Recombinant Sulfolobus solfataricus Putative 5-amino-6- (5-phosphoribosylamino)uracil reductase

What is 5-amino-6-(5-phosphoribosylamino)uracil reductase and what reaction does it catalyze?

5-amino-6-(5-phosphoribosylamino)uracil reductase (EC 1.1.1.193) is an enzyme that catalyzes the reduction of 5-amino-6-(5-phosphoribosylamino)uracil using NADPH as a cofactor. Specifically, it converts 5-amino-6-(5-phosphoribitylamino)uracil and NADP+ to 5-amino-6-(5-phosphoribosylamino)uracil, NADPH, and H+ . This enzyme belongs to the oxidoreductase family, acting on the CH-OH group of donors with NAD+ or NADP+ as acceptor . Its systematic name is 5-amino-6-(5-phosphoribitylamino)uracil:NADP+ 1'-oxidoreductase, and it is also known as aminodioxyphosphoribosylaminopyrimidine reductase . The enzyme plays a crucial role in riboflavin metabolism pathways.

Why is Sulfolobus solfataricus an important source organism for enzyme research?

Sulfolobus solfataricus is a hyperthermophilic archaeon that grows optimally above 80°C . This extremophile has garnered significant research interest because its enzymes (extremozymes) exhibit remarkable stability under harsh conditions that would denature most mesophilic proteins. S. solfataricus enzymes maintain functionality at high temperatures, extreme pH values, in the presence of organic solvents, and during long-term storage . These hyperthermostable enzymes often demonstrate resistance to proteolysis, detergents, and chemical denaturants, making them excellent candidates for industrial applications . Additionally, S. solfataricus utilizes unusual metabolic pathways, such as the non-phosphorylative Entner-Doudoroff pathway for glucose and galactose metabolism, featuring enzymes with broad substrate specificity .

What is the relationship between the enzyme and riboflavin metabolism?

5-amino-6-(5-phosphoribosylamino)uracil reductase is an essential enzyme in the riboflavin (vitamin B2) biosynthetic pathway . Riboflavin serves as a precursor for flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), critical cofactors for numerous redox enzymes. In the riboflavin biosynthesis pathway, this reductase catalyzes the conversion of intermediates that ultimately lead to the formation of the riboflavin molecule. The substrate, 5-amino-6-(5-phosphoribosylamino)uracil, is a key metabolite in this pathway, consisting of 5,6-diaminouracil with one hydrogen on the 6-amino function substituted by a 5-phospho-beta-D-ribosyl residue . This N-glycosyl compound participates in the complex series of reactions that transform guanosine triphosphate (GTP) and ribulose-5-phosphate into riboflavin.

What expression systems are most effective for recombinant production of S. solfataricus enzymes?

Escherichia coli is the predominant expression system for recombinant production of S. solfataricus enzymes due to its simplicity, cost-effectiveness, and high yield potential. The search results specifically mention successful expression of S. solfataricus proteins using E. coli BL21(DE3)-RIL strain . This strain contains extra copies of genes encoding tRNAs that recognize rare codons, facilitating expression of archaeal genes with different codon usage patterns.

For optimal expression of S. solfataricus proteins, including putative 5-amino-6-(5-phosphoribosylamino)uracil reductase, the following conditions are typically employed:

For S. solfataricus enzymes, including DNA polymerases and dehydrogenases, induction at 16°C for 12 hours with 0.2 mM IPTG has proven effective for maintaining protein solubility . This approach reduces inclusion body formation while allowing sufficient protein synthesis.

What purification strategies are recommended for hyperthermostable archaeal enzymes?

Purification of hyperthermostable enzymes from S. solfataricus benefits from their inherent thermal stability, allowing for heat treatment steps that denature most mesophilic host proteins. A comprehensive purification strategy typically includes:

• Heat treatment: Incubating cell lysates at 70-80°C for 10-30 minutes precipitates most E. coli host proteins while leaving the thermostable target enzyme in solution.

