Recombinant Aliivibrio salmonicida Ferrochelatase (hemH)

What is Aliivibrio salmonicida Ferrochelatase and what is its biological role?

Aliivibrio salmonicida (formerly known as Vibrio salmonicida) is a psychrophilic bacterium that causes cold-water vibriosis, a fatal bacterial septicemia primarily affecting farmed salmonid fish . Ferrochelatase (hemH) catalyzes the terminal step of heme biosynthesis - the insertion of ferrous iron into protoporphyrin IX to produce heme . In proteobacteria like A. salmonicida, the protoporphyrin-dependent pathway is utilized for heme biosynthesis, unlike some Gram-positive bacteria that employ a coproporphyrin-dependent pathway . This enzyme is critical for producing heme-containing proteins essential for respiration and other cellular processes.

How does Ferrochelatase structure relate to its function?

Bacterial ferrochelatases typically consist of two Rossmann-type domains that interact to form the active site . The enzyme contains a characteristic π helix with several conserved residues critical for catalysis . Based on structural studies of other bacterial ferrochelatases, A. salmonicida ferrochelatase likely shares structural similarity with B. subtilis ferrochelatase, functioning as a monomer rather than the dimeric structure seen in eukaryotic ferrochelatases . The active site architecture creates a distorted binding pocket for the porphyrin substrate, facilitating metal insertion through precise positioning of conserved catalytic residues.

What is the relationship between iron regulation and ferrochelatase in A. salmonicida?

In A. salmonicida, the Ferric uptake regulator (Fur) functions as a global transcription factor that regulates bacterial gene expression in an iron-dependent manner . Electrophoretic mobility shift assays have demonstrated that A. salmonicida Fur (AS-Fur) binds to the vibrio Fur box consensus and multiple promoter regions containing Fur boxes . While direct regulation of ferrochelatase by Fur in A. salmonicida hasn't been explicitly demonstrated in the provided search results, the connection between iron availability sensing (via Fur) and iron utilization (via ferrochelatase) represents an important area for investigation in understanding the bacterium's iron homeostasis and virulence mechanisms.

What expression systems are most effective for producing recombinant A. salmonicida Ferrochelatase?

When expressing recombinant A. salmonicida ferrochelatase, researchers should consider several factors specific to this psychrophilic enzyme:

• Temperature optimization: Lower expression temperatures (15-20°C) should be employed to maintain proper folding of this cold-adapted enzyme

• Expression vectors: pET-based systems with T7 promoters under IPTG control have been successful for similar bacterial ferrochelatases

• Host selection: E. coli BL21(DE3) strains or derivatives lacking endogenous proteases are recommended

• Induction conditions: Gentle induction with lower IPTG concentrations (0.1-0.5 mM) and extended expression times (overnight) at reduced temperatures

• Buffer composition: Including reducing agents to prevent oxidation of iron-binding sites is essential

The psychrophilic nature of A. salmonicida demands modifications to standard protocols to ensure proper folding and activity of the recombinant enzyme .

How can researchers accurately measure Ferrochelatase activity in vitro?

Ferrochelatase activity can be measured through several complementary approaches:

• Spectrofluorometric assay: Monitor the decrease in protoporphyrin IX fluorescence (excitation 410 nm, emission 630 nm) as it's converted to non-fluorescent heme

• Zinc substitution assay: Using Zn²⁺ instead of Fe²⁺ to avoid oxidation issues, measuring the formation of zinc-protoporphyrin (which is fluorescent)

• HPLC analysis: Separating substrate and product to quantify conversion rates

• Coupled enzyme assays: When measuring activity in complex mixtures

For A. salmonicida ferrochelatase specifically, assays should be conducted at lower temperatures (10-15°C) to mimic the natural conditions of this psychrophilic bacterium and maximize enzymatic activity .

What purification strategies yield the highest purity and activity for recombinant A. salmonicida Ferrochelatase?

