Recombinant Serratia proteamaculans Succinyl-CoA ligase [ADP-forming] subunit beta (sucC)

What is the role of Succinyl-CoA ligase (ADP-forming) in bacterial metabolism?

Succinyl-CoA ligase (ADP-forming) (EC 6.2.1.5) is a critical enzyme that catalyzes the reversible reaction: ATP + succinate + CoA ⟶ ADP + phosphate + succinyl-CoA. This enzyme plays a pivotal role in multiple metabolic pathways, most notably in the tricarboxylic acid (TCA) cycle where it catalyzes the only substrate-level phosphorylation step. In S. proteamaculans, as in other bacteria, the enzyme participates in at least four distinct metabolic pathways: the citric acid cycle, propanoate metabolism, C5-branched dibasic acid metabolism, and the reductive carboxylate cycle for CO₂ fixation . The beta subunit (sucC) specifically contributes to the nucleotide-binding domain of the enzyme, facilitating the ADP-forming function that distinguishes this variant from the GDP-forming isozyme.

How does Serratia proteamaculans Succinyl-CoA ligase differ structurally from other bacterial homologs?

While the search results don't provide specific structural information about S. proteamaculans Succinyl-CoA ligase, comparative analysis with other bacterial Succinyl-CoA ligases indicates that the enzyme is likely a heterodimer composed of alpha (sucD) and beta (sucC) subunits. The beta subunit contains the nucleotide-binding domain responsible for ADP specificity. Structural studies of Succinyl-CoA ligase across various organisms have resulted in multiple solved structures (PDB accession codes: 1CQI, 1CQJ, 1JKJ, 1JLL, 1OI7, 1SCU, 2NU6, 2NU7, 2NU8, 2NU9, 2NUA, and 2SCU) . These structures provide valuable templates for homology modeling of S. proteamaculans SucC and can guide research into species-specific structural features that may influence enzyme activity or regulatory interactions.

What are the optimal conditions for heterologous expression of recombinant S. proteamaculans sucC?

For optimal heterologous expression of recombinant S. proteamaculans sucC, a methodological approach should account for codon usage bias, protein solubility, and potential toxicity. Based on comparable studies with related bacterial enzymes, the following protocol is recommended:

• Clone the sucC gene into a pET-based expression vector with an N-terminal His-tag for purification

• Transform the construct into E. coli BL21(DE3) or Rosetta(DE3) strains to address potential codon bias

• Grow cultures at 37°C until OD₆₀₀ reaches 0.6-0.8

• Induce expression with 0.5-1.0 mM IPTG

• Shift temperature to 18-20°C for overnight expression to enhance protein solubility

• Harvest cells and lyse in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol, and protease inhibitors

This approach maximizes yield while maintaining enzyme activity, which can be subsequently confirmed using the enzymatic assays described in section 3.1.

What purification strategy yields the highest activity for recombinant S. proteamaculans Succinyl-CoA ligase?

A multi-step purification strategy is essential for obtaining high-activity recombinant S. proteamaculans Succinyl-CoA ligase. The recommended protocol incorporates:

• Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin with a gradient elution (20-250 mM imidazole)

• Ion exchange chromatography (IEX) with a Q-Sepharose column (pH 8.0) to separate based on charge properties

• Size exclusion chromatography using Superdex 200 to ensure heterodimer formation with the alpha subunit if co-expressed

For functional studies, co-expression or reconstitution with the alpha subunit (sucD) is critical, as the catalytically active enzyme is a heterodimer. The purification buffer should contain 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM MgCl₂, and 1 mM DTT to maintain enzyme stability and activity. The addition of 10% glycerol can further enhance stability during storage at -80°C.

What are the established methods for measuring Succinyl-CoA ligase activity in recombinant preparations?

Several established methods exist for measuring Succinyl-CoA ligase activity, each with specific advantages depending on the research question:

Method 1: Spectrophotometric coupled assay
This assay measures the formation of succinyl-CoA by coupling it to the reduction of NAD⁺ through pyruvate kinase and lactate dehydrogenase:

• Reaction mixture: 50 mM HEPES (pH 7.5), 10 mM MgCl₂, 0.1 mM CoA, 0.5 mM ATP, 20 mM succinate, 0.2 mM NADH, 0.5 mM phosphoenolpyruvate, 2 U pyruvate kinase, 2 U lactate dehydrogenase

• Monitor decrease in absorbance at 340 nm (NADH oxidation)

• Calculate activity using extinction coefficient of NADH (6,220 M⁻¹cm⁻¹)

Method 2: Direct assay measuring ADP formation
This method directly quantifies ADP production using HPLC:

• Reaction mixture: 50 mM Tris-HCl (pH 8.0), 10 mM MgCl₂, 0.5 mM CoA, 1 mM ATP, 10 mM succinate

• Incubate at 37°C and quench at various time points with perchloric acid

• Analyze nucleotides by HPLC with a C18 reverse-phase column

• Quantify ADP formation relative to ATP standard curve

Method 3: Radioactive assay
For highest sensitivity:

• Reaction mixture containing [¹⁴C]-succinate or [³²P]-ATP

• Separate products by thin-layer chromatography

• Quantify radioactive product formation by autoradiography or scintillation counting

The choice of method depends on available equipment and the specific aspect of enzyme function being investigated.

