would require specific investigation targeting this gene .

How can genetic code expansion techniques be applied to study Shewanella baltica sucC function and interactions?

While the search results don't specifically mention genetic code expansion for S. baltica sucC, research on the related Shewanella oneidensis MR-1 provides valuable insights. Genetic code expansion techniques enable the site-specific incorporation of noncanonical amino acids (ncAAs) into proteins, which has been successfully demonstrated in S. oneidensis MR-1 .

This technology could be adapted to study S. baltica sucC by:

• Incorporating ncAAs at specific sites in the sucC protein to introduce bioorthogonal functional groups

• Using these functional groups for site-selective fluorescent labeling to track protein localization and interactions

• Introducing photocrosslinking amino acids to capture transient protein-protein interactions between sucC and other metabolic enzymes

• Introducing environmental sensors in the form of ncAAs that change properties based on local conditions

Importantly, research has confirmed that the biosynthetic machinery for ncAA incorporation is compatible with the endogenous pathways of Shewanella for protein synthesis and maturation, suggesting that similar techniques could be applied to study sucC in S. baltica .

What are the optimal conditions for reconstitution and storage of Recombinant Shewanella baltica Succinyl-CoA ligase?

For optimal handling of Recombinant Shewanella baltica Succinyl-CoA ligase [ADP-forming] subunit beta:

Reconstitution protocol:

• Briefly centrifuge the vial before opening to bring contents to the bottom

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

• Add glycerol to a final concentration of 5-50% (50% is recommended as default)

• Aliquot for long-term storage

Storage guidelines:

• For short-term use: Store working aliquots at 4°C for up to one week

• For long-term storage: Store at -20°C, or preferably -80°C for extended periods

• Avoid repeated freezing and thawing cycles

Shelf life considerations:

• Liquid form: Approximately 6 months at -20°C/-80°C

• Lyophilized form: Approximately 12 months at -20°C/-80°C

Note that shelf life depends on multiple factors including buffer ingredients, storage temperature, and the intrinsic stability of the protein itself .

What enzymatic assays can be used to measure the activity of Shewanella baltica Succinyl-CoA ligase?

Although specific assays for S. baltica Succinyl-CoA ligase are not detailed in the search results, standard enzymatic assays for Succinyl-CoA ligase activity can be adapted for this protein:

Forward reaction assay (succinyl-CoA → succinate):

• Measure the production of ATP using a coupled assay with pyruvate kinase and lactate dehydrogenase

• Monitor the decrease in NADH absorbance at 340 nm as ATP is produced and utilized

Reverse reaction assay (succinate → succinyl-CoA):

• Monitor the formation of succinyl-CoA by coupling with a reaction that consumes it

• Measure CoA release using thiol-reactive probes

Respirometric method:
Based on the studies of S. baltica KB30 metabolism, respirometric methods can be used to indirectly assess TCA cycle activity, including Succinyl-CoA ligase function, by measuring oxygen consumption rates during substrate utilization .

These assays should be performed under optimal conditions for the enzyme, typically at pH 7.2-7.8 and at temperatures suitable for a psychrotropic organism like S. baltica (15-25°C).

How can researchers troubleshoot expression and purification issues with Recombinant Shewanella baltica sucC?

When encountering challenges with the expression and purification of Recombinant S. baltica sucC, researchers should consider the following systematic troubleshooting approaches:

Expression optimization:

• Test multiple E. coli expression strains (BL21(DE3), Rosetta, Arctic Express) that may better accommodate the codon usage of S. baltica genes

• Vary induction conditions (IPTG concentration, temperature, duration) to improve soluble protein yield

• Consider co-expression with chaperones if protein misfolding occurs

• Test expression with different fusion tags (His, GST, MBP) if the default tag yields poor results

Purification troubleshooting:

• Adjust lysis buffer conditions (salt concentration, pH, reducing agents) to improve protein solubility

• Use multiple chromatography steps (affinity, ion exchange, size exclusion) to achieve higher purity

• Include protease inhibitors to prevent degradation during purification

• Test different elution conditions to maximize yield while maintaining purity

Protein quality assessment:

• Verify protein identity using mass spectrometry

• Confirm protein integrity using western blotting with anti-tag or anti-sucC antibodies

• Assess protein folding using circular dichroism or limited proteolysis

• Test enzymatic activity to ensure functional protein is obtained

How does Shewanella baltica Succinyl-CoA ligase differ from homologous enzymes in related species?

The Succinyl-CoA ligase [ADP-forming] from S. baltica belongs to a conserved family of enzymes found across many bacterial species, but with specific adaptations to the ecological niche of S. baltica. While detailed comparative information is not provided in the search results, several inferences can be made:

• Substrate specificity: The beta subunit of S. baltica Succinyl-CoA ligase determines its specificity for ADP rather than GDP, distinguishing it from GDP-forming variants found in some other bacterial species .

