Recombinant Geobacillus thermodenitrificans Ferrochelatase (hemH)

What expression systems are recommended for recombinant G. thermodenitrificans ferrochelatase production?

For recombinant expression of G. thermodenitrificans ferrochelatase, Escherichia coli remains the most widely used heterologous host due to its well-established genetic tools, rapid growth, and high protein yields. Several E. coli strains have proven successful for the expression of thermophilic proteins from Geobacillus species:

Methodology for expression typically involves:

• PCR amplification of the hemH gene from G. thermodenitrificans genomic DNA

• Cloning into an expression vector with appropriate restriction sites (e.g., NdeI and XhoI)

• Transformation into the chosen E. coli strain

• Induction with IPTG (typically 0.1-1.0 mM) at optimal temperature (often 30°C rather than 37°C to enhance solubility)

For thermostable enzymes like those from G. thermodenitrificans, a heat treatment step (60-70°C) can be incorporated during purification to remove heat-labile E. coli proteins .

What purification strategies are most effective for recombinant G. thermodenitrificans ferrochelatase?

Based on studies with other recombinant proteins from G. thermodenitrificans, the following purification protocol would be effective:

• Cell lysis: Sonication or high-pressure homogenization in a buffer containing 50 mM phosphate buffer (pH 7.5-8.0), 300 mM NaCl, and 10% glycerol.

• Heat treatment: Exploiting the thermostability of G. thermodenitrificans proteins by heating the cell lysate at 60-70°C for 10-20 minutes to precipitate E. coli proteins while keeping the target protein soluble .

• Immobilized metal affinity chromatography (IMAC): Using a His-tag incorporated into the recombinant design for purification with Ni-NTA or similar matrices.

• Size exclusion chromatography: As a polishing step to remove aggregates and obtain homogeneous protein.

A typical purification yield for recombinant G. thermodenitrificans proteins is around 40% recovery with 20-25 fold purification, as seen with other enzymes from this organism . For ferrochelatase specifically, it's important to consider the potential presence of an iron-sulfur cluster, which might necessitate anaerobic conditions during purification to maintain enzyme activity .

How can co-expression strategies improve the yield and activity of recombinant heme-binding proteins?

Co-expression of ferrochelatase with target heme-binding proteins represents an innovative approach to overcome incomplete heme incorporation, which is a common challenge in recombinant heme protein expression. Research has demonstrated that this strategy significantly enhances the production of properly folded, heme-incorporated proteins .

Methodology for co-expression system implementation:

• Construct a dual-expression system using vectors such as pACYCduet, which contains two multiple cloning sites .

• Clone the G. thermodenitrificans hemH gene into one site and the target heme protein gene into the second site.

• Transform into E. coli BL21(DE3) or similar expression strains.

• Supplement the growth medium with δ-aminolevulinic acid (δ-ALA) at approximately 60 µM (10 mg/L) to increase heme biosynthesis .

Comparative analysis of expression methods:

The co-expression approach addresses the limitation where protein folding outpaces heme delivery, resulting in a population of proteins incorporating protoporphyrin IX instead of heme . This is particularly significant as protoporphyrin IX-incorporated proteins have similar spectral characteristics to heme-loaded targets and are therefore difficult to detect even in purified samples.

What are the kinetic and thermodynamic properties of G. thermodenitrificans ferrochelatase, and how do they compare to mesophilic counterparts?

While specific kinetic data for G. thermodenitrificans ferrochelatase is not directly available in the provided search results, extrapolation from other thermophilic enzymes from this organism suggests the following properties:

Expected kinetic parameters:

• Optimal temperature: 60-70°C (compared to 37°C for mesophilic counterparts)

• Thermal stability: Likely maintains >50% activity after incubation at 70°C for 60 minutes

• pH optimum: Expected to be 7.0-9.0, similar to other G. thermodenitrificans enzymes

Structural features contributing to thermostability:

• Increased number of salt bridges and hydrogen bonds

• Higher proportion of charged amino acids

• Shorter surface loops

• More compact packing of the hydrophobic core

For comparison, other enzymes from G. thermodenitrificans have demonstrated remarkable thermostability. For example, recombinant β-xylosidase from this organism exhibits optimal activity at 60°C with a half-life (T₁/₂) of 58 minutes at 70°C .

