Recombinant Neurospora crassa 1- (5-phosphoribosyl)-5-[ (5-phosphoribosylamino)methylideneamino] imidazole-4-carboxamide isomerase (his-6)

What is the function of His6 enzyme in Neurospora crassa?

His6 enzyme in Neurospora crassa catalyzes a critical isomerization step in the histidine biosynthesis pathway. Specifically, it performs the Amadori rearrangement that converts N'-[(5′-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PRFAR) to 5′-ProFAR (5′-phosphoribulosylformimino-5-aminoimidazole-4-carboxamide ribonucleotide) . This isomerization involves opening the ribofuranose ring, forming a Schiff base intermediate, and subsequent proton abstraction that yields an enolamine that converts to the keto product . This reaction is followed by condensation with ammonia and cleavage to form 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and imidazole glycerol phosphate (ImGP) .

What are the conserved residues in His6 enzyme critical for its function?

Sequence alignment studies of His6/HisA proteins reveal that only a limited number of residues are completely conserved across all sequences. These include seven glycine residues, Asp9, His57, Leu61, and Trp152 . Among these, Asp9 appears to play a particularly crucial role in catalysis, likely functioning as both a proton acceptor and donor during the isomerization reaction. Unlike the related TIM enzyme which uses distinct residues for acid and base functions during enolization, the His6 enzyme appears to employ the Asp9 carboxylate in a dual role - positioned to both take the proton from C2′ and donate it to C1′ during the catalytic cycle .

What are the recommended methods for cloning and expressing recombinant Neurospora crassa His6 enzyme?

For cloning and expression of recombinant Neurospora crassa His6 enzyme, a proven approach involves PCR amplification of the coding region with primers designed to incorporate a C-terminal HIS6 tag. The general strategy includes:

• Design forward and reverse primers that amplify the complete coding region with the HIS6 tag at the C-terminus .

• PCR-amplify the region and purify the product using gel extraction .

• Clone the purified product into an intermediate vector (such as pJET) .

• Subclone into an expression vector containing a strong promoter (such as ccg-1/grg-1 promoter for Neurospora expression) .

• Transform into the appropriate host system.

For enhanced expression in E. coli systems, consider using a dual hexahistidine-maltose-binding protein (His6-MBP) affinity tag, which can significantly improve yield and solubility of the recombinant protein .

What purification protocols yield the highest purity for recombinant His6 enzyme?

A two-step immobilized metal affinity chromatography (IMAC) approach is recommended for obtaining crystallization-grade His6 enzyme. The protocol includes:

• Initial purification using Ni-NTA resin to capture the His-tagged protein .

• Cleavage of the fusion protein with a His6-tagged tobacco etch virus (TEV) protease to separate the tag from the target protein .

• Second IMAC step where unwanted byproducts and the cleaved tag bind to the Ni-NTA resin, while the pure target protein flows through .

This dual IMAC approach effectively removes both the affinity tag and endogenous proteins that may bind non-specifically to the Ni-NTA resin during the first purification step, resulting in highly pure protein suitable for crystallographic and enzymatic studies .

How can researchers effectively characterize the enzymatic activity of purified His6 enzyme?

Characterization of His6 enzyme activity requires assays that can detect the isomerization of PRFAR to ProFAR. A comprehensive approach includes:

• Spectrophotometric assays that monitor changes in absorbance associated with substrate conversion

• HPLC-based assays that can separate and quantify substrate and product

• Coupled enzyme assays where the product of the His6 reaction feeds into a subsequent reaction with a more easily detectable output

For kinetic characterization, researchers should determine:

• Km values for PRFAR substrate

• kcat values under varying pH and temperature conditions

• Effects of potential inhibitors or activators

Additionally, differential scanning fluorimetry (DSF) can be used to assess thermal stability of the enzyme and the effects of ligand binding on protein stability.

What crystallization conditions are optimal for obtaining high-resolution structures of Neurospora crassa His6?

Based on successful crystallization of homologous His6 enzymes, the following conditions may be effective for Neurospora crassa His6:

• Protein concentration: 10-15 mg/mL in a buffer containing 20 mM Tris-HCl pH 7.5 and 150 mM NaCl

• Crystallization method: Hanging drop vapor diffusion with 1:1 protein:reservoir ratio

• Promising reservoir solutions:

  • 100 mM sodium citrate pH 5.5-6.5 with 15-25% PEG 3350

  • 100 mM HEPES pH 7.0-8.0 with 1.5-2.0 M ammonium sulfate

For co-crystallization with substrates or substrate analogs, incubate the protein with 5-10 mM ligand for 1 hour prior to setting up crystallization drops.