• Environmental adaptation: As S. baltica is a psychrotropic organism found in marine environments like the Baltic Sea, its Succinyl-CoA ligase likely has adaptations for function at lower temperatures compared to mesophilic bacteria .

• Genetic plasticity: Given the substantial horizontal gene transfer observed in S. baltica populations, its sucC gene may show greater sequence diversity compared to more genetically isolated species .

A comparative analysis with the related Shewanella oneidensis MR-1, which has been more extensively studied, would likely reveal structural and functional similarities while potentially highlighting adaptations specific to the Baltic Sea environment where S. baltica thrives .

What can metabolic modeling reveal about the role of Succinyl-CoA ligase in Shewanella baltica energy metabolism?

Metabolic modeling of S. baltica growth on different substrates provides insights into the central role of Succinyl-CoA ligase in cellular energetics:

• Carbon source utilization: S. baltica can metabolize diverse carbon sources including carbohydrates, carboxylic acids, amino acids, and lipids. The kinetic modeling of S. baltica KB30 revealed different growth patterns on sodium acetate, glucose, tween 80, and peptone, with each following different kinetic models (Monod for sodium acetate and tween 80; Contois for glucose and peptone) .

• Metabolic flux distribution: As a key enzyme in the TCA cycle, Succinyl-CoA ligase represents a critical node in carbon flux distribution. Its activity directly impacts:

  • ATP production through substrate-level phosphorylation

  • Replenishment of TCA cycle intermediates

  • Balancing of redox cofactors

• Environmental adaptation: The substrate degradation rates observed in S. baltica (with peptone being consumed faster than other substrates, exceeding 60 mg O₂ L⁻¹ h⁻¹) suggest that proteolytic metabolism is favored. This indicates that Succinyl-CoA ligase activity may be particularly important when amino acids serve as carbon sources, which could be an adaptation to protein-rich marine environments .

A comprehensive metabolic model would position Succinyl-CoA ligase at the intersection of catabolic and anabolic pathways, highlighting its role in both energy generation and biosynthetic precursor formation.

How might studying Shewanella baltica sucC contribute to understanding bacterial adaptation in marine environments?

Research on S. baltica sucC can provide significant insights into bacterial adaptation mechanisms in marine environments:

• Cold adaptation mechanisms: As a psychrotropic organism prevalent in the Baltic Sea, S. baltica's central metabolic enzymes including Succinyl-CoA ligase must function efficiently at low temperatures. Studying the structural and kinetic properties of sucC could reveal how this essential metabolic enzyme has adapted to function in cold marine environments .

• Metabolic flexibility: S. baltica's ability to utilize diverse carbon sources (at least 37 documented substrates) suggests that its central metabolic pathways, including those involving sucC, have evolved to accommodate variable nutrient conditions in marine ecosystems. Understanding this flexibility could illuminate bacterial survival strategies in changing marine environments .

• Ecological succession: Studies have shown that different Shewanella species, including S. baltica, dominate H₂S-producing bacterial populations during different seasons and storage conditions of marine fish. This succession pattern suggests that metabolic adaptations, including those involving central enzymes like Succinyl-CoA ligase, play a role in determining which species thrive under specific environmental conditions .

• Horizontal gene transfer implications: The high levels of genetic exchange observed in S. baltica populations may include transfers affecting metabolic genes like sucC. Studying sequence variation and recombination patterns in sucC could provide insights into how horizontal gene transfer contributes to metabolic adaptation in marine bacterial communities .

What potential biotechnological applications might emerge from research on Shewanella baltica Succinyl-CoA ligase?

Research on S. baltica Succinyl-CoA ligase could lead to several innovative biotechnological applications:

• Bioremediation enhancements: Understanding the role of Succinyl-CoA ligase in S. baltica's metabolic network could inform strategies for optimizing the bacterium's ability to degrade various pollutants in marine environments, particularly in cold waters where other bioremediation organisms may be less effective.

• Cold-active enzyme development: The enzyme's adaptations for function at lower temperatures could inspire the engineering of cold-active enzymes for industrial applications where low-temperature processes are desirable (reducing energy costs and preserving heat-sensitive compounds).

• Bioelectrochemical systems: Related Shewanella species like S. oneidensis MR-1 are known for their electron transfer capabilities. Insights from S. baltica's central metabolism, including Succinyl-CoA ligase function, could inform the development of bioelectrochemical systems tailored for cold marine environments .

• Synthetic biology applications: The genetic code expansion techniques demonstrated in S. oneidensis MR-1 could potentially be applied to S. baltica, allowing for the engineering of novel metabolic pathways involving modified versions of Succinyl-CoA ligase with enhanced or altered functions .

