Recombinant Cysteine desulfurase (sufS)

What is the role of SufS in bacterial iron-sulfur cluster biogenesis?

SufS functions as a cysteine desulfurase in the SUF-like pathway, which is essential for iron-sulfur (Fe-S) cluster biogenesis, particularly in Gram-positive bacteria. SufS catalyzes the removal of sulfur from L-cysteine, generating a protein-bound persulfide intermediate that serves as the sulfur source for Fe-S cluster assembly. In bacteria such as Staphylococcus aureus, SufS works in conjunction with the sulfurtransferase SufU to form the transient SufSU complex, which executes the first stage of Fe-S cluster biogenesis . This pathway is particularly critical in Gram-positive bacteria because they typically lack redundant Fe-S cluster biogenesis pathways, making the SUF-like pathway essential for survival .

How are Type I and Type II cysteine desulfurases distinguished structurally?

Type I and Type II cysteine desulfurases have distinct structural characteristics:

Type II cysteine desulfurases (like SufS):

• Lack the flexible loop region present in Type I enzymes

• Contain interfacial β-hook regions

• Have conserved active site residues including the catalytic cysteine (Cys389 in S. aureus SufS), the lysine that covalently harbors PLP (Lys250), and the histidine that acts as a base in the sulfur acquisition reaction (His147)

The SUF-like systems of Fe-S cluster biogenesis typically possess type II cysteine desulfurases. Sequence analysis of S. aureus SufS confirms its classification as a type II cysteine desulfurase, showing high sequence identity (61%) with B. subtilis SufS, another confirmed type II enzyme .

What is the relationship between SufS and SufU in bacterial systems?

SufS and SufU form a functional complex in which SufU acts as a sulfurtransferase that accepts the persulfide from SufS. This interaction varies significantly between bacterial species:

The surprising finding that S. aureus SufS is significantly less stimulated by SufU compared to other bacterial species suggests species-specific differences in the regulation of Fe-S cluster biogenesis . Cross-species activity analysis revealed that cysteine desulfurase activity levels are dictated by the SufS homologue, while SufU proteins from different species are functionally interchangeable .

How do kinetic parameters differ between SufS from various bacterial species?

Kinetic analysis reveals significant differences in SufS activity and stimulation across bacterial species. For S. aureus and B. subtilis, the following comparative data has been observed:

These results demonstrate that S. aureus SufS is intrinsically less responsive to stimulation by SufU than its B. subtilis counterpart. The fact that the stimulation factor is determined by the SufS homologue rather than the SufU partner suggests fundamental differences in the catalytic mechanism or structural interactions between these proteins across bacterial species .

What are the proposed mechanisms for persulfide formation and transfer in cysteine desulfurases?

The persulfide formation and transfer mechanism in cysteine desulfurases involves several discrete steps that can be analyzed using radiolabeling and stopped-flow kinetics:

• Initial binding of L-cysteine to the PLP cofactor at the active site

• Formation of an external aldimine intermediate

• Abstraction of the α-hydrogen to form a quinonoid intermediate

• Nucleophilic attack by the catalytic cysteine on the substrate cysteine's β-carbon

• Formation of persulfide on the catalytic cysteine and release of alanine

• Transfer of the persulfide to a recipient protein (e.g., SufU or ISCU2)

In human systems, frataxin (FXN) accelerates both the persulfide formation on the cysteine desulfurase (NFS1) and the interprotein sulfur transfer to scaffold proteins . Kinetic models suggest that the persulfide transfer reaction is reversible with an equilibrium constant near 1, and the rate-limiting step can vary depending on the specific system and substrate conditions .

What factors might explain the reduced stimulation of S. aureus SufS by SufU compared to other bacterial systems?

Several hypotheses have been proposed to explain the reduced stimulation of S. aureus SufS by SufU:

• Role of additional protein partners: In the E. coli SUF pathway, SufS activity is stimulated 8-fold by SufE alone, but 32-fold when the SufBCD scaffold complex is also present . The S. aureus SUF-like pathway may require additional protein partners for full stimulation.

• Mechanism of persulfide transfer: The underlying mechanism of persulfide transfer from S. aureus SufS to SufU may differ from other bacterial systems, resulting in altered stimulation patterns .

• Structural variations: Despite high sequence identity (61%) between S. aureus and B. subtilis SufS, subtle structural differences may affect the SufS-SufU interaction interface.

• Regulatory differences: S. aureus may employ different regulatory mechanisms for Fe-S cluster biogenesis, possibly reflecting adaptations to its pathogenic lifestyle or specific niche requirements.

These differences highlight the importance of species-specific characterization of Fe-S cluster biogenesis pathways rather than relying solely on homology-based predictions .

