Recombinant Thermus thermophilus Threonine--tRNA ligase (thrS), partial

What is the basic structure of Thermus thermophilus threonyl-tRNA synthetase?

Thermus thermophilus threonyl-tRNA synthetase (ThrRS) is composed of two identical subunits with a molecular mass of approximately 77,000 Da each, similar to E. coli ThrRS. The enzyme contains a polypeptide chain of 659 amino acids with a molecular weight of 75,550 Da. Comparative analysis with other subclass IIa synthetases reveals that ThrRS is organized into four distinct structural modules: two N-terminal modules specific to Thr-tRNA synthetases, a catalytic core, and a C-terminal anticodon-binding module. Each monomer contains one tightly bound zinc atom that is essential for catalytic activity, as confirmed by atomic absorption spectrometry .

How does the primary sequence of T. thermophilus ThrRS compare to E. coli ThrRS?

The primary sequence comparison between T. thermophilus and E. coli threonyl-tRNA synthetases shows limited homology, with only about 29% identity in the first 45 amino acid residues. Despite this relatively low sequence identity, both enzymes maintain similar functional capabilities in their homologous systems. This divergence in sequence while preserving function demonstrates the evolutionary plasticity of aminoacyl-tRNA synthetases while maintaining their essential catalytic functions .

What are the conserved domains in T. thermophilus ThrRS that are critical for function?

T. thermophilus ThrRS shares its modular organization with other class IIa synthetases but has distinctive N-terminal extensions. The eubacterial and eukaryotic enzymes possess a large extension folded into two structural domains, N1 and N2, which differ significantly from the shorter extension found in archaebacterial enzymes. Research with truncated ThrRS demonstrates that domain N1 is not essential for tRNA charging activity. The catalytic core and C-terminal anticodon-binding modules contain highly conserved residues involved in substrate binding and catalytic activity, which can be identified through comparison with the three-dimensional structure of E. coli ThrRS in complex with tRNA^Thr .

What are the most effective approaches for recombinant expression of T. thermophilus ThrRS?

Based on successful expression strategies used for other T. thermophilus proteins, the optimal approach for expressing recombinant T. thermophilus ThrRS involves cloning the gene into an expression vector with a strong promoter (such as T7) and transformation into an E. coli expression strain. While specific conditions for ThrRS are not detailed in the provided search results, similar thermophilic proteins from T. thermophilus, such as DNA polymerase, have been successfully expressed in E. coli systems. The overexpression system for T. thermophilus DNA polymerase was very efficient, yielding 700,000 units of activity from 1 liter of induced culture, suggesting that similar high-yield expression may be achievable for ThrRS .

What purification methods yield the highest purity and activity for recombinant T. thermophilus ThrRS?

The most effective purification strategy for native T. thermophilus ThrRS involves a combination of column chromatography techniques. The enzyme can be purified to electrophoretic homogeneity using sequential chromatography on DEAE-Sepharose, S-Sepharose, ACA-44 Ultrogel, and HA-Ultrogel. This method yields approximately 17 mg of purified enzyme from 1 kg of biomass. For recombinant His-tagged versions, Ni^2+ affinity chromatography would be the method of choice, as demonstrated with other T. thermophilus proteins like DNA polymerase, where a polyhistidine tag at the N-terminus enabled single-step purification with high recovery of enzymatic activity .

How do different buffer systems affect the stability and activity of purified T. thermophilus ThrRS?

While specific buffer optimization for T. thermophilus ThrRS is not detailed in the provided search results, general principles for thermostable enzymes from T. thermophilus apply. Buffers that maintain stability at high temperatures (50-80°C) are essential. Buffer systems containing divalent cations (particularly Mg^2+) are typically required for aminoacyl-tRNA synthetase activity. Since T. thermophilus ThrRS contains a zinc atom essential for activity, buffers should avoid chelating agents that might remove this metal. Additionally, the inclusion of polyamines in the buffer can enhance activity, as demonstrated in the reconstituted T. thermophilus translation system where tetraamine was most effective for translation at both high and low temperatures .

What are the optimal conditions for measuring T. thermophilus ThrRS aminoacylation activity?

The optimal conditions for measuring T. thermophilus ThrRS aminoacylation activity include temperatures between 55-75°C (reflecting the thermophilic nature of the organism), though activity has been observed at temperatures as low as 37°C in reconstituted systems. The reaction requires ATP, threonine, and appropriate tRNA substrates. The presence of polyamines, particularly tetraamine, enhances enzymatic activity. For maximum specificity, homologous T. thermophilus tRNA^Thr should be used as the substrate, as the enzyme shows significantly reduced activity (approximately 700-fold lower) with heterologous E. coli tRNA^Thr .

What kinetic parameters (Km, kcat, kcat/Km) characterize the activity of T. thermophilus ThrRS compared to mesophilic homologs?