• Affinity chromatography: His-tagged recombinant proteins can be purified using immobilized metal affinity chromatography (IMAC). The search results indicate successful cloning of S. solfataricus genes into pET vectors, which often incorporate His-tags .

• Ion exchange chromatography: Further purification can be achieved based on the protein's isoelectric point, using either anion or cation exchange chromatography.

• Size exclusion chromatography: A final polishing step to separate proteins based on molecular size and ensure homogeneity.

The intrinsic stability of S. solfataricus enzymes also allows for more aggressive washing conditions during affinity chromatography, including higher imidazole concentrations and elevated temperatures, which can improve purity while maintaining activity.

How can I optimize recombinant expression to improve solubility and yield?

Optimizing recombinant expression of S. solfataricus putative 5-amino-6-(5-phosphoribosylamino)uracil reductase requires addressing several factors that affect protein solubility and yield:

• Temperature optimization: Lower induction temperatures (16°C) significantly improve solubility of archaeal proteins in E. coli, as demonstrated with Dpo2 and Dpo3 from S. solfataricus .

• Codon optimization: Adjusting codons to match E. coli preference or using strains with rare tRNA genes (like BL21(DE3)-RIL) can enhance translation efficiency.

• Fusion partners: Adding solubility-enhancing tags such as SUMO, MBP, or GST may improve folding and solubility.

• Induction strategy: Using lower IPTG concentrations (0.2 mM) and longer induction times promotes slower, more accurate protein folding .

• Media composition: Enriched media formulations or auto-induction media can increase biomass and protein yield.

• Co-expression with chaperones: E. coli chaperone proteins can assist in correct folding of archaeal proteins.

The specific strategy employed for Dpo2 and Dpo3 from S. solfataricus—expression in BL21(DE3)-RIL with 0.2 mM IPTG induction at 16°C for 12 hours—provides a validated starting point for optimizing expression of the putative 5-amino-6-(5-phosphoribosylamino)uracil reductase .

What methods are appropriate for assessing the enzymatic activity of 5-amino-6-(5-phosphoribosylamino)uracil reductase?

Enzymatic activity of 5-amino-6-(5-phosphoribosylamino)uracil reductase can be assessed through several complementary approaches:

• Spectrophotometric assays: The most direct method involves monitoring NADPH oxidation or NADP+ reduction at 340 nm (ε = 6220 M-1 cm-1), similar to the approach used for xylose dehydrogenase from S. solfataricus . This method works because the enzyme catalyzes a reaction involving NADP+ as a substrate, producing NADPH .

• HPLC analysis: Quantification of substrate depletion or product formation through high-performance liquid chromatography provides precise measurement of enzyme activity.

• Coupled enzyme assays: For complex kinetic studies, the reductase can be coupled with other enzymes in the riboflavin pathway to monitor flux through multiple steps.

For S. solfataricus enzymes, activity assays should be performed at elevated temperatures (typically 70-80°C) to reflect their optimal conditions . A typical spectrophotometric assay could be structured as follows:

One unit of enzyme activity would be defined as the amount of enzyme required to produce 1 μmol of NADPH per minute under these conditions .

How does the structure of the enzyme contribute to its thermostability?

Though specific structural information for S. solfataricus putative 5-amino-6-(5-phosphoribosylamino)uracil reductase is not directly provided in the search results, insights can be drawn from related S. solfataricus enzymes. Thermostability in archaeal enzymes typically results from several structural features:

• Increased ionic interactions: Higher number of salt bridges stabilizes the tertiary structure at elevated temperatures.

• Enhanced hydrophobic core packing: More compact hydrophobic cores reduce thermal motion and unfolding.

• Reduced surface loops and flexibility: Shorter surface loops and reduced flexibility in non-catalytic regions increase rigidity.

• Higher proportion of α-helices: Secondary structures with more extensive hydrogen bonding networks resist thermal denaturation.

• Disulfide bonds: Strategic disulfide bonds can significantly enhance thermostability.

