Recombinant Ignicoccus hospitalis Succinyl-CoA ligase [ADP-forming] subunit beta (sucC)

How does the structure of I. hospitalis Succinyl-CoA ligase compare to other archaeal homologs?

The structure of I. hospitalis Succinyl-CoA ligase [ADP-forming] likely shares common features with other archaeal homologs while possessing unique adaptations for hyperthermophilic environments. Based on studies of similar enzymes in I. hospitalis, such as the AMP-forming acetyl-CoA synthetase (ACS), which exhibits a molecular mass of ~690 kDa with a monomeric mass of 77 kDa , we can predict that succinyl-CoA ligase may also form higher-order oligomeric structures. Structure prediction analyses of related CoA-utilizing enzymes in I. hospitalis suggest conservation of key catalytic domains while differing in oligomerization patterns. Notably, while most characterized ACSs form homo-oligomeric structures, some ADP-forming acetyl-CoA synthetases in hyperthermophiles like Pyrococcus furiosus or Pyrobaculum aerophilum consist of two different subunits , potentially providing insights into I. hospitalis sucC organization.

What is known about the subcellular localization of CoA-utilizing enzymes in I. hospitalis?

Research on I. hospitalis has revealed remarkable compartmentalization of metabolic processes. Surprisingly, CoA-utilizing enzymes show distinct localization patterns that challenge conventional understanding of archaeal cell organization. For example, immunolabeling studies have demonstrated that the AMP-forming acetyl-CoA synthetase (ACS) is localized primarily at the outermost membrane of I. hospitalis cells, with minimal presence in the inner membrane or cytoplasm . This finding coincides with the localization of ATP synthase and H₂:sulfur oxidoreductase complexes to the outermost membrane . Given these patterns, there is significant research interest in determining whether succinyl-CoA ligase follows similar localization or differs in its cellular distribution. The intermembrane compartment (IMC) appears to be not only the site of ATP synthesis but potentially involved in primary steps of carbon fixation , suggesting that CoA-related metabolism may be compartmentalized in this unique archaeon.

How is the expression of sucC regulated in I. hospitalis during different growth conditions?

The regulation of sucC expression in I. hospitalis likely responds to metabolic demands and environmental conditions. Studies of related CoA-utilizing enzymes provide some insights into potential regulatory mechanisms. For instance, the ACS in I. hospitalis appears to be constitutively expressed rather than induced by substrate availability; experiments with I. hospitalis cultures grown with different acetate concentrations showed similar ACS levels across conditions . This suggests that some CoA-metabolizing enzymes may be maintained at relatively constant levels regardless of substrate availability.

For sucC regulation, several potential mechanisms may exist based on findings in related organisms:

• Post-translational modification: In some bacteria, acetyl-CoA synthetase activity is regulated through reversible acetylation of conserved lysine residues .

• Transcriptional control: Changes in transcription factors have been observed in I. hospitalis under different conditions, including co-culture with Nanoarchaeum equitans .

• Metabolic regulation: Global regulators like the P-II signal transduction protein (GlnK) show altered abundance under different metabolic states , potentially affecting CoA-metabolizing enzyme expression.

Understanding sucC regulation would require specific expression studies across different growth phases, temperatures, and substrate availabilities.

What methods are most effective for purifying recombinant I. hospitalis Succinyl-CoA ligase while maintaining enzymatic activity?

Purification of recombinant I. hospitalis Succinyl-CoA ligase requires specialized approaches to maintain the stability and activity of this hyperthermophilic enzyme. Based on successful protocols for related enzymes, the following methodological considerations are recommended:

• Expression system selection: E. coli BL21(DE3) with a pET-based expression system using codon optimization for archaeal genes typically yields better expression.

• Temperature modulation: Induction at lower temperatures (16-20°C) for extended periods (16-24 hours) often improves solubility of hyperthermophilic proteins.

• Purification protocol: A successful approach for related I. hospitalis membrane-associated proteins involves:

  • Cell lysis in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 5 mM MgCl₂, and 1 mM DTT

  • Initial purification via Ni-NTA affinity chromatography for His-tagged constructs

  • Secondary purification via size exclusion chromatography

  • For membrane-associated proteins like ACS, inclusion of mild detergents (0.1% Triton X-100 or 0.05% DDM) helps maintain native structure

• Activity preservation: Hyperthermophilic enzymes often retain activity better when purified at elevated temperatures (40-60°C) and in the presence of stabilizing agents such as glycerol (10-20%).

