Recombinant Pseudomonas putida Cysteine desulfurase (iscS)

What is cysteine desulfurase (IscS) and what is its primary function in Pseudomonas species?

Cysteine desulfurase (IscS) is an essential enzyme (EC 2.8.1.7) involved in iron-sulfur (Fe-S) cluster biosynthesis in Pseudomonas species. In P. aeruginosa, IscS is encoded by the PA3814 gene located within the isc operon. The enzyme catalyzes the removal of sulfur from L-cysteine, generating alanine and a protein-bound persulfide intermediate that serves as the sulfur source for Fe-S cluster assembly. IscS plays a critical role in various cellular processes by supporting the formation of Fe-S clusters, which are essential cofactors for numerous proteins involved in electron transfer, catalysis, and gene regulation .

How is IscS structurally characterized in Pseudomonas compared to other bacterial species?

IscS in Pseudomonas aeruginosa shares high protein sequence identity (approximately 75%) with Salmonella enterica IscS (STM2543). The enzyme contains a conserved PLP-binding domain, with residue Q183 being particularly important for its function. Structural studies based on homology with E. coli and S. enterica IscS suggest that the Pseudomonas variant maintains the characteristic homodimeric structure with each monomer containing a pyridoxal phosphate (PLP) cofactor at the active site. The conserved architecture of the active site contains a catalytic cysteine residue that forms the persulfide intermediate during the desulfuration reaction .

What is the genomic context of the iscS gene in Pseudomonas putida?

In Pseudomonas putida, similar to P. aeruginosa, the iscS gene is located within the isc operon, which encodes proteins involved in Fe-S cluster biosynthesis. This operon typically includes other genes such as iscR (a transcriptional regulator), iscU (a scaffold protein for Fe-S cluster assembly), iscA (an alternative scaffold or iron donor), and hscB and hscA (chaperone proteins that facilitate cluster transfer). The genomic organization of the isc operon in P. putida reflects its functional role in the coordinated production of proteins required for Fe-S cluster biosynthesis .

How does 2-aminoacrylate (2AA) affect IscS activity in Pseudomonas and what are the molecular mechanisms underlying this interaction?

2-Aminoacrylate (2AA) is a reactive metabolite that can damage specific PLP-dependent enzymes, including IscS in Pseudomonas aeruginosa. Research demonstrates that IscS is a significant target of 2AA in P. aeruginosa, and damage to this enzyme causes substantial growth defects during enamine stress. The molecular mechanism involves 2AA reacting with the PLP cofactor in IscS, thereby inactivating the enzyme.

The interaction between 2AA and IscS occurs at the enzyme's PLP-binding domain, likely interfering with the coordination of the PLP cofactor essential for catalysis. This inhibition disrupts Fe-S cluster biosynthesis, which has downstream effects on multiple cellular processes dependent on Fe-S proteins. Notably, a Q183P substitution in IscS decreases its sensitivity to 2AA damage, suggesting this residue plays a crucial role in the interaction between 2AA and the enzyme's active site .

What is the relationship between RidA protein and IscS function in Pseudomonas species?

RidA (Reactive Intermediate Deaminase A) proteins, such as PA5339 in P. aeruginosa, are responsible for deaminating reactive enamine species like 2AA, preventing them from damaging PLP-dependent enzymes. In the absence of RidA (ridA mutants), 2AA accumulates and targets various enzymes, with IscS being identified as a critical target in P. aeruginosa.

The relationship between RidA and IscS is hierarchical - RidA functions upstream by neutralizing 2AA before it can damage IscS. Without this protective mechanism, 2AA-mediated damage to IscS leads to defects in Fe-S cluster biosynthesis, resulting in growth and motility deficiencies. This relationship was demonstrated by the finding that an IscS Q183P variant, which is less sensitive to 2AA, suppressed the phenotypic defects of ridA mutants. This represents a novel mechanism of phenotypic suppression and highlights the significant functional relationship between these two proteins in Pseudomonas metabolism .