The crystal structure of glucose dehydrogenase from S. solfataricus (PDB: 2CD9) provides an example of structural adaptations in thermophilic enzymes . This enzyme demonstrates substrate promiscuity while maintaining stability at high temperatures. Similar structural features likely contribute to the thermostability of putative 5-amino-6-(5-phosphoribosylamino)uracil reductase from the same organism.

What are the kinetic properties of the enzyme at various temperatures?

Hyperthermophilic enzymes from S. solfataricus typically display unique temperature-dependent kinetic profiles. While specific kinetic data for the putative 5-amino-6-(5-phosphoribosylamino)uracil reductase is not provided in the search results, general patterns observed in other S. solfataricus enzymes can be informative:

• Temperature optimum: Typically between 70-90°C, corresponding to the optimal growth temperature of S. solfataricus.

• Activity at lower temperatures: Contrary to common assumptions, hyperthermophilic enzymes can retain significant activity at lower temperatures. The search results mention that SsoPox, another enzyme from S. solfataricus, shows potential to be active at subzero temperatures .

• Thermodynamic parameters: Activation energy (Ea) values for thermophilic enzymes are often higher than mesophilic counterparts, resulting in steeper temperature-activity profiles.

• Substrate binding: KM values may increase with temperature due to weaker substrate binding at higher temperatures, but this is often compensated by higher kcat values.

Expected kinetic behavior of the putative reductase might follow this pattern observed for xylose dehydrogenase from S. solfataricus :

This table represents a typical pattern rather than specific values for the putative 5-amino-6-(5-phosphoribosylamino)uracil reductase.

How can directed evolution be applied to enhance specific properties of the recombinant enzyme?

Directed evolution represents a powerful approach for enhancing specific properties of recombinant 5-amino-6-(5-phosphoribosylamino)uracil reductase from S. solfataricus. The search results mention that directed evolution has driven the "third wave of biocatalysis," highlighting its importance in enzyme engineering . A comprehensive directed evolution strategy would include:

• Mutant library generation:

  • Error-prone PCR with controlled mutation rates

  • Site-saturation mutagenesis at active site or substrate-binding residues

  • DNA shuffling between homologous reductases from different thermophiles

• High-throughput screening methods:

  • Colorimetric assays monitoring NADPH production

  • Growth-based selection in auxotrophic strains requiring functional riboflavin pathway

  • Fluorescence-activated cell sorting (FACS) using fluorogenic substrate analogs

• Iterative improvement:

  • Multiple rounds of mutation and selection

  • Combining beneficial mutations through site-directed mutagenesis

The search results describe a successful example: SsoPox-W263I, a variant with increased lactonase and phosphotriesterase activities . This demonstrates the feasibility of improving S. solfataricus enzymes through targeted mutations. Similar approaches could enhance the putative 5-amino-6-(5-phosphoribosylamino)uracil reductase for specific activities, substrate range, or stability under different conditions.

What interactions does the enzyme have with other components of the riboflavin biosynthetic pathway?

The putative 5-amino-6-(5-phosphoribosylamino)uracil reductase operates within the complex riboflavin biosynthetic pathway, where coordination between multiple enzymes is crucial. While specific interaction data isn't provided in the search results, we can infer potential interactions based on pathway organization:

• Metabolic channeling: The enzyme likely participates in substrate channeling complexes where intermediates are passed directly between enzymes without diffusing into the bulk cytosol.

• Protein-protein interactions: Physical interactions with upstream and downstream enzymes in the pathway could regulate activity or enhance efficiency.

• Regulatory interactions: Feedback inhibition by riboflavin or its derivatives might modulate enzyme activity.

• Cofactor sharing: Coordination of NADP+/NADPH utilization with other redox enzymes in the pathway might occur.

To investigate these interactions, researchers could employ:

• Pull-down assays with tagged recombinant enzyme

• Crosslinking studies followed by mass spectrometry

• Surface plasmon resonance (SPR) to measure binding kinetics

• Isothermal titration calorimetry (ITC) for thermodynamic parameters of interactions

Understanding these interactions could provide insights into the regulation of riboflavin biosynthesis in S. solfataricus and potentially reveal novel regulatory mechanisms unique to archaea.

