Recombinant Nitrosomonas europaea Proline--tRNA ligase (proS), partial

What is Proline--tRNA ligase (ProS) and what is its role in protein biosynthesis?

Proline--tRNA ligase, commonly referred to as prolyl-tRNA synthetase (ProRS), is a critical enzyme in protein biosynthesis that attaches proline to its cognate tRNA (tRNAPro). ProRS belongs to the Class II aminoacyl-tRNA synthetase family, characterized by three distinctive structural motifs (Motifs I, II, and III) in its active site. Specifically, it falls within Subclass IIa, which also includes SerRS, ThrRS, GlyRS, and HisRS. These Class IIa enzymes typically exist as α2 homodimers with cross-subunit tRNA binding .

The aminoacylation reaction catalyzed by ProRS occurs in two steps:

• Activation of proline using ATP to form prolyl-adenylate (Pro-AMP)

• Transfer of the prolyl group to the 3'-end of tRNAPro

This process is essential for accurate translation of genetic information, ensuring proline is incorporated correctly into nascent polypeptide chains during protein synthesis .

How are the prokaryotic and eukaryotic forms of ProRS different?

There are two distinct types of ProRS enzymes with structural differences that generally correlate with taxonomic divisions:

Prokaryote-like ProRS:

• Found primarily in eubacteria and mitochondria

• Composed of three domains:

  • N-terminal catalytic domain

  • Central insertion domain (INS)

  • C-terminal anticodon-binding domain

• Contains specific editing mechanisms to prevent misacylation

Eukaryote-like ProRS:

• Found in the cytoplasm of eukaryotes and in archaebacteria

• Also composed of three domains, but differently organized:

  • N-terminal catalytic domain

  • Central anticodon-binding domain

  • C-terminal zinc-binding domain

Interestingly, the classification of ProRS does not always follow taxonomic boundaries. For example, though Thermus thermophilus is taxonomically classified as a bacterium, it contains a "eukaryote-like" ProRS . Nitrosomonas europaea, being a bacterium, would typically be expected to have a prokaryote-like ProRS, but specific structural analysis is required for confirmation.

What are the optimal conditions for expressing recombinant N. europaea ProRS in E. coli?

Based on experience with other bacterial ProRS enzymes, the expression of recombinant N. europaea ProRS in E. coli often requires optimization to prevent the formation of insoluble protein. Drawing from similar work with P. aeruginosa ProRS, the following approach is recommended:

• Clone the proS gene into an appropriate expression vector with a histidine tag for purification

• Transform the construct into an E. coli expression strain (e.g., BL21(DE3))

• Optimize expression conditions:

  • Growth temperature: 30°C is typically optimal (lower temperatures reduce inclusion body formation)

  • IPTG concentration: 25-50 μM (lower concentrations often improve solubility)

  • Growth medium: Rich media such as LB or 2xYT supplemented with appropriate antibiotics

  • Induction time: 4-6 hours after reaching mid-log phase (OD600 of 0.6-0.8)

Expression optimization should be monitored via SDS-PAGE analysis of both soluble and insoluble fractions to maximize the yield of soluble protein .

What purification strategy yields the highest purity and activity for recombinant N. europaea ProRS?

A multi-step purification protocol typically yields the best results for recombinant ProRS:

• Initial capture:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged protein

  • Buffer conditions: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole

  • Elution with imidazole gradient (50-250 mM)

• Intermediate purification:

  • Ion exchange chromatography (typically Q-Sepharose)

  • Buffer: 20 mM Tris-HCl pH 8.0, 50 mM NaCl

  • Elution with NaCl gradient (50-500 mM)

• Polishing step:

  • Size exclusion chromatography (Superdex 200)

  • Buffer: 20 mM HEPES pH 7.5, 100 mM NaCl, 5 mM MgCl2, 1 mM DTT

Throughout purification, it's essential to include protease inhibitors in all buffers and maintain the temperature at 4°C to prevent degradation. Activity assays should be performed at each purification step to track specific activity and ensure the purification process preserves enzyme function .