Optimal purification of recombinant A. salmonicida ferrochelatase typically involves:

• Affinity chromatography: His-tagged constructs purified via Ni-NTA or similar matrices

• Ion exchange chromatography: Separating based on charge properties, often using DEAE or Q-Sepharose

• Size exclusion chromatography: Final polishing step to achieve high purity

Critical considerations include:

• Maintaining reducing conditions throughout purification (typically 1-5 mM DTT or β-mercaptoethanol)

• Using buffer systems that maintain stability at lower temperatures

• Including glycerol (10-20%) to enhance stability

• Avoiding metal chelators like EDTA that might strip essential metals

• Working quickly and keeping the protein cold throughout the process

These approaches maximize both purity and specific activity of the enzyme .

What active site features distinguish A. salmonicida Ferrochelatase from other bacterial ferrochelatases?

While specific structural information for A. salmonicida ferrochelatase is limited in the available research, comparison with other bacterial ferrochelatases suggests several key features:

• Conserved histidine and glutamate residues that coordinate the metal ion

• A distorted porphyrin binding pocket that facilitates metal insertion

• Specific residues involved in proton abstraction from the porphyrin

• Potential psychrophilic adaptations including increased flexibility around the active site

• Conservation of the characteristic π helix containing catalytically important residues

Structural modeling and molecular dynamics simulations would provide valuable insights into the specific interactions between A. salmonicida ferrochelatase and its substrates .

How does the psychrophilic nature of A. salmonicida influence Ferrochelatase properties?

The psychrophilic nature of A. salmonicida likely confers several adaptations to its ferrochelatase:

• Increased structural flexibility, particularly around the active site

• Higher catalytic efficiency at lower temperatures compared to mesophilic homologs

• Lower thermal stability as a trade-off for cold-temperature activity

• Modified substrate binding interactions that are less temperature-dependent

• Altered surface charge distribution that may enhance enzyme dynamics at low temperatures

These adaptations would enable A. salmonicida ferrochelatase to maintain sufficient catalytic activity in the cold marine environments where the bacterium naturally thrives .

What cofactors or interacting partners influence A. salmonicida Ferrochelatase activity?

Ferrochelatase function likely depends on several cofactors and interacting partners:

• Iron delivery proteins: Systems that provide ferrous iron in a bioavailable form

• Porphyrin synthesis enzymes: Potential metabolon formation with preceding enzymes in the heme biosynthesis pathway

• Regulatory proteins: Including Fur and other iron-responsive factors

• Membrane components: If membrane-associated, as in some bacterial systems

• Redox maintenance systems: To ensure the iron substrate remains in the ferrous state

In eukaryotic systems, ferrochelatase is part of a larger complex of heme biosynthesis enzymes, and similar associations might exist in A. salmonicida, although with structural differences reflecting its prokaryotic nature .

How can recombinant A. salmonicida Ferrochelatase be used to study cold-water vibriosis?

Recombinant A. salmonicida ferrochelatase provides several research applications for studying cold-water vibriosis:

• Target validation: Gene knockout/complementation studies to confirm the role of heme biosynthesis in virulence

• Drug discovery: Screening for specific inhibitors of A. salmonicida ferrochelatase as potential antimicrobial agents

• Pathogenesis studies: Understanding how iron acquisition and heme synthesis contribute to bacterial survival during infection

• Environmental adaptation: Investigating how temperature affects heme biosynthesis capacity

• Vaccine development: Exploring ferrochelatase or associated pathways as potential vaccine targets

These approaches could advance our understanding of A. salmonicida pathogenesis and potentially lead to new control strategies for cold-water vibriosis .

What advantages does A. salmonicida Ferrochelatase offer as a model for studying psychrophilic enzymes?

A. salmonicida ferrochelatase presents several advantages as a model psychrophilic enzyme:

• Biological relevance: Essential metabolic enzyme with clear phenotypic consequences when altered

• Comparative potential: Well-characterized mesophilic and thermophilic homologs available for comparison

• Structural complexity: Sufficient complexity to reveal meaningful adaptations without being too large for detailed analysis

• Catalytic mechanism: Well-defined reaction allowing precise kinetic measurements

• Environmental significance: Connection to bacterial adaptations in cold marine environments

These characteristics make it a valuable model for understanding molecular adaptations enabling enzyme function at low temperatures .

How can researchers use A. salmonicida Ferrochelatase to develop selective antimicrobial strategies?