What are the optimal conditions for heterologous expression of recombinant IscS in Pseudomonas putida?

Based on successful heterologous expression strategies in P. putida, the optimal conditions for recombinant IscS expression would include:

Expression System Parameters:

• Host strain: P. putida KT2440 (GRAS-certified strain with robust expression capabilities)

• Vector design: Integration-based expression using transposon-mediated chromosomal insertion

• Promoter selection: Strong native P. putida promoters for constitutive expression rather than inducible T7 system

• Growth temperature: 20-30°C, with lower temperatures (20°C) often yielding better protein folding

• Medium composition: Rich medium under high aeration conditions

• Carbon source: Glucose or other preferred carbon sources for P. putida

Optimization Approaches:

• Screening multiple chromosomal integration sites to identify high-expression loci

• Codon optimization of the iscS gene for P. putida expression

• Co-expression of chaperones if necessary for proper folding

• Fine-tuning fermentation parameters including dissolved oxygen levels, pH, and nutrient feeding strategies

These conditions have been shown to support high-level expression of complex heterologous proteins in P. putida, with yields potentially reaching 94 mg/L or higher under optimized conditions .

What purification strategies are most effective for isolating recombinant IscS from Pseudomonas putida?

An effective purification strategy for recombinant IscS from P. putida would involve:

Initial Processing:

• Cell harvesting by centrifugation (6,000 × g, 10 min, 4°C)

• Cell lysis using either sonication or high-pressure homogenization in buffer containing:

  • 50 mM Tris-HCl (pH 8.0)

  • 300 mM NaCl

  • 5% glycerol

  • 1 mM DTT (to protect cysteine residues)

  • Protease inhibitor cocktail

• Clarification of lysate by ultracentrifugation (100,000 × g, 1 hour, 4°C)

Chromatographic Purification Sequence:

• Immobilized Metal Affinity Chromatography (IMAC) - Using His-tagged IscS:

  • Ni-NTA column equilibrated with lysis buffer

  • Step gradients of imidazole (20 mM, 50 mM, 250 mM) for wash and elution

• Ion Exchange Chromatography:

  • Anion exchange (Q-Sepharose) using pH gradient based on IscS theoretical pI

  • Buffer system: 20 mM Tris-HCl (pH 8.0), with NaCl gradient from 50-500 mM

• Size Exclusion Chromatography:

  • Superdex 200 column to isolate the dimeric form of IscS

  • Running buffer: 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM DTT

Critical Considerations:

• Maintain reducing conditions throughout purification to protect the catalytic cysteine

• Include PLP (0.1 mM) in buffers to ensure cofactor saturation

• Process samples rapidly at 4°C to minimize protein degradation

• Verify enzyme activity after each purification step using established cysteine desulfurase assays

This purification scheme should yield >95% pure IscS suitable for biochemical and structural studies, with typical yields of 5-10 mg purified protein per liter of culture .

What methods can be used to assess the enzymatic activity of recombinant IscS?

Several complementary methods can be employed to assess the enzymatic activity of recombinant IscS:

1. Methylene Blue Assay for Sulfide Production:

• Principle: Quantification of H₂S produced from cysteine by IscS

• Procedure:

  • Reaction mixture contains purified IscS, L-cysteine, DTT, and PLP in buffer

  • Incubate at 37°C for 15-30 minutes

  • Add N,N-dimethyl-p-phenylenediamine and FeCl₃ to form methylene blue

  • Measure absorbance at 670 nm

  • Calculate specific activity (nmol sulfide/min/mg protein)

2. Coupled Enzyme Assay with IscU:

• Principle: Monitoring Fe-S cluster formation on the IscU scaffold protein

• Procedure:

  • Reaction mixture contains IscS, IscU, L-cysteine, Fe²⁺, DTT, and PLP

  • Monitor absorbance changes at 456 nm (characteristic of [2Fe-2S] cluster formation)

