Recombinant Oceanobacillus iheyensis Thiol-disulfide oxidoreductase resA (resA)

What is Oceanobacillus iheyensis Thiol-disulfide oxidoreductase resA (resA) and what is its biological function?

Oceanobacillus iheyensis Thiol-disulfide oxidoreductase resA (resA) is a protein belonging to the family of thiol-disulfide oxidoreductases found in the extremely halotolerant and alkaliphilic bacterium Oceanobacillus iheyensis. This organism was originally isolated from deep-sea sediment at a depth of 1,050 meters on the Iheya Ridge . Functionally, ResA proteins are involved in cytochrome c maturation (CCM) through the reduction of disulfide bonds in the CXXCH motif of apocytochrome c, which is necessary for the covalent attachment of heme .

Similar to its homologs in other bacteria, O. iheyensis ResA likely functions as a control point for directing electrons into specific protein maturation pathways. ResA reduces oxidized apocytochrome c, thus preparing it for heme attachment in the process of cytochrome c maturation . This specificity ensures that reducing equivalents are not lost to random disulfides in the extracellular environment, making ResA a crucial component of the bacterium's redox control system.

What is the taxonomic context of Oceanobacillus iheyensis as a source organism?

Oceanobacillus iheyensis is classified under the following taxonomic hierarchy:

• Domain: Bacteria

• Kingdom: Bacillati

• Phylum: Bacillota

• Class: Bacilli

• Order: Bacillales

• Family: Amphibacillaceae

• Genus: Oceanobacillus

• Species: O. iheyensis

O. iheyensis is the type species of its genus and has been fully characterized as an extremely halotolerant (able to grow in NaCl concentrations of 0-21% at pH 7.5) and alkaliphilic bacterium (able to thrive in alkaline environments) . It is particularly significant in ecological studies of deep-sea environments and has potential biotechnological applications due to its extremophilic nature .

What methodologies are recommended for expressing and purifying recombinant O. iheyensis ResA?

Recombinant O. iheyensis ResA can be efficiently expressed using an E. coli expression system with a His-tag to facilitate purification. Based on established protocols for similar proteins, the following methodology is recommended:

• Expression Vector Design: Clone the O. iheyensis resA gene into a pET or similar expression vector with an N-terminal or C-terminal His-tag.

• Expression Conditions: Transform the construct into E. coli BL21(DE3) or a similar strain. Culture cells in LB medium supplemented with the appropriate antibiotic at 37°C until OD600 reaches 0.6-0.8. Induce protein expression with 0.5-1 mM IPTG and continue incubation at a reduced temperature (16-25°C) for 16-18 hours to maximize soluble protein yield.

• Cell Lysis: Harvest cells by centrifugation and resuspend in lysis buffer (typically 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM DTT). Lyse cells by sonication or high-pressure homogenization.

• Purification:

  • Perform immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

  • Apply a gradient of imidazole (10-250 mM) to elute the protein

  • Follow with size exclusion chromatography for higher purity

  • Consider ion exchange chromatography as an additional purification step

• Storage: Store the purified protein in Tris-based buffer with 50% glycerol at -20°C for extended storage . Avoid repeated freeze-thaw cycles.

The expression and purification protocol may need optimization based on specific research requirements and the desired redox state of the protein.

How can the redox activity of O. iheyensis ResA be experimentally measured?

The redox activity of O. iheyensis ResA can be assessed using several complementary approaches:

• Substrate Reduction Assay: Similar to methods used for B. subtilis ResA, the reduction of oxidized substrates can be monitored spectrophotometrically. For example, the reduction rate of a synthetic peptide containing the C-X-X-C-H motif versus a non-specific substrate like oxidized glutathione (GSSG) can be compared . The relative rates provide insight into substrate specificity.

• Enzyme Kinetics Analysis: Varying substrate concentrations can be used to determine kinetic parameters (Km, kcat, kcat/Km). For B. subtilis ResA, this approach revealed higher activity toward a mimetic peptide compared to GSSG, as shown in Table 1:

This methodology can be adapted to characterize O. iheyensis ResA .

• Redox Potential Determination: The midpoint redox potential can be determined using fluorescence of aromatic residues (if present) or by direct electrochemical methods. For comparison, B. subtilis ResA has a midpoint potential (Em) of -345 mV .

• Thiol Reactivity Assays: The reactivity of the active site thiols can be assessed using thiol-reactive probes such as DTNB (5,5'-dithiobis-(2-nitrobenzoic acid)) or fluorescent maleimides.

What techniques can be used to investigate structural changes in O. iheyensis ResA between oxidized and reduced states?

