Recombinant Synechococcus sp. Imidazole glycerol phosphate synthase subunit hisF1 (hisF1)

What is the basic structure and function of HisF1 in Synechococcus sp.?

HisF1 functions as the cyclase subunit of the imidazole glycerol phosphate synthase (HisFH) complex. The HisFH heterodimer consists of two subunits: the glutaminase subunit (HisH) and the cyclase subunit (HisF). HisF adopts a (βα)8-barrel fold, also known as a TIM barrel structure, with the active site located at the C-terminal face of the barrel. The protein contains a central tunnel that allows for ammonia transport from the HisH active site to the HisF active site, spanning approximately 25 Å across the beta-strand barrel .

The primary function of HisF is to catalyze the cyclization reaction using ammonia (generated by HisH) and N′-[(5′-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PrFAR) to produce imidazole glycerol phosphate (ImGP) and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) .

How can I confirm the proper folding and activity of recombinant HisF1?

To confirm proper folding of recombinant HisF1:

• Spectroscopic Methods: Circular dichroism (CD) spectroscopy can verify secondary structure content consistent with the (βα)8-barrel fold.

• Thermal Shift Assays: Differential scanning fluorimetry can assess thermal stability and proper folding.

• Size Exclusion Chromatography: This technique can confirm the monomeric state of isolated HisF1 or its ability to form the heterodimeric complex with HisH.

• Activity Assays: Functional validation can be performed through:

  • Ammonia-dependent cyclase activity using PrFAR as substrate

  • Monitoring ImGP and AICAR formation using HPLC or coupled enzymatic assays

  • Testing allosteric activation of HisH glutaminase activity when HisF binds its substrate

What expression systems are suitable for recombinant HisF1 production?

Based on the characteristics of both HisF proteins and Synechococcus sp., several expression systems can be utilized:

Synechococcus sp. PCC 11901 offers particular advantages as it grows rapidly (doubling time ≈2 hours) and can reach high cell densities (≈33 g dry cell weight per liter) . For heterologous expression in this system, the gene can be integrated at neutral genomic sites that don't affect growth even at high cell densities .

How can I assess the binding affinity between recombinant HisF1 and HisH?

Several methodological approaches can determine the binding affinity and interaction characteristics between HisF1 and HisH:

• Tryptophan Fluorescence: HisH contains a tryptophan residue (hW123) that becomes buried in the interface when the heterodimer forms. Monitoring changes in tryptophan fluorescence upon complex formation provides a simple method to determine binding affinities and kinetics .

• Isothermal Titration Calorimetry (ITC): This technique can provide thermodynamic parameters (ΔH, ΔS, KD) of binding by measuring heat changes during titration of one protein into another.

• Surface Plasmon Resonance (SPR): Real-time binding kinetics can be determined by immobilizing one protein partner on a sensor chip and flowing the other partner across the surface.

• Microscale Thermophoresis (MST): This technique measures changes in the movement of molecules along microscopic temperature gradients, allowing determination of binding affinities in solution.

The tryptophan fluorescence method is particularly useful as it leverages the natural fluorophore (hW123) that becomes shielded from solvent upon binding to HisF, causing changes in fluorescence intensity and emission maximum .

What structural changes occur in HisF1 upon substrate binding that contribute to allosteric activation?

When both substrates (glutamine and ProFAR/PrFAR) bind to the HisFH complex, four significant structural changes convert the inactive conformation to the catalytically active form:

• β1α1 loop (fL1) Movement: This loop undergoes a dramatic 25 Å displacement from a position near helix fα1 to a conformation covering the HisF active site. This substantial conformational change is evidenced by a large root-mean-square deviation (RMSD) when comparing inactive and active structures .

• Active Site Interactions: In the closed state, several highly conserved residues form extensive contacts:

  • fK19 forms a salt bridge with fD176 and the phosphate group of ProFAR

  • fF23 establishes π-stacking interactions with the imidazole ring of ProFAR

  • fN22 forms hydrogen bonds with backbone carbonyls of fI173, fD174, and fD176

• Oxyanion Hole Formation: The allosteric activation establishes an oxyanion hole in the HisH active site, which is essential for efficient catalysis and explains the observed 4500-fold increase in activity compared to the inactive conformation .

• Ammonia Tunnel Formation: Proper alignment of residues creates a continuous tunnel for ammonia transport from HisH to HisF active sites.

These conformational changes demonstrate that the catalytically active HisFH complex only forms when both substrates are bound, preventing wasteful glutamine hydrolysis in the absence of the HisF substrate .

What are the optimal conditions for expressing soluble and active HisF1 in Synechococcus sp. PCC 11901?

