Recombinant Picrophilus torridus Phosphoribosylformylglycinamidine synthase 2 (purL), partial

What is Phosphoribosylformylglycinamidine synthase (FGARAT) and its role in purine biosynthesis?

Phosphoribosylformylglycinamidine synthase (FGARAT), encoded by the purL gene, catalyzes the fourth step in the purine biosynthetic pathway. Specifically, FGARAT transforms formylglycinamide ribonucleotide (FGAR), ATP, and glutamine into formylglycinamidine ribonucleotide (FGAM), ADP, inorganic phosphate, and glutamate . This enzyme is essential for the synthesis of purines, which are nitrogenous bases (adenine and guanine) that compose DNA and RNA, making it critical for cellular function across all domains of life. In extremophiles like Picrophilus torridus, this enzyme must function under harsh conditions, including high temperatures (55-60°C) and extremely acidic environments (pH 0.7) . Understanding FGARAT's structure and function provides insights into how essential cellular processes adapt to extreme environments.

How does P. torridus purL likely compare structurally to other known forms of FGARAT?

FGARAT exists in three main structural forms across different organisms. The monomeric form (lgpurL) is approximately 140 kDa with about 1300 amino acids and is found primarily in gamma and beta proteobacteria and eukaryotes . The heterotetrameric form (smpurL) is smaller at about 80 kDa with approximately 750 amino acids and requires two additional subunits (PurQ and PurS) to function properly . A heterodimeric form has also been identified where PurL and PurS subunits are fused to create one large "PurL" subunit, with a separate polypeptide for PurQ . Although the specific structure of P. torridus purL is not fully characterized in the provided research, as an archaeal organism with a typical replication apparatus, its purL likely has adaptations that enable function in extreme acidic and high-temperature environments. Comparative structural analysis would be valuable for understanding how this enzyme maintains catalytic activity under such challenging conditions.

What are the optimal growth conditions for P. torridus cultures when studying purL expression?

Picrophilus torridus thrives at 55 to 60°C and pH 0.7, making it one of the most acidophilic organisms known . For laboratory culture, researchers typically harvest P. torridus cells from cultures at an optical density at 600 nm (OD600) of approximately 0.4, which represents mid-logarithmic growth phase . The organism was originally isolated from a dry solfataric field in northern Japan . When studying purL expression, maintaining these extreme conditions is crucial for obtaining physiologically relevant results. Culture media must be carefully formulated to maintain the acidic pH required by this organism. Temperature control during incubation is also critical to ensure optimal growth and proper protein expression. Shifts from these optimal conditions could alter gene expression patterns and potentially affect the structure and function of the recombinant purL protein.

What is the recommended protocol for cloning the purL gene from P. torridus?

Based on established protocols for cloning genes from P. torridus, the following methodological approach is recommended for purL:

• Genomic DNA isolation: Extract genomic DNA from P. torridus culture using a freeze-thaw method. Cells should be collected from liquid medium and subjected to alternating freezing (in liquid nitrogen or at -80°C) and thawing cycles to disrupt the cell walls .

• PCR amplification: Design primers targeting the purL gene based on the genomic sequence available in databases like NCBI. Include appropriate restriction sites (such as NdeI and HindIII) to facilitate subsequent cloning steps. The PCR reaction should use a high-fidelity polymerase suitable for GC-rich templates, such as Phusion DNA polymerase .

• Initial cloning: Clone the PCR product into an intermediate vector such as pCR2.1 TOPO for sequence verification . This step is crucial to confirm the accuracy of the amplified sequence before proceeding to expression constructs.

• Subcloning into expression vector: Following sequence verification, subclone the purL gene into an appropriate expression vector such as pET-28a for protein production in E. coli, or pASK-IBA43plus which has been successfully used for other P. torridus proteins .

• Sequence verification: Confirm the final construct by restriction digestion and DNA sequencing to ensure the gene is correctly inserted and contains no mutations .

Which expression systems have proven effective for recombinant extremophile proteins like P. torridus purL?

For recombinant expression of extremophile proteins such as those from P. torridus, several expression systems have been demonstrated as effective, each with specific advantages:

What purification strategy is most suitable for obtaining high-purity recombinant P. torridus purL?

A multi-step purification strategy is recommended for obtaining high-purity recombinant P. torridus purL, based on successful approaches with other extremophile proteins:

• Initial affinity chromatography: If expressed with an MBP tag, use amylose resin affinity chromatography as demonstrated for P. torridus Orc1/Cdc6 . Alternatively, if expressed with a His-tag, use nickel affinity chromatography.