Notably, ACS from I. hospitalis was successfully identified using two-dimensional native PAGE/SDS-PAGE, followed by MALDI-TOF tandem mass spectrometry and N-terminal sequencing , suggesting similar approaches may work for sucC characterization.

How does the interaction between I. hospitalis and N. equitans affect the expression and activity of Succinyl-CoA ligase?

The unique symbiotic/parasitic relationship between I. hospitalis and N. equitans creates a fascinating system for studying metabolic adaptation and interaction. Proteomic analyses have revealed significant remodeling of I. hospitalis metabolism when associated with N. equitans, potentially affecting CoA-utilizing enzymes like Succinyl-CoA ligase. Gene Set Enrichment Analysis (GSEA) of I. hospitalis proteins shows that in co-culture with N. equitans, there is increased energy generation and membrane biogenesis accompanied by decreased transcription and replication functions .

Given these broad metabolic shifts, several hypotheses regarding sucC can be formulated:

• Upregulation hypothesis: Succinyl-CoA ligase may be upregulated to support increased energy demands imposed by N. equitans.

• Metabolic rewiring hypothesis: The dicarboxylate/4-hydroxybutyrate cycle intermediates may be diverted to support N. equitans, affecting sucC function and regulation.

• Compartmentalization hypothesis: The localization of sucC may be altered in co-culture to facilitate metabolite transfer or retention.

Notably, stress response proteins in I. hospitalis show upregulation in co-culture, including oxidoreductases and AAA-ATPases (2-fold increase) . This suggests that the presence of N. equitans may impose metabolic stress, potentially affecting CoA-metabolizing enzyme function. Research examining specifically how sucC expression and activity changes in response to N. equitans would provide valuable insights into this archaeal interaction.

How does the Succinyl-CoA ligase integrate with the unique dicarboxylate/4-hydroxybutyrate CO₂ fixation pathway in I. hospitalis?

The dicarboxylate/4-hydroxybutyrate cycle represents a novel CO₂ fixation pathway in I. hospitalis, where Succinyl-CoA ligase likely plays a critical role. Based on metabolic pathway reconstructions, this enzyme would function at the interface between energy metabolism and carbon fixation.

Integration of sucC in this pathway likely includes:

• Metabolic role: Catalyzing the reversible conversion of succinyl-CoA to succinate coupled with ADP phosphorylation to generate ATP.

• Cycle positioning: Potentially serving as a key connection point between the 4-hydroxybutyrate portion of the cycle and central carbon metabolism.

• Energetic contribution: The ADP-forming activity would contribute to substrate-level phosphorylation, supplementing ATP generated through chemiosmotic mechanisms.

• Regulatory significance: Likely serving as a control point for carbon flux through the cycle based on energy status.

The compartmentalization of this enzyme is particularly intriguing, as research indicates that the intermembrane compartment of I. hospitalis is involved in primary steps of CO₂ fixation . Understanding how sucC contributes to this spatial organization would provide insights into the unique metabolic architecture of this archaeon.

What are the most effective approaches for studying protein-protein interactions involving I. hospitalis Succinyl-CoA ligase?

Investigating protein-protein interactions involving I. hospitalis Succinyl-CoA ligase requires specialized approaches due to the hyperthermophilic nature and unique cellular architecture of the organism. Based on successful studies of related systems, the following methodological approaches are recommended:

• Native co-immunoprecipitation: Using antibodies against sucC to pull down interaction partners, followed by mass spectrometry identification.

  • Critical modification: Performed at elevated temperatures (40-60°C) using buffers containing stabilizing agents

• Two-dimensional native PAGE/SDS-PAGE: Successfully used to identify the ACS complex in I. hospitalis .

  • First dimension: Blue native PAGE or high-resolution Clear Native PAGE

  • Second dimension: Standard SDS-PAGE

  • Visualization: Silver staining followed by mass spectrometry identification

• Crosslinking mass spectrometry (XL-MS): Chemical crosslinking combined with MS to identify spatial proximity of proteins in complexes.