What are the key structural elements of ProRS that determine its specificity for proline?

The specificity of ProRS for proline is determined by several key structural elements in its active site:

• Proline binding pocket:

  • Hydrophobic residues that accommodate proline's cyclic side chain

  • A network of hydrogen bonds that position the α-amino and α-carboxyl groups

  • Key conserved residues that typically include threonine, histidine, and phenylalanine

• ATP binding region:

  • Three conserved motifs characteristic of Class II aminoacyl-tRNA synthetases

  • Motif 1: A long α-helix followed by a β-strand

  • Motif 2: Two antiparallel β-strands connected by a loop

  • Motif 3: A β-strand followed by a loop

• Editing domain:

  • Prokaryotic ProRS contains editing mechanisms to discriminate against similar amino acids (particularly alanine and cysteine)

  • The editing site is spatially distinct from the active site

Crystallographic studies of ProRS from other bacteria show these elements are highly conserved, suggesting similar structural features would be present in N. europaea ProRS .

How do editing mechanisms in N. europaea ProRS prevent misacylation with non-cognate amino acids?

Bacterial ProRS enzymes employ sophisticated editing mechanisms to ensure aminoacylation accuracy, which are likely conserved in N. europaea ProRS:

• Pre-transfer editing:

  • Hydrolysis of misactivated aminoacyl-adenylate (aa-AMP) before transfer to tRNA

  • Occurs when non-cognate amino acids (particularly alanine) form unstable adenylates

  • Does not require tRNA binding

• Post-transfer editing:

  • Hydrolysis of the ester bond in mischarged tRNAPro

  • Requires translocation of the 3' end of the tRNA from the synthetic site to the editing site

  • Specific for amino acids that are structurally similar to proline but lack its cyclic structure

These mechanisms are crucial because proline's cyclic structure makes it challenging to discriminate from other small amino acids based solely on size. The double-sieve mechanism (coarse sieve at activation step, fine sieve at editing step) ensures high fidelity in protein synthesis .

What are the recommended assays for measuring the aminoacylation activity of N. europaea ProRS?

Two primary assays are recommended for characterizing N. europaea ProRS activity:

• ATP-PPi Exchange Assay:

  • Measures the first step of the aminoacylation reaction (amino acid activation)

  • Principle: Reversible formation of aminoacyl-adenylate allows incorporation of [32P] from PPi into ATP

  • Components: ProRS, proline, ATP, [32P]PPi, Mg2+, appropriate buffer

  • Quantification: Measure radioactive ATP through TLC or charcoal adsorption

  • Advantages: Simple, rapid, can be used to determine kinetic parameters for ATP and proline

• Aminoacylation Assay:

  • Measures the complete aminoacylation reaction (transfer of proline to tRNAPro)

  • Principle: Formation of [3H]Pro-tRNAPro using [3H]proline

  • Components: ProRS, [3H]proline, ATP, tRNAPro, Mg2+, appropriate buffer

  • Quantification: TCA precipitation of aminoacylated tRNA and scintillation counting

  • Advantages: Measures complete reaction, provides information about tRNA recognition

For high-throughput applications, a scintillation proximity assay (SPA) can be developed, similar to that described for P. aeruginosa ProRS .

What kinetic parameters can be expected for N. europaea ProRS compared to other bacterial ProRS enzymes?

While specific kinetic parameters for N. europaea ProRS have not been reported in the provided references, we can provide reasonable expectations based on data from other bacterial ProRS enzymes:

Variations in kinetic parameters can be attributed to differences in experimental conditions and evolutionary adaptations specific to each organism. When determining kinetic parameters for N. europaea ProRS, it is important to use standardized conditions (pH 7.5-8.0, 37°C, 5-10 mM Mg2+) to enable meaningful comparisons with values reported for other ProRS enzymes .