• Ion exchange chromatography: Given the acidophilic nature of P. torridus proteins, a carefully pH-controlled ion exchange step can separate the target protein from contaminants with different charge profiles.

• Size exclusion chromatography: As a final polishing step, size exclusion can separate aggregates and further purify the protein based on molecular weight.

• Stability considerations: Throughout purification, maintain buffer conditions that stabilize the protein. For acidophilic proteins, this may require buffers at lower pH than typically used for mesophilic proteins.

• Quality assessment: Analyze protein purity by SDS-PAGE and confirm identity by Western blotting using specific antibodies or mass spectrometry.
  The purification protocol may need adjustment based on the specific properties of P. torridus purL and the expression system used. Monitoring enzyme activity throughout purification is advisable to ensure the protein remains functional.

What analytical methods are most informative for characterizing the structure of P. torridus purL?

Multiple complementary analytical methods should be employed to fully characterize the structure of P. torridus purL:

How can researchers effectively measure the enzymatic activity of recombinant P. torridus purL?

The enzymatic activity of recombinant P. torridus purL can be measured using several approaches:

• Coupled enzyme assay: Monitor the conversion of FGAR to FGAM coupled to the consumption of ATP using auxiliary enzymes that produce a detectable signal.

• HPLC-based assay: Quantify the conversion of substrate (FGAR) to product (FGAM) using high-performance liquid chromatography, which allows direct measurement of reaction progress.

• Radioactive assay: Use radiolabeled substrates (e.g., 14C-glutamine) to track the incorporation of the amide group into the product.

• ADP formation assay: Measure the production of ADP, which is stoichiometrically coupled to FGAM formation, using commercially available kits.
  Key experimental considerations include:

• Ensuring proper pH (may need acidic conditions reflecting P. torridus' natural environment)

• Testing activity across a range of temperatures (particularly 55-60°C, P. torridus' optimal growth temperature)

• Including appropriate controls to account for non-enzymatic reactions under extreme conditions

• Verifying substrate stability under the assay conditions
  These assays should be performed under various pH and temperature conditions to determine the optimal parameters for enzyme activity, which are expected to reflect P. torridus' acidophilic and thermophilic nature.

What approaches can determine how P. torridus purL maintains stability and function in extreme conditions?

Several experimental approaches can elucidate the mechanisms by which P. torridus purL maintains stability and function in extreme conditions:

• Comparative sequence analysis: Identify unique amino acid compositions and motifs by comparing P. torridus purL with homologs from mesophilic and other extremophilic organisms. Look for enrichment of specific residues associated with acid stability (e.g., decreased number of acid-sensitive residues) and thermostability (e.g., increased hydrophobic interactions, disulfide bonds).

• Thermal shift assays: Measure protein unfolding temperatures using differential scanning fluorimetry (DSF) or differential scanning calorimetry (DSC) across a range of pH values to quantify thermostability.

• Limited proteolysis: Assess structural rigidity and flexibility by subjecting the protein to controlled proteolytic digestion under various conditions.

• Site-directed mutagenesis: Systematically mutate residues hypothesized to contribute to extreme condition adaptation and measure the impact on stability and activity.

• Molecular dynamics simulations: Model protein behavior in silico under extreme pH and temperature conditions to identify key stabilizing interactions.

• Long-term stability studies: Monitor activity retention during prolonged incubation at various temperatures and pH values to quantify kinetic stability.
  These approaches would provide insight into the molecular adaptations that allow P. torridus purL to function at pH 0.7 and temperatures of 55-60°C , potentially identifying principles that could be applied to engineer stability in other proteins.

How might the study of P. torridus purL inform our understanding of enzyme evolution in extreme environments?

The study of P. torridus purL provides a valuable model for understanding enzyme evolution in extreme environments for several reasons. First, comparing the P. torridus purL sequence and structure with homologs from mesophiles and other extremophiles can reveal convergent and divergent evolutionary strategies for adaptation to acidic, high-temperature environments. Second, the purine biosynthesis pathway is highly conserved across all domains of life, making purL an excellent candidate for studying how essential metabolic functions are maintained despite environmental challenges .
Research has observed a high rate of horizontal gene transfer among archaeal genomes to prokaryotes, which may contribute to the spread of adaptive features . Examining whether P. torridus acquired its purL through horizontal gene transfer or through gradual adaptation could provide insights into the evolutionary mechanisms of extremophile adaptation. Additionally, analyzing the different forms of purL (monomeric, heterotetrameric, and heterodimeric) across different organisms can help trace the evolutionary history of this essential enzyme and determine whether structural simplification or complexification correlates with adaptation to extreme environments .

