Recombinant Thermotoga maritima Protein PyrBI (pyrBI), partial

What is the Thermotoga maritima PyrBI protein and what is its function?

The Thermotoga maritima PyrBI protein is an aspartate carbamoyltransferase (ATCase) that catalyzes a key step in pyrimidine biosynthesis. Unlike many bacterial ATCases, the T. maritima enzyme combines both catalytic and regulatory functions on a single polypeptide that assembles into trimers . The enzyme has EC number 2.1.3.2 and is also known as aspartate transcarbamylase . It catalyzes the conversion of aspartate and carbamoyl phosphate to N-carbamoylaspartate and phosphate, representing the first committed step in the pyrimidine biosynthetic pathway.

What is unique about Thermotoga maritima as an organism?

Thermotoga maritima is a hyperthermophilic, anaerobic bacterium first discovered in marine geothermal sediments near Vulcano, Italy . It represents one of the deepest and most slowly evolving lineages within the bacterial domain . Key characteristics include:

• Growth temperature range of 55-90°C with an optimal temperature of 80°C

• Rod-shaped, gram-negative morphology with a distinctive sheath-like envelope (toga)

• Ability to produce hydrogen by fermenting various carbohydrates including cellulose and xylan

• Despite being classified as an anaerobe, it can tolerate low oxygen concentrations (up to 0.5% v/v)

• Possesses adaptations for surviving extreme temperatures, including specialized enzymes and protective mechanisms

How is the T. maritima pyrBI gene organized compared to other bacteria?

In Thermotoga maritima, the catalytic and regulatory functions that are typically carried out by specialized polypeptides in class B ATCases (such as those from Enterobacteriaceae, Vibrio sp., and Archaea) are fused into a single polypeptide . Alignments with 31 ATCase genes revealed that T. maritima PyrBI protein has the highest identity (51.7%) with Treponema denticola ATCase . This structural similarity between T. maritima and T. denticola (which is not a thermophile) suggests possible horizontal gene transfer between these organisms .

The gene organization differs from E. coli, where pyrB and pyrI are separate genes encoding the catalytic and regulatory chains, respectively. In T. maritima, these functions are encoded by a single gene .

What expression systems are effective for producing recombinant T. maritima PyrBI?

Based on the research literature, the following expression systems have been successfully used for T. maritima proteins:

• E. coli expression systems: Most studies utilize E. coli as the heterologous host for T. maritima protein expression . For instance, a mutant E. coli strain with a pyrBI deletion was successfully complemented with the T. maritima pyrBI gene in the pKK 223-3 vector .

• Inducible expression systems: For controlled expression, the T7 RNA polymerase system has been effective for other T. maritima proteins, such as dihydrofolate reductase (DHFR) . The gene was cloned into pET3a vector under control of the T7 RNA polymerase promoter, resulting in high-level expression in E. coli BL21(DE3) cells .

• Yeast expression systems: While not specifically mentioned for PyrBI, some T. maritima proteins have been successfully expressed in yeast systems, which may provide appropriate post-translational modifications .

What are the recommended storage and handling conditions for recombinant T. maritima PyrBI?

For optimal stability and activity, recombinant T. maritima PyrBI should be handled according to these guidelines:

• Store at -20°C; for extended storage, conserve at -20°C or -80°C

• Avoid repeated freezing and thawing; prepare working aliquots and store at 4°C for up to one week

• Reconstitute lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Consider adding glycerol (5-50% final concentration) when preparing aliquots for long-term storage

• The protein demonstrates high thermostability, which can be leveraged during purification to remove heat-labile contaminants

The shelf life is typically 6 months at -20°C/-80°C for liquid formulations and 12 months for lyophilized preparations .

What purification strategies are most effective for T. maritima proteins?

While specific purification protocols for PyrBI are not detailed in the search results, successful approaches for other T. maritima proteins include:

• Heat treatment: Exploiting the thermostability of T. maritima proteins by heating crude extracts (e.g., 20 min at 60-80°C) to denature and precipitate E. coli host proteins while leaving the thermostable target protein in solution

• Chromatographic techniques: Following heat treatment, further purification can be achieved through:

  • Ion exchange chromatography

  • Affinity chromatography (particularly for tagged proteins)

  • Size exclusion chromatography (which can also provide information about oligomeric state)

• Folate-affinity chromatography: For specific proteins like DHFR, specialized affinity matrices have been used successfully

What is known about the oligomeric structure of T. maritima PyrBI?

