Recombinant Photobacterium profundum Cysteine desulfurase (iscS)

What is Photobacterium profundum IscS and what is its primary function?

Photobacterium profundum cysteine desulfurase (IscS) is a pyridoxal 5′-phosphate (PLP)-dependent homodimeric enzyme that catalyzes the conversion of L-cysteine into L-alanine and sulfur. Like other bacterial IscS proteins, it plays a critical role in transferring sulfur from L-cysteine to numerous cellular pathways, particularly in the biosynthesis of iron-sulfur clusters and other sulfur-containing cofactors . P. profundum is a deep-sea bacterium that lives under high-pressure conditions, making its IscS particularly interesting for studying adaptations to extreme environments.

The catalytic mechanism involves the formation of a PLP-substrate complex followed by several intermediates, including Cys-aldimine, Cys-ketimine, Cys-quinonoid, Ala-ketimine, and Ala-aldimine, culminating in the formation of a persulfide group on a conserved cysteine residue. This persulfide serves as the sulfur donor for downstream biosynthetic pathways .

How does the PLP cofactor interact with P. profundum IscS?

In P. profundum IscS, as in other cysteine desulfurases, the PLP cofactor is anchored in the active site pocket through several key interactions:

• Formation of an internal aldimine Schiff base with a conserved lysine residue (equivalent to Lys206 in E. coli IscS)

• Hydrogen bonding between the phenolate oxygen of PLP and a conserved glutamine residue (equivalent to Gln183 in E. coli)

• Hydrogen bonding between the pyridine N1 of PLP and a conserved aspartate residue (equivalent to Asp180 in E. coli)

• Additional polar and nonpolar interactions that stabilize the cofactor in the active site pocket

These interactions are critical for proper positioning of the PLP cofactor and ensuring catalytic efficiency. When purified, P. profundum IscS typically displays a characteristic yellow color due to the presence of the PLP cofactor, with a distinctive absorption peak at approximately 395 nm in UV-visible spectroscopy .

What conserved active site residues are essential for P. profundum IscS function?

Based on homology to well-characterized bacterial IscS proteins, P. profundum IscS contains several critical active site residues:

Mutation of these conserved residues typically results in altered spectroscopic properties and significantly reduced or abolished enzymatic activity .

What are the optimal conditions for expressing recombinant P. profundum IscS?

Successful expression of active recombinant P. profundum IscS requires specific conditions:

• Expression System: E. coli BL21(DE3) or similar strains are recommended due to their reduced protease activity and efficient T7 RNA polymerase expression

• Expression Vector: pET series vectors with a His-tag or SUMO fusion for purification

• Temperature: Lower induction temperatures (16-20°C) are preferred to enhance proper folding

• Induction: IPTG concentration of 0.1-0.5 mM is typically sufficient

• Media Supplementation: Adding 50-100 μM PLP to the media during expression ensures proper cofactor incorporation

• Growth Phase: Induce at mid-log phase (OD600 ~0.6-0.8)

For P. profundum proteins, which naturally exist under high-pressure conditions, expression may benefit from specialized equipment that can maintain pressure during growth, though standard atmospheric pressure conditions can still yield functional protein .

How can site-directed mutagenesis be used to study P. profundum IscS catalytic mechanisms?

Site-directed mutagenesis is a powerful approach for investigating the catalytic mechanism of P. profundum IscS:

• Target residues based on sequence alignment with other well-characterized cysteine desulfurases (E. coli IscS, human NFS1)

• Design mutations that:

  • Replace catalytic residues with nonreactive counterparts (H→Q, C→S, K→A)

  • Alter hydrogen bonding networks (D→G, Q→E)

  • Modify surface charge distribution (R→K)

• Characterize mutants by:

  • UV-visible spectroscopy to observe shifts in PLP absorption (typically at 395 nm in wild-type)

  • Activity assays using methods like Siegel's sulfide detection

  • Analyzing the appearance of new absorption peaks corresponding to reaction intermediates

For example, mutagenesis studies of E. coli IscS revealed that mutations H104Q, Q183E, and K206A generate new absorption peaks at 340 nm and 350 nm, potentially corresponding to Cys-ketimine and Cys-aldimine intermediates, respectively .

What spectroscopic techniques can identify enzymatic intermediates in P. profundum IscS reactions?

Several spectroscopic techniques are valuable for characterizing P. profundum IscS reaction intermediates:

• UV-Visible Spectroscopy:

  • Wild-type enzyme: ~395 nm (PLP internal aldimine)

  • Cys-ketimine intermediate: ~340 nm

  • Cys-aldimine intermediate: ~350 nm

  • Cys-quinonoid intermediate: ~510 nm

  • Ala-ketimine intermediate: ~325 nm

  • Ala-aldimine intermediate: ~345 nm

• Stopped-Flow Spectroscopy:

  • Captures rapid changes in absorption spectra during catalysis

  • Enables determination of individual rate constants for intermediate formation and decay

• Resonance Raman Spectroscopy:

  • Identifies vibrational modes of PLP-intermediate complexes

  • Distinguishes between different protonation states

• Mass Spectrometry:

  • Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) can identify covalent intermediates

  • Particularly useful for detecting persulfide formation on the catalytic cysteine

How does high pressure adaptation affect P. profundum IscS structure and function?

