Recombinant Edwardsiella ictaluri Ferrochelatase (hemH)

What is Edwardsiella ictaluri Ferrochelatase (hemH) and what is its function?

Ferrochelatase (hemH) catalyzes the insertion of Fe²⁺ into protoporphyrin IX to generate protoheme, representing the terminal step in the heme biosynthesis pathway . In Edwardsiella ictaluri, a host-restricted pathogen of channel catfish (Ictalurus punctatus), ferrochelatase plays a crucial role in the bacterium's ability to acquire and utilize iron, which is essential for its pathogenesis and survival within the host . The enzyme is part of a sophisticated iron uptake system that is typically regulated by the ferric uptake regulator (Fur) protein, allowing the bacterium to overcome iron limitation imposed by the host during infection .

How does the Fur protein regulate hemH expression in E. ictaluri?

The Fur (ferric uptake regulator) protein in E. ictaluri tightly controls the expression of genes involved in iron uptake, including those related to heme utilization . Under iron-replete conditions, Fur binds to specific sequences in the promoter regions of target genes, repressing their transcription. When iron becomes limited, Fur dissociates from these sequences, allowing transcription to proceed.

Interestingly, the E. ictaluri Fur protein lacks the N-terminal region found in the majority of pathogen-encoded Fur proteins. Despite this structural difference, it remains fully functional in regulating genes encoding iron uptake proteins, including those involved in the heme uptake system . This regulation is evidenced by the observation that under iron-limited conditions, E. ictaluri upregulates an outer membrane protein (HemR) that shows heme-hemoglobin transport activity and is tightly regulated by Fur .

What is the relationship between E. ictaluri ferrochelatase and bacterial pathogenesis?

Ferrochelatase activity is critical for E. ictaluri virulence as it enables the bacterium to synthesize heme, which is essential for various cellular processes including energy production and defense against oxidative stress. Research has demonstrated that disruption of iron acquisition pathways, which are interconnected with heme metabolism, significantly impacts bacterial virulence .

In vivo studies have shown that an E. ictaluri Δfur mutant (with disrupted iron regulation) is attenuated and immune protective in both zebrafish (Danio rerio) and catfish (Ictalurus punctatus), triggering systemic immunity . This indicates that proper regulation of iron and heme metabolism, in which ferrochelatase plays a key role, is essential for full virulence of the pathogen. The attenuation of virulence in fur mutants has led researchers to suggest that an E. ictaluri Δfur mutant could serve as an effective component of an immersion-oral vaccine for the catfish industry .

What are the optimal expression conditions for recombinant E. ictaluri Ferrochelatase?

Optimal expression of recombinant E. ictaluri ferrochelatase requires careful consideration of several factors:

• Expression System Selection: While the search results do not specify conditions specific to E. ictaluri ferrochelatase, studies with other bacterial ferrochelatases suggest that E. coli expression systems such as BL21(DE3) are commonly used .

• Vector Design: Vectors containing appropriate promoters (T7 or similar inducible promoters) and affinity tags (His-tag) facilitate controlled expression and subsequent purification .

• Induction Parameters: Optimization of IPTG concentration, induction temperature, and duration is critical. Lower temperatures (16-25°C) often improve the solubility of recombinant ferrochelatase.

• Iron Supplementation: Since ferrochelatase catalyzes the insertion of Fe²⁺ into protoporphyrin IX, ensuring adequate iron availability in the growth medium may improve enzyme activity.

• Co-expression Strategy: As demonstrated with other heme-binding proteins, co-expression of ferrochelatase with the protein of interest can significantly improve heme incorporation and functional protein yield .

How can co-expression of ferrochelatase improve heme incorporation in recombinant proteins?

Co-expression of ferrochelatase represents a strategic approach to enhance heme incorporation in recombinant heme-binding proteins. Studies have demonstrated that overexpression of heme-binding proteins in E. coli often results in sub-optimal heme incorporation, with the amount of heme-bound protein varying considerably depending on the specific protein .

The mechanism behind this improvement involves:

• Enhanced Conversion of Porphyrin to Heme: Recombinant proteins expressed in E. coli often contain less than a full complement of heme because they are partially incorporated with free-base porphyrin instead. Co-expressed ferrochelatase catalyzes the insertion of iron into the accumulated porphyrin, increasing the available heme pool .

• Timing of Heme Availability: Co-expression ensures that heme synthesis occurs concurrently with target protein production, optimizing the chances for proper incorporation.

• Effectiveness Across Protein Types: This method has been shown to be effective for proteins containing either Cysteine-ligated or Histidine-ligated hemes, demonstrating its broad applicability .

This approach provides a straightforward and inexpensive solution to the problem of incomplete heme incorporation, which is crucial for biochemical characterization, spectroscopy, structural studies, and the production of homogeneous proteins with high activity .