What are the optimal conditions for assaying recombinant SufS cysteine desulfurase activity?

Based on published protocols, optimal conditions for assaying recombinant SufS activity include:

Buffer conditions:

• 50 mM HEPES, pH 8.0

• 300 mM NaCl

• Reducing agent (DTT or TCEP, with TCEP being more effective)

Assay components:

• 4 μM SufS

• 140 μM SufU (if measuring stimulated activity)

• 500 μM L-cysteine (substrate)

• 420 μM DTT or equivalent reducing agent

Important considerations:

• Substrate inhibition can occur at ≥1 mM cysteine for some SufS systems

• The choice of reducing agent significantly affects activity measurements (TCEP has been shown to be more effective in regenerating cysteine desulfurases for subsequent substrate turnover)

• Reaction time needs to be carefully controlled, as different systems exhaust the substrate supply at different rates

For accurate comparative studies, it's essential to use consistent conditions across different protein systems and to consider the linear range of product formation for the specific system being studied.

How can one track the formation and transfer of persulfide species in SufS-mediated reactions?

Several complementary techniques can be used to track persulfide formation and transfer:

• Acid-quench radiolabeling assay:

  • Use L-[35S]cysteine as substrate

  • Quench reactions at various time points with acid

  • Separate proteins under quench conditions

  • Quantify radiolabel on individual proteins

  This approach allows direct measurement of persulfide formation on SufS and transfer to acceptor proteins like SufU .

• Stopped-flow kinetics:

  • Monitor changes in PLP absorbance (typically at 300-500 nm)

  • Can detect formation and decay of reaction intermediates

  • Particularly useful for determining rate constants for individual steps in the reaction mechanism

• Pulse-chase experiments:

  • Incubate proteins with labeled cysteine, then chase with excess unlabeled cysteine

  • Monitor loss of label from different proteins

  • Provides insights into the reversibility of persulfide transfer and the stability of persulfide species

These methods have revealed that persulfide transfer between cysteine desulfurase and scaffold proteins is often reversible, with equilibrium constants near 1 .

What expression systems are most effective for producing active recombinant SufS?

For successful production of active recombinant SufS:

Expression system considerations:

• E. coli BL21(DE3) or derivative strains are commonly used

• Co-expression with chaperones may improve solubility

• For S. aureus SufS, expression at lower temperatures (16-20°C) after IPTG induction may yield more soluble protein

Critical factors for activity:

• Ensuring proper incorporation of the PLP cofactor (supplementation of the growth medium or reconstitution post-purification may be necessary)

• Maintaining reducing conditions throughout purification to prevent oxidation of the catalytic cysteine

• Including stabilizing agents (glycerol, reducing agents) in storage buffers

Purification strategy:

• Initial capture using affinity tags (His-tag is common)

• Intermediate purification with ion exchange chromatography

• Final polishing step with size exclusion chromatography

• Yellow color (due to PLP) can be used as a visual indicator of properly folded protein during purification

Active SufS should demonstrate the characteristic absorption spectrum of PLP-containing enzymes, with peaks at approximately 280 nm (protein) and 420 nm (PLP cofactor).

How should researchers address the apparent contradiction between SufS activity measurements across different studies?

When addressing contradictions in SufS activity measurements, researchers should consider:

• Methodological variations:

  • Different assay methods (direct sulfide detection vs. alanine production)

  • Varied reaction conditions (pH, salt concentration, temperature)

  • Different reducing agents (DTT vs. TCEP)

• Substrate concentration effects:

  • Substrate inhibition at high cysteine concentrations

  • Non-linear kinetics at certain substrate ranges

• Protein preparation differences:

  • Variations in PLP content

  • Oxidation state of the catalytic cysteine

  • Presence of contaminating proteins or inhibitors

• Experimental time frame:

  • Linear range of product formation varies between systems

  • Differential substrate exhaustion rates

For example, the observed basal activity for B. subtilis SufS can differ by an order of magnitude depending on the reducing agent used, with TCEP providing more effective regeneration of the enzyme for subsequent turnover compared to DTT .

When comparing activities across studies, standardize conditions where possible or include internal controls (such as a well-characterized homolog) to provide a reference point for relative activity measurements.

What are the key considerations for designing cross-species activity experiments with SufS and SufU proteins?