How do mutations in the sucC gene affect the catalytic properties of S. proteamaculans Succinyl-CoA ligase?

While specific mutational studies on S. proteamaculans sucC are not directly addressed in the search results, comparative analysis with related Succinyl-CoA ligases suggests several critical residues likely impact catalytic function:

Mutations in the nucleotide-binding domain of the beta subunit (sucC) would particularly affect ADP/ATP specificity. Site-directed mutagenesis targeting these regions provides valuable insights into structure-function relationships and can reveal potential allosteric regulation mechanisms specific to S. proteamaculans metabolism.

How is sucC expression regulated in S. proteamaculans under different environmental conditions?

In S. proteamaculans, sucC expression is likely regulated in response to changes in carbon source availability, oxygen levels, and growth phase. While specific data for sucC regulation in S. proteamaculans is not provided in the search results, comparative analysis with related bacterial systems suggests:

• Carbon source regulation: Expression increases during growth on gluconeogenic substrates and decreases in the presence of preferred carbon sources due to catabolite repression.

• Oxygen-dependent regulation: Under anaerobic conditions, sucC expression may be altered to accommodate shifts in metabolic flux through the TCA cycle. The reversibility of the enzyme reaction becomes particularly important under low oxygen conditions when the TCA cycle may operate non-cyclically.

• Growth phase-dependent expression: Similar to other TCA cycle genes, sucC expression likely peaks during late exponential and early stationary phases when metabolic demands shift.

• Quorum sensing influence: Given that S. proteamaculans employs a LuxI/LuxR type Quorum Sensing system (with SprR and SprI components), and this system regulates various metabolic functions, it may also influence sucC expression in a population density-dependent manner . This would coordinate metabolic activities with bacterial population density.

Experimental approaches to study this regulation include qRT-PCR analysis of sucC expression under various conditions, reporter gene fusions, and chromatin immunoprecipitation studies to identify transcription factor binding sites.

What is the relationship between Succinyl-CoA ligase activity and virulence in S. proteamaculans?

The relationship between Succinyl-CoA ligase activity and virulence in S. proteamaculans represents a complex intersection of central metabolism and pathogenicity. While direct evidence is limited in the search results, several mechanistic connections can be inferred:

• Metabolic adaptation during infection: Succinyl-CoA ligase, as a key TCA cycle enzyme, likely supports metabolic flexibility required during the transition from environmental to host conditions. Its role in energy production would be critical for supporting virulence factor synthesis and secretion.

• Connection to protease regulation: S. proteamaculans virulence is linked to proteolytic enzymes such as the 32-kDa thermostable protealysin that cleaves filamentous actin and matrix metalloprotease MMP2 in human cells . The energy metabolism supported by Succinyl-CoA ligase may influence the expression and secretion of these proteases.

• Quorum sensing intersection: Research shows that in S. proteamaculans 94, the quorum sensing system regulates invasive activity . If sucC expression is influenced by quorum sensing (as many metabolic genes are), this would establish an indirect link between Succinyl-CoA ligase activity and virulence regulation.

• Role in cellular invasion: S. proteamaculans strain 94 can invade human larynx carcinoma HEp-2 cells . This invasive capability requires significant metabolic support, potentially involving altered Succinyl-CoA ligase activity to accommodate energy demands during cellular invasion.

Research approaches to further elucidate this relationship include creating sucC knockout or knockdown strains and assessing their virulence in appropriate models, as well as measuring Succinyl-CoA ligase activity during different stages of infection.

What are the critical factors to consider when designing experiments to study recombinant S. proteamaculans sucC function?

When designing experiments to study recombinant S. proteamaculans sucC function, researchers should consider:

• Heterodimer requirement: Succinyl-CoA ligase functions as a heterodimer of alpha (sucD) and beta (sucC) subunits. Experimental designs must account for this by either co-expressing both subunits or reconstituting the complex from separately purified components.