P. profundum is a piezophilic (pressure-loving) bacterium that thrives under high hydrostatic pressure in deep-sea environments. This adaptation likely influences its IscS enzyme:

• Structural Adaptations:

  • Increased flexibility in loop regions

  • Modified amino acid composition with more charged residues on the protein surface

  • Potentially altered oligomerization interfaces to maintain proper quaternary structure under pressure

• Functional Implications:

  • May exhibit broader substrate specificity compared to shallow-water bacterial homologs

  • Potentially different optimal temperature and pH profiles

  • Could display pressure-dependent changes in catalytic efficiency

• Experimental Approaches to Study Pressure Effects:

  • High-pressure enzyme activity assays using specialized equipment

  • Comparative analysis of kinetic parameters at different pressures

  • Molecular dynamics simulations to predict pressure effects on protein structure

Researchers studying P. profundum IscS should consider these adaptations when designing experiments and interpreting results in the context of the organism's natural high-pressure environment.

What are the preferred methods for purifying recombinant P. profundum IscS?

Purification of recombinant P. profundum IscS typically follows a multi-step protocol:

• Affinity Chromatography:

  • For His-tagged constructs: Ni-NTA or TALON resin

  • For SUMO-tagged constructs: Ni-NTA followed by SUMO protease digestion and reverse purification

• Ion Exchange Chromatography:

  • Anion exchange (Q-Sepharose) at pH 8.0, as the protein's pI is typically acidic

  • Gradual salt gradient (0-500 mM NaCl) for optimal separation

• Size Exclusion Chromatography:

  • Final polishing step using Superdex 200 or similar matrix

  • Buffer typically contains 20 mM Tris-HCl pH 8.0, 500 mM NaCl

• Buffer Considerations:

  • Maintain 20-50 μM PLP in all buffers to minimize cofactor loss

  • Include reducing agent (1-5 mM DTT or TCEP) to prevent oxidation of the catalytic cysteine

  • Consider adding 5-10% glycerol for long-term storage stability

What assays can be used to measure P. profundum IscS enzymatic activity?

Several complementary methods can assess P. profundum IscS activity:

When interpreting activity data, researchers should consider that different assays may yield different apparent activity values due to their specific detection mechanisms .

How can researchers troubleshoot poor expression or activity of recombinant P. profundum IscS?

When facing challenges with P. profundum IscS expression or activity, consider the following troubleshooting approaches:

• Poor Expression:

  • Optimize codon usage for E. coli expression

  • Try different fusion tags (His, GST, SUMO, MBP)

  • Test expression in different E. coli strains (BL21, Rosetta, Arctic Express)

  • Reduce induction temperature to 16-18°C

  • Use auto-induction media instead of IPTG induction

• Inclusion Body Formation:

  • Express as fusion with solubility-enhancing partners (SUMO, MBP)

  • Add low concentrations of non-denaturing detergents to lysis buffer

  • Consider refolding protocols if necessary

• Low Enzymatic Activity:

  • Ensure sufficient PLP incorporation by adding PLP during expression and purification

  • Check for oxidation of the catalytic cysteine (add reducing agents)

  • Verify protein folding using circular dichroism or fluorescence spectroscopy

  • Assess oligomerization state using size exclusion chromatography

• Unstable Protein:

  • Optimize buffer conditions (pH, salt concentration)

  • Add stabilizing agents (glycerol, trehalose)

  • Test different storage conditions (4°C, -20°C, -80°C, flash freezing)

  • Consider chemical chaperones during expression (osmolytes, arginine)

How can researchers investigate P. profundum IscS protein-protein interactions?

Studying the interaction network of P. profundum IscS is crucial for understanding its physiological role:

• Pull-Down Assays:

  • Use affinity-tagged IscS as bait to capture interacting partners

  • Analyze recovered proteins by mass spectrometry

  • Validate specific interactions with co-immunoprecipitation

• Surface Plasmon Resonance (SPR):

  • Immobilize IscS or potential partners on sensor chips

  • Determine binding kinetics and affinities (ka, kd, KD)

  • Test effects of mutations on binding properties

• Isothermal Titration Calorimetry (ITC):

  • Measure thermodynamic parameters of binding (ΔH, ΔS, ΔG)

  • Determine stoichiometry of complex formation

  • Works well with purified components in solution

• Bacterial Two-Hybrid System:

  • Screen for novel interaction partners in vivo

  • Confirm interactions detected by other methods

• Crosslinking Mass Spectrometry:

  • Use chemical crosslinkers to capture transient interactions

  • Identify crosslinked peptides to map interaction interfaces

  • Provides structural information about the complex

Expected interaction partners include IscU (scaffold protein for Fe-S cluster assembly), IscA (alternative scaffold or iron donor), and other components of the iron-sulfur cluster biosynthesis machinery .