  • Recommended crosslinkers: Thermostable crosslinkers such as DSS or BS3

• Fluorescence microscopy with immunolabeling: Successfully applied to localize ACS in I. hospitalis .

  • Primary antibodies against sucC

  • Fluorophore-conjugated secondary antibodies

  • SYTO9 for DNA counterstaining

• In silico approaches:

  • Comparative genomic neighborhood analysis

  • Protein-protein interaction prediction based on co-expression patterns

Each approach has specific advantages, and a combination of methods typically provides the most robust results. The high coverage of cellular proteomes achieved in studies of I. hospitalis (amongst the highest reported for any organism) suggests that proteomics-based approaches are particularly powerful for this system.

What are the key considerations for designing kinetic studies of I. hospitalis Succinyl-CoA ligase?

Designing rigorous kinetic studies for I. hospitalis Succinyl-CoA ligase requires careful consideration of several factors specific to hyperthermophilic enzymes:

• Temperature considerations:

  • Reactions should be conducted at or near physiological temperature (80-90°C)

  • Equipment must accommodate high-temperature reactions (sealed vessels, high-pressure systems)

  • Temperature gradient studies (70-100°C) to determine activation energy

• Assay development:

  • Coupled spectrophotometric assays linking ADP formation to NADH oxidation

  • Direct measurement of CoA via thiol-reactive fluorescent probes

  • Radiometric assays using labeled substrates for highest sensitivity

• Reaction conditions optimization:

  • Buffer stability at high temperatures (phosphate or PIPES buffers recommended)

  • pH adjustment accounting for temperature effects on pKa

  • Inclusion of stabilizing agents (glycerol, trehalose)

  • Careful consideration of ion effects, particularly divalent cations

• Experimental design for determining kinetic parameters:

  • Initial velocity measurements at varying substrate concentrations

  • Product inhibition studies

  • Forward and reverse reaction characterization

  • Effect of potential allosteric regulators

• Data analysis considerations:

  • Non-linear regression for determining Km and Vmax

  • Evaluation of different kinetic models (ordered, random, ping-pong mechanisms)

  • Temperature effects modeled using Arrhenius plots

The bidirectional nature of the reaction catalyzed by Succinyl-CoA ligase necessitates careful consideration of thermodynamic parameters and reaction directionality under physiological conditions. Comparison with mesophilic homologs can provide valuable insights into adaptations specific to the hyperthermophilic enzyme.

How can advanced mass spectrometry techniques be applied to characterize post-translational modifications of I. hospitalis Succinyl-CoA ligase?

Advanced mass spectrometry techniques offer powerful approaches for characterizing post-translational modifications (PTMs) of I. hospitalis Succinyl-CoA ligase, which may play crucial roles in regulating its activity. Based on findings with related enzymes, several strategic approaches are recommended:

• Sample preparation considerations:

  • Rapid quenching methods to preserve labile modifications

  • Enrichment strategies for specific PTMs (phosphopeptides, acetylated peptides)

  • Both top-down (intact protein) and bottom-up (peptide) approaches

• MS instrumentation and methods:

  • High-resolution instruments (Orbitrap or QTOF) for accurate mass determination

  • Parallel reaction monitoring (PRM) for targeted analysis of suspected modification sites

  • Electron transfer dissociation (ETD) fragmentation to preserve labile modifications

• Key PTMs to investigate:

  • Acetylation: Related ACS enzymes are regulated by acetylation of conserved lysine residues

  • Phosphorylation: Potential regulation by protein kinases/phosphatases

  • Methylation: May contribute to thermostability

  • S-glutathionylation or other redox modifications

• Comparative studies design:

  • PTM analysis under different growth conditions

  • Comparison of PTM patterns between pure culture and co-culture with N. equitans

  • Correlation of modification status with enzymatic activity

• Bioinformatic analysis:

  • Site localization algorithms to precisely identify modified residues

  • Evolutionary conservation analysis of potential modification sites

  • Structural mapping of PTMs to predict functional consequences

The high cellular proteome coverage achieved in previous studies of I. hospitalis suggests that comprehensive PTM profiling is feasible. Given the extensive literature on acetylation-based regulation of ACS enzymes in bacteria and eukaryotes , investigation of similar regulatory mechanisms in I. hospitalis sucC may prove particularly insightful.