• Metal ion dependency: The enzyme requires divalent metal ions (typically Mg²⁺) for activity. Experimental buffers must contain appropriate concentrations of MgCl₂ (5-10 mM), and experiments should control for potential interference from other metal ions.

• Substrate concentrations: The kinetic parameters of the enzyme dictate appropriate substrate concentrations. Initial experiments should include substrate saturation curves to determine Km values for ATP, succinate, and CoA.

• Directionality of the reaction: Succinyl-CoA ligase catalyzes a reversible reaction. Experimental conditions (substrate concentrations, pH) will influence the directional equilibrium, which should be considered when interpreting results.

• pH optimization: Activity is typically pH-dependent, with optimal activity for bacterial Succinyl-CoA ligases generally occurring between pH 7.0-8.0. A pH profile should be established early in experimental characterization.

• Temperature sensitivity: Though bacterial enzymes often exhibit thermostability, activity assays should be conducted at physiologically relevant temperatures (28-37°C for S. proteamaculans).

• Appropriate controls: Experiments should include negative controls (heat-inactivated enzyme, missing substrate) and positive controls (commercially available Succinyl-CoA ligase from a related organism).

• Storage conditions: The enzyme typically requires stabilizing agents (glycerol, reducing agents) for long-term storage at -80°C to prevent activity loss between experiments.

How can researchers effectively compare wild-type and mutant forms of S. proteamaculans Succinyl-CoA ligase?

Effective comparison of wild-type and mutant forms of S. proteamaculans Succinyl-CoA ligase requires a systematic approach:

• Standardized expression and purification: Use identical expression systems, growth conditions, and purification protocols to minimize variation unrelated to the mutation. Verify protein purity by SDS-PAGE and confirm protein concentration using consistent methods (Bradford assay or absorbance at 280 nm with calculated extinction coefficients).

• Structural integrity assessment: Employ circular dichroism (CD) spectroscopy to verify that mutations haven't caused major conformational changes that would indirectly affect activity. Thermal shift assays can also identify stability differences between variants.

• Comprehensive kinetic analysis: Determine full kinetic parameters (Km, Vmax, kcat, kcat/Km) for all substrates to distinguish between effects on binding versus catalysis. This should include:

  • Initial velocity measurements at varying substrate concentrations

  • Product inhibition studies

  • pH-rate profiles to identify potential changes in catalytic mechanism

• Biophysical characterization: Techniques such as isothermal titration calorimetry (ITC) can quantify changes in substrate binding affinity and thermodynamics, while size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) can identify changes in oligomeric state.

• Functional complementation: Test the ability of mutant variants to complement sucC knockout strains in vivo, providing insights into physiological significance beyond in vitro studies.

• Data presentation: Present data in standardized formats including:

  Enzyme Variant	kcat (s⁻¹)	Km ATP (μM)	Km Succinate (μM)	Km CoA (μM)	kcat/Km ATP (M⁻¹s⁻¹)
  Wild-type	x.xx	xxx	xxx	xx	x.xx × 10⁶
  Mutant K45A	x.xx	xxx	xxx	xx	x.xx × 10⁶
  Mutant D57N	x.xx	xxx	xxx	xx	x.xx × 10⁶

This systematic approach enables meaningful interpretation of how specific mutations affect enzyme function at the molecular level.

How can recombinant S. proteamaculans Succinyl-CoA ligase be utilized in metabolic engineering applications?

Recombinant S. proteamaculans Succinyl-CoA ligase offers several valuable applications in metabolic engineering:

• Enhanced succinate production: Overexpression of sucC along with other appropriate TCA cycle enzymes can redirect carbon flux toward succinate production in industrial strains. This approach is particularly valuable because Succinyl-CoA ligase can operate in both directions of the TCA cycle, potentially enhancing succinate yields under optimized conditions.

• Metabolic flux analysis: Isotopically labeled substrates combined with activity assays of recombinant Succinyl-CoA ligase can help quantify carbon flux through the TCA cycle in engineered organisms. This information is crucial for optimizing production strains for various metabolites.

• Biosensor development: The enzyme can be incorporated into biosensors for succinate or succinyl-CoA detection in fermentation processes, allowing real-time monitoring of metabolite levels.

• Modifying TCA cycle regulation: Given that SDH/CII dysfunction leads to TCA cycle arrest and altered respiration , strategic modifications of Succinyl-CoA ligase activity could be used to manipulate metabolic flux for desired outcomes.

• Cross-species metabolic engineering: The S. proteamaculans enzyme may offer unique properties (stability, catalytic efficiency, or regulatory features) that make it advantageous for expression in other organisms compared to their native enzyme.