The thermostability of G. thermodenitrificans ferrochelatase would make it particularly valuable for biotechnological applications requiring high-temperature reactions or increased stability during storage.

What structural features are conserved in ferrochelatases across species, and what insights might this provide about G. thermodenitrificans ferrochelatase?

Ferrochelatases across bacterial and mammalian species share several conserved structural elements that are critical for function:

• Cleft structure: The enzyme typically contains a cleft formed by structural elements from two similar domains, each with a four-stranded parallel β-sheet flanked by α-helices. This cleft houses the active site and accommodates the porphyrin substrate .

• Metal-binding site: A conserved histidine residue (His183 in B. subtilis ferrochelatase) is involved in metal coordination. This residue is located in the cleft and is critical for ferrous ion binding .

• FC signature sequence: A highly conserved sequence motif found in all ferrochelatases that is involved in substrate binding and catalysis .

• Iron-sulfur cluster: Many ferrochelatases contain an iron-sulfur cluster, with approximately 2 iron atoms per molecule, which contributes to structural integrity and possibly function .

Structural alignment analysis would likely show that G. thermodenitrificans ferrochelatase maintains these core features while incorporating thermostabilizing elements. The B. subtilis ferrochelatase structure (PDB: 1AK1) provides a valuable template for homology modeling of G. thermodenitrificans ferrochelatase, given that both organisms are gram-positive thermophiles .

Expected differences in G. thermodenitrificans ferrochelatase would include:

• Additional salt bridges at the domain interface

• Higher content of charged residues on the protein surface

• Potentially shorter loops connecting secondary structure elements

What spectroscopic methods can differentiate between properly heme-incorporated proteins and those containing protoporphyrin IX in recombinant expression systems?

A significant challenge in recombinant heme protein production is distinguishing between proteins containing proper heme versus those incorporating protoporphyrin IX (the free-base porphyrin without iron). The following spectroscopic methods can be used to make this critical distinction:

1. UV-Visible Spectroscopy:

• Heme-incorporated proteins typically show a characteristic Soret peak at approximately 403 nm

• The ratio of Abs403/Abs280 provides a quantitative measure of heme incorporation

• Complete heme incorporation in recombinant systems typically yields an Abs403/Abs280 ratio of ~0.6, while incomplete incorporation shows lower ratios (0.25-0.40)

2. Fluorescence Spectroscopy:

• Iron-bound heme is non-fluorescent when excited at ~400 nm

• Protoporphyrin IX exhibits strong fluorescence when excited at this wavelength

• This difference provides a clear method to detect incomplete heme incorporation

3. Resonance Raman Spectroscopy:

• Can detect the presence of free-base porphyrin in protein samples

• Samples with complete heme incorporation show no evidence of porphyrin in their Raman spectra

Methodological approach for assessment:

• Purify the recombinant protein using standard chromatographic techniques

• Perform UV-Vis spectroscopy to determine the Abs403/Abs280 ratio

• Conduct fluorescence emission spectroscopy with excitation at 400-410 nm

• If available, confirm results with resonance Raman spectroscopy

These spectroscopic methods provide complementary information and should be used in combination for comprehensive characterization of recombinant heme proteins.

How might the genomic context of the hemH gene in G. thermodenitrificans provide insights into its regulation and function?

The genomic context of the hemH gene can provide valuable insights into its regulation and functional relationships within the cell. While specific information about the G. thermodenitrificans hemH genomic context is not directly provided in the search results, analysis of the complete genome sequence would reveal:

• Operon structure: Whether hemH is part of a larger operon containing other genes involved in heme biosynthesis or related processes.

• Regulatory elements: Presence of promoter regions, transcription factor binding sites, and other regulatory elements that control hemH expression.

• Two-component systems: Potential regulation by two-component systems similar to those observed in other bacteria. For example, in Corynebacterium glutamicum, the two-component systems ChrSA and HrrSA are involved in heme-dependent gene regulation .

Predicted regulatory network:
Based on studies in other organisms, G. thermodenitrificans hemH gene expression likely responds to:

• Iron availability (repression under iron limitation)

• Oxygen levels (potential upregulation under aerobic conditions)

• Temperature stress (given its thermophilic nature)

The recent availability of the complete genome sequence for G. thermodenitrificans subsp. calidus DSM 22629T (3,408,575 bp, 48.94% GC content) provides an important resource for such genomic context analysis . This genome contains 3,615 predicted genes, which would include the complete hemH gene and its regulatory elements.