When designing cross-species activity experiments with SufS and SufU proteins:

• Protein purity and integrity:

  • Ensure comparable purity of proteins from different species

  • Verify structural integrity using techniques like circular dichroism

  • Confirm PLP content for SufS proteins

• Standardized conditions:

  • Use identical buffer conditions, substrate concentrations, and protein concentrations

  • Control temperature precisely

  • Maintain consistent reaction times

• Control experiments:

  • Include measurements of basal activity (SufS alone)

  • Include homologous SufS-SufU pairs as internal controls

  • Test multiple protein concentrations to ensure saturation

• Analysis approach:

  • Calculate stimulation factors (ratio of activity with SufU to basal activity)

  • Determine which component (SufS or SufU) dictates activity in cross-species pairs

  • Consider kinetic modeling to extract mechanistic information

Previous cross-species experiments between S. aureus and B. subtilis SufS/SufU revealed that cysteine desulfurase activity levels are dictated by the SufS homologue, while SufU proteins are functionally interchangeable . This unexpected finding challenges assumptions about conserved mechanisms across related bacterial systems.

What techniques can be used to investigate the structural basis for functional differences between SufS homologs?

To investigate structural differences underlying functional variation between SufS homologs:

• X-ray crystallography:

  • Determine high-resolution structures of SufS alone and in complex with SufU

  • Compare active site architecture and substrate binding pockets

  • Identify differences in flexible loops or interface regions

• Small-angle X-ray scattering (SAXS):

  • Characterize protein conformation in solution

  • Detect conformational changes upon complex formation

  • Complement crystallographic data for dynamic regions

• Site-directed mutagenesis:

  • Target residues that differ between homologs

  • Create chimeric proteins with swapped domains

  • Analyze the impact on activity and stimulation

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Map regions with differential dynamics or solvent accessibility

  • Identify conformational changes upon SufU binding

  • Detect allosteric networks within the protein

• Molecular dynamics simulations:

  • Model protein dynamics and conformational changes

  • Investigate the energetics of protein-protein interactions

  • Predict the impact of mutations or environmental conditions

These approaches can reveal why, despite high sequence identity (61%) and structural similarity, S. aureus SufS and B. subtilis SufS exhibit dramatically different responses to stimulation by SufU .

How does the PLP chemistry in cysteine desulfurases differ from other PLP-dependent enzymes?

Cysteine desulfurases utilize PLP chemistry that is distinct from other PLP-dependent enzymes in several ways:

• Dual catalytic mechanism:

  • Initial PLP-dependent α,β-elimination reaction similar to other PLP enzymes

  • Subsequent nucleophilic attack by the catalytic cysteine, which is unique to desulfurases

• Persulfide intermediate formation:

  • The catalytic cysteine forms a persulfide intermediate (Cys-S-SH)

  • This sulfur is subsequently transferred to a recipient protein rather than released as H₂S

• Mobile S-transfer loop:

  • Contains the catalytic cysteine residue

  • Functions in substrate binding, general acid catalysis, nucleophilic attack, and sulfur carrier roles at different steps of the reaction

  • Undergoes significant conformational changes during catalysis

• Reaction acceleration by protein partners:

  • Activity can be significantly enhanced by sulfur acceptor proteins

  • In human systems, frataxin (FXN) accelerates PLP chemistry by enhancing the rates of both persulfide formation and decay of PLP intermediates

Stopped-flow kinetic studies have identified up to 7 accumulating intermediates in the cysteine desulfurase reaction, allowing detailed mechanistic mapping of this complex enzymatic process .

What is the evidence for reversibility in the persulfide transfer reaction between SufS and SufU?

Evidence supporting the reversibility of persulfide transfer between SufS and SufU includes:

• Kinetic modeling:

  • Models incorporating reversible sulfur transfer better fit experimental data compared to irreversible transfer models

  • Equilibrium constants near 1 have been observed for several systems, indicating similar forward and reverse rates

• Pulse-chase experiments:

  • In experiments where labeled persulfide-containing proteins are chased with excess unlabeled cysteine, label is lost from both SufS and sulfur acceptor proteins

  • The similar rates of label loss from both proteins in some systems suggest equilibration of the label through reversible transfer

• Cross-species compatibility:

  • The functional interchangeability of SufU proteins from different species with various SufS enzymes suggests a conserved transfer mechanism that can operate in both directions

This reversibility has important implications for understanding the regulation of Fe-S cluster biogenesis, as it suggests that the direction of sulfur flow can be modulated by the relative concentrations and states of the participating proteins.

How might the SufBCD scaffold complex influence SufS activity in complete Fe-S cluster biogenesis systems?

The SufBCD scaffold complex likely influences SufS activity through several mechanisms:

Future studies examining complete reconstituted systems containing SufS, SufU, and SufBCD components will be essential for understanding the full regulatory network controlling bacterial Fe-S cluster biogenesis and explaining species-specific differences in SufS stimulation .