Synechococcus sp. PCC 11901 offers significant advantages for recombinant protein expression due to its rapid growth and high biomass production. For optimal expression of soluble and active HisF1:

• Growth Medium: Use A+ medium supplemented with:

  • 20 mM NaHCO3

  • 1 μg/mL cobalamin (vitamin B12) - essential as PCC 11901 is auxotrophic for cobalamin

  • 30 g/L sea salts (Marine mixture)

• Expression System:

  • Utilize neutral genomic integration sites that have been characterized for stability

  • For constitutive expression, the P cpt promoter (truncated cpcB promoter) provides strong expression

  • For inducible expression, the DAPG-inducible PhlF repressor system offers tight regulation with a 228-fold dynamic range

• Cultivation Conditions:

  • Temperature: 38°C (optimal for PCC 11901)

  • Light intensity: Up to 660 μmol photons m-2 s-1 (PCC 11901 tolerates high light)

  • CO2: 1% for enhanced growth

  • Aeration: Vigorous shaking or bubbling with air/CO2 mixture

• Harvest Time:

  • For constitutive expression, harvest at late exponential phase

  • For inducible expression, induce at OD730 of 1.0-2.0 and harvest 24 hours post-induction

PCC 11901 can accumulate up to 33 g dry cell weight per liter in optimized conditions, providing excellent biomass for protein extraction .

How can I optimize purification protocols to maintain the allosteric properties of HisF1?

Maintaining the allosteric properties of HisF1 during purification requires careful consideration of buffer conditions and handling procedures:

• Cell Lysis:

  • Use gentle mechanical disruption methods (sonication with cooling intervals or high-pressure homogenization)

  • Include protease inhibitors to prevent degradation

  • Maintain reducing conditions (1-5 mM DTT or 2-10 mM β-mercaptoethanol) to preserve cysteine residues

• Buffer Composition:

  • Base buffer: 50 mM Tris-HCl or HEPES, pH 7.5-8.0

  • Salt: 100-300 mM NaCl to maintain solubility while minimizing dissociation of protein complexes

  • Additives: 5-10% glycerol for stability and 1 mM EDTA to chelate metal ions that could promote oxidation

• Purification Strategy:

  • Affinity chromatography: His-tag or GST-tag depending on the construct design

  • Size exclusion chromatography: Critical for separating monomeric HisF1 from aggregates

  • Ion exchange chromatography: As a polishing step to remove contaminants

• Functional Validation:

  • Perform activity assays immediately after purification

  • Assess allosteric activation by measuring HisH activity in the presence and absence of ProFAR

  • Verify that the purified protein exhibits the expected 4500-fold allosteric activation

• Storage Conditions:

  • Flash-freeze aliquots in liquid nitrogen

  • Store at -80°C with 10-20% glycerol as a cryoprotectant

  • Avoid repeated freeze-thaw cycles

What methods can be used to investigate the ammonia tunneling mechanism in the HisF1 barrel?

Investigating the ammonia tunneling mechanism within the HisF1 barrel requires specialized techniques that can probe the molecular details of this fascinating process:

• Site-Directed Mutagenesis:

  • Systematically mutate conserved residues lining the predicted tunnel

  • Focus on residues with hydrophobic side chains that create the hydrophobic tunnel environment

  • Analyze effects on both HisF activity and coupled HisH-HisF activity

• X-ray Crystallography with Tunnel Probes:

  • Co-crystallize HissFH with ammonia mimics or tunnel-binding molecules

  • Use heavy atom derivatives to map the exact path of the tunnel

  • Capture different conformational states that represent tunnel opening and closing

• Molecular Dynamics Simulations:

  • Model ammonia movement through the 25 Å tunnel under various conditions

  • Calculate energy profiles and identify potential bottlenecks or gates

  • Predict effects of mutations on tunnel properties

• Chemical Rescue Experiments:

  • Block the tunnel with site-specific mutations

  • Attempt to rescue activity by providing alternative ammonia sources or creating alternative access routes

  • Measure kinetic parameters to quantify the efficiency of different tunneling paths

• Isotope Labeling and Tracing:

  • Use 15N-labeled glutamine and track the labeled ammonia through the reaction

  • Employ rapid quench techniques to trap intermediates

  • Analyze products using mass spectrometry to determine if tunnel leakage occurs

The ammonia tunnel spans approximately 25 Å from the HisH active site through the beta-strand barrel of HisF to reach the cyclase active site at the opposite face of the barrel . This molecular tunnel prevents the loss of reactive ammonia to the bulk solvent and ensures efficient coupling between the two enzymatic activities.