What are the kinetic parameters of recombinant bacterial ferrochelatases and how do they compare?

While specific kinetic parameters for E. ictaluri ferrochelatase are not provided in the search results, we can examine data from related bacterial ferrochelatases for comparison. For example, a cucumber ferrochelatase with similar function showed the following kinetic parameters:

• Optimum pH: 7.7

• Apparent Km values:

  • For deuteroporphyrin IX: 14.4 μM

  • For Fe²⁺: 4.7 μM

The activity was inhibited by N-methylprotoporphyrin IX with an I50 value of 4 nM .

For E. ictaluri ferrochelatase, researchers would typically determine similar parameters through enzymatic assays. Commonly measured parameters include:

• Km values for both porphyrin substrate and iron

• Vmax (maximum reaction velocity)

• kcat (turnover number)

• kcat/Km (catalytic efficiency)

• Inhibition constants for various inhibitors

These parameters can vary significantly between bacterial species based on structural differences in the enzyme and the specific physiological roles they play in their respective organisms.

How does mutation of the fur gene affect E. ictaluri virulence and hemH regulation?

Mutation of the fur gene in E. ictaluri has profound effects on both virulence and iron uptake regulation:

• Attenuated Virulence: In vivo studies have shown that an E. ictaluri Δfur mutant is attenuated in zebrafish (Danio rerio) and catfish (Ictalurus punctatus) . This attenuation is likely due to disruptions in multiple cellular processes regulated by Fur, including iron acquisition and utilization.

• Dysregulation of Iron Uptake Genes: Without functional Fur protein, genes involved in iron uptake and heme utilization become constitutively expressed rather than being regulated in response to iron availability . This likely includes the ferrochelatase gene and associated heme biosynthesis pathways.

• Immune Protection: Despite its attenuated virulence, the Δfur mutant triggers systemic immunity in fish hosts, suggesting its potential use as a live attenuated vaccine . This immune protection indicates that the mutant still expresses important antigenic determinants despite its reduced pathogenicity.

• HemR Expression: The Δfur mutation leads to upregulation of HemR, an outer membrane protein involved in heme-hemoglobin transport, even under iron-replete conditions where it would normally be repressed . This demonstrates the direct regulatory link between Fur and components of the heme utilization pathway in which ferrochelatase participates.

What expression systems are most suitable for the production of functional recombinant E. ictaluri Ferrochelatase?

Based on the information available and general principles of recombinant protein expression, several expression systems can be considered for E. ictaluri ferrochelatase:

• E. coli Expression Systems:

  • BL21(DE3) strain with T7 promoter-based vectors is commonly used for recombinant ferrochelatase expression

  • pET vector systems offer tight control of expression through IPTG induction

  • For co-expression strategies, compatible vectors such as pACYCduet can be employed

• Expression Parameters:

  • Temperature: Lower induction temperatures (16-20°C) often improve solubility

  • Induction time: Extended induction periods (overnight) at lower temperatures

  • Media supplements: Addition of δ-aminolevulinic acid (ALA) as a heme precursor and iron supplements

• Fusion Tags:

  • N-terminal His6-tag for affinity purification

  • Cleavable tags with TEV or thrombin protease sites if tag-free protein is required

• Co-expression Strategy:

  • As demonstrated for other heme proteins, co-expression with ferrochelatase improves heme incorporation

  • This can be achieved using dual-plasmid systems with compatible origins of replication and different antibiotic resistance markers

The exact methodology would involve cloning the E. ictaluri hemH gene into an appropriate vector, transforming into an expression host, optimizing induction conditions, and developing a purification strategy that maintains enzyme activity.

How can one design experiments to study the regulatory relationship between Fur and hemH in E. ictaluri?

To investigate the regulatory relationship between Fur and hemH in E. ictaluri, researchers can employ several experimental approaches:

• Promoter Analysis Studies:

  • Identify putative Fur-binding sites in the hemH promoter region using bioinformatic approaches

  • Perform electrophoretic mobility shift assays (EMSA) with purified Fur protein and labeled hemH promoter fragments

  • Create reporter gene fusions (e.g., hemH promoter-lacZ) to quantitatively measure expression under varying iron conditions

• Gene Expression Analysis:

  • Compare hemH transcript levels in wild-type vs. Δfur mutant strains using qRT-PCR

  • Conduct RNA-seq analysis to identify all genes co-regulated with hemH in response to iron limitation and Fur mutation

  • Perform Northern blot analysis to determine if hemH expression is light-responsive or tissue-specific, as observed in other organisms

• Protein Expression Studies:

  • Use Western blot analysis with antibodies against ferrochelatase to compare protein levels under different conditions

  • Develop a ferrochelatase activity assay to correlate transcriptional changes with functional enzyme levels

• Complementation Studies:

  • Construct plasmids containing the fur gene with its own promoter (as done in the study with pEZ136 and pEZ191)

  • Transform these plasmids into Δfur mutants to determine if normal regulation of hemH is restored

• Iron Limitation Experiments:

  • Culture bacteria under iron-replete and iron-limited conditions (using iron chelators)

  • Compare hemH expression and ferrochelatase activity between these conditions in both wild-type and Δfur backgrounds

These approaches would provide comprehensive insights into how Fur regulates hemH expression in E. ictaluri and how this regulation contributes to the bacterium's iron homeostasis and virulence.