  • Calculate initial rates of cluster assembly

3. Alanine Production Assay:

• Principle: Measuring alanine produced as a byproduct of cysteine desulfuration

• Procedure:

  • Reaction mixture contains IscS, L-cysteine, and PLP

  • After incubation, derivatize amino acids

  • Analyze alanine production by HPLC or a coupled enzymatic assay

4. PLP Binding Assessment:

• UV-visible spectroscopy to monitor the characteristic absorbance of PLP-bound enzyme (~420 nm)

• Titration experiments to determine PLP binding affinity

Comparative Activity Table:

These methods provide a comprehensive assessment of IscS functionality, from cofactor binding to product formation and physiological activity .

How does P. putida IscS compare structurally and functionally to IscS from other bacterial species?

The structural and functional comparison of IscS across bacterial species reveals both conservation and specialization:

Sequence and Structural Comparison:

P. putida IscS shares highest similarity with other Pseudomonas species IscS proteins, particularly P. aeruginosa (>95% identity). The protein maintains the characteristic homodimeric architecture with each monomer containing a PLP cofactor at the active site. The catalytic cysteine residue that forms the persulfide intermediate during desulfuration is strictly conserved across species.

Functional Comparison:
While the core cysteine desulfurase function is conserved, species-specific differences exist in:

• Substrate specificity: P. putida IscS may have distinct preferences for protein partners during Fe-S cluster assembly

• Regulatory mechanisms: The control of IscS expression and activity varies between species

• Sensitivity to inhibitors: P. putida IscS shows species-specific sensitivity to reactive metabolites like 2AA

• Protein-protein interactions: The interaction network of IscS with other components of the Fe-S cluster assembly machinery may differ

These comparative insights highlight that while the fundamental enzymatic mechanism is preserved across species, the evolutionary adaptations in P. putida IscS likely reflect its specific metabolic requirements and environmental niche .

What insights can be gained from studying the sensitivity of IscS to 2-aminoacrylate (2AA) across different bacterial species?

Comparative analysis of IscS sensitivity to 2AA across bacterial species provides valuable insights into:

1. Evolutionary Adaptation of Metabolic Damage Control:
Different bacterial species show varying degrees of IscS sensitivity to 2AA, suggesting evolutionary adaptation to specific metabolic challenges. While P. aeruginosa IscS is significantly affected by 2AA, the sensitivity profile may differ in P. putida and other species, reflecting their ecological niches and metabolic networks.

2. Structure-Function Relationships:
By examining which residues confer resistance or sensitivity to 2AA across species, researchers can identify critical structural determinants of enzyme function. For example, the Q183P variant in P. aeruginosa IscS decreased sensitivity to 2AA damage, suggesting this position is crucial for interaction with the reactive metabolite. Mapping such residues across homologs provides insight into potential "hotspots" for enzyme engineering.

3. RidA-IscS Relationship Across Species:
The relationship between RidA and IscS appears to differ across bacterial species:

• In P. aeruginosa, IscS is a principal target of 2AA in ridA mutants

• In Salmonella, other PLP-dependent enzymes have been identified as primary targets

• The specific consequences of IscS damage likely depend on the organism's metabolic network

4. Metabolic Network Organization:
The phenotypic outputs of IscS damage vary across species due to differences in metabolic network architecture:

• P. aeruginosa ridA mutants show both growth and motility defects

• S. enterica ridA mutants exhibit different nutritional requirements

• E. coli shows another pattern of metabolic disruption

These differences highlight how the same enzymatic damage can propagate differently through metabolic networks based on species-specific pathway organization .

How do the isc operon structure and regulation differ between Pseudomonas putida and other bacterial species?