• Expanding biochemical production capabilities: As Succinyl-CoA ligase participates in multiple pathways beyond the TCA cycle, including propanoate metabolism and C5-branched dibasic acid metabolism , the enzyme can be leveraged to engineer new or enhanced pathways for specialty chemical production.

For these applications, it's critical to understand not only the basic catalytic properties of the enzyme but also its regulatory features and how it interacts with other components of central metabolism.

What is the relationship between S. proteamaculans Succinyl-CoA ligase and the regulation of oxidative stress response?

The relationship between S. proteamaculans Succinyl-CoA ligase and oxidative stress response, while not directly addressed in the search results, can be analyzed through comparative metabolic understanding:

Experimental approaches to investigate this relationship could include:

• Measuring ROS levels in wild-type versus sucC mutant strains under oxidative stress conditions

• Analyzing changes in sucC expression in response to various oxidative stressors

• Determining how Succinyl-CoA ligase activity affects the cellular redox state (NADH/NAD⁺ ratio) during oxidative stress

• Investigating potential post-translational modifications of the enzyme under oxidative conditions that might alter its activity or substrate preference

What are common challenges in expressing active recombinant S. proteamaculans Succinyl-CoA ligase and how can they be addressed?

Researchers frequently encounter several challenges when expressing active recombinant S. proteamaculans Succinyl-CoA ligase:

• Subunit co-expression imbalance: As a heterodimeric enzyme, unequal expression of alpha and beta subunits can result in reduced active enzyme recovery.
  Solution: Design bicistronic expression constructs with optimized ribosome binding sites for each subunit, or adjust induction conditions to balance expression levels.

• Protein insolubility: Overexpression often leads to inclusion body formation.
  Solution: Lower induction temperature (16-20°C), reduce IPTG concentration (0.1-0.2 mM), use solubility-enhancing fusion tags (SUMO, MBP), or employ specialized E. coli strains like Arctic Express or SHuffle.

• Loss of activity during purification: The enzyme may lose activity due to subunit dissociation or cofactor loss.
  Solution: Include stabilizing agents (glycerol 10-20%, 1-5 mM MgCl₂) in all buffers, minimize freeze-thaw cycles, and consider adding low concentrations of substrates during purification.

• Improper folding: Recombinant expression may not recapitulate native folding pathways.
  Solution: Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE) to assist proper folding.

• Post-translational modifications: If critical for activity, these may be absent in heterologous systems.
  Solution: Express in bacterial hosts more closely related to S. proteamaculans or identify and engineer modifications if critical.

• Enzyme instability: The purified enzyme may exhibit poor stability during storage.
  Solution: Optimize storage buffer (typically 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM DTT), store at high concentration (>1 mg/ml), and add stabilizing ligands (e.g., ADP, succinate at low concentrations).

The most effective approach often combines multiple strategies, optimized through systematic testing for the specific recombinant construct being used.

How can researchers address unexpected data inconsistencies when studying S. proteamaculans sucC function?

When faced with data inconsistencies in S. proteamaculans sucC function studies, researchers should implement a systematic troubleshooting approach:

• Enzyme preparation variability:

  • Problem: Different preparations yield inconsistent specific activities.

  • Solution: Standardize expression and purification protocols; perform quality control testing on each preparation (SDS-PAGE, dynamic light scattering for aggregation); establish minimum purity and activity criteria.

• Assay interference:

  • Problem: Components in the reaction mixture interfere with activity measurements.

  • Solution: Test for interference by comparing different assay methods (e.g., spectrophotometric vs. HPLC-based); include appropriate blank reactions; consider using alternative coupling enzymes if interference is detected.

• Buffer composition effects:

  • Problem: Minor differences in buffer composition significantly impact activity.

  • Solution: Prepare master buffer stocks for consistency; test sensitivity to specific buffer components (ions, pH, additives); document exact buffer composition with each experiment.

• Substrate quality variation:

  • Problem: Commercial substrate lots vary in purity affecting kinetic parameters.

  • Solution: Use high-quality substrates from reliable sources; characterize new lots against known standards; prepare internal reference standards for quality control.

• Data analysis inconsistencies:

  • Problem: Different data processing methods yield varying interpretations.

  • Solution: Establish standardized data analysis workflows; use multiple fitting methods to ensure robust parameter determination; report raw data alongside processed results.

• Environmental variables:

  • Problem: Temperature fluctuations or oxidation during assays.

  • Solution: Control assay temperature precisely (±0.1°C); minimize exposure to oxygen for sensitive components; include internal standards in each assay run.

For unexpected results that persist despite these measures, consider fundamental biological hypotheses:

• Is there post-translational regulation occurring?

