Recombinant Putative thiosulfate sulfurtransferase sseA (sseA)

What is thiosulfate sulfurtransferase sseA and what is its biochemical function?

Thiosulfate sulfurtransferase sseA belongs to the rhodanese family of sulfurtransferases that catalyze the transfer of sulfur atoms. These enzymes form an active-site cysteine persulfide during their reaction cycle and can play a role in protein persulfidation. In Mycobacterium tuberculosis, the protein CysA2 (encoded by Rv0815c) functions as a putative thiosulfate sulfurtransferase that is secreted during infection and participates in the essential sulfur assimilation pathway .

SseA catalyzes the transfer of sulfur from thiosulfate to various acceptors, with the general reaction:
$\text{S}_2\text{O}_3^{2-} + \text{CN}^- \rightarrow \text{SO}_3^{2-} + \text{SCN}^-$

SseA functions include:

• Participation in sulfur energy metabolism

• Protection against oxidative stress

• Involvement in macrophage infection and immune response

• Potential role in cyanide detoxification

What are the structural characteristics of thiosulfate sulfurtransferase sseA?

Thiosulfate sulfurtransferases typically have the following structural features:

• α/β protein with two rhodanese-like domains

• Conserved catalytic cysteine residue in the active site

• Characteristic TST motif CRXGX[R/T] in many cases

• Forms a persulfide intermediate during catalysis

In Mycobacterium tuberculosis, the SseA protein (CysA2) maintains functional motifs that are critical for its activity as a dual sulfurtransferase . The protein forms a covalent enzyme-sulfur intermediate (E-S) characterized by a persulfide bond at the sulfhydryl group of the reactive cysteine in the active site .

Structural studies using homology modeling and experimental approaches have helped elucidate the functional domains and critical residues involved in catalysis .

What expression systems are optimal for producing recombinant sseA?

Escherichia coli BL21(DE3) is the most commonly used expression host for recombinant sulfurtransferases including sseA. The methodology typically involves:

• Vector selection: pET vectors (pET28a(+), pET30a) are commonly used with N-terminal His-tags for purification

• Induction conditions: IPTG induction (typically 0.1-1.0 mM) at reduced temperatures (25-30°C) often increases soluble expression

• Media selection: Rich media like LB or TB supplemented with appropriate antibiotics

For enhanced expression, consider:

• Co-expression with chaperones to improve folding

• Using thermostable protein nanoparticles (tES) to improve expression and stabilization

• Optimization of culture conditions using multivariate experimental design approaches

A multivariate experimental design approach is recommended to identify optimal conditions for soluble expression rather than testing one variable at a time .

What is the recommended purification protocol for His-tagged recombinant sseA?

The following protocol is recommended for purifying His-tagged recombinant sseA:

• Cell lysis: Sonicate cells in binding buffer containing:

  • 50 mM phosphate buffer (pH 8.0)

  • 100 mM NaCl

  • 10 mM MgCl₂

  • 1 mg/ml lysozyme

  • 5 mM β-mercaptoethanol

  • 5% (v/v) glycerol

• Immobilized Metal Affinity Chromatography (IMAC):

  • Pack column with Ni-NTA His·Bind Resin (2 ml bed volume)

  • Equilibrate with 5-column volumes of binding buffer

  • Bind protein by passing supernatant through the column multiple times

  • Wash with 2-column volumes of wash buffer (binding buffer + 20 mM imidazole)

  • Elute with increasing gradient of imidazole (250-500 mM)

• Quality control:

  • Verify purity by 12% SDS-PAGE

  • Confirm activity by enzyme assay

  • Assess folding state by circular dichroism or thermal stability assays

For enhanced stability during purification, consider including reducing agents and protease inhibitors throughout the process .

What methods are used to measure thiosulfate sulfurtransferase activity?

Several complementary assays can be used to characterize thiosulfate sulfurtransferase activity:

• Thiosulfate:cyanide sulfurtransferase (TST) activity:

  • Measures the formation of thiocyanate from thiosulfate and cyanide

  • Colorimetric detection using Fe³⁺ ions, which form a red complex with thiocyanate

  • Typically performed at pH 8.0, 30-37°C

• Thiosulfate:thioredoxin sulfurtransferase activity:

  • Measures transfer of sulfur from thiosulfate to thioredoxin

  • Requires thioredoxin reductase and NADPH

  • Can determine Km values for both thiosulfate and thioredoxin

• GSSH:sulfite sulfurtransferase activity:

  • Measures transfer of sulfur from glutathione persulfide (GSSH) to sulfite

  • Can be performed under steady-state or single-turnover conditions

• Persulfide detection:

  • Biotin thiol assay (BTA) to detect protein persulfidation

  • Distinguishes between persulfides and other oxidative cysteine modifications

The choice of assay depends on the specific research question and the acceptors being studied.