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

Several established methods can be employed to assess the enzymatic activity of recombinant E. ictaluri ferrochelatase:

• Spectrophotometric Assays:

  • Measure the decrease in absorbance of the porphyrin substrate (typically at around 380-420 nm)

  • Monitor the appearance of metalloporphyrin product (around 400-450 nm)

  • These assays can be performed in plate reader format for high-throughput screening

• Fluorescence-Based Assays:

  • Utilize the fact that metallation of porphyrins results in fluorescence quenching

  • Measure the decrease in fluorescence as ferrochelatase catalyzes iron insertion into the porphyrin substrate

• HPLC Analysis:

  • Separate and quantify substrate and product using reverse-phase HPLC

  • This method allows for precise quantification and detection of side products

• Optimizing Assay Conditions:

  • pH optimization: Determine optimal buffer pH (cucumber ferrochelatase showed optimal activity at pH 7.7)

  • Temperature dependence: Establish the temperature profile for enzymatic activity

  • Metal ion specificity: Test various metal ions as substrates (Fe²⁺, Zn²⁺, Co²⁺, etc.)

  • Substrate kinetics: Determine Km and Vmax values for both porphyrin and metal ion substrates

• Inhibition Studies:

  • Test known ferrochelatase inhibitors such as N-methylprotoporphyrin IX (reported I50 = 4 nM for cucumber ferrochelatase)

  • Determine inhibition constants and mechanisms (competitive, non-competitive, etc.)

These methods, when combined, provide a comprehensive assessment of ferrochelatase activity and allow for comparison with enzymes from other organisms.

How should researchers interpret discrepancies in ferrochelatase activity across different experimental conditions?

When analyzing ferrochelatase activity data, researchers should consider several factors that might explain variability:

• Enzyme Structural Integrity:

  • Incomplete heme incorporation affects structural integrity and catalytic efficiency

  • Presence of free-base porphyrin instead of heme can lead to misleading spectral characteristics that are difficult to detect even in purified samples

• Expression System Variations:

  • Different expression hosts may produce enzymes with varying post-translational modifications

  • Co-expression with ferrochelatase significantly improves heme incorporation in recombinant proteins, which may explain activity differences across expression systems

• Assay Condition Variables:

  • pH dependency (optimal pH reported as 7.7 for cucumber ferrochelatase)

  • Metal ion availability and speciation

  • Reducing environment and oxygen sensitivity

  • Buffer composition effects on enzyme stability

• Data Normalization Approaches:

  • Normalize activity to protein concentration determined by multiple methods

  • Use internal standards to account for day-to-day variations

  • Apply appropriate statistical tests to determine significance of observed differences

• Control Experiments:

  • Include positive controls (known active ferrochelatase) and negative controls

  • Perform inhibition studies with established inhibitors like N-methylprotoporphyrin IX

  • Test activity with multiple substrate analogs to confirm enzyme specificity

By systematically analyzing these factors, researchers can identify the source of discrepancies and determine whether they represent meaningful biological differences or technical artifacts.

What is the potential for E. ictaluri ferrochelatase in vaccine development?

While ferrochelatase itself has not been directly implicated as a vaccine target in the search results, the information about fur mutants provides valuable insights into potential vaccine strategies:

• Attenuated Live Vaccines:

  • E. ictaluri Δfur mutants are attenuated yet immune protective in zebrafish and catfish

  • These mutants could serve as effective components of immersion-oral vaccines for the catfish industry

  • The disruption of iron metabolism pathways (including heme biosynthesis) contributes to this attenuation

• Recombinant Subunit Vaccines:

  • Proteins involved in iron/heme acquisition are often immunogenic

  • HemR, an outer membrane protein involved in heme transport and regulated by Fur, could be a potential vaccine antigen

  • Understanding ferrochelatase's role in the heme utilization pathway could identify additional vaccine targets

• Experimental Approaches to Vaccine Development:

  • Comparative immunization studies using wild-type, Δfur, and ΔhemH strains

  • Analysis of immune responses to purified recombinant HemR and other components of the heme uptake system

  • In vivo challenge studies in fish models to determine protective efficacy

• Delivery Systems:

  • Immersion-oral vaccine delivery approaches appear particularly relevant for fish pathogens like E. ictaluri

  • Live attenuated strains with mutations in fur or related genes could serve as delivery vehicles for heterologous antigens

These applications highlight the importance of understanding the molecular biology of E. ictaluri's iron/heme acquisition systems, including ferrochelatase, for developing effective control strategies against this economically important fish pathogen.