Development of selective antimicrobial strategies using A. salmonicida ferrochelatase involves:

• Structural comparison with human ferrochelatase to identify differences that can be exploited for selective inhibition

• High-throughput screening for compounds that specifically inhibit the bacterial enzyme

• Structure-based drug design targeting unique features of A. salmonicida ferrochelatase

• Development of prodrugs activated by the bacterial enzyme but not by host ferrochelatase

• Investigation of combination approaches targeting multiple steps in bacterial heme biosynthesis

These approaches could lead to new treatments for cold-water vibriosis with minimal impact on the host fish .

How does iron delivery to Ferrochelatase occur in A. salmonicida?

The mechanism of iron delivery to ferrochelatase in A. salmonicida represents an important research question:

• Potential iron chaperones: Proteins that might specifically deliver iron to ferrochelatase

• Connection to Fur regulation: How the Fur system might coordinate iron acquisition with ferrochelatase activity

• Redox control: Mechanisms maintaining iron in the ferrous state for enzyme activity

• Compartmentalization: Potential localization of ferrochelatase near iron acquisition systems

• Adaptation to low iron environments: Strategies for maintaining heme synthesis during iron limitation

Understanding these mechanisms could reveal potential vulnerabilities in the bacterium's iron metabolism pathways.

What molecular mechanisms explain temperature adaptation in A. salmonicida Ferrochelatase?

The temperature adaptation of A. salmonicida ferrochelatase likely involves several molecular mechanisms:

• Enhanced flexibility: Reduced number of rigid structural elements and fewer stabilizing interactions

• Surface modifications: Altered surface charge distribution and exposed hydrophobic residues

• Active site adaptations: More accessible active site with fewer geometric constraints

• Loop modifications: Longer or more flexible loops connecting secondary structure elements

• Decreased arginine/proline content: Fewer stabilizing interactions in the protein core

Detailed structural and molecular dynamics studies comparing A. salmonicida ferrochelatase with mesophilic homologs would provide insights into these cold-adaptation mechanisms .

How does substrate specificity of A. salmonicida Ferrochelatase compare with other bacterial ferrochelatases?

*Values for A. salmonicida are predicted based on its psychrophilic nature and proteobacterial classification, as specific experimental data is not available in the search results.

This comparative analysis highlights the expected differences between protoporphyrin-dependent and coproporphyrin-dependent ferrochelatases, as well as potential adaptations in the psychrophilic A. salmonicida enzyme .

How does A. salmonicida Ferrochelatase research integrate with aquaculture disease management?

Research on A. salmonicida ferrochelatase has important implications for aquaculture disease management:

• Diagnostic development: Creating molecular or biochemical tests targeting ferrochelatase or its products

• Vaccine strategies: Using recombinant ferrochelatase or attenuated strains with modified ferrochelatase activity

• Treatment optimization: Developing compounds that inhibit bacterial heme synthesis

• Environmental monitoring: Understanding how environmental conditions affect bacterial virulence factors

• Integrated management: Combining insights about iron metabolism with other disease control approaches

These applications could contribute to reducing the economic impact of cold-water vibriosis in salmonid aquaculture .

What insights from human Ferrochelatase research can be applied to A. salmonicida Ferrochelatase studies?

Human ferrochelatase research provides valuable perspectives for bacterial ferrochelatase studies:

• Inhibitor design: Approaches used to develop selective inhibitors of human ferrochelatase

• Disease mechanisms: Understanding how ferrochelatase deficiency leads to pathology (porphyrias)

• Structure-function relationships: Detailed analyses of residues critical for substrate binding and catalysis

• Regulatory mechanisms: Control of enzyme activity through protein-protein interactions

• Assay technologies: Sophisticated methods for measuring enzyme activity and inhibition

While significant structural differences exist between human and bacterial ferrochelatases, these insights can inform experimental design and interpretation .

How might environmental factors influence A. salmonicida Ferrochelatase expression and activity in natural settings?

Environmental factors likely influence A. salmonicida ferrochelatase in natural marine settings:

• Temperature: Affecting enzyme activity, stability, and expression levels

• Iron availability: Modulating expression through Fur-dependent regulation

• Salinity: Potentially impacting protein folding and stability

• pH fluctuations: Altering optimal activity conditions

• Host factors: Presence of iron-binding proteins and immune responses

Understanding these interactions could help predict conditions favoring bacterial virulence and inform environmental management approaches to disease prevention .