The isc operon structure and regulation show both conservation and divergence across bacterial species:

Operon Structure Comparison:

Regulatory Mechanisms:

• IscR Regulation:

  • P. putida likely employs IscR as the primary transcriptional regulator of the isc operon

  • IscR activity is modulated by its own Fe-S cluster status, creating feedback regulation

  • P. putida may have evolved specific operator sequences that affect binding affinity of IscR

• Environmental Response Elements:

  • The isc operon in Pseudomonas species contains unique promoter elements that respond to environmental signals relevant to their ecological niches

  • P. putida, as a soil bacterium, may have additional regulatory mechanisms related to rhizosphere adaptation

• Cross-regulation with Other Systems:

  • In P. putida, potential cross-talk between the isc system and other iron-responsive systems may exist

  • Integration with oxidative stress response differs from enteric bacteria

  • Connection to biofilm formation or secondary metabolite production pathways may be unique to Pseudomonas

These differences in operon organization and regulation reflect the evolutionary adaptation of each species to its environmental niche and metabolic requirements. Understanding these distinctions is crucial for optimizing heterologous expression strategies and metabolic engineering approaches in P. putida .

What genetic engineering approaches are most effective for expressing recombinant IscS in Pseudomonas putida?

Several genetic engineering approaches have proven effective for recombinant protein expression in P. putida, which can be applied to IscS expression:

1. Chromosomal Integration Strategies:

• Transposon-mediated random integration (e.g., Tn5-based systems) with screening for high-expression loci

• Site-specific integration using CRISPR-Cas9 or recombinase-based systems targeting transcriptionally active regions

• Integration of expression cassettes into multiple genomic locations for gene dosage increase

2. Expression Control Elements:

• Strong constitutive promoters native to P. putida (e.g., promoters from highly expressed housekeeping genes)

• Inducible systems adapted for P. putida, including:

  • XylS/Pm system (m-toluate induction)

  • Cumate-inducible system

  • Cyclohexanone-inducible system

3. Genetic Stability Enhancement:

• Elimination of mobile genetic elements from expression constructs

• Removal of recognition sites for native restriction-modification systems

• Codon optimization specific for P. putida bias

4. Protein Engineering Approaches:

• Addition of affinity tags (His6, Strep-tag II) for purification

• Fusion to solubility-enhancing partners (e.g., SUMO, MBP) with protease cleavage sites

• Inclusion of signal peptides for potential periplasmic localization if beneficial

Comparative Effectiveness Table:

Research indicates that for efficient P. putida expression, a combination of chromosomal integration at highly transcribed loci with strong native promoters provides the best balance of stability and expression levels, as demonstrated in successful heterologous protein production systems .

How can researchers troubleshoot common issues with recombinant IscS expression in P. putida?

Common Issue #1: Low Expression Levels

Common Issue #2: Inclusion Body Formation

Common Issue #3: Loss of Enzymatic Activity

Common Issue #4: Genetic Instability

Methodological Workflow for Troubleshooting:

• Expression analysis:

  • Monitor protein levels using SDS-PAGE, Western blotting, and activity assays

  • Check mRNA levels by RT-qPCR to distinguish transcriptional from translational issues

• Solubility assessment:

  • Analyze soluble vs. insoluble fractions after cell lysis

  • Microscopy to detect potential inclusion bodies

• Activity testing:

  • Apply multiple activity assays to detect partial activity

  • Test enzyme under various buffer conditions

• Optimization iterations:

  • Implement changes based on findings

  • Re-test expression in a systematic manner

This structured approach allows researchers to methodically identify and resolve issues with recombinant IscS expression in P. putida .

What strategies can be used to improve the solubility and stability of recombinant IscS in P. putida?

