Recombinant Pasteurella multocida Cysteine desulfurase (iscS)

What is cysteine desulfurase (IscS) and what role does it play in bacterial metabolism?

Cysteine desulfurase (IscS) is a pyridoxal 5′-phosphate (PLP)-dependent homodimeric enzyme that catalyzes the conversion of L-cysteine into L-alanine and sulfur. This enzyme plays a critical role in bacterial metabolism by transferring sulfur from L-cysteine to numerous cellular pathways . In bacteria like Escherichia coli, IscS modifies basal metabolism through this sulfur transfer mechanism.
The catalytic mechanism involves:

• Formation of an internal aldimine between PLP and a conserved lysine residue (Lys206 in E. coli IscS)

• Substrate binding and formation of external aldimine with L-cysteine

• Cleavage of the C-S bond and persulfide formation on a catalytic cysteine residue (Cys328 in E. coli)

• Transfer of the sulfur to various acceptor proteins or pathways

• Release of L-alanine and regeneration of the enzyme
  While P. multocida IscS has not been extensively characterized, based on homology with other bacterial species, it likely serves similar functions in sulfur metabolism and iron-sulfur cluster biosynthesis.

What structural features are essential for cysteine desulfurase activity?

Based on structural studies of bacterial cysteine desulfurases, several key features are critical for enzymatic activity:

• PLP cofactor: Positioned inside the active site pocket near the protein surface

• Active site pocket: Composed of charged or polar amino acid side chains, including His104, Lys105, Asn155, Glu156, Tyr337, and Arg354 (in E. coli IscS)

• Catalytic residues:

  • His104: Acts as an acid-base catalyst in protonation/deprotonation processes

  • Lys206: Forms an internal aldimine Schiff base with PLP

  • Cys328: Forms a persulfide intermediate via nucleophilic attack

• PLP-binding interactions:

  • Phenolate oxygen forms hydrogen bonds with Gln183

  • Pyridine N1 interacts with Asp180

  • Asp79-Asp180 hydrogen bonding affects electron-withdrawing interaction with pyridine N1
    Mutations of these conserved active site residues typically result in loss of enzymatic activity, highlighting their essential roles in catalysis.

What methodologies are recommended for expression and purification of recombinant P. multocida IscS?

While specific protocols for P. multocida IscS are not detailed in the available literature, effective methodologies for bacterial cysteine desulfurases typically include:

• Cloning strategy:

  • PCR amplification of the iscS gene from P. multocida genomic DNA

  • Insertion into an expression vector with an appropriate tag (His-tag, SUMO-tag)

  • Transformation into an E. coli expression strain (BL21, Rosetta)

• Expression conditions:

  • Induction with IPTG (typically 0.1-0.5 mM)

  • Growth at lower temperatures (16-25°C) to enhance solubility

  • Supplementation with PLP (100 μM) during expression to ensure cofactor incorporation

• Purification protocol:

  • Cell lysis by sonication in buffer containing 500 mM NaCl and 20 mM Tris-HCl (pH 8.0)

  • Affinity chromatography (Ni-NTA for His-tagged proteins)

  • Size exclusion chromatography for further purification

  • Treatment with PLP prior to purification can increase holo-enzyme yield

• Quality control:

  • SDS-PAGE to assess purity

  • UV-visible spectroscopy to confirm PLP incorporation (absorption peak at ~395 nm)

  • Activity assays using Siegel's sulfide detection method

What spectroscopic features characterize the reaction intermediates of cysteine desulfurase?

The enzymatic reaction of cysteine desulfurase progresses through several intermediates, each with distinctive spectroscopic signatures that can be observed using UV-visible spectroscopy:

• IscS variants (H104Q, Q183E, K206A, K206A&C328S) show new absorption peaks at 420-430 nm

• These altered spectral properties are associated with PLP migration within the active site pocket
  These spectroscopic features provide valuable tools for monitoring enzyme-substrate interactions and reaction progress in real-time studies.

How do specific active site mutations affect the catalytic mechanism of cysteine desulfurase?

Site-directed mutagenesis studies have revealed critical insights into the roles of conserved active site residues:

• His104 mutations:

  • H104Q variant shows altered absorption spectrum with peaks at 340 and 350 nm

  • These peaks correspond to trapped Cys-ketimine and Cys-aldimine intermediates

  • Loss of desulfurase activity indicates His104's essential role in acid-base catalysis

• Asp180 mutations:

  • D180G variant affects PLP binding

  • Critical for modulating electron withdrawal at pyridine N1 of PLP

  • Impairs the stability of reaction intermediates

• Gln183 mutations:

  • Q183E variant shows new absorption peaks at 340 and 350 nm

  • Disrupts hydrogen bonding with phenolate oxygen of PLP

  • Loses desulfurase activity

• Lys206 mutations:

  • K206A variant has absorption peaks at 338 and 428 nm

  • Cannot form the internal aldimine with PLP

  • Complete loss of enzymatic activity

• Cys328 mutations:

  • C328S variant cannot form the persulfide intermediate

  • Essential for the nucleophilic attack on PLP-bound cysteine

  • Required for red IscS formation in iron-depleted conditions
    These findings demonstrate how specific residues contribute to different steps of the catalytic cycle, providing a framework for rational enzyme engineering.

