Recombinant Escherichia coli O8 Cysteine desulfurase (iscS), partial,Yeast

What is cysteine desulfurase IscS and what is its primary function in bacterial systems?

Cysteine desulfurase IscS is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that plays a critical role in sulfur mobilization. Its primary function is to transfer sulfur from L-cysteine to various cellular pathways, with particular importance in the biosynthesis of iron-sulfur (Fe-S) clusters. IscS catalyzes the conversion of L-cysteine to L-alanine while generating a protein-bound persulfide intermediate that serves as the sulfur donor for Fe-S cluster assembly .

This enzyme has been demonstrated to have a major role in vivo for Fe-S cluster formation, as evidenced by significant decreases in the activities of various Fe-S cluster-containing proteins in IscS deletion strains. These affected proteins include both soluble proteins (aconitase B, 6-phosphogluconate dehydratase, glutamate synthase, fumarase A, and FNR) and membrane-bound proteins (NADH dehydrogenase I and succinate dehydrogenase) .

How does the functional relationship between E. coli IscS and yeast Nfs1 (its homologue) differ?

While E. coli IscS and yeast Nfs1 are homologues that share the same fundamental cysteine desulfurase activity, they exhibit significant differences in their functional characteristics and requirements:

• Essentiality: Unlike E. coli IscS which can be deleted (though resulting in growth defects), the IscS homologue (Nfs1) in yeast is essential and cannot be disrupted without lethal consequences .

• Activity modulation: E. coli IscS can modify basal metabolism by independently transferring sulfur from L-cysteine to numerous cellular pathways. In contrast, human NFS1 (which shares characteristics with yeast Nfs1) is only fully active when it forms part of the [Acp]2:[ISD11]2:[NFS1]2 complex .

• Absorption characteristics: When the N-terminus of IscS is fused with the C-terminus of NFS1, the resulting chimeric protein exhibits PLP absorption peaks at 395 nm, indicating structural differences in how the enzymes bind their cofactor .

What is the recommended approach for generating and validating an IscS deletion strain?

Based on established methodologies, the following approach is recommended for generating and validating an IscS deletion strain:

Generation protocol:

• Replace the iscS coding region with an antibiotic resistance gene (e.g., kanamycin resistance gene KnR) through homologous recombination.

• Select transformants on appropriate antibiotic-containing media.

Validation methods:

• PCR verification using gene-specific primers to confirm replacement of the iscS coding region.

• Genetic mapping using P1 generalized transduction with markers tightly linked to iscS (e.g., Tn10 at zff-208, 57.4 min, which is closely linked to iscS at 57.3 min on the E. coli chromosome).

• Phenotypic confirmation by assaying the activities of Fe-S cluster-containing enzymes, which should show decreased specific activities in the deletion strain .

This genetically verified ΔiscS::KnR strain provides a powerful tool for studying the in vivo function of IscS and its role in Fe-S cluster assembly pathways.

What spectroscopic methods are most informative for analyzing IscS reaction intermediates?

UV-visible absorption spectroscopy provides valuable insights into the formation and accumulation of reaction intermediates during the IscS catalytic cycle. The following absorption peaks correspond to specific intermediates:

Additional analytical methods that complement spectroscopic analysis include high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS), which can be used to identify and quantify reaction products and intermediates .

Which conserved active site residues are critical for IscS function, and how do mutations affect catalytic activity?

Several conserved active site residues play crucial roles in IscS function, with mutations leading to specific biochemical consequences:

The K206A&C328S double mutation has particularly pronounced effects, demonstrating the synergistic importance of proper PLP binding and persulfide formation in the catalytic mechanism.

What is the proposed chemical mechanism for IscS-catalyzed cysteine desulfuration?

The chemical mechanism of IscS involves multiple intermediate steps:

• Initial complex formation: The amine group of L-cysteine performs a nucleophilic attack on the PLP cofactor, forming a gem-diamine complex.

• Aldimine-ketimine equilibrium: The reaction proceeds through Cys-aldimine and Cys-ketimine forms that exist in rapid equilibrium.

• Quinonoid intermediate: Cys-aldimine converts to a short-lived Cys-quinonoid intermediate (absorbing at 510 nm), followed by formation of Cys-ketimine.

• C-S bond cleavage: The active site cysteine (Cys328) performs a nucleophilic attack, cleaving the C-S bond and forming a persulfide (Cys328-S-SH). This persulfide serves as the sulfur donor for iron-sulfur cluster biosynthesis.

• Product release and regeneration: Alanine is released as a product, and the internal aldimine between PLP and Lys206 is regenerated.