How do variations in the active site architecture affect substrate specificity?

Active site architecture plays a crucial role in determining substrate specificity of enzymes. While specific structural information about the putative 5-amino-6-(5-phosphoribosylamino)uracil reductase is not provided in the search results, insights can be drawn from the crystal structure of glucose dehydrogenase from S. solfataricus, which exhibits substrate promiscuity .

In glucose dehydrogenase, substrate promiscuity is linked to specific structural features:

• The enzyme selectively binds the beta-anomeric, pyranose form of sugars

• It requires specific stereochemistry at C2 and C3 positions

• It shows activity with diverse sugars including glucose, galactose, xylose, and L-arabinose

For the putative 5-amino-6-(5-phosphoribosylamino)uracil reductase, key active site residues likely include:

• NADP+ binding pocket: Residues that interact with the nicotinamide ring, ribose, and phosphate groups

• Substrate binding region: Residues recognizing the uracil moiety and phosphoribosylamino group

• Catalytic residues: Typically includes acidic or basic amino acids that facilitate hydride transfer

Structural studies, such as X-ray crystallography of enzyme-substrate complexes, would be necessary to fully characterize how active site architecture influences specificity. Site-directed mutagenesis of predicted key residues could then validate their roles and potentially alter substrate preferences.

What are the potential biotechnological applications of this enzyme?

The putative 5-amino-6-(5-phosphoribosylamino)uracil reductase from S. solfataricus has several potential biotechnological applications stemming from its unique properties as a hyperthermostable enzyme:

• Riboflavin production: The enzyme could be employed in engineered pathways for enhanced production of riboflavin, an important vitamin with applications in food fortification, animal feed supplements, and pharmaceuticals.

• Biocatalysis: Like other thermostable enzymes from S. solfataricus, this reductase could serve as a robust biocatalyst for industrial redox reactions under harsh conditions. Hyperthermophilic enzymes are particularly valuable for industrial applications due to their resistance to organic solvents, high temperatures, and long-term storage stability .

• Biosensors: The enzyme's NADP+/NADPH-dependent activity could be harnessed in biosensor development for detecting specific metabolites.

• Synthetic biology: As part of engineered metabolic pathways, the enzyme could contribute to the production of novel compounds through its redox capabilities.

• Model system: The enzyme serves as an excellent model for studying enzyme adaptation to extreme conditions and for exploring the relationship between thermostability and activity.

The extreme stability of S. solfataricus enzymes makes them particularly valuable for industrial processes requiring prolonged reactions under harsh conditions .

How does the archaeal enzyme differ from its bacterial or eukaryotic counterparts?

While specific comparative information is not provided in the search results, archaeal enzymes typically exhibit distinct characteristics compared to their bacterial and eukaryotic counterparts:

The search results indicate that S. solfataricus enzymes often show activity at a broad temperature range, including unexpectedly high activity at lower temperatures despite being from hyperthermophiles . This characteristic might also apply to the putative 5-amino-6-(5-phosphoribosylamino)uracil reductase, distinguishing it from mesophilic homologs.

The enzyme from E. coli is known to participate in riboflavin metabolism , but its structural and kinetic properties likely differ significantly from the archaeal version, reflecting adaptation to different cellular environments and evolutionary pressures.

What are common challenges in expressing archaeal enzymes in bacterial hosts and how can they be overcome?

Expressing archaeal enzymes in bacterial hosts presents several challenges, with specific solutions based on the search results and general recombinant protein techniques:

• Codon usage differences:

  • Challenge: Archaeal genes often contain codons rarely used in E. coli

  • Solution: Use E. coli strains like BL21(DE3)-RIL that contain extra copies of rare tRNA genes or perform codon optimization of the gene sequence

• Protein folding issues:

  • Challenge: Archaeal proteins may misfold in mesophilic hosts

  • Solution: Lower induction temperature (16°C) and extend induction time (12 hours) as demonstrated for Dpo2 and Dpo3