How should kinetic parameters be determined for recombinant sseA?

Determination of kinetic parameters for recombinant sseA should follow these methodological steps:

• Substrate range determination:

  • Test various concentrations of thiosulfate (typically 0.1-50 mM)

  • For dual-activity characterization, also test 3-mercaptopyruvate (0.1-50 mM)

• Reaction conditions optimization:

  • Determine optimal pH (usually 7.5-8.5)

  • Identify temperature optimum (typically 30-37°C for mesophilic enzymes)

  • Establish linear range for time and enzyme concentration

• Data collection and analysis:

  • Perform at least three independent replicates

  • Use Michaelis-Menten kinetic model to determine Km and Vmax

  • Calculate kcat and catalytic efficiency (kcat/Km)

Example kinetic parameters for M. tuberculosis CysA2:

Note: These values demonstrate the dual TST/MST activity of this enzyme .

How can substrate specificity of sseA be comprehensively characterized?

To comprehensively characterize substrate specificity of sseA, implement the following experimental approach:

• Sulfur donor screening:

  • Test thiosulfate (S₂O₃²⁻)

  • Test 3-mercaptopyruvate

  • Test glutathione persulfide (GSSH)

  • Compare activity with each donor using standardized assay conditions

• Acceptor specificity analysis:

  • Test small molecule acceptors: cyanide, sulfite

  • Test protein acceptors: thioredoxin, glutaredoxin

  • Determine Km values for each acceptor

• Inhibition studies:

  • Test product inhibition (e.g., thiosulfate inhibition of GSSH:sulfite activity)

  • Determine inhibition constants and mechanisms (competitive, non-competitive)

• Data presentation format:

How can recombinant sseA be used to study protein persulfidation?

Recombinant sseA can serve as a valuable tool for studying protein persulfidation through these methodological approaches:

• In vitro persulfidation system:

  • Use purified sseA with thiosulfate as sulfur donor

  • Add target proteins as potential persulfidation substrates

  • Detect persulfidation using the biotin thiol assay (BTA)

  • Compare with control reactions lacking sseA or thiosulfate

• Identification of persulfidation targets:

  • Perform pull-down assays with sseA to identify interacting proteins

  • Use mass spectrometry to identify persulfidated proteins and specific cysteine residues

  • Validate results using site-directed mutagenesis of target cysteines

• Functional consequences of persulfidation:

  • Compare enzymatic activity, protein-protein interactions, or localization before and after persulfidation

  • Investigate the role of persulfidation in redox regulation and oxidative stress response

The ability of sulfurtransferases to form persulfides makes them excellent tools for studying this emerging post-translational modification that plays roles in signaling and stress responses.

How can contradictory results in sseA research be effectively analyzed and reconciled?

When faced with contradictory results in sseA research, apply the following systematic approach:

• Methodological assessment:

  • Compare experimental protocols in detail: buffers, pH, temperature, assay conditions

  • Evaluate enzyme preparation: expression system, purification method, storage conditions

  • Assess assay methods: detection limits, interference, sensitivity

• Data contradiction analysis framework:

  • Use a dialectical approach that identifies opposing findings

  • Frame contradictions in practical terms while preserving multiple perspectives

  • Consider regulation frameworks from ergonomics of activity

• Experimental verification:

  • Design definitive experiments that directly address contradictions

  • Include appropriate controls that can distinguish between competing hypotheses

  • Perform replication studies in independent laboratories

• Synthesis of findings:

  • Develop explanatory models that can accommodate seemingly contradictory results

  • Consider context-dependent enzyme behavior (e.g., substrate availability, cellular conditions)

  • Evaluate whether contradictions reflect genuine biological complexity rather than experimental error

An example framework for addressing contradictory kinetic parameters:

What experimental design approaches optimize recombinant sseA production and characterization?