• Marine food preservation: Given S. baltica's role in fish spoilage, understanding its metabolism could lead to targeted approaches for inhibiting its growth in seafood, potentially contributing to improved preservation methods that specifically target key metabolic enzymes like Succinyl-CoA ligase .

What are the current challenges in studying the structure-function relationship of Shewanella baltica Succinyl-CoA ligase?

Researchers face several challenges when investigating the structure-function relationship of S. baltica Succinyl-CoA ligase:

• Structural characterization limitations: While the amino acid sequence is known, detailed three-dimensional structural information specific to S. baltica Succinyl-CoA ligase is not readily available in the search results. Obtaining crystal structures is challenging due to:

  • Protein purification in active conformation

  • Crystallization conditions for psychrotropic proteins

  • Capturing different conformational states during catalysis

• Functional analysis in native context: Studying the enzyme's function within the living S. baltica cell presents challenges:

  • Creating gene knockouts without polar effects on adjacent genes

  • Distinguishing direct effects from compensatory metabolic responses

  • Maintaining physiologically relevant conditions during in vitro assays

• Environmental adaptations: Understanding how the enzyme's structure and function are adapted to S. baltica's marine environment requires:

  • Comparative studies with homologs from different environments

  • Analysis of temperature-dependent kinetic parameters

  • Investigation of potential allosteric regulators specific to marine conditions

• Integration with metabolic networks: Contextualizing Succinyl-CoA ligase within S. baltica's broader metabolic network requires:

  • Comprehensive metabolic modeling specific to S. baltica

  • Flux analysis under various growth conditions

  • Understanding regulatory mechanisms controlling enzyme expression and activity

Advanced techniques combining genetic manipulation, structural biology, and metabolomics will be necessary to overcome these challenges and fully elucidate the structure-function relationship of this important metabolic enzyme.

What experimental design would be optimal for studying the effects of environmental conditions on Shewanella baltica sucC expression and activity?

An optimal experimental design to investigate environmental effects on S. baltica sucC expression and activity would include:

Experimental conditions matrix:

Methodological approach:

• Gene expression analysis:

  • qRT-PCR to quantify sucC mRNA levels

  • Reporter gene constructs (e.g., sucC promoter fused to GFP)

  • RNA-seq to place sucC regulation in whole-transcriptome context

• Protein level analysis:

  • Western blotting with anti-sucC antibodies

  • Proteomics to quantify enzyme abundance

  • Pulse-chase experiments to determine protein turnover rates

• Enzyme activity measurements:

  • In vitro assays using purified enzyme under varying conditions

  • In vivo metabolic flux analysis using isotope labeling

  • Respirometric measurements correlating with TCA cycle activity

• Structural analysis:

  • Circular dichroism to assess secondary structure under different conditions

  • Thermal shift assays to determine protein stability

  • Activity assays coupled with structural probes

This comprehensive experimental design would allow researchers to determine how environmental parameters affect sucC at the transcriptional, translational, and post-translational levels, providing insights into the adaptation mechanisms of S. baltica to its natural marine habitat .

How can researchers effectively compare the kinetic properties of Shewanella baltica Succinyl-CoA ligase with homologs from other bacterial species?

To effectively compare kinetic properties of S. baltica Succinyl-CoA ligase with homologs from other species, researchers should employ a systematic approach:

Protein preparation protocol:

• Express and purify homologous enzymes from multiple species using identical tags and purification methods

• Verify protein quality through SDS-PAGE, mass spectrometry, and circular dichroism

• Standardize protein concentrations and storage conditions

• Perform activity tests to ensure functional enzyme preparations

Kinetic characterization methodology:

Comparative analysis framework:

• Correlate kinetic differences with amino acid sequence variations

• Relate parameters to ecological niches of source organisms

• Use structural modeling to predict the molecular basis of kinetic differences

• Apply statistical methods to identify significant patterns across phylogenetic relationships

This approach would provide valuable insights into how S. baltica Succinyl-CoA ligase has evolved kinetic properties suited to its ecological niche compared to homologs from bacteria inhabiting different environments .

How should researchers interpret discrepancies in experimental results when working with Recombinant Shewanella baltica Succinyl-CoA ligase?