Based on studies of related proteins, several techniques can be employed to investigate redox-dependent structural changes in O. iheyensis ResA:

• X-ray Crystallography: Determine high-resolution structures of both oxidized and reduced forms to identify conformational differences. For B. subtilis ResA, this approach revealed changes in the active site helix (α1), the β7-α4 loop, and a surface cavity that is present in the reduced state but not in the oxidized state .

• NMR Spectroscopy: 2D 1H/15N heteronuclear single quantum coherence (HSQC) spectra can detect significant backbone chemical shift changes between reduced and oxidized states, indicating conformational changes. For B. subtilis ResA, NMR analysis revealed more extensive structural changes than those observed by X-ray crystallography .

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): This technique can identify regions with altered solvent accessibility or hydrogen bonding patterns between the two redox states.

• Fluorescence Spectroscopy: If appropriately positioned tryptophan or tyrosine residues are present, changes in their fluorescence emission can indicate conformational changes.

• Small-Angle X-ray Scattering (SAXS): This technique can detect larger-scale conformational changes in solution.

How do mutations in key residues affect the function of thiol-disulfide oxidoreductases like ResA?

Mutational analysis of key residues in thiol-disulfide oxidoreductases provides valuable insights into their function. Based on studies of similar proteins, the following effects might be expected:

What is the significance of O. iheyensis ResA in cytochrome c maturation compared to other thiol-disulfide oxidoreductases?

The specificity of O. iheyensis ResA for cytochrome c maturation highlights its role as a controlled electron transfer system. Unlike many thiol-disulfide oxidoreductases that react with a wide range of substrates, ResA proteins show specificity for the CXXCH motif in apocytochrome c .

This specificity represents an example of a "control point" mechanism that ensures electrons are directed specifically to cytochrome maturation rather than being lost to random disulfides. Key points about this specificity include:

• Substrate Preference: Similar to B. subtilis ResA, O. iheyensis ResA likely has a higher activity toward the CXXCH motif of apocytochrome c compared to non-specific substrates like glutathione, despite having a less favorable redox potential difference .

• Conformational Selection: The redox-dependent conformational changes observed in ResA proteins are believed to be the mechanism by which they select appropriate interaction partners. In the reduced state, ResA specifically recognizes oxidized apocytochrome c, while in the oxidized state, it interacts with membrane thiol-disulfide oxidoreductases to become re-reduced .

• Physiological Implications: This specificity ensures that electrons are siphoned off from the general reducing pool only when needed for cytochrome maturation, forming an elegant control system that responds to the availability of the substrate .

• Evolutionary Context: The maintained specificity of ResA across different bacterial species suggests strong evolutionary pressure to preserve this control mechanism, highlighting its importance in cellular redox homeostasis .

What spectroscopic methods can be used to study O. iheyensis ResA structure and function?

Several spectroscopic techniques can provide valuable insights into the structure and function of O. iheyensis ResA:

• Circular Dichroism (CD) Spectroscopy: Useful for monitoring secondary structure content and structural changes upon substrate binding or redox state changes. Far-UV CD (190-250 nm) provides information about secondary structure, while near-UV CD (250-350 nm) reports on tertiary structure.

• UV-Visible Absorption Spectroscopy: Can be used to monitor the formation or reduction of disulfide bonds, especially when coupled with appropriate thiol-reactive chromophores.

• Fluorescence Spectroscopy: Intrinsic tryptophan or tyrosine fluorescence can report on conformational changes. Fluorescent probes can also be used to monitor substrate binding or activity.

• Nuclear Magnetic Resonance (NMR) Spectroscopy: 2D and 3D NMR experiments can provide atomic-level information about protein structure, dynamics, and interactions. For B. subtilis ResA, NMR revealed significant chemical shift changes between oxidized and reduced states, indicating widespread conformational changes .

• Raman Spectroscopy: Single-cell Raman spectroscopy has been used to identify and classify Oceanobacillus species, including O. iheyensis . Similar techniques could potentially be applied to study the protein in cellular contexts.

How can molecular dynamics simulations complement experimental studies of O. iheyensis ResA?

Molecular dynamics (MD) simulations can significantly enhance experimental studies of O. iheyensis ResA by providing insights that may be difficult to obtain experimentally:

• Redox-State Transitions: MD simulations can model the transition between oxidized and reduced states, revealing intermediate conformations and energy barriers that are challenging to capture experimentally.

• Water Networks: Simulations can identify and characterize networks of water molecules that may be crucial for substrate binding and catalysis. For OiMacroD, five fixed water molecules were identified that play significant roles in substrate binding .

• Conformational Flexibility: MD can reveal the dynamic behavior of loop regions and substrate binding sites, identifying transient conformations that may be important for function.

• Substrate Recognition: Docking and MD simulations with potential substrates can predict binding modes and interaction energies, guiding experimental studies on substrate specificity.

• Mutation Effects: In silico mutations can predict the effects of amino acid substitutions on protein stability, dynamics, and function before experimental validation.