What are the challenges in crystallizing recombinant P. torridus purL, and how might they be overcome?

Crystallizing proteins from extremophiles like P. torridus presents unique challenges due to their adapted biophysical properties. The following table outlines major challenges and potential solutions specific to P. torridus purL crystallization:

How does the intermediate size of purL in certain organisms inform our understanding of protein domain evolution?

The existence of purL proteins of intermediate size, between the large monomeric form (lgpurL, ~140 kDa) and the small form requiring additional subunits (smpurL, ~80 kDa), provides valuable insights into protein domain evolution . The research indicates that the purL gene can encode proteins with different subunit structures: a monomeric form with all components fused together, a heterotetrameric form with separate polypeptides for each subunit, and a heterodimeric form where some but not all subunits are fused .
This domain architecture diversity suggests an evolutionary pathway for complex enzymes. The heterodimeric form, where PurL and PurS are fused while PurQ remains separate, represents a potential intermediate stage in the evolution from a multi-subunit complex to a fully fused single polypeptide . This supports the hypothesis that complex proteins may have evolved through gene fusion events, where genes encoding separate subunits gradually combined.
Functional analysis of these intermediate forms could reveal whether such fusions confer advantages in certain environments, such as increased stability in extreme conditions or improved catalytic efficiency through proximity effects. Additionally, studying the interfaces between fused domains may provide insights into protein-protein interaction mechanisms that are essential for function in the non-fused forms.

What biotechnological applications might benefit from P. torridus purL research?

Research on P. torridus purL has several potential biotechnological applications:

• Biocatalysis under extreme conditions: Enzymes from extremophiles like P. torridus can catalyze reactions under harsh industrial conditions where conventional enzymes would denature. Understanding how purL maintains function at pH 0.7 and high temperatures could inform the development of robust biocatalysts for acid-mediated chemical processes .

• Protein engineering: Identifying the specific structural features that confer acid stability to P. torridus purL could provide principles for engineering acid resistance into other proteins of industrial importance.

• Biosynthetic pathway enhancement: The purine biosynthetic pathway is crucial not only for nucleic acid synthesis but also for producing precursors for various biomolecules. Enhanced understanding of FGARAT could enable metabolic engineering of microorganisms to increase production of purines or related compounds.

• Therapeutic applications: Research has linked purL function to toxin production in certain bacteria, such as Clostridium difficile . Understanding the conserved motifs and regulatory mechanisms of purL could potentially inform new antimicrobial strategies targeting purine biosynthesis in pathogenic organisms.

• Structural biology advancements: The unique properties of extremophile proteins like P. torridus purL challenge and expand our understanding of protein folding and stability principles, potentially leading to improved computational prediction tools and protein design algorithms.
  These applications highlight the value of basic research on extremophile enzymes like P. torridus purL beyond their immediate biochemical characterization.

What strategies can address low expression yields of recombinant P. torridus purL?

Low expression yields of recombinant P. torridus purL can be addressed through multiple optimization strategies:

• Codon optimization: Adapt the P. torridus purL coding sequence to the codon usage preference of the expression host, particularly since extremophile genes often have different codon biases compared to standard expression hosts like E. coli.

• Expression vector selection: Test multiple expression vectors with different promoter strengths. For challenging proteins, vectors that enable tight control over expression (such as pASK-IBA series used successfully for other P. torridus proteins) may be preferable .

• Fusion partners: Express the protein with solubility-enhancing fusion partners such as MBP, SUMO, or thioredoxin. MBP fusions have been successfully used for other P. torridus proteins .

• Host strain selection: Screen multiple E. coli strains designed for expressing difficult proteins, such as C41(DE3), C43(DE3), or Rosetta strains that supply rare tRNAs.

• Culture conditions optimization: Systematically vary:

  • Induction temperature (typically lower temperatures improve folding)

  • Induction duration

  • Inducer concentration

  • Media composition (rich vs. minimal media)

  • Cell density at induction time

• Chaperone co-expression: Co-express molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE) to assist protein folding.

• Periplasmic or secretory expression: Direct the protein to the periplasmic space or culture medium to potentially improve folding and reduce proteolytic degradation.
  Maintaining detailed records of expression trials and systematically optimizing these parameters will help identify conditions that yield functional P. torridus purL.