While the search results do not provide specific kinetic parameters for T. thermophilus ThrRS, comparative functional data indicates that the enzyme has similar efficiency to E. coli ThrRS in homologous systems. When aminoacylating its native T. thermophilus tRNA^Thr, the efficiency is comparable to that of E. coli ThrRS with E. coli tRNA^Thr. Interestingly, E. coli ThrRS maintains this efficiency when charging T. thermophilus tRNA^Thr, but T. thermophilus ThrRS shows a 700-fold decrease in efficiency when charging E. coli tRNA^Thr. This asymmetry in cross-species aminoacylation efficiency suggests significant differences in tRNA recognition mechanisms between the thermophilic and mesophilic enzymes that would be reflected in their kinetic parameters, particularly in kcat/Km values for heterologous substrates .

How does the zinc-binding domain contribute to T. thermophilus ThrRS catalytic activity?

The zinc-binding domain in T. thermophilus ThrRS plays a critical role in its catalytic activity. Atomic absorption spectrometry analysis revealed that each monomer contains one tightly bound zinc atom that is essential for enzymatic function. The zinc ion likely participates in proper substrate positioning and may contribute to the discrimination of threonine from structurally similar amino acids like valine and serine. The metal ion probably coordinates with specific amino acid residues in the active site to create the appropriate microenvironment for catalysis. The essential nature of this zinc atom is consistent with findings for other threonyl-tRNA synthetases, suggesting a conserved mechanistic role across species despite sequence divergence .

What structural features contribute to the thermostability of T. thermophilus ThrRS?

Although the search results don't specifically address the thermostability features of T. thermophilus ThrRS, general principles observed in other T. thermophilus proteins can be applied. Thermostability in proteins from extreme thermophiles typically involves increased hydrophobic interactions, additional salt bridges, higher content of charged amino acids relative to uncharged polar residues, and reduced flexibility in loop regions. The enzyme likely possesses a more rigid core structure compared to its mesophilic counterparts, with strategic stabilizing interactions that maintain proper folding and function at elevated temperatures. This thermostability allows T. thermophilus ThrRS to operate efficiently at temperatures that would denature most mesophilic proteins .

How does T. thermophilus ThrRS differ from other aminoacyl-tRNA synthetases in the same organism?

T. thermophilus ThrRS is categorized as a eubacterial-type enzyme, which is notable because some other aminoacyl-tRNA synthetases from T. thermophilus exhibit archaebacterial characteristics. This distinction highlights the complex evolutionary history of T. thermophilus, suggesting potential lateral gene transfer events in its ancestry. Unlike some other T. thermophilus aminoacyl-tRNA synthetases, ThrRS does not appear to have archaeal features in its sequence or structure. Additionally, T. thermophilus possesses a single ThrRS, unlike its aspartyl-tRNA synthetase system, where a second genetically distinct enzyme (AspRS2) exists alongside the primary one .

What are the key differences in tRNA recognition between T. thermophilus ThrRS and E. coli ThrRS?

The most striking difference in tRNA recognition between T. thermophilus and E. coli ThrRS is revealed in cross-species aminoacylation experiments. While E. coli ThrRS efficiently aminoacylates both E. coli and T. thermophilus tRNA^Thr with similar efficiency, T. thermophilus ThrRS shows a dramatic 700-fold decrease in efficiency when charging E. coli tRNA^Thr compared to its homologous substrate. This asymmetry suggests that T. thermophilus ThrRS has more stringent recognition requirements for tRNA substrates, possibly involving specific interactions with identity elements in the tRNA structure that differ between the two species. These differences likely reflect adaptations to the distinct environmental conditions in which these organisms evolved .

How has T. thermophilus ThrRS contributed to our understanding of translation system evolution?

T. thermophilus ThrRS provides valuable insights into the evolution of translation systems, particularly regarding the adaptation of protein synthesis machinery to extreme environments. The enzyme exhibits typical eubacterial characteristics despite the fact that T. thermophilus possesses some translation components with archaeal features. This mosaic pattern suggests complex evolutionary dynamics, potentially involving horizontal gene transfer events. The preservation of ThrRS's essential function despite significant sequence divergence from mesophilic homologs (only 29% identity in the N-terminal region between T. thermophilus and E. coli) demonstrates the plasticity of aminoacyl-tRNA synthetases throughout evolution. Additionally, the finding that T. thermophilus translation components can function at temperatures below the organism's minimal growth temperature (37°C) suggests that translation machinery adaptation to extreme temperatures involves systems beyond the individual components themselves .

What advantages does recombinant T. thermophilus ThrRS offer for in vitro translation systems?