What approaches can be used to design chimeric cysteine desulfurases with enhanced properties?

Research on E. coli IscS provides valuable insights into designing chimeric cysteine desulfurases with enhanced properties, which could be applied to P. multocida IscS:

• Domain fusion strategy:

  • Fusion of the N-terminus of IscS with the C-terminus of human NFS1 (EH-IscS)

  • This chimeric construct exhibited significant growth recovery and NADH-dehydrogenase I activity in iscS mutant cells

  • The EH-IscS chimera displayed a PLP absorption peak at 395 nm, indicating proper cofactor binding

• Performance metrics:

  • Enzymatic activity using Siegel's sulfide detection method

  • Complementation of growth defects in iscS mutant strains

  • Restoration of NADH dehydrogenase I activity in vivo

• Optimization considerations:

  • Active site pocket architecture preservation

  • PLP-binding residue conservation

  • Sulfur transfer efficiency
    These approaches could be applied to create P. multocida IscS variants with improved stability, activity, or substrate specificity for biotechnological applications or vaccine development.

How might cysteine desulfurase be integrated into multicomponent vaccine strategies against P. multocida?

Current vaccine development strategies against P. multocida have focused on recombinant outer membrane and lipoprotein antigens. Integrating cysteine desulfurase into these approaches could provide additional benefits:

• Multi-antigen formulations:

  • Research has shown that vaccine formulations combining multiple recombinant P. multocida proteins (VacJ, PlpE, OmpH) with appropriate adjuvants provided 100% protection in duck models

  • Including IscS as part of a multi-antigen formulation could enhance protective coverage

• Adjuvant selection:

  • Water-in-oil adjuvants have proven effective for subunit vaccines

  • Oil-coated adjuvants have been used successfully with killed vaccines

• Protection assessment:

  • Challenge studies with virulent P. multocida (typically 20 LD50)

  • Histopathological examination to evaluate tissue damage reduction

  • Bacterial load detection to assess colonization inhibition

• Potential advantages of IscS-based vaccines:

  • Metabolic essentiality constrains mutation of target epitopes

  • Conserved across different strains of P. multocida

  • Potential cross-protection against related pathogens
    Integration of IscS into existing vaccine platforms could potentially address some of the challenges in developing effective protection against P. multocida infections in both humans and animals.

What are the current challenges in characterizing PLP-dependent enzymes like cysteine desulfurase?

Researchers face several technical challenges when working with PLP-dependent enzymes like cysteine desulfurase:

• Cofactor incorporation:

  • Recombinant overexpression often leads to incomplete PLP incorporation

  • "Because the recombinant proteins are overexpressed while PLP synthesised by the host strain is limited, some of the purified proteins are apoproteins"

  • Supplementation with exogenous PLP can increase holo-enzyme yield

• Intermediate stabilization:

  • Reaction intermediates are often transient and difficult to characterize

  • Specific mutations may trap intermediates but also alter their properties

  • Time-resolved spectroscopy and rapid-mixing techniques are often necessary

• Spectroscopic interpretation:

  • Multiple overlapping absorbance peaks can complicate analysis

  • Distinguishing enzyme-bound species from free PLP or reaction byproducts

  • Environmental effects on peak positions and intensities

• Functional redundancy:

  • Many bacteria possess multiple sulfur transfer systems (like ISC and SUF)

  • Complementation effects can mask phenotypes in genetic studies

  • Studies need to account for potential compensatory mechanisms

• Structural dynamics:

  • Conformational changes during catalysis affect substrate binding and product release

  • Crystallographic structures may not capture the full range of dynamic states

  • Advanced techniques like hydrogen-deuterium exchange mass spectrometry may be needed
    Addressing these challenges requires integrated approaches combining spectroscopy, structural biology, genetic manipulation, and biochemical assays.

What assays are recommended for measuring cysteine desulfurase activity in recombinant P. multocida IscS?

Several complementary methods can be employed to assess the activity of recombinant cysteine desulfurase:

• Siegel's sulfide detection method:

  • Standard approach for quantifying sulfide production

  • Measures the formation of methylene blue from the reaction of N,N-dimethyl-p-phenylenediamine with H2S under acidic conditions

  • Allows for spectrophotometric detection at ~670 nm

• UV-visible spectroscopic monitoring:

  • Real-time observation of changes in absorption peaks

  • Tracks the conversion of the 395 nm PLP peak to various intermediate species

  • Particularly useful for kinetic studies of wild-type and mutant enzymes

• Functional complementation assays:

  • Transformation of recombinant IscS into ΔiscS mutant cells

  • Assessment of growth recovery in minimal media

  • Evaluation of NADH dehydrogenase I activity as a proxy for iron-sulfur cluster biogenesis

• Chromatographic analysis:

  • HPLC and UPLC-MS to detect and quantify reaction products

  • Identification of reaction intermediates and potential side products

  • Measurement of substrate consumption rates
    A combination of these methods provides comprehensive characterization of enzymatic properties and mechanisms.