• Proton Transfer Mechanisms: Quantum mechanical/molecular mechanical (QM/MM) simulations can model the electronic details of catalysis, including proton transfer events that are critical for the redox function.

What analytical methods are available for detecting O. iheyensis in environmental or experimental samples?

Several analytical methods can be used to detect O. iheyensis in environmental or experimental samples:

• Single-Cell Raman Spectroscopy (SCRS): This label-free, non-destructive approach has been successfully applied to identify O. iheyensis in complex microbial communities. SCRS coupled with machine learning algorithms achieved high classification accuracy for distinguishing O. iheyensis from other Oceanobacillus species .

• 16S rRNA Gene Amplicon Sequencing: This technique has been used to detect and identify O. iheyensis in samples such as Daqu starter for Chinese baijiu production. The 16S rRNA gene sequence can be compared to reference databases for taxonomic classification .

• Species-Specific PCR: Primers targeting unique regions of the O. iheyensis genome can be designed for specific detection using PCR methods.

• Metaproteomics: This approach can identify proteins from O. iheyensis in complex samples and has been used to study the role of O. iheyensis in regulating the metabolism of five-member heterocyclic amino acids in Daqu microbiota .

• Culture-Based Methods: For cultivation of O. iheyensis, media containing peptone (1.0%), beef extract (0.3%), NaCl (0.5%), and agar (1.5%) have been used successfully . The extreme halotolerance of O. iheyensis allows selective isolation using high-salt media.

How can protein-protein interactions of O. iheyensis ResA be studied experimentally?

Understanding protein-protein interactions of O. iheyensis ResA is crucial for elucidating its functional role. Several experimental approaches can be employed:

• Co-Immunoprecipitation (Co-IP): Using antibodies against ResA or a tag (such as His-tag) on recombinant ResA to pull down interaction partners from cell lysates.

• Surface Plasmon Resonance (SPR): This technique can measure binding kinetics and affinities between ResA and potential interaction partners, such as apocytochrome c or membrane thiol-disulfide oxidoreductases.

• Isothermal Titration Calorimetry (ITC): Provides thermodynamic parameters of binding interactions, including binding stoichiometry, affinity, and enthalpy changes.

• Crosslinking Mass Spectrometry: Chemical crosslinking followed by mass spectrometry analysis can identify interaction interfaces between ResA and its partners.

• Bacterial Two-Hybrid Systems: Can be used to screen for potential interaction partners in vivo.

• Fluorescence Resonance Energy Transfer (FRET): By tagging ResA and potential interaction partners with appropriate fluorophores, FRET can detect interactions in solution or in cells.

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): Can identify regions of ResA that become protected upon binding to interaction partners.

• NMR Titration Experiments: Can map the interaction surface on ResA when titrated with a binding partner through chemical shift perturbations.

How can structural data from multiple techniques be integrated to build a comprehensive model of O. iheyensis ResA function?

Integrating structural data from multiple experimental techniques provides a more comprehensive understanding of O. iheyensis ResA function:

What bioinformatics approaches can identify potential substrates and interaction partners of O. iheyensis ResA?

Various bioinformatics approaches can help identify potential substrates and interaction partners:

• Sequence-Based Methods:

  • Pattern matching to identify proteins with CXXCH motifs (potential substrates)

  • Genomic context analysis to identify genes co-localized with resA

  • Phylogenetic profiling to identify proteins with similar evolutionary patterns

• Structure-Based Approaches:

  • Molecular docking to predict binding of potential substrates

  • Binding site comparison with homologous proteins of known specificity

  • Electrostatic surface analysis to identify complementary interaction surfaces

• Network-Based Methods:

  • Protein-protein interaction network analysis

  • Text mining of scientific literature for reported interactions

  • Co-expression analysis to identify genes with similar expression patterns

• Machine Learning Applications:

  • Train models on known thiol-disulfide oxidoreductase interactions

  • Use feature extraction from protein sequences and structures

  • Apply deep learning approaches to predict protein-protein interactions

• Systems Biology Integration:

  • Metabolic pathway analysis to identify processes requiring thiol-disulfide exchange

  • Flux balance analysis to predict the impact of ResA on cellular redox state

  • Multi-omics data integration to contextualize ResA function

What are the recommended approaches for studying O. iheyensis ResA in the context of the organism's extreme halotolerance?