Notably, when the PLP cofactor is occupied by a second L-cysteine molecule, persulfide cleavage can be inhibited, leading to substrate inhibition under steady-state conditions .

How can fusion proteins be designed to restore IscS function in deletion strains?

Chimeric fusion proteins can be strategically designed to restore IscS function in deletion strains and provide insights into domain requirements for activity. Research has demonstrated success with the following approach:

Fusing the N-terminus of IscS with the C-terminus of human NFS1 creates a chimeric cysteine desulfurase (SUMO-EH-IscS) that:

• Exhibits a PLP absorption peak at 395 nm, indicating proper cofactor binding

• Demonstrates significant growth recovery when expressed in iscS mutant cells

• Restores NADH-dehydrogenase I activity, a key Fe-S dependent enzyme

• Maintains substantial cysteine desulfurase activity comparable to native IscS

This chimeric approach not only validates functional complementation but also provides a system for investigating the structural elements that confer activity differences between bacterial and eukaryotic cysteine desulfurases.

What experimental approaches can distinguish between direct and indirect effects of IscS deficiency?

Distinguishing between direct and indirect effects of IscS deficiency requires multiple complementary approaches:

• Specific Fe-S independent controls: Utilize Fe-S cluster independent variants of proteins that normally contain Fe-S clusters. For example, research has demonstrated that an Fe-S cluster independent mutant of FNR remains unaffected by the lack of IscS, confirming that activity losses in Fe-S proteins are specifically due to deficient cluster assembly rather than general metabolic effects .

• Activity profiling: Systematically measure the activities of various Fe-S cluster-containing enzymes with different cluster types ([2Fe-2S], [3Fe-4S], [4Fe-4S]) to establish patterns of dependency.

• Time-course analysis: Monitor the decline in activities of various Fe-S proteins following IscS depletion to distinguish immediate (direct) from delayed (indirect) effects.

• Targeted complementation: Express wild-type IscS or specific variants in iscS deletion strains and measure the restoration of activities for different Fe-S dependent pathways.

What factors contribute to the accumulation of a red-colored IscS intermediate, and how does this affect experimental interpretation?

The accumulation of red-colored IscS is associated with specific biochemical conditions and has important implications for experimental interpretation:

The red coloration corresponds to the Cys-quinonoid intermediate, which absorbs at approximately 510 nm in the visible spectrum . This intermediate accumulates under several conditions:

• Iron deficiency: Red-colored IscS has been observed in E. coli cells as a result of deficiency in accessible iron, suggesting a regulatory feedback mechanism between Fe-S cluster assembly and IscS activity.

• Specific mutations: Alterations to active site residues can disrupt the normal catalytic cycle, leading to accumulation of the quinonoid intermediate.

• Substrate/product imbalances: Disruptions in the normal ratio of substrate (cysteine) to product (alanine) can affect the reaction equilibrium and promote intermediate accumulation.

Experimental implications:

• The presence of red coloration indicates altered catalytic kinetics and potentially reduced sulfur transfer efficiency

• Spectroscopic measurements should account for the additional 510 nm absorption peak when characterizing enzyme preparations

• The quinonoid intermediate accumulation may serve as a visible indicator of iron availability in the cellular environment

How can researchers address contradictory results in IscS activity assays?

When confronting contradictory results in IscS activity assays, consider the following methodological factors and troubleshooting approaches:

Common sources of variability:

• Substrate inhibition effects: At high concentrations, L-cysteine can inhibit the IscS reaction by occupying the PLP cofactor and preventing persulfide cleavage. Researchers should perform careful concentration-dependent activity measurements to identify optimal assay conditions .

• PLP cofactor status: The activity of IscS is critically dependent on properly bound PLP. The PLP:protein ratio should be verified spectroscopically (395 nm peak), and additional PLP may need to be supplied in reaction buffers to ensure full enzyme activation.

• Oxidation state control: The active site cysteine (Cys328) is susceptible to oxidation, which can inactivate the enzyme. Including reducing agents in buffers and conducting assays under anaerobic conditions may be necessary for consistent results.

• Protein partner effects: In vivo, IscS functions in complexes with other proteins. The presence or absence of these partners can significantly affect activity measurements in different experimental setups.

Standardization recommendations:

• Use multiple complementary activity assays (e.g., sulfide production, alanine formation, and persulfide formation)

• Include positive controls with well-characterized IscS preparations

• Document and control all reaction parameters (pH, temperature, buffer composition)

• Consider the physiological context when interpreting in vitro activity measurements