How can researchers distinguish between apo- and holo-forms of recombinant cysteine desulfurase?

Distinguishing between apo- (without PLP) and holo- (PLP-bound) forms of cysteine desulfurase is critical for accurate characterization:

• Spectroscopic identification:

  • Holo-IscS shows a characteristic absorption peak at 395 nm

  • Apo-IscS lacks this distinctive spectral feature

  • The ratio of A280/A395 can be used to estimate the proportion of holo-enzyme

• Reconstitution protocol:

  • Incubation with excess PLP (typically 100 μM)

  • Removal of unbound PLP by dialysis or gel filtration

  • Confirmation of successful reconstitution by increased absorption at 395 nm

• Activity correlation:

  • Apo-enzyme typically shows minimal catalytic activity

  • Activity increases proportionally with PLP reconstitution

  • Allows for quantitative assessment of functional reconstitution

• Structural assessment:

  • Circular dichroism spectroscopy to detect secondary structure differences

  • Thermal stability assays (DSF/DSC) often show increased stability in the holo-form

  • Native gel electrophoresis may reveal mobility differences
    These approaches ensure that enzymatic characterization reflects the properties of the catalytically competent holo-enzyme.

What are effective strategies for investigating the role of P. multocida IscS in pathogenesis?

To investigate the potential role of IscS in P. multocida pathogenesis, researchers should consider these approaches:

• Gene deletion studies:

  • Construction of iscS knockout or conditional mutants

  • Phenotypic characterization under various stress conditions

  • Virulence assessment in appropriate animal models

• Transcriptomic analysis:

  • Expression profiling during infection or under host-mimicking conditions

  • Identification of co-regulated genes in sulfur metabolism pathways

  • Comparison with expression patterns of known virulence factors

• Protein interaction mapping:

  • Identification of IscS interaction partners unique to P. multocida

  • Comparative analysis with non-pathogenic bacteria

  • Functional characterization of pathogen-specific interactions

• Infection model studies:

  • Testing iscS mutants in models relevant to P. multocida infections

  • Assessment of bacterial survival and dissemination in vivo

  • Evaluation of host immune responses

• Inhibitor studies:

  • Development of specific IscS inhibitors

  • Testing effects on bacterial growth and virulence

  • Potential therapeutic applications in P. multocida infections like those described in clinical cases
    These approaches would help establish whether IscS represents a viable target for therapeutic intervention in P. multocida infections.

How might comparative analysis of cysteine desulfurases inform novel antimicrobial strategies?

Comparative analysis of cysteine desulfurases from different species offers promising avenues for antimicrobial development:

• Structural divergence exploitation:

  • Identification of pathogen-specific features in the active site architecture

  • Design of selective inhibitors targeting P. multocida IscS over mammalian counterparts

  • Structure-based drug design informed by the catalytic mechanism

• Species-specific interaction targeting:

  • Mapping differences in protein-protein interactions between bacterial and mammalian systems

  • Disruption of pathogen-specific complexes required for virulence

  • Combination therapies targeting multiple components of sulfur transfer systems

• Metabolic vulnerability identification:

  • Assessment of sulfur metabolism essentiality across different bacterial species

  • Identification of compensatory mechanisms that might confer resistance

  • Design of suicide substrates that generate toxic products in pathogen-specific contexts

• Translation to clinical applications:

  • Development of narrow-spectrum antimicrobials for targeted therapy of P. multocida infections

  • Potential application in treating resistant cases of pasteurellosis

  • Relevance to severe clinical manifestations like sepsis and osteomyelitis reported in human cases
    This approach could lead to novel treatments for P. multocida infections that cause significant morbidity in both humans and animals.

What potential exists for engineering cysteine desulfurase variants with enhanced vaccine properties?

Engineering cysteine desulfurase variants specifically for vaccine applications presents several promising opportunities:

• Epitope enhancement:

  • Identification of immunodominant epitopes in P. multocida IscS

  • Surface-exposure modification of conserved epitopes

  • Integration into multicomponent vaccine formulations with other proven antigens like VacJ, PlpE, and OmpH

• Stability optimization:

  • Engineering thermostable variants for improved vaccine shelf-life

  • Reduction of PLP dependency for greater stability in formulation

  • Modification of solvent-exposed residues to enhance antigen processing

• Adjuvant compatibility:

  • Design of variants optimized for specific adjuvant systems

  • Testing with water-in-oil adjuvants proven effective in P. multocida vaccine studies

  • Co-expression with immunostimulatory molecules

• Efficacy testing framework:

  • Challenge studies with virulent P. multocida strains

  • Assessment of protection levels compared to existing vaccines

  • Evaluation of cross-protection against multiple serotypes

• Delivery system integration:

  • Adaptation for various delivery platforms (subunit, DNA, viral vector)

  • Optimization for mucosal immunity induction

  • Formulation for single-dose effectiveness These approaches could lead to next-generation vaccines against P. multocida with broader protection and improved efficacy in both veterinary and human applications.