Genetic and Expression Strategies

• Fusion Partners:

  • N-terminal solubility tags such as MBP (maltose-binding protein), SUMO, or Trx (thioredoxin)

  • Add TEV or PreScission protease sites for tag removal

  • C-terminal stabilizing tags that do not interfere with N-terminal folding

• Expression Rate Control:

  • Use weaker promoters or lower copy number vectors

  • Implement auto-induction media for gradual protein accumulation

  • Lower cultivation temperature to 16-20°C during expression phase

• Co-expression Approaches:

  • Co-express molecular chaperones (GroEL/ES, DnaK/DnaJ/GrpE)

  • Co-express other Isc proteins like IscU to facilitate native interactions

  • Include specific P. putida chaperones that may have evolved for optimal folding in the host

Biochemical Optimization

• Buffer Composition:

  • Include PLP (0.1-0.5 mM) in all buffers to maintain cofactor saturation

  • Add reducing agents (2-5 mM DTT or TCEP) to protect the catalytic cysteine

  • Optimize salt concentration (typically 100-300 mM NaCl)

  • Include glycerol (5-10%) for stabilization

• Additives for Stability:

  • Add osmolytes like trehalose or sucrose (5-10%)

  • Include amino acids like arginine (50-100 mM) to prevent aggregation

  • Test metal ions (Fe²⁺) at low concentrations to stabilize enzyme structure

Processing Modifications

• Gentle Lysis Methods:

  • Use enzymatic lysis (lysozyme + DNase) instead of sonication

  • Employ gentle detergents (0.1% Triton X-100) to aid solubilization

  • Perform all operations at 4°C with protease inhibitors

• Refolding Protocols:

  • If inclusion bodies form, develop a refolding protocol using gradual dialysis

  • Include PLP and reducing agents in refolding buffers

  • Test on-column refolding during purification

Experimental Optimization Table:

By systematically optimizing these parameters, researchers can achieve significantly improved solubility and stability of recombinant IscS in P. putida expression systems .

How can recombinant P. putida IscS be used to study the mechanisms of Fe-S cluster assembly?

Recombinant P. putida IscS provides a valuable tool for investigating Fe-S cluster assembly mechanisms through several experimental approaches:

1. In vitro Reconstitution Systems:

• Purified recombinant IscS can be combined with other Fe-S cluster assembly components (IscU, IscA, ferredoxin, frataxin) to reconstitute the assembly process in a controlled environment

• The kinetics and efficiency of cluster formation can be monitored spectroscopically (UV-visible absorption at ~420 nm)

• Variations in reaction conditions (iron source, reducing equivalents, pH) can reveal mechanistic details of the assembly process

2. Structure-Function Studies:

• Site-directed mutagenesis of conserved residues in P. putida IscS can identify critical amino acids involved in:

  • Substrate binding (cysteine)

  • PLP cofactor coordination

  • Protein-protein interactions with scaffold proteins

  • Persulfide formation and transfer

• Crystallization of recombinant IscS alone or in complex with partner proteins can provide structural insights into the assembly mechanism

3. Protein-Protein Interaction Analysis:

• Pull-down assays using tagged recombinant IscS to identify interaction partners in P. putida

• Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

• Surface plasmon resonance or isothermal titration calorimetry to determine binding affinities and kinetics

• Crosslinking coupled with mass spectrometry to capture transient interactions

4. Real-time Assembly Monitoring:

• Development of FRET-based sensors using fluorescently labeled IscS and IscU to monitor their interaction dynamics

• Time-resolved techniques to capture intermediate states during Fe-S cluster assembly

• Single-molecule approaches to observe heterogeneity in assembly mechanisms

These approaches can address fundamental questions about the P. putida Fe-S cluster assembly pathway, including species-specific features, regulation mechanisms, and integration with other metabolic networks .

What insights can studies of recombinant IscS provide about the metabolic interactions between RidA and Fe-S cluster biosynthesis?