Studying O. iheyensis ResA in the context of extreme halotolerance requires specialized approaches:

• Salt-Dependent Structural Analysis:

  • Perform structural studies (X-ray, NMR, CD) at varying salt concentrations

  • Analyze the effects of salt on protein stability and conformation

  • Investigate salt-dependent changes in dynamic properties

• Functional Assays Under Halophilic Conditions:

  • Measure enzyme activity across a range of salt concentrations (0-21% NaCl)

  • Compare activity at different pH values (7.5 vs. 9.5) with varying salt

  • Determine salt effects on substrate specificity and kinetic parameters

• Adaptation Analysis:

  • Compare sequence and structural features with homologs from non-halophilic organisms

  • Identify halophilic adaptation signatures (e.g., increased acidic residues on surface)

  • Study the co-evolution of ResA with other components of the cytochrome maturation system

• Molecular Dynamics in High Salt:

  • Simulate protein behavior in high salt environments

  • Analyze ion binding sites and their effects on protein dynamics

  • Model the influence of salt on water networks critical for catalysis

• Physiological Context Studies:

  • Investigate ResA expression levels under varying salt conditions

  • Examine the impact of salt stress on cytochrome maturation

  • Study the redox balance in O. iheyensis under halophilic conditions

• Protein Engineering Applications:

  • Identify features conferring salt tolerance for transfer to other proteins

  • Design salt-resistant variants of non-halophilic ResA homologs

  • Develop halophilic ResA as a potential biotechnological tool

What are promising avenues for applying O. iheyensis ResA in biotechnology or synthetic biology?

Several promising applications for O. iheyensis ResA in biotechnology and synthetic biology exist:

• Engineered Electron Transfer Systems:

  • Design synthetic redox pathways with controlled electron flow

  • Create modular components for synthetic cellular redox networks

  • Develop biosensors for specific redox states or substrates

• Protein Engineering Applications:

  • Create salt-tolerant variants of industrially relevant oxidoreductases

  • Design ResA variants with altered substrate specificity

  • Develop temperature-stable or pH-resistant variants

• Biocatalysis Under Extreme Conditions:

  • Utilize ResA's halotolerance for biocatalysis in high-salt environments

  • Develop processes for disulfide bond formation/reduction in non-conventional media

  • Create immobilized ResA systems for continuous biocatalytic applications

• Therapeutic Applications:

  • Develop ResA-based strategies for controlling cellular redox states

  • Design inhibitors targeting bacterial ResA for potential antimicrobial applications

  • Explore the potential of ResA in protein folding applications

• Structural Biology Tools:

  • Use the redox-dependent conformational changes as molecular switches

  • Develop ResA-based structural probes for monitoring cellular redox state

  • Create fusion proteins for site-specific redox modifications

What unresolved questions remain about the catalytic mechanism of ResA proteins?

Despite significant research, several key questions about ResA catalytic mechanisms remain unresolved:

• Detailed Catalytic Mechanism:

  • The exact role of the conserved aspartate requires further clarification, as mutation doesn't completely abolish activity

  • The precise mechanism of proton transfer during catalysis needs further investigation

  • The role of water molecules in catalysis requires additional study

• Substrate Recognition Determinants:

  • The molecular basis for distinguishing between specific and non-specific substrates

  • Factors determining the preference for oxidized apocytochrome c

  • Structural elements responsible for protein-protein specificity

• Conformational Changes:

  • The complete pathway of conformational changes between oxidized and reduced states

  • The energetics of these conformational transitions

  • How conformational changes couple to substrate binding and product release

• Physiological Regulation:

  • Mechanisms controlling ResA expression and activity in vivo

  • Integration of ResA function with cellular redox homeostasis

  • Environmental factors affecting ResA function in extremophiles

• Evolutionary Aspects:

  • How substrate specificity evolved in ResA compared to more promiscuous thiol-disulfide oxidoreductases

  • The co-evolution of ResA with its substrate proteins

  • Adaptation of ResA function in extremophilic organisms like O. iheyensis

How can systems biology approaches advance our understanding of O. iheyensis ResA in cellular redox networks?

Systems biology approaches can significantly enhance our understanding of O. iheyensis ResA within cellular redox networks:

• Integrated Redox Network Modeling:

  • Construct comprehensive models of cellular redox systems

  • Integrate ResA function with other redox enzymes and substrates

  • Simulate the flow of electrons through multiple pathways

• Multi-Omics Integration:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Identify condition-dependent changes in ResA expression and activity

  • Correlate ResA function with global cellular redox state

• Perturbation Analysis:

  • Study system-wide effects of ResA deletion or mutation

  • Analyze cellular responses to redox stress in wild-type vs. ResA-modified strains

  • Identify compensatory mechanisms when ResA function is compromised

• Network Control Theory:

  • Identify control points in redox networks

  • Analyze how ResA specificity contributes to network robustness

  • Develop predictive models for cellular responses to redox perturbations

• Comparative Systems Biology:

  • Compare redox networks across organisms with different ResA homologs

  • Analyze adaptations in halophilic vs. non-halophilic organisms

  • Identify conserved and variable components of cytochrome maturation systems