Recombinant Mycobacterium gilvum Ferrochelatase (hemH)

What is Mycobacterium gilvum Ferrochelatase (hemH) and what is its primary function?

Mycobacterium gilvum Ferrochelatase (hemH) is an enzyme (EC 4.99.1.1) that catalyzes the insertion of ferrous iron (Fe²⁺) into protoporphyrin IX to generate protoheme, the final step in heme biosynthesis. This enzyme is also known by alternative names including Heme synthase and Protoheme ferro-lyase . The enzyme belongs to the ferrochelatase family and plays an essential role in the synthesis of heme, which serves as a critical cofactor for numerous proteins involved in electron transfer, oxygen transport, and enzymatic reactions.

What are the structural characteristics of recombinant M. gilvum Ferrochelatase?

Recombinant M. gilvum Ferrochelatase is a full-length protein comprising 340 amino acids. Its primary sequence begins with MTFDALLLLS and continues through a series of amino acid residues as documented in product specifications . While the complete three-dimensional structure of M. gilvum Ferrochelatase has not been explicitly detailed in the available search results, research on homologous ferrochelatases indicates that these enzymes typically possess a conserved active site where metal binding and porphyrin interaction occur. Studies on other ferrochelatases have shown that residues such as M76 play crucial roles in active site metal binding, while residues like E343 participate in proton abstraction and product release .

What are the optimal conditions for enzyme activity of M. gilvum Ferrochelatase?

While specific optimal conditions for M. gilvum Ferrochelatase are not directly stated in the search results, studies on similar ferrochelatases provide valuable reference points. For instance, cucumber ferrochelatase demonstrates optimal activity at pH 7.7, with apparent Km values of 14.4 μM for deuteroporphyrin IX and 4.7 μM for Fe²⁺ . This suggests that M. gilvum Ferrochelatase likely functions optimally in slightly alkaline conditions and requires micromolar concentrations of its substrates. Temperature optima and cofactor requirements would need to be experimentally determined specifically for the M. gilvum enzyme.

How does the catalytic mechanism of M. gilvum Ferrochelatase compare to other bacterial and eukaryotic ferrochelatases?

The catalytic mechanism of ferrochelatases across species follows similar principles but with important variations. Based on studies of homologous enzymes, M. gilvum Ferrochelatase likely operates through a mechanism where metal binding and insertion occur from the opposite side to where pyrrole proton abstraction takes place . Evidence from human ferrochelatase studies suggests that conserved residues form weak iron-protein ligands that are necessary for product release after catalysis.

Key differences between bacterial ferrochelatases like that from M. gilvum and eukaryotic counterparts often involve the presence of iron-sulfur clusters in some eukaryotic versions and variations in active site architecture. These differences can affect substrate specificity, catalytic efficiency, and regulation. The exact mechanistic details specific to M. gilvum Ferrochelatase would require targeted kinetic and structural studies.

What role do specific active site residues play in M. gilvum Ferrochelatase catalysis?

While specific information about M. gilvum Ferrochelatase active site residues is limited in the search results, studies on homologous ferrochelatases provide insight into likely critical residues. Research on human ferrochelatase has identified that residues analogous to M76 play crucial roles in active site metal binding . Similarly, residues corresponding to E343 appear to be involved in proton abstraction and product release.

Additionally, studies suggest the presence of a peptide loop composed of residues similar to Q302, S303, and K304 that functions as a metal sensor and coordinates with other residues during product release . These active site residues likely form a specialized environment that facilitates porphyrin distortion, metal coordination, and the precise positioning needed for efficient catalysis.

How does the M. gilvum ecological niche influence its ferrochelatase properties?

M. gilvum is an environmental rapidly growing mycobacterium found in soil and water environments, measuring approximately 1.4 ± 0.5 μm in size . This ecological niche may influence its ferrochelatase properties in several ways. As a soil organism, M. gilvum must adapt to fluctuating iron availability, potentially impacting the metal-binding properties of its ferrochelatase.

Interestingly, M. gilvum has been observed to survive within amoeba trophozoites without multiplying or killing the host . This survival strategy might relate to specialized adaptations in iron acquisition and heme synthesis pathways, including possible unique features of its ferrochelatase. The ability to maintain functional heme biosynthesis under the nutrient-limited conditions within a host cell could be crucial for its survival strategy.

What expression systems are most effective for producing recombinant M. gilvum Ferrochelatase?

Based on commercial protein production information, E. coli appears to be an effective expression system for producing recombinant M. gilvum Ferrochelatase . When expressing this enzyme, researchers should consider the following methodological aspects:

• Vector selection: Choose expression vectors with appropriate promoters (T7, tac, etc.) for controlled expression

• Fusion tags: Consider His-tags or other affinity tags to facilitate purification

• Growth conditions: Optimize temperature, induction time, and IPTG concentration to maximize soluble protein yield

• Codon optimization: Adjust codon usage to match the expression host if needed

After expression, purification typically achieves >85% purity as assessed by SDS-PAGE . The recombinant protein can be stored at -20°C for regular use, with extended storage recommended at -20°C or -80°C to maintain stability and activity.