What strategies can overcome inclusion body formation when expressing thermophilic ferrochelatase in mesophilic hosts?

Expression of thermophilic proteins like G. thermodenitrificans ferrochelatase in mesophilic hosts such as E. coli often results in inclusion body formation. The following strategies can mitigate this challenge:

1. Optimization of expression conditions:

• Lower induction temperature (16-25°C)

• Reduced IPTG concentration (0.1-0.3 mM)

• Extended expression time (overnight instead of 3-4 hours)

2. Use of solubility-enhancing fusion tags:

3. Co-expression with molecular chaperones:

• GroEL/GroES system

• DnaK/DnaJ/GrpE system

• Trigger factor

4. Post-solubilization refolding protocol for inclusion bodies:

• Isolate inclusion bodies by centrifugation after cell lysis

• Wash with buffer containing low concentrations of denaturants (0.5-1% Triton X-100)

• Solubilize in buffer containing 8M urea or 6M guanidine hydrochloride

• Perform refolding by gradual dilution or dialysis against buffer containing metal cofactors (Fe²⁺)

• Purify refolded protein by affinity chromatography

5. Optimization of codon usage:

• Analyze codon bias between G. thermodenitrificans and E. coli

• Synthesize a codon-optimized gene for improved expression

• Consider using E. coli Rosetta strains that supply rare tRNAs

Implementation of these strategies, particularly the combination of lower temperature expression with solubility-enhancing tags, has been successful for other thermophilic enzymes from Geobacillus species .

How can researchers assess and maintain the iron-sulfur cluster integrity in recombinant ferrochelatase?

Ferrochelatases often contain iron-sulfur clusters that are essential for their structural integrity and potentially their function . Maintaining and assessing the integrity of these clusters in recombinant ferrochelatase presents several challenges:

Methods for assessing iron-sulfur cluster integrity:

• UV-Visible spectroscopy:

  • Iron-sulfur clusters typically exhibit characteristic absorbance near 320 nm

  • The ratio of this peak to protein absorbance (280 nm) provides a quantitative measure of cluster incorporation

• Electron Paramagnetic Resonance (EPR) spectroscopy:

  • Can identify the oxidation state and environment of iron in the cluster

  • Particularly useful for distinguishing between different types of iron-sulfur clusters (2Fe-2S, 4Fe-4S)

• Metal content analysis:

  • Inductively coupled plasma mass spectrometry (ICP-MS) to determine iron:protein ratio

  • Expected ratio of approximately 2 iron atoms per molecule for ferrochelatase

Strategies to maintain iron-sulfur cluster integrity:

• Expression conditions:

  • Supplement growth media with iron (50-100 μM ferric citrate)

  • Include cysteine in the medium to facilitate iron-sulfur cluster assembly

• Purification considerations:

  • Use anaerobic or low-oxygen conditions during cell lysis and protein purification

  • Include reducing agents (2-5 mM DTT or β-mercaptoethanol) in all buffers

  • Add iron-chelating agents (0.1-0.5 mM EDTA) to prevent non-specific iron binding

• Storage conditions:

  • Store purified enzyme under anaerobic conditions or with oxygen scavengers

  • Add glycerol (10-20%) to stabilize the protein structure

  • Maintain at -80°C for long-term storage

These approaches have proven effective for maintaining iron-sulfur cluster integrity in recombinant mammalian ferrochelatase expressed in E. coli and would be applicable to G. thermodenitrificans ferrochelatase as well.

How can recombinant G. thermodenitrificans ferrochelatase be applied in in vitro heme reconstitution for structural biology studies?