• Post-translational modifications:

  • Challenge: Archaea may have different modification patterns than bacteria

  • Solution: Consider eukaryotic expression systems for enzymes requiring specific modifications

• Protein toxicity:

  • Challenge: Archaeal enzyme expression may be toxic to bacterial host

  • Solution: Use tightly controlled expression systems and lower IPTG concentrations (0.2 mM)

• Inclusion body formation:

  • Challenge: High expression levels can lead to aggregation

  • Solution: Fusion with solubility-enhancing tags or co-expression with chaperones

The successful expression of multiple S. solfataricus proteins as described in the search results provides validated approaches for overcoming these challenges . Specifically, the use of BL21(DE3)-RIL cells, appropriate antibiotic selection, reduced induction temperature (16°C), and moderate IPTG concentration (0.2 mM) has proven effective for S. solfataricus enzymes.

How can I detect and troubleshoot enzyme inactivation during experimental procedures?

Detecting and troubleshooting inactivation of the putative 5-amino-6-(5-phosphoribosylamino)uracil reductase requires systematic analysis:

• Activity monitoring: Regular activity assays during purification and storage are essential. For this enzyme, following NADPH production spectrophotometrically at 340 nm provides a direct measure of activity retention.

• Thermal stability assays: Differential scanning calorimetry (DSC) or thermal shift assays can detect changes in thermal stability, which often correlate with activity loss.

• Reactivation attempts: The search results mention that bacterial secreted materials could reactivate heat-inactivated SsoPox-W263I . This suggests testing various buffer additives for reactivation potential:

  • Divalent metal ions (Mg2+, Mn2+, Ca2+)

  • Reducing agents (DTT, β-mercaptoethanol)

  • Osmolytes (glycerol, trehalose)

  • Bacterial lysates or specific cellular components

• Structural analysis: Circular dichroism (CD) spectroscopy can detect significant changes in secondary structure associated with inactivation.

• Storage optimization: Testing various storage conditions can prevent activity loss:

  • Different buffers and pH values

  • Addition of stabilizing agents (glycerol, BSA)

  • Lyophilization versus liquid storage

  • Various temperatures (-80°C, -20°C, 4°C)

The exceptional stability of S. solfataricus enzymes under harsh conditions suggests that the putative reductase may resist typical inactivation factors, but proper experimental design remains crucial for maintaining activity .

What strategies can be employed to improve enzyme stability for long-term storage and repeated use?

Improving stability of the putative 5-amino-6-(5-phosphoribosylamino)uracil reductase for long-term storage and repeated use can be approached through several strategies:

• Buffer optimization: Testing various buffer compositions for maximal stability:

  • Buffer type (phosphate, HEPES, Tris)

  • pH optimization (typically 7.0-8.0 for long-term storage)

  • Ionic strength adjustment

• Stabilizing additives: Several compounds can enhance enzyme stability:

  • Glycerol (20-50%) prevents freezing damage

  • Reducing agents (DTT, β-mercaptoethanol) maintain cysteine residues

  • Bovine serum albumin (BSA) as a carrier protein

  • Metal ions if required for structural integrity

• Immobilization: Enzyme immobilization can dramatically improve stability:

  • Covalent attachment to solid supports

  • Entrapment in polymeric matrices

  • Cross-linked enzyme aggregates (CLEAs)

• Storage conditions: Optimizing physical storage parameters:

  • Flash freezing in liquid nitrogen before -80°C storage

  • Lyophilization with appropriate cryoprotectants

  • Small aliquots to avoid freeze-thaw cycles

• Protein engineering: Introducing stabilizing mutations:

  • Disulfide bonds at strategic positions

  • Surface charge optimization

  • Rigidifying flexible regions

The search results indicate that SsoPox from S. solfataricus showed remarkable resistance to harsh conditions, including various organic solvents, sterilization processes, and storage conditions . Similar properties might be expected for the putative 5-amino-6-(5-phosphoribosylamino)uracil reductase, making it potentially suitable for biotechnological applications requiring robust enzymes.