How does the tissue localization of ferrochelatase influence experimental design?

Understanding the tissue localization of ferrochelatase is crucial for experimental design, as demonstrated in studies of other organisms:

• Differential Expression Patterns:

  • In cucumber, ferrochelatase expression varied significantly between tissues, with the hemH gene expressed mainly in hypocotyls and roots but little in cotyledons

  • The level of hemH transcripts was not light-responsive, suggesting tissue-specific regulation

• Implications for E. ictaluri Research:

  • Experiments should examine ferrochelatase expression across different growth conditions that might mimic various host environments

  • Tissue-specific expression in the pathogen during infection might reveal important regulatory mechanisms

• Subcellular Localization Considerations:

  • In plants, ferrochelatase activity was mainly associated with thylakoid membranes of chloroplasts

  • For bacterial ferrochelatase, determining membrane association or cytosolic localization would inform purification strategies

• Experimental Approaches:

  • Western blot analysis with specific antibodies to detect the enzyme in different fractions

  • Immunolocalization studies in bacterial cells under various growth conditions

  • Construction of reporter gene fusions to monitor expression patterns in vivo

  • Subcellular fractionation to determine the enzyme's location within the bacterial cell

• Functional Redundancy:

  • Evidence from cucumber suggests the presence of multiple types of ferrochelatase with different localizations

  • Researchers should investigate whether E. ictaluri possesses multiple ferrochelatase isoforms with distinct functions or locations

These considerations highlight the importance of understanding both the expression patterns and subcellular localization of ferrochelatase when designing experiments to study its function in E. ictaluri.

How does E. ictaluri Ferrochelatase compare to ferrochelatases from other bacterial species?

A comparative analysis of E. ictaluri ferrochelatase with those from other species reveals important differences and similarities:

• Structural Comparisons:

  Species	Molecular Weight	Cofactors	Metal Specificity	Special Features
  E. ictaluri	Not specified in results	Fe²⁺	Fe²⁺ → heme	Regulated by Fur
  Cucumber	40 kDa	Fe²⁺	Fe²⁺ → protoheme	Tissue-specific expression
  E. coli	Not specified in results	Fe²⁺	Fe²⁺ → heme	Used in co-expression systems

• Regulatory Mechanisms:

  • E. ictaluri ferrochelatase is likely regulated by the Fur protein, which controls iron uptake genes

  • Unlike many photosynthetic organisms, the E. ictaluri homolog would not be expected to show light-responsive regulation

• Functional Context:

  • E. ictaluri ferrochelatase functions within a heme-hemoglobin uptake system that is important for iron acquisition during infection

  • This contrasts with cucumber ferrochelatase, which functions in non-photosynthetic tissues for heme biosynthesis

• Gene Structure:

  • The E. ictaluri Fur protein, which regulates hemH, lacks the N-terminal region found in most pathogen-encoded Fur proteins

  • This structural difference doesn't impair function but represents an interesting evolutionary adaptation

Understanding these comparisons helps researchers contextualize their findings and design experiments that account for the unique features of E. ictaluri ferrochelatase.

What innovations in ferrochelatase research could advance our understanding of E. ictaluri pathogenesis?

Several innovative approaches could significantly advance our understanding of E. ictaluri ferrochelatase and its role in pathogenesis:

• Structural Biology Approaches:

  • X-ray crystallography or cryo-EM studies of E. ictaluri ferrochelatase to reveal unique structural features

  • Structure-guided drug design targeting ferrochelatase or related enzymes in the heme biosynthesis pathway

• Systems Biology Integration:

  • Multi-omics approaches combining transcriptomics, proteomics, and metabolomics to understand the broader context of ferrochelatase function

  • Network analysis to identify all interacting partners and regulatory connections

• Advanced Genetic Tools:

  • CRISPR-Cas9 technology for precise genome editing in E. ictaluri

  • Inducible gene expression systems to study the effects of ferrochelatase levels on pathogenesis

• Host-Pathogen Interaction Studies:

  • Real-time imaging of iron/heme acquisition during infection using fluorescent reporters

  • Dual RNA-seq to simultaneously monitor host and pathogen responses related to iron/heme metabolism

• Translational Applications:

  • Development of ferrochelatase inhibitors as potential antimicrobials

  • Engineering of attenuated strains with modified heme metabolism as live vaccines

  • Design of diagnostic tools based on unique aspects of E. ictaluri iron metabolism

These innovative approaches would not only advance our understanding of E. ictaluri ferrochelatase but could also lead to practical applications in controlling infections caused by this important fish pathogen.