What assays can be used to measure M. gilvum Ferrochelatase activity?

Several assay methods can be employed to measure ferrochelatase activity, which can be adapted for the M. gilvum enzyme:

Spectrophotometric Assays:
The most common approach involves monitoring the decrease in porphyrin substrate absorbance or the increase in metalloporphyrin product absorbance over time. For example, the conversion of protoporphyrin IX to protoheme can be monitored by following changes in the characteristic absorption spectra.

Fluorescence-based Assays:
Since porphyrins are highly fluorescent while metalloporphyrins exhibit reduced fluorescence, the enzymatic reaction can be monitored by the decrease in fluorescence as the reaction proceeds.

Inhibition Studies:
N-methylprotoporphyrin IX has been identified as a potent inhibitor of ferrochelatase with an I₅₀ value of approximately 4 nM in cucumber ferrochelatase . Similar inhibition studies could be performed with M. gilvum Ferrochelatase to characterize its inhibition profile and compare it with other ferrochelatases.

How can researchers determine kinetic parameters for M. gilvum Ferrochelatase?

Determining reliable kinetic parameters for M. gilvum Ferrochelatase requires careful experimental design:

Substrate Concentration Series:
For accurate Km determination, researchers should use a wide range of substrate concentrations spanning at least 0.2-5× the expected Km value. Based on data from similar enzymes, starting ranges might include 1-50 μM for porphyrin substrates and 0.5-20 μM for Fe²⁺ .

Reaction Conditions Table:

What structural features distinguish M. gilvum Ferrochelatase from other bacterial ferrochelatases?

The complete M. gilvum Ferrochelatase consists of 340 amino acids . While detailed structural comparisons are not provided in the search results, analysis of the protein sequence and homology to other ferrochelatases suggests several key structural features:

• The enzyme likely adopts a bilobed structure with the active site located at the cleft between domains

• Conserved histidine residues probably participate in metal coordination

• Hydrophobic residues likely form a pocket that accommodates the porphyrin macrocycle

Structural studies of homologous ferrochelatases have revealed that these enzymes undergo conformational changes during catalysis, including movement of conserved loops that may control substrate access and product release . The M. gilvum enzyme likely shares these general features while possessing unique structural elements that reflect its adaptation to its ecological niche.

How can site-directed mutagenesis inform our understanding of M. gilvum Ferrochelatase?

Site-directed mutagenesis represents a powerful approach for investigating the functional roles of specific residues in M. gilvum Ferrochelatase. Based on studies of other ferrochelatases, researchers might target:

• Residues corresponding to M76, which appears to play a role in active site metal binding

• Residues homologous to E343, implicated in proton abstraction and product release

• Residues that form the peptide loop (Q302, S303, K304 equivalents) that may function as a metal sensor

By creating systematic variants and characterizing their kinetic properties, researchers can establish structure-function relationships specific to the M. gilvum enzyme. Crystal structures of these variants, coupled with biophysical studies like hydrogen-deuterium exchange, can further illuminate how specific residues contribute to substrate binding, catalysis, and product release.

How does the M. gilvum Ferrochelatase contribute to the organism's survival in its ecological niche?

M. gilvum has been found to survive within amoeba trophozoites without multiplying or killing the host during extended co-culture periods . This suggests a specialized adaptation that may involve unique properties of its essential enzymes, including ferrochelatase. The ability to maintain heme biosynthesis under the potentially iron-limited conditions within amoeba could be crucial for survival.

Additionally, as an environmental organism measuring approximately 1.4 ± 0.5 μm, M. gilvum must adapt to diverse soil and water environments where iron availability and oxygen levels fluctuate . The properties of its ferrochelatase, including substrate affinity, catalytic efficiency, and regulation, likely reflect adaptations to these ecological challenges.

What evolutionary insights can be gained from comparing M. gilvum Ferrochelatase with homologs from other species?

Comparative analysis of ferrochelatases across species can provide valuable evolutionary insights. While specific comparative data for M. gilvum Ferrochelatase is limited in the search results, general patterns observed in ferrochelatase evolution suggest:

• Conservation of catalytic residues across diverse species, reflecting the essential nature of the enzyme's function

• Diversification in regulatory domains and metal-binding properties, reflecting adaptation to different ecological niches

• Potential horizontal gene transfer events among soil bacteria, influencing enzyme properties

By conducting phylogenetic analyses and comparing key functional properties of ferrochelatases from different mycobacterial species and other bacteria, researchers can better understand how this enzyme family has evolved and adapted to diverse environmental challenges.