When collecting diffraction data, consider using the anomalous signal from sulfur atoms for phase determination, especially if the protein contains multiple cysteine and methionine residues . This approach was successfully used for the yeast His6 enzyme, resulting in high-quality electron density maps at 1.3 Å resolution .

How does the structure of Neurospora crassa His6 compare to homologous enzymes from other species?

The His6 enzyme from Neurospora crassa can be expected to show structural similarities to its homologs from other species, particularly the well-characterized yeast (S. cerevisiae) enzyme. Both likely feature the conserved TIM barrel architecture with parallel β-strands connected by long loops at their C-terminal end .

Analysis of structural matches using the MSD server of the European Bioinformatics Institute would provide quantitative assessment of structural similarity between Neurospora crassa His6 and homologs from other species .

What is the proposed catalytic mechanism of His6 enzyme and how might it be experimentally verified?

The proposed catalytic mechanism for His6 enzyme involves several key steps:

• Binding of the PRFAR substrate in the active site

• Opening of the ribofuranose ring and formation of a Schiff base intermediate

• Proton abstraction to yield an enolamine

• Conversion to the keto product (ProFAR)

Based on structural analysis, Asp9 appears to play a dual role in catalysis, functioning as both a proton acceptor and donor . Unlike TIM, which uses separate residues (His95 and Glu165) for acid-base catalysis, His6 likely employs the Asp9 carboxylate for both functions .

This proposed mechanism could be experimentally verified through:

• Site-directed mutagenesis of Asp9 and other conserved residues to assess their role in catalysis

• pH-rate profile studies to determine ionization states of key catalytic groups

• Solvent isotope effect experiments to probe proton transfer steps

• Trapping and characterization of reaction intermediates using rapid-quench techniques

• Computational approaches such as QM/MM simulations to model the energetics of the proposed reaction pathway

What structure-function relationships exist in the His6 enzyme active site?

The active site of His6 enzyme contains several key structural elements that contribute to its function:

• Conserved Asp9 - Critical for proton transfer during catalysis

• Asp134 - Likely involved in substrate binding and positioning

• Conserved glycine residues - Provide conformational flexibility required for enzyme function

A deeper understanding of structure-function relationships could be gained through:

• Co-crystallization with substrate analogs or transition state mimics

• Hydrogen-deuterium exchange mass spectrometry to identify regions with altered dynamics during catalysis

• Systematic alanine scanning of active site residues to quantify their contribution to catalysis

• Comparative analysis with related enzymes that catalyze similar reactions

How can recombinant His6 enzyme be engineered for enhanced stability or altered substrate specificity?

Engineering the His6 enzyme for improved properties could focus on several strategies:

• Stability Enhancement:

  • Introduction of disulfide bonds at positions identified through computational analysis

  • Consensus design approach based on alignment of His6 sequences from thermophilic organisms

  • Rigidification of flexible loops through targeted mutations

• Substrate Specificity Modification:

  • Targeted mutations of residues lining the substrate binding pocket

  • Directed evolution using error-prone PCR and selection for activity on alternative substrates

  • Chimeric approaches that combine elements from related enzymes with different specificities

What are the common difficulties in expressing soluble and active Neurospora crassa His6 enzyme?

Researchers frequently encounter several challenges when working with recombinant Neurospora crassa His6 enzyme:

• Insolubility issues - The enzyme may form inclusion bodies in heterologous expression systems, particularly in E. coli. This can be addressed by:

  • Using fusion tags like MBP that enhance solubility

  • Expressing at lower temperatures (16-18°C)

  • Co-expressing with chaperone proteins

• Protein instability - Site-directed mutagenesis attempts to incorporate additional methionines (for crystallographic phasing) have resulted in unstable proteins that are lost during purification . This suggests sensitivity to certain modifications and highlights the need for careful design when introducing mutations.

• Proper folding - The TIM barrel fold requires correct formation of multiple β-strands and α-helices in the proper orientation. Misfolding can lead to inactive protein despite apparent solubility.

How can researchers overcome challenges in obtaining high-resolution structural data for His6 enzyme?