The T. maritima PyrBI protein assembles into trimers, which is a distinctive feature compared to many other bacterial ATCases . This trimeric organization combines both catalytic and regulatory functions on each monomer.

While specific information about PyrBI oligomerization is limited in the search results, studies on other T. maritima proteins provide insights into potential factors affecting oligomerization:

• Buffer pH can significantly impact quaternary structure, as demonstrated with T. maritima DHFR, which exists as a dimer at neutral pH but as a monomer at low pH (4.3) in the absence of NaCl

• Salt concentration affects oligomerization; NaCl promotes dimer formation in DHFR at both low and high pH values

• The C-terminal domain often plays a crucial role in maintaining the oligomeric state of T. maritima proteins

How does the thermal stability of T. maritima PyrBI compare to similar enzymes from mesophilic organisms?

As a protein from a hyperthermophilic organism, T. maritima PyrBI exhibits remarkable thermal stability compared to mesophilic counterparts. While specific comparative data for PyrBI are not provided in the search results, insights from other T. maritima proteins suggest:

• Enhanced structural stabilization: T. maritima proteins often contain extensive intra- and inter-subunit salt bridges that contribute to thermostability

• Activity at high temperatures: Unlike mesophilic enzymes that denature at elevated temperatures, T. maritima enzymes maintain activity at temperatures approaching the organism's optimal growth temperature (~80°C)

• Thermostability as a purification advantage: The significant thermostability difference between T. maritima proteins and E. coli host proteins allows for simple purification by heat treatment

What specific molecular adaptations contribute to the thermostability of T. maritima proteins?

Several structural features contribute to the exceptional thermostability of T. maritima proteins:

• Extensive salt bridge networks: Intra- and inter-subunit salt bridges provide electrostatic stabilization that maintains protein structure at elevated temperatures

• Compact folding: Thermophilic proteins often display more compact structures with fewer and smaller cavities

• Increased hydrophobic interactions: The hydrophobic core is often strengthened in thermophilic proteins

• Domain organization: In some T. maritima proteins, like SurE, the C-terminal domain is primarily important for maintaining oligomeric state, while the N-terminal domain serves as the functional domain

• Protection mechanisms: T. maritima also upregulates proteins involved in reactive oxygen species detoxification, iron-sulfur center synthesis/repair, and cysteine biosynthesis pathways when exposed to environmental stressors

How can researchers study the regulatory mechanisms of T. maritima ATCase?

To investigate the regulatory mechanisms of T. maritima ATCase, researchers can employ several approaches:

• Site-directed mutagenesis: Identify and mutate conserved residues in the regulatory domain to assess their impact on enzyme activity and allosteric regulation

• Kinetic analysis: Measure enzyme activity under varying substrate concentrations and in the presence of potential allosteric regulators (such as nucleotides) to determine kinetic parameters and regulatory models

• Structural biology approaches: X-ray crystallography or cryo-EM to resolve the protein structure in different states (e.g., with and without allosteric effectors)

• Comparative genomics: Analyze the sequence and structure of T. maritima PyrBI in relation to ATCases from other species to identify conserved regulatory motifs

• Heterologous reconstitution experiments: Mix protein components from different species to identify which domains contribute to specific regulatory properties, as demonstrated with RNase P studies

What insights does T. maritima PyrBI provide about horizontal gene transfer and bacterial evolution?