Recombinant G. thermodenitrificans ferrochelatase offers significant advantages for in vitro heme reconstitution in structural biology studies due to its expected thermostability and high activity. The following methodological approach can be implemented:

Protocol for in vitro heme reconstitution:

• Preparation of apo-protein:

  • Express the target heme protein in E. coli under conditions that limit heme incorporation

  • Alternatively, remove heme from purified proteins using acidified acetone extraction

  • Confirm heme removal by UV-visible spectroscopy

• Reconstitution reaction setup:

  • Mix apo-protein (10-50 μM) with protoporphyrin IX (5-20 μM) in buffer containing 50 mM Tris-HCl pH 8.0, 100 mM NaCl

  • Add ferrous ammonium sulfate (10-50 μM) under anaerobic conditions

  • Add purified recombinant G. thermodenitrificans ferrochelatase (0.1-1 μM)

  • Incubate at 50-60°C for 30-60 minutes

• Monitoring the reconstitution:

  • Follow the reaction by UV-visible spectroscopy, tracking the appearance of the characteristic heme Soret peak

  • Confirm complete reconstitution by the absence of fluorescence from free protoporphyrin IX

The thermostability of G. thermodenitrificans ferrochelatase provides distinct advantages:

• The reaction can be performed at elevated temperatures (50-60°C), potentially increasing reaction rates

• The enzyme remains stable during extended incubation periods

• The higher temperature may enhance the solubility of hydrophobic substrates like protoporphyrin IX

This approach is particularly valuable for structural biology studies requiring homogeneous heme incorporation for crystallography, cryo-EM, or NMR spectroscopy .

What insights can comparative genomics provide about the evolution of ferrochelatase in thermophilic organisms like G. thermodenitrificans?

Comparative genomics analysis of ferrochelatase genes across thermophilic and mesophilic organisms can provide valuable insights into the evolutionary adaptations of this enzyme to high-temperature environments:

Methodological approach for comparative genomics analysis:

• Sequence retrieval and alignment:

  • Extract hemH gene sequences from diverse bacterial genomes, including thermophiles, mesophiles, and psychrophiles

  • Perform multiple sequence alignment using MUSCLE or MAFFT algorithms

  • Identify conserved regions and thermophile-specific substitutions

• Phylogenetic analysis:

  • Construct maximum likelihood or Bayesian phylogenetic trees

  • Assess whether thermophilic ferrochelatases form monophyletic groups or evolved independently

  • Calculate evolutionary rates to identify acceleration or constraints in thermophiles

• Structural comparison:

  • Create homology models of ferrochelatases from different temperature adaptations

  • Compare with known structures (e.g., B. subtilis ferrochelatase, PDB: 1AK1)

  • Identify structural features associated with thermostability

Expected evolutionary insights:

• Amino acid composition shifts:

  • Higher content of charged residues (Arg, Glu, Lys) in thermophilic ferrochelatases

  • Decreased frequency of thermolabile residues (Asn, Gln, Cys) in loop regions

  • Increased hydrophobicity in the protein core

• Conserved vs. variable regions:

  • Complete conservation of active site residues across temperature adaptations

  • Variable surface-exposed loops, particularly in substrate entrance regions

  • Conservation of metal-binding motifs involving histidine residues

• Genomic context evolution:

  • Potential differences in operon organization between thermophiles and mesophiles

  • Evolution of regulatory elements controlling hemH expression

  • Horizontal gene transfer events contributing to thermophilic adaptations

The complete genome sequence of G. thermodenitrificans provides an excellent starting point for such comparative analyses, offering insights into thermophilic adaptations that may inform protein engineering efforts for enhanced thermostability.

How can structure-function studies of G. thermodenitrificans ferrochelatase contribute to protein engineering for enhanced thermostability?

Structure-function studies of G. thermodenitrificans ferrochelatase can provide valuable insights for protein engineering applications aimed at enhancing thermostability in industrial enzymes:

Methodological approach for structure-function studies:

• Homology modeling and structural analysis:

  • Generate a homology model using the B. subtilis ferrochelatase crystal structure (PDB: 1AK1) as a template

  • Identify unique structural features potentially contributing to thermostability

  • Perform molecular dynamics simulations at different temperatures to identify flexible regions

• Site-directed mutagenesis experiments:

  • Create single-point mutations at positions differing between thermophilic and mesophilic ferrochelatases

  • Express and purify mutant proteins to assess thermostability changes

  • Measure half-life at elevated temperatures and compare to wild-type enzyme

• Domain swapping experiments:

  • Exchange domains between G. thermodenitrificans ferrochelatase and mesophilic counterparts

  • Assess thermostability of chimeric proteins

  • Identify domains or subdomains critical for thermostability

Principles for engineering enhanced thermostability:

Based on studies of other thermophilic proteins, including those from G. thermodenitrificans , the following strategies may prove effective:

• Surface charge optimization:

  • Introduce additional salt bridges on the protein surface

  • Replace neutral residues with charged ones in surface-exposed positions

  • Create ion pair networks spanning critical domains

• Core packing enhancement:

  • Increase hydrophobic packing in the protein core

  • Replace small hydrophobic residues with larger ones where cavities exist

  • Introduce disulfide bonds at strategic positions

• Loop stabilization:

  • Shorten surface loops connecting secondary structure elements

  • Introduce proline residues in loop regions to reduce flexibility

  • Add hydrogen bonding networks to stabilize loop conformations

These approaches have been successfully applied to other enzymes and could be particularly valuable for engineering ferrochelatase variants with enhanced thermostability for biotechnological applications.

What are the potential causes and solutions for low activity of recombinant G. thermodenitrificans ferrochelatase?

When recombinant G. thermodenitrificans ferrochelatase exhibits unexpectedly low activity, several factors may be responsible. The following troubleshooting guide addresses common issues and their solutions:

Specific considerations for G. thermodenitrificans ferrochelatase:

• Temperature sensitivity:

  • While thermostable, the enzyme may have an optimal temperature range

  • Activity assays should be performed at various temperatures (40-80°C)

  • Thermal activation may be required before maximum activity is observed

• C-terminal integrity:

  • Studies with mammalian ferrochelatase have shown that the C-terminal region is critical for activity

  • Elimination of the C-terminal 30 amino acid residues results in an inactive enzyme

  • Verify the integrity of the C-terminus in the recombinant protein

• Metal analysis:

  • Perform metal analysis to confirm the expected iron content (~2 iron atoms per molecule)

  • ICP-MS or atomic absorption spectroscopy can quantify iron content

  • Compare with properly functioning ferrochelatase samples

These troubleshooting approaches can help identify and address the specific factors limiting the activity of recombinant G. thermodenitrificans ferrochelatase in experimental settings.

How can researchers distinguish between enzyme inactivation and substrate limitations in ferrochelatase activity assays?

When conducting activity assays with recombinant G. thermodenitrificans ferrochelatase, distinguishing between enzyme inactivation and substrate limitations is essential for accurate characterization. The following methodological approaches can help make this distinction:

1. Time-course analysis with substrate replenishment:

• Monitor activity over time with regular sampling

• At various time points, add fresh substrate to reaction aliquots

• If activity recovers with fresh substrate, limitation is substrate-related

• If activity remains low despite substrate addition, enzyme inactivation is likely

2. Enzyme concentration dependence:

• Perform assays with increasing enzyme concentrations

• Plot reaction velocity versus enzyme concentration

• Linear relationship indicates enzyme stability

• Non-linear relationship (plateauing) suggests substrate limitation

3. Product inhibition analysis:

• Add increasing concentrations of reaction product (heme)

• Measure initial reaction rates

• If rates decrease with added product, product inhibition is occurring

• This can be distinguished from enzyme inactivation by dilution experiments

4. Thermal stability time course:

• Pre-incubate enzyme at assay temperature for various time periods

• Start reactions with substrate addition after pre-incubation

• Plot remaining activity versus pre-incubation time

• Calculate inactivation rate constant (kinact)

5. Substrate solubility assessment:

• Monitor substrate (protoporphyrin IX) concentration over time

• Assess precipitation or aggregation using light scattering

• Add solubilizing agents (detergents, organic solvents) at sub-inhibitory concentrations

• Compare reaction rates with and without solubilizing agents

For G. thermodenitrificans ferrochelatase specifically, the following considerations are important:

• Assays should be conducted at the optimal temperature (likely 60-70°C)

• Thermal stability may be significantly higher than mesophilic counterparts

• Iron oxidation from Fe²⁺ to Fe³⁺ may occur more rapidly at elevated temperatures

• Oxygen solubility decreases at higher temperatures, which may actually benefit assays with this oxygen-sensitive enzyme

These approaches provide complementary information that can help researchers accurately characterize the kinetic properties of recombinant G. thermodenitrificans ferrochelatase and distinguish between different factors affecting observed activity.