Recombinant T. thermophilus ThrRS offers several significant advantages for in vitro translation systems, particularly for high-temperature applications. As a component of reconstituted T. thermophilus translation systems, it enables protein synthesis at temperatures up to 65°C with yields reaching 60 μg/ml. The enzyme's thermostability makes it suitable for applications requiring elevated temperatures, such as the expression of proteins that aggregate or misfold at lower temperatures. Additionally, research has shown that T. thermophilus translation components, including ThrRS, can function at lower temperatures (37°C) as well, providing flexibility in experimental design. The enzyme's stringent tRNA recognition properties may also be advantageous in systems requiring high fidelity of amino acid incorporation .

How can T. thermophilus ThrRS be integrated into orthogonal translation systems?

T. thermophilus ThrRS can be integrated into orthogonal translation systems by exploiting its distinct tRNA recognition properties. The enzyme's high selectivity for its homologous tRNA substrate (showing 700-fold lower activity with E. coli tRNA^Thr) provides a natural orthogonality that can be further engineered. For development of orthogonal pairs, researchers could modify specific regions of the enzyme and its cognate tRNA to maintain their interaction while preventing cross-talk with endogenous components. The N-terminal domains, particularly the non-essential N1 domain, offer potential sites for engineering without disrupting core function. Additionally, the ability of T. thermophilus translation components to function across a wide temperature range (37-65°C) provides flexibility for deploying such orthogonal systems in various host organisms or in vitro conditions .

What approaches can be used to engineer T. thermophilus ThrRS for enhanced properties or novel functions?

Engineering T. thermophilus ThrRS for enhanced properties or novel functions can be approached through several strategies. Structure-guided mutagenesis targeting the active site or tRNA-binding regions can alter substrate specificity, potentially creating variants that recognize non-canonical amino acids or modified tRNAs. Domain swapping with other aminoacyl-tRNA synthetases could generate chimeric enzymes with hybrid functionalities. The non-essential N1 domain provides an opportunity for substantial modifications without disrupting core catalytic activity. For genomic integration of engineered variants, researchers could utilize approaches similar to those demonstrated for T. thermophilus, such as the counterselection system that has been successfully applied to generate unmarked mutants in this organism. Directed evolution approaches, incorporating screens or selections at elevated temperatures, could identify variants with enhanced thermostability, broader substrate scope, or other desirable properties .

What are common challenges in expressing active recombinant T. thermophilus ThrRS in E. coli, and how can they be addressed?

When expressing T. thermophilus ThrRS in E. coli, researchers may encounter several challenges. Protein misfolding can occur due to different chaperone systems between thermophilic and mesophilic hosts; this can be addressed by co-expressing thermophilic chaperones or using E. coli strains with enhanced folding capabilities. Codon bias differences may lead to poor translation efficiency, which can be overcome by codon optimization of the thrS gene for E. coli expression. Proteolytic degradation of the recombinant protein is another potential issue; adding protease inhibitors during purification and using protease-deficient host strains can mitigate this problem. Additionally, the essential zinc cofactor might not be properly incorporated in recombinant systems; supplementing growth media with zinc and avoiding chelating agents during purification can help ensure retention of this critical cofactor. These approaches reflect successful strategies used for other T. thermophilus proteins expressed in E. coli .

How can the aminoacylation activity assay for T. thermophilus ThrRS be optimized for different experimental conditions?

Optimization of the aminoacylation activity assay for T. thermophilus ThrRS requires careful consideration of several parameters. Temperature optimization is critical - while the enzyme functions optimally at 55-75°C, assays may need to be conducted at lower temperatures depending on experimental requirements. The addition of polyamines, particularly tetraamine, significantly enhances activity and should be included in reaction buffers. For maximum sensitivity, using homologous T. thermophilus tRNA^Thr as substrate is recommended, given the enzyme's poor activity with heterologous tRNAs. ATP concentration and pH should be optimized, with pH typically maintained around 7.5-8.0 for aminoacyl-tRNA synthetases. For detection methods, traditional approaches using radioactive amino acids offer high sensitivity, while newer non-radioactive methods such as malachite green detection of released pyrophosphate or fluorescently labeled tRNAs provide safer alternatives with comparable sensitivity .

What strategies can address issues of protein stability during purification and storage of T. thermophilus ThrRS?

To maintain protein stability during purification and storage of T. thermophilus ThrRS, several strategies can be employed. Taking advantage of the enzyme's thermostability, a heat treatment step (65-70°C for 10-20 minutes) can be included early in the purification process to denature most E. coli proteins while leaving the thermostable ThrRS intact. Storage buffers should contain glycerol (20-30%) to prevent freeze-thaw damage during storage at -20°C or -80°C. Since the enzyme contains an essential zinc atom, avoiding chelating agents in buffers is crucial. Addition of reducing agents like DTT or β-mercaptoethanol helps maintain the redox state of cysteine residues involved in zinc coordination. For long-term storage, lyophilization may be preferable to freezing, as the enzyme's thermostable nature suggests it would retain structure and activity upon rehydration. Dividing purified enzyme into single-use aliquots prevents repeated freeze-thaw cycles that could lead to activity loss .