To optimize recombinant sseA production and characterization, implement these experimental design strategies:

• Multivariate experimental design for expression optimization:

  • Use factorial or response surface methodology rather than one-factor-at-a-time approaches

  • Test combinations of variables: temperature, IPTG concentration, induction time, media composition

  • Analyze responses mathematically to identify significant variables and interactions

  • Apply statistical tools to characterize experimental error

• Data table design for clear result presentation:

  • Place independent variables (what you purposefully change) in the left column

  • Include dependent variables (what you measure) with different trials in subsequent columns

  • Add derived or calculated values (often averages) in the rightmost column

  • Use clear titles stating the purpose of the experiment3

Example data table format for expression optimization:

This approach allows systematic optimization and statistical analysis to achieve high levels of soluble, functional sseA protein (potentially up to 250 mg/L) .

How do sseA interaction partners modulate its activity and what are the structural determinants?

Recent research has revealed that sseA activity can be significantly modulated by interaction partners:

• Partner identification approach:

  • Use genomic neighborhood analysis to identify potential functional partners

  • Perform pull-down assays with tagged recombinant sseA

  • Validate interactions using techniques like surface plasmon resonance or isothermal titration calorimetry

• Activity modulation characterization:

  • Compare sseA activity alone versus with potential partners

  • Determine kinetic parameters in the presence and absence of interacting proteins

  • Analyze the effect on substrate specificity and reaction rates

For example, in Mycobacterium tuberculosis, a SufE-like protein (SufEMtb, encoded by Rv3284) was identified as an sseA interaction partner that significantly enhances its catalytic activity . The SufEMtb protein:

• Increases sulfur transfer activity of sseA

• May facilitate conformational changes in sseA structure

• Contributes to more efficient catalysis

Structural studies suggest that "displacement of the water molecules at the entrance of the active site cavity and formation of the sulfur adduct at Cys245 of SseA may contribute to trigger displacement of Arg246" which then facilitates complex formation with SufEMtb .

What roles do sseA and related sulfurtransferases play in microbial sulfur metabolism networks?

Thiosulfate sulfurtransferases like sseA participate in complex sulfur metabolism networks with multiple roles:

• Integration in sulfur energy metabolism:

  • In Aquifex aeolicus, the rhodanese SbdP functions as a sulfur carrier to key enzymes like sulfur reductase and sulfur oxygenase reductase

  • SbdP can load long sulfur chains and transfer them to enzyme partners

  • This enables channeling of sulfur substrate in the cell and greater efficiency of sulfur energy metabolism

• Overlap between sulfate and thiosulfate assimilation:

  • In Saccharomyces cerevisiae, rhodanese (Rdl2p) is the only additional enzyme needed for thiosulfate utilization compared to sulfate assimilation

  • Rdl2p exhibits thiosulfate sulfurtransferase activity, producing sulfite and releasing H₂S in the presence of glutathione

  • The reaction proceeds through formation of glutathione persulfide (GSSH) as an intermediate

• Methodological approach for studying metabolic networks:

  • Use gene knockout studies to assess enzyme essentiality

  • Employ isotope tracing to follow sulfur atom movement through metabolic pathways

  • Apply metabolomics to identify pathway intermediates

  • Measure flux through alternative pathways under different growth conditions

The common presence of rhodanese in most organisms including Bacteria, Archaea, and Eukarya suggests that most organisms with sulfate assimilation systems also use thiosulfate, providing metabolic flexibility .

What are the latest strategies for engineering modified sseA proteins with enhanced properties?

Advanced protein engineering strategies can be employed to enhance sseA properties for research and potential applications:

• Thermostabilization approaches:

  • Engineer thermostable protein nanoparticles (tES) that can fold and stabilize recombinant sseA

  • These nanoparticles provide steric accommodation and charge complementation

  • Enzymes encapsulated in tES retain activity while gaining resistance to thermal, organic, chaotropic, and proteolytic denaturation

• Fusion protein strategies:

  • Create TAT-HA-tagged fluorescent sseA fusions for tracking cellular localization

  • Incorporate enhanced green fluorescent protein (EGFP) or mCherry for visualization

  • Include 6×His tags for purification

  • Express in E. coli BL21(DE3) and purify by immobilized metal affinity chromatography

• Application in transduction technology:

  • Use penetrating peptides like TAT-HA to introduce recombinant sseA into mammalian cells

  • Monitor uptake and localization using fluorescent tags

  • Study function in cellular contexts

• Expression optimization:

  • Apply experimental design methodology to develop optimal process conditions

  • Aim for high levels of soluble expression (potentially 250 mg/L) to reduce operational costs

  • Recover protein in active form with high homogeneity (≥75%)