When encountering discrepancies in experimental results with Recombinant S. baltica Succinyl-CoA ligase, researchers should consider a systematic approach to interpretation:

Common sources of experimental variability:

Systematic troubleshooting approach:

• Validate enzyme quality:

  • Verify purity by SDS-PAGE (should be >85%)

  • Confirm correct reconstitution according to recommended protocols

  • Check for protein degradation using western blotting

• Assess experimental conditions:

  • Verify optimal assay conditions (pH, temperature, ionic strength)

  • Test for interfering compounds in buffers or substrate preparations

  • Evaluate potential inhibitors in the experimental system

• Consider biological relevance:

  • Compare results with known biological parameters of S. baltica

  • Evaluate whether discrepancies reflect natural variability or experimental artifacts

  • Consider whether observed differences might reflect undocumented regulatory mechanisms

• Statistical analysis:

  • Apply appropriate statistical tests to determine significance of discrepancies

  • Use sufficient replicates to distinguish random variation from systematic differences

  • Consider Bayesian approaches to incorporate prior knowledge about enzyme behavior

By systematically addressing these factors, researchers can determine whether discrepancies represent genuine biological phenomena or experimental variables requiring control .

What analytical methods are most appropriate for studying the integration of Succinyl-CoA ligase activity within the broader metabolic network of Shewanella baltica?

To effectively study how Succinyl-CoA ligase integrates within S. baltica's broader metabolic network, researchers should employ a multi-faceted analytical approach:

Metabolic flux analysis techniques:

• Growth phenotype analysis:

  • Respirometric measurements under different carbon sources

  • Growth rate analysis in defined media compositions

  • Metabolic inhibitor studies targeting specific pathways

  • Comparison of wild-type and sucC-modified strains (if available)

This integrated analytical approach would provide a comprehensive understanding of how Succinyl-CoA ligase activity influences and is influenced by the broader metabolic network in S. baltica, particularly in the context of its ability to utilize diverse carbon sources and adapt to changing environmental conditions .

What emerging technologies could advance our understanding of Shewanella baltica Succinyl-CoA ligase structure and function?

Several cutting-edge technologies show promise for advancing our understanding of S. baltica Succinyl-CoA ligase:

• Cryo-electron microscopy (Cryo-EM):

  • Enables visualization of protein structures without crystallization

  • Could capture Succinyl-CoA ligase in multiple conformational states

  • May reveal interactions with other TCA cycle enzymes in larger complexes

• Genetic code expansion and bioorthogonal chemistry:

  • Site-specific incorporation of noncanonical amino acids (ncAAs)

  • Introduction of biophysical probes at specific positions

  • Capture of transient protein-protein interactions

  • The successful application of these techniques in S. oneidensis MR-1 suggests feasibility in S. baltica

• Advanced computational approaches:

• Single-molecule techniques:

  • FRET-based approaches to monitor conformational changes during catalysis

  • Optical tweezers to study enzyme mechanics

  • Single-molecule enzymology to detect heterogeneity in enzyme behavior

• Genome editing with CRISPR-Cas systems:

  • Precise genome modifications to study sucC function in vivo

  • Creation of conditional knockdowns to assess metabolic impact

  • Introduction of sequence variations to test evolutionary hypotheses

  • Engineering of reporter fusions to monitor expression in real-time

These emerging technologies, particularly when used in combination, could provide unprecedented insights into the structure, dynamics, and function of S. baltica Succinyl-CoA ligase within its cellular and ecological context .

How might understanding the evolutionary adaptation of Shewanella baltica Succinyl-CoA ligase inform research on bacterial metabolism in changing marine environments?

Understanding the evolutionary adaptation of S. baltica Succinyl-CoA ligase provides valuable insights into bacterial metabolic adaptation in marine environments facing environmental change:

• Cold adaptation mechanisms:

  • S. baltica as a psychrotropic organism has evolved enzymes that function effectively at low temperatures

  • Studying the structural and kinetic adaptations of its Succinyl-CoA ligase could reveal molecular mechanisms of cold adaptation

  • These insights could help predict how marine bacterial communities might respond to ocean temperature changes

• Metabolic flexibility and ecological succession:

  • S. baltica's ability to utilize diverse carbon sources suggests metabolic plasticity

  • The dominance of S. baltica among H₂S-producing bacteria during specific seasonal conditions indicates ecological specialization

  • Understanding how central metabolic enzymes like Succinyl-CoA ligase contribute to this flexibility could inform models of bacterial community dynamics in changing marine ecosystems

• Genetic exchange and adaptive evolution:

  • The extensive horizontal gene transfer observed in S. baltica populations provides a mechanism for rapid adaptation

  • Studying how genetic exchange affects metabolic genes like sucC could illuminate how bacterial populations adapt to novel environmental conditions

  • This understanding could help predict evolutionary trajectories of marine bacterial communities under changing conditions

• Comparative analysis across marine gradients:

  • Comparing Succinyl-CoA ligase from S. baltica strains isolated from different marine environments (varying in temperature, salinity, oxygen levels)

  • Identifying adaptive signatures in enzyme sequence and function

  • Correlating enzyme properties with environmental parameters to develop predictive models