The T. maritima PyrBI protein presents an intriguing case study for horizontal gene transfer (HGT) and bacterial evolution:

• Unexpected sequence similarity: Despite T. maritima being a hyperthermophile, its PyrBI protein shows highest identity (51.7%) with Treponema denticola ATCase, a non-thermophilic organism, suggesting HGT between these evolutionary distant bacteria

• Genomic evidence of HGT: Studies have found compelling evidence for recombination between different Thermotoga lineages despite substantial ecological and genetic differences

• Plasmid transfer: A plasmid from T. petrophila RKU1 was found to be 99% identical to one from Thermotoga sp. strain RQ7, despite the strains being distant in the rRNA tree, indicating recent horizontal transfer

• Comparative genomics insights: Analysis of eight Thermotoga members revealed they differ by 3-20% in gene content despite occupying physically distinct environments globally

• Ecological adaptation: Differences in carbohydrate utilization genes between Thermotoga species suggest adaptation to varying substrate availability in their respective environments

How does analyzing protein-substrate interactions inform the design of enzyme activity assays for T. maritima PyrBI?

Developing effective enzyme activity assays for T. maritima PyrBI requires understanding its protein-substrate interactions:

• Temperature considerations: Assays should be performed at temperatures that maintain enzyme stability while providing measurable activity. While T. maritima grows optimally at 80°C, some of its enzymes show maximum activity at lower temperatures

• Buffer optimization: pH and salt concentration significantly affect T. maritima protein stability and activity, as demonstrated with DHFR where buffer composition impacted both quaternary structure and enzymatic function

• Substrate preparation: For ATCase assays, the preparation and stability of substrates (aspartate and carbamoyl phosphate) at elevated temperatures must be considered

• Activity measurement approaches:

  • Spectrophotometric methods tracking the formation of N-carbamoylaspartate

  • Coupled enzyme assays that link product formation to a detectable signal

  • Methods that monitor phosphate release using colorimetric detection

• Control experiments: Include heat-treated E. coli extracts as negative controls when working with recombinant proteins to ensure measured activity is from the thermostable T. maritima enzyme

What are the current limitations in expressing and studying T. maritima PyrBI?

Researchers face several challenges when working with T. maritima PyrBI:

• Expression toxicity: High-level expression of some T. maritima proteins in E. coli can be toxic to the host, as observed with DHFR where cell growth ceased within 30 minutes of induction

• Protein solubility: Despite the thermostable nature of T. maritima proteins, achieving high yields of soluble protein can be challenging

• Reconstitution of native conditions: Creating experimental conditions that accurately reflect the hyperthermophilic, anaerobic native environment of T. maritima is technically demanding

• Structural analysis challenges: High-resolution structural studies may be complicated by protein flexibility or the requirement for specific cofactors or binding partners

• Limited comparative data: The relative scarcity of characterized hyperthermophilic enzymes makes comparative analyses challenging

How might T. maritima PyrBI be utilized in biotechnological applications?

The unique properties of T. maritima PyrBI suggest several potential biotechnological applications:

• Thermostable biocatalysts: The enzyme's stability at high temperatures makes it potentially valuable for industrial processes requiring elevated temperatures or increased resistance to denaturation

• Structural scaffolds: The robust thermostable structure could serve as a scaffold for protein engineering to introduce new catalytic functions while maintaining stability

• Model system for evolution studies: The evidence of horizontal gene transfer involving T. maritima genes provides opportunities to study evolutionary mechanisms and adaptation

• Educational tools: The distinct structural features of T. maritima PyrBI make it an excellent model for teaching protein structure-function relationships and adaptations to extreme environments

• Biosensors: The specificity of enzyme-substrate interactions could be harnessed for the development of biosensors operational under harsh conditions

What unanswered questions remain about the structure-function relationship of T. maritima PyrBI?

Despite existing research, several important questions about T. maritima PyrBI remain unanswered:

• Regulatory mechanisms: How does the fusion of catalytic and regulatory functions on a single polypeptide affect allosteric regulation compared to the separated functions in E. coli?

• Thermal adaptation vs. activity: What molecular trade-offs exist between thermal stability and catalytic efficiency in this enzyme?

• Evolutionary origins: Did the fusion of catalytic and regulatory domains in T. maritima PyrBI occur before or after adaptation to hyperthermophilic conditions?

• Conformational dynamics: How do temperature-dependent conformational changes influence substrate binding and catalysis?

• Interactome context: Does T. maritima PyrBI interact with other proteins in metabolic complexes, and how do these interactions influence its function in vivo?