How does P. profundum IscS contribute to cellular stress responses?

P. profundum IscS likely plays important roles in bacterial stress responses, particularly under conditions relevant to deep-sea environments:

• Oxidative Stress:

  • Iron-sulfur clusters are highly sensitive to oxidative damage

  • IscS activity increases during oxidative stress to repair damaged Fe-S clusters

  • May interact with redox-sensing transcription factors

• Iron Limitation:

  • Previous studies with E. coli IscS showed that iron depletion leads to accumulation of "red IscS"

  • This form has an additional absorption peak at approximately 528 nm

  • May represent a regulatory mechanism to manage iron-sulfur cluster biosynthesis during iron starvation

• Pressure Stress:

  • P. profundum experiences varying hydrostatic pressures in its native environment

  • IscS activity may be modulated by pressure changes

  • Could serve as a pressure-sensing component in cellular adaptation mechanisms

• Cold Adaptation:

  • Deep-sea environments are typically cold

  • P. profundum IscS likely possesses adaptations for activity at low temperatures

  • May show distinct temperature-activity profiles compared to mesophilic homologs

What approaches can resolve discrepancies between in vitro and in vivo studies of P. profundum IscS?

When in vitro biochemical data conflict with in vivo functional studies of P. profundum IscS, consider these approaches to resolve discrepancies:

• Protein Expression Validation:

  • Confirm protein levels and folding state in vivo using antibodies or tagged constructs

  • Verify subcellular localization matches expectations

• Environmental Factors:

  • Recreate physiologically relevant conditions in vitro:

    • Adjust pH, salt concentration, and temperature

    • Include molecular crowding agents (PEG, Ficoll)

    • Consider applying hydrostatic pressure to mimic deep-sea conditions

• Complementation Experiments:

  • Test if P. profundum IscS can complement growth defects in E. coli iscS mutants

  • Compare activity of chimeric constructs (as done with E. coli IscS and human NFS1)

  • Assess restoration of specific cellular functions (e.g., NADH dehydrogenase I activity)

• Multi-Protein Complex Reconstitution:

  • In vivo activity may depend on protein partners absent in vitro

  • Reconstitute minimal functional complexes with key interaction partners

  • Test activity in the presence of potential regulatory factors

• In-Cell Assays:

  • Develop assays to measure IscS activity directly in living cells

  • Use genetic approaches (suppressor screens, synthetic lethality) to identify functional interactions

How can structural biology advance our understanding of P. profundum IscS?

Structural studies would significantly enhance our understanding of P. profundum IscS:

• X-ray Crystallography:

  • Determine high-resolution structures of:

    • P. profundum IscS with bound PLP

    • Enzyme-substrate complexes

    • Catalytic intermediates trapped by mutation or substrate analogs

  • Compare with structures from non-piezophilic organisms to identify pressure adaptations

• Cryo-Electron Microscopy:

  • Visualize IscS in complex with partner proteins

  • Study conformational changes during the catalytic cycle

  • May be particularly valuable for larger complexes

• Nuclear Magnetic Resonance (NMR):

  • Analyze protein dynamics during catalysis

  • Study changes in protein structure under varying pressure conditions

  • Identify residues involved in substrate binding and catalysis

• Molecular Dynamics Simulations:

  • Model effects of high pressure on protein structure and dynamics

  • Predict conformational changes during catalysis

  • Design mutations to test computational predictions experimentally

What role might P. profundum IscS play in novel biotechnological applications?

The unique properties of P. profundum IscS offer several potential biotechnological applications:

• Biocatalysis:

  • Development of pressure-stable enzyme systems for industrial processes

  • Engineering IscS for production of sulfur-containing compounds

  • Creation of chimeric enzymes with enhanced catalytic properties

• Biosensors:

  • Using the spectroscopic properties of IscS (particularly "red IscS") as biosensors for iron availability or oxidative stress

  • Developing high-pressure biological sensing systems

• Synthetic Biology:

  • Engineering pressure-responsive cellular pathways using components from P. profundum

  • Creating microorganisms with enhanced ability to function under high pressure

  • Designing novel sulfur metabolism pathways for bioremediation or chemical production

• Structural Biology Tools:

  • Using piezophilic proteins as models for studying pressure effects on protein structure

  • Developing pressure-stabilized protein scaffolds for biotechnology applications