How does the conformational equilibrium between inactive and active states affect HisF1 catalytic efficiency?

The conformational equilibrium between inactive and active states is a critical determinant of HisF1 catalytic efficiency:

• Population Distribution:

  • In solution, the inactive and active conformations exist in a dynamic equilibrium

  • The population of the active conformation directly correlates with observed turnover rates

  • This alignment with the ensemble model of allostery suggests that substrate binding shifts the equilibrium rather than inducing a completely new conformation

• Rate-Limiting Steps:

  • The conversion from inactive to active conformation may become rate-limiting under certain conditions

  • The transition involves significant structural rearrangements, particularly in the β1α1 loop (fL1) which moves by 25 Å

  • Factors that stabilize the active conformation can enhance catalytic efficiency

• Quantitative Analysis:

  • The allosteric activation increases the turnover number (kcat) of the glutaminase reaction by approximately 4500-fold

  • This enhancement correlates with the formation of the oxyanion hole in the HisH active site

  • Mathematical modeling of the conformational equilibrium can predict activity under various substrate concentrations

• Engineering Implications:

  • Mutations that shift the equilibrium toward the active conformation could create constitutively active variants

  • Conversely, stabilizing the inactive conformation could create switchable enzyme variants for biotechnological applications

  • Understanding this equilibrium is essential for rational enzyme engineering

What CRISPR-based tools are available for engineering HisF1 in Synechococcus sp. PCC 11901?

Recent developments have established powerful CRISPR-based tools specifically optimized for Synechococcus sp. PCC 11901:

• DAPG-Inducible CRISPRi System:

  • A dCas9-based CRISPR interference system that provides tunable gene repression

  • Controlled by the 2,4-diacetylphloroglucinol (DAPG)-inducible PhlF repressor system

  • Shows tight regulation with a 228-fold dynamic range of induction

  • Highly responsive to repression targeting various genomic loci

• CRISPR-Cas12a Genome Editing:

  • Modular method for generating markerless mutants

  • High efficiency for single insertion (31-81%)

  • Capable of multiplex double insertion with 25% efficiency

  • Based on CyanoGate MoClo system for standardized assembly of genetic parts

• Integration Site Selection:

  • Characterized neutral genomic sites suitable for stable integration

  • Sites that do not affect growth even at high cell densities

  • Enable consistent expression of engineered HisF1 variants

• Promoter Options:

  • Suite of constitutive promoters with varying strengths

  • Inducible systems including the novel DAPG-inducible PhlF system

  • Various terminators characterized specifically in PCC 11901

These tools enable precise engineering of HisF1 for various applications, including structure-function studies, enzyme optimization, and biosynthetic pathway engineering in Synechococcus sp. PCC 11901.

How can HisF1 be engineered to enhance its catalytic properties or substrate specificity?

Engineering HisF1 to enhance catalytic properties or modify substrate specificity can be approached through several strategies:

• Structure-Guided Mutagenesis:

  • Target residues that directly interact with the substrate

  • Focus on fK19, fF23, and fN22 which form extensive contacts with ProFAR and the active site residues

  • Modify residues in the fL1 loop to alter its mobility and potentially affect the inactive-active transition

• Allosteric Interface Engineering:

  • Modify residues at the HisF-HisH interface to enhance or alter allosteric communication

  • Target residues in the β1α1 loop (hL1) and adjacent β3α2 loop (hL4) of HisH which show significant chemical shift perturbations upon allosteric activation

  • Engineer the oxyanion hole formation mechanism to enhance catalytic efficiency

• Ammonia Tunnel Modifications:

  • Alter the hydrophobicity or dimensions of the ammonia tunnel

  • Engineer tunnel residues to accommodate alternative nucleophiles for novel reactions

  • Create switchable tunnel gates for controlled reactivity

• Directed Evolution Approaches:

  • Develop high-throughput screening systems based on growth complementation or colorimetric assays

  • Utilize the CRISPR-Cas12a system in PCC 11901 to generate diverse variant libraries

  • Select for desired properties such as enhanced thermostability, altered substrate specificity, or increased catalytic efficiency

• Domain Swapping and Chimeras:

  • Create hybrid enzymes by swapping domains between HisF homologs

  • Design chimeric proteins combining the (βα)8-barrel of HisF with other catalytic domains

  • Engineer novel allosteric connections to create switchable multi-enzyme complexes

These engineering approaches can be implemented efficiently using the genetic tools developed for Synechococcus sp. PCC 11901, which include markerless genome editing, inducible gene expression systems, and high-efficiency transformation methods .