How can researchers distinguish between the different forms of purL when analyzing genomic data?

To distinguish between different forms of purL (large monomeric form, small heterotetrameric form, or intermediate heterodimeric form) when analyzing genomic data, researchers should employ a systematic bioinformatic approach:

• Sequence length analysis: The monomeric form (lgpurL) typically contains approximately 1300 amino acids, while the small form (smpurL) contains approximately 750 amino acids . Intermediate forms may have lengths between these values, with the example in the research containing 974 amino acids .

• Domain architecture analysis: Use tools like PFAM, InterPro, or CDD to identify characteristic domains:

  • Monomeric form: Contains regions corresponding to all subunits (PurS, PurL, PurQ) in a single polypeptide

  • Small form: Contains only the core catalytic domain

  • Intermediate form: May show fusion of some but not all domains

• Genomic context examination: Check for nearby genes encoding potential partner proteins:

  • For small purL forms, look for adjacent purQ and purS genes which encode necessary partner proteins

  • Absence of these genes suggests a monomeric or intermediate form that doesn't require separate subunits

• Phylogenetic analysis: Construct phylogenetic trees of purL sequences to place the query sequence in evolutionary context:

  • Large forms typically appear in gamma and beta proteobacteria and eukaryotes

  • Small forms are common in many other prokaryotes

• Structural homology modeling: Generate models based on solved structures of known purL forms to predict the likely quaternary structure of the query protein.
  This multi-faceted approach allows researchers to classify purL genes accurately and predict their functional requirements in different organisms.

What key knowledge gaps remain in our understanding of purL genes in extremophiles?

Several significant knowledge gaps remain in our understanding of purL genes in extremophiles like Picrophilus torridus:

• Structure-function relationships: The precise structural adaptations that allow P. torridus purL to function at extremely low pH (0.7) and high temperatures (55-60°C) remain poorly characterized . How do these adaptations differ from those in thermophiles that operate at neutral pH?

• Evolutionary history: The evolutionary path leading to different forms of purL (monomeric, heterotetrameric, and heterodimeric) across different extremophiles is not fully mapped . Did these different forms evolve independently in response to similar environmental pressures, or through horizontal gene transfer?

• Regulatory mechanisms: How extremophiles regulate purL expression and activity in response to environmental changes remains largely unknown. Do they employ unique regulatory mechanisms compared to mesophiles?

• Protein-protein interactions: For heterodimeric and heterotetrameric forms, the specific interactions between purL and its partner proteins (PurQ, PurS) in extreme conditions are not well characterized . How do these interactions differ from those in mesophilic organisms?

• Metabolic integration: How the purine biosynthesis pathway integrates with other metabolic networks in extremophiles under stress conditions is poorly understood. Does pathway regulation differ significantly from that in mesophiles?

• Comparative performance: Systematic comparisons of catalytic efficiency, substrate specificity, and stability between purL enzymes from different extremophiles are lacking.
  Addressing these gaps would significantly enhance our understanding of how essential metabolic functions adapt to extreme environments.

What potential applications exist for engineered purL variants based on extremophile research?

Research on P. torridus purL and other extremophile variants provides inspiration for engineering novel enzymes with enhanced properties. Potential applications include:

• pH-resistant biocatalysts: Engineering acid-stable enzymes for industrial processes that operate under acidic conditions, such as biofuel production from lignocellulosic biomass pretreatment or food processing.

• Thermostable enzymes for industrial processes: Developing heat-resistant variants that function efficiently at elevated temperatures, reducing cooling requirements and microbial contamination risks in industrial bioreactors.

• Designer purL enzymes for metabolic engineering: Creating optimized purL variants for synthetic biology applications to enhance purine biosynthesis in production strains, potentially improving yields of nucleotide-derived products.

• Biosensors for extreme environments: Utilizing engineered purL proteins as components of biosensors designed to function in challenging environments such as acid mine drainage, hot springs, or industrial waste streams.

• Therapeutic enzyme development: Applying extremophile-derived stability principles to engineer therapeutic enzymes with enhanced shelf-life and resistance to gastrointestinal conditions.

• Agricultural applications: Developing enzymes for soil amendment in acidic soils or for crop protection formulations that must withstand challenging environmental conditions.

• Space and astrobiology research: Creating biological systems capable of functioning in extraterrestrial environments characterized by extreme conditions. These applications demonstrate how fundamental research on extremophile enzymes like P. torridus purL can translate into practical biotechnological innovations addressing significant industrial and environmental challenges.