Studies of recombinant IscS can provide critical insights into the metabolic interface between RidA function and Fe-S cluster biosynthesis:

1. Molecular Mechanism of 2AA Damage to IscS:

• In vitro studies with purified recombinant IscS and chemically synthesized 2AA can elucidate:

  • The binding mode of 2AA to IscS

  • The chemical mechanism of enzyme inactivation

  • Potential conformational changes induced by 2AA binding

  • Specific modifications to the PLP cofactor or catalytic residues

2. Comparative Susceptibility Analysis:

• Wild-type IscS versus the resistant Q183P variant can be compared to understand:

  • Structural features that confer 2AA resistance

  • Changes in enzyme kinetics associated with the resistance-conferring mutation

  • Potential trade-offs between 2AA resistance and normal catalytic function

  • Conformational dynamics differences using hydrogen-deuterium exchange

3. Systems-Level Metabolism Investigation:

• Recombinant IscS can be used to reconstitute Fe-S cluster assembly in the presence of:

  • RidA (functional)

  • Inactive RidA variants

  • 2AA at various concentrations

  • Threonine dehydratase (as a 2AA generator)

4. Metabolic Flux Integration:

• Isotope labeling experiments using recombinant enzymes can track:

  • The flow of sulfur atoms from cysteine through IscS to Fe-S clusters

  • How this flux is affected by 2AA accumulation

  • Potential compensatory metabolic rerouting when IscS is inhibited

Key Findings Table from Published Research:

These studies demonstrate how recombinant IscS can serve as a key tool for understanding the complex interaction between primary metabolism (amino acid processing), reactive intermediate detoxification (RidA function), and essential cofactor biosynthesis (Fe-S clusters), providing insights into the integration of these systems in bacterial metabolism .

What are promising future research directions for studying recombinant P. putida IscS in the context of bacterial metabolism and stress responses?

Several promising research directions can advance our understanding of P. putida IscS function in metabolism and stress response:

1. Systems Biology Integration:

• Global proteomic and metabolomic analysis comparing wild-type and iscS mutant P. putida under various stress conditions

• Flux balance analysis incorporating IscS function and Fe-S cluster-dependent enzymes

• Network modeling to predict metabolic adaptations to IscS impairment

• Multi-omics data integration to identify compensatory pathways activated during Fe-S cluster deficiency

2. Environmental Adaptation Mechanisms:

• Investigation of IscS regulation and activity under conditions relevant to P. putida's ecological niche:

  • Oxidative stress (relevant to rhizosphere colonization)

  • Heavy metal exposure (common in contaminated soils where P. putida thrives)

  • Carbon source shifts (reflecting environmental versatility)

  • Biofilm formation conditions

• Comparison of IscS function across Pseudomonas species adapted to different environments

3. Synthetic Biology Applications:

• Engineering IscS variants with enhanced stability or activity for biotechnological applications

• Creating biosensors based on Fe-S cluster assembly to detect specific environmental conditions

• Integration of optimized IscS expression with production pathways requiring Fe-S enzymes

• Development of P. putida as a chassis for Fe-S protein production

4. Structural and Mechanistic Evolution:

• Comprehensive structure-function analysis across bacterial phyla to understand evolutionary adaptations

• Identification of species-specific regulatory mechanisms controlling IscS expression and activity

• Investigation of potential moonlighting functions of IscS beyond canonical Fe-S cluster assembly

• Exploration of potential IscS involvement in stress signaling cascades

5. Therapeutic Target Exploration:

• Comparative analysis of IscS between pathogens and non-pathogenic species like P. putida

• Structure-guided design of potential inhibitors targeting pathogen-specific features

• Investigation of IscS as a potential antibiotic target given its essential function

• Understanding of host-microbe interactions involving bacterial Fe-S proteins

Research Methodology Advancements:

• Development of in vivo Fe-S cluster assembly tracking methods

• Application of cryo-EM for capturing dynamic assembly intermediates

• Advanced computational modeling of IscS conformational changes during catalysis

• Integration of machine learning approaches to predict Fe-S protein interactions and regulatory networks

This multifaceted research agenda would significantly advance our understanding of IscS function in P. putida while providing broader insights into bacterial metabolism, stress adaptation, and potential biotechnological applications .