Obtaining high-resolution structural data for His6 enzyme presents several challenges that can be addressed through specialized approaches:

• Phase determination challenges:

  • When methionine incorporation is problematic (as seen with yeast His6), consider using the anomalous signal from native sulfur atoms for phasing

  • Collect highly redundant diffraction data (>20-fold) at longer wavelengths (approximately 1.77 Å) to maximize the anomalous signal from sulfur atoms

  • Ensure excellent signal-to-noise ratio in the diffraction data (reported as 30.3 in the highest resolution shell for successful sulfur-SAD phasing of yeast His6)

• Crystal quality improvements:

  • Screen additives that might stabilize the protein, particularly those that mimic substrate components

  • Try microseeding to improve crystal nucleation and growth

  • Consider surface entropy reduction mutations to promote crystal contacts

• Dealing with flexible regions:

  • The regions comprising residues 18-37 and 180-186 showed missing electron density in yeast His6 structures, indicating flexibility

  • Consider co-crystallization with substrate analogs that might stabilize these regions

  • Design constructs with these flexible regions modified or removed if they're not essential for catalysis

How does the catalytic efficiency of Neurospora crassa His6 compare to homologous enzymes from other organisms?

A comprehensive comparison of His6 enzymes from different organisms should examine several kinetic parameters:

*Note: Exact values would be determined through experimental studies comparing these enzymes under standardized conditions.

Factors affecting catalytic efficiency differences may include:

• Variations in active site architecture

• Organism-specific adaptations to growth conditions

• Different regulatory mechanisms affecting enzyme dynamics

• Co-evolution with other enzymes in the histidine biosynthesis pathway

What evolutionary insights can be gained from comparing His6 enzymes across different fungal species?

Evolutionary analysis of His6 enzymes across fungal species can provide valuable insights into:

• Conservation patterns:

  • The seven glycine residues, Asp9, His57, Leu61, and Trp152 show high conservation across His6/HisA sequences

  • This conservation pattern suggests essential roles in maintaining the structural integrity and catalytic function

• Adaptation signatures:

  • Analysis of sequence variations in non-conserved regions might reveal adaptation signatures related to different ecological niches

  • Comparison of thermophilic and mesophilic fungal His6 enzymes could highlight mechanisms of temperature adaptation

• Evolutionary relationships:

  • Phylogenetic analysis based on His6 sequences may complement existing fungal taxonomy

  • This could help resolve evolutionary relationships, especially in closely related fungal species

• Functional divergence:

  • Identification of residues showing evidence of positive selection might indicate sites involved in specialized functions

  • These residues could be targets for functional studies to understand their contribution to species-specific properties

What ethical considerations and regulatory requirements apply to research with recombinant Neurospora crassa His6?

Research involving recombinant Neurospora crassa His6 enzyme must comply with institutional biosafety guidelines and relevant regulations:

• Biosafety level classification:

  • Neurospora crassa is typically classified as a Biosafety Level 1 (BSL-1) organism

  • Recombinant DNA experiments involving Neurospora must follow institutional Institutional Biosafety Committee (IBC) protocols

• Laboratory practices:

  • Standard microbiological practices for handling fungal cultures

  • Proper containment to prevent environmental release

  • Decontamination procedures for materials and waste

• Human subjects considerations:

  • If the research involves human subjects (such as testing enzyme inhibitors for potential therapeutic application), additional IRB approval would be needed

  • Research with children would require special considerations under 45 CFR part 46, subpart D

• Data sharing and research integrity:

  • Proper documentation of experimental methods to ensure reproducibility

  • Transparent reporting of all results, including negative findings

  • Sharing of plasmids and strains according to institutional material transfer agreements

How should researchers approach contradictory findings in His6 enzyme research?

When encountering contradictory findings in His6 enzyme research, a systematic approach is recommended:

• Critical evaluation of methodological differences:

  • Examine differences in protein preparation (tags, purification methods)

  • Compare assay conditions (buffer composition, pH, temperature)

  • Assess protein quality metrics (purity, activity, stability)

• Reproducibility assessment:

  • Repeat key experiments using multiple batches of protein

  • Have different researchers perform the same experiments

  • Validate findings using complementary techniques

• Collaborative resolution:

  • Engage with researchers reporting contradictory findings

  • Exchange materials and protocols to identify variables causing discrepancies

  • Consider joint publications addressing and resolving contradictions

• Biological context consideration:

  • Evaluate whether contradictions might reflect genuine biological variability

  • Consider strain differences, growth conditions, or post-translational modifications

  • Investigate whether contradictory results reflect different functional states of the enzyme

By following these approaches, researchers can transform contradictory findings into opportunities for deeper understanding of His6 enzyme structure and function.