• Are there unidentified cofactors or binding partners?

• Could there be alternative catalytic mechanisms under specific conditions?

Documenting all experimental conditions thoroughly and creating standardized protocols can significantly reduce inconsistencies and improve reproducibility across research groups.

How might studying S. proteamaculans Succinyl-CoA ligase contribute to understanding bacterial adaptations to extreme environments?

Investigating S. proteamaculans Succinyl-CoA ligase offers unique insights into bacterial adaptations to extreme environments through several research avenues:

• Metabolic flexibility in nutrient limitation: Serratia proteamaculans inhabits diverse environments including plant surfaces, soil, water, and occasionally animal hosts, requiring metabolic adaptability. Succinyl-CoA ligase's critical position in the TCA cycle makes it a potential regulatory point for metabolic reprogramming during environmental transitions. Comparative studies of enzyme kinetics across growth conditions could reveal how central metabolism adjusts to resource availability.

• Cold adaptation mechanisms: S. proteamaculans is known to grow at lower temperatures than many other enterobacteria. Structural and functional characterization of its Succinyl-CoA ligase could reveal adaptations that maintain catalytic efficiency at lower temperatures, potentially including:

  • Altered amino acid composition favoring flexibility in cold conditions

  • Modified substrate binding affinities to compensate for reduced reaction rates

  • Temperature-dependent regulatory mechanisms

• Stress response integration: Under extreme conditions, the TCA cycle often shifts from its canonical operation. Succinyl-CoA ligase may serve as a metabolic switch point, potentially:

  • Redirecting carbon flux toward alternative pathways during stress

  • Contributing to energy conservation strategies under nutrient limitation

  • Participating in the production of secondary metabolites that enhance survival

• Host-microbe interaction dynamics: As S. proteamaculans can be both free-living and associated with hosts, Succinyl-CoA ligase might reveal metabolic adaptations that facilitate this lifestyle transition, particularly in relation to invasive capability which is regulated by the sprI gene through modifications in protease activity and outer membrane protein expression .

Research approaches should include:

• Comparative enzymology across growth conditions mimicking environmental extremes

• In vivo metabolic flux analysis using isotope labeling

• Expression studies correlating sucC regulation with environmental stress responses

• Structure-function analyses identifying unique features compared to homologs from non-adaptable bacteria

What are the implications of S. proteamaculans Succinyl-CoA ligase research for understanding bacterial evolution and horizontal gene transfer?

Research on S. proteamaculans Succinyl-CoA ligase has significant implications for understanding bacterial evolution and horizontal gene transfer (HGT):

• Evolutionary conservation versus divergence: Succinyl-CoA ligase represents an evolutionary ancient enzyme essential for central metabolism. Comparative genomic analysis of sucC across diverse bacterial species can reveal:

  • Highly conserved catalytic domains indicating functional constraints

  • Divergent regulatory regions suggesting adaptive evolution

  • Species-specific structural features that may confer specialized functions

• Horizontal gene transfer detection: The sucC gene's evolutionary history can provide evidence of HGT events:

  • Anomalous GC content or codon usage compared to the core genome would suggest recent acquisition

  • Phylogenetic incongruence between sucC and species trees could indicate HGT

  • Synteny analysis examining gene neighborhood conservation across related species

• Metabolic pathway evolution: The ADP-forming specificity of S. proteamaculans Succinyl-CoA ligase (versus GDP-forming variants in some bacteria) represents an evolutionary choice with consequences for energy metabolism. This specificity influences:

  • Integration with other metabolic pathways

  • Adaptation to specific ecological niches

  • Potential for metabolic innovation following HGT events

• Accessory gene integration: How transferred genes integrate with core metabolic functions is a fundamental question in bacterial evolution. sucC provides an excellent model to study:

  • Co-evolution of acquired genes with central metabolism

  • Regulatory network integration following HGT

  • Functional constraints that limit or enhance HGT events involving central metabolic genes

• Pathogenicity evolution: Given the relationship between central metabolism and virulence in Serratia species, studying sucC evolution provides insights into how metabolic capabilities influence pathogenic potential. S. proteamaculans invasive activity correlates with changes in protease activity , suggesting complex interactions between metabolism and virulence factors.

Research approaches should include:

• Comprehensive phylogenetic analysis of sucC across diverse bacterial phyla

• Experimental evolution studies tracking sucC adaptation under selective pressures

• Functional characterization of recombinant Succinyl-CoA ligase from diverse bacterial sources

• Systems biology approaches examining metabolic network evolution

This research direction connects fundamental biochemistry to broader evolutionary questions about bacterial adaptation and specialization.