How does N. europaea ProRS compare structurally and functionally with ProRS from other bacterial species?

N. europaea ProRS is expected to share structural and functional similarities with other bacterial ProRS enzymes, particularly those from proteobacteria. A comparative analysis would likely reveal:

• Domain organization:

  • N. europaea ProRS, as a prokaryotic enzyme, likely contains the three-domain structure: N-terminal catalytic domain, central insertion domain, and C-terminal anticodon-binding domain

  • Sequence conservation is typically highest in the catalytic domain and lowest in the anticodon-binding domain

• Active site conservation:

  • Key residues involved in proline and ATP binding are generally highly conserved

  • The ATP-binding pocket formed by the three Class II motifs would show strong sequence and structural conservation

  • The proline-binding pocket would contain conserved hydrophobic residues adapted to recognize proline's cyclic side chain

• Editing mechanisms:

  • Being an ammonia-oxidizing bacterium with a specialized metabolism, N. europaea might have specific adaptations in its editing mechanisms

  • Comparative analysis of the editing domain could reveal adaptations related to the ecological niche of N. europaea

• tRNA recognition elements:

  • Recognition of tRNAPro typically involves interactions with the acceptor stem and the anticodon

  • Species-specific variations may exist in the anticodon-binding domain

Structural analysis through homology modeling based on crystal structures of ProRS from organisms like P. aeruginosa can provide insights into these similarities and differences .

What can be learned from studying the evolutionary relationships between N. europaea ProRS and other aminoacyl-tRNA synthetases?

Evolutionary analysis of N. europaea ProRS in relation to other aminoacyl-tRNA synthetases can provide valuable insights:

• Phylogenetic relationships:

  • N. europaea belongs to the beta-proteobacteria, a group with interesting evolutionary positions

  • Comparing its ProRS with those from diverse bacterial phyla can reveal evolutionary trajectories

  • Analysis may show whether N. europaea ProRS evolved through vertical inheritance or horizontal gene transfer

• Functional adaptations:

  • N. europaea's specialized ammonia-oxidizing metabolism may have driven specific adaptations

  • Comparative analysis can reveal amino acid substitutions that might be linked to metabolic specialization

  • Coevolution of ProRS with its cognate tRNAPro can provide insights into adaptation mechanisms

• Domain architecture evolution:

  • Analysis of domain boundaries and interdomain connections can reveal evolutionary events

  • The insertion domain, which is specific to prokaryotic ProRS, may show adaptations specific to N. europaea

• Connection to translation fidelity:

  • Comparing editing mechanisms across species can reveal how translation fidelity mechanisms evolved

  • This is particularly relevant for understanding how organisms adapt to different environmental stresses

These evolutionary insights can guide experimental approaches and help interpret experimental results in a broader biological context .

How can recombinant N. europaea ProRS be used as a tool for studying aminoacylation mechanisms?

Recombinant N. europaea ProRS can serve as a valuable research tool for investigating fundamental aspects of aminoacylation mechanisms:

• Structure-function studies:

  • Site-directed mutagenesis of conserved residues to probe their roles in catalysis

  • Creation of chimeric enzymes by domain swapping with other ProRS enzymes

  • Investigation of substrate specificity determinants through rational design of active site variants

• Editing mechanism investigation:

  • Analysis of pre- and post-transfer editing pathways using modified substrates

  • Comparison of editing efficiency with non-cognate amino acids

  • Engineering of editing-deficient variants to study the impact on translation fidelity

• tRNA recognition studies:

  • Investigation of identity elements in tRNAPro recognized by N. europaea ProRS

  • Cross-species aminoacylation assays to evaluate tRNA specificity

  • Structural studies of ProRS-tRNA complexes to understand molecular recognition

• Development of inhibitors:

  • Screening for selective inhibitors of bacterial ProRS

  • Structure-based design of compounds targeting N. europaea ProRS

  • Development of biochemical assays for inhibitor characterization

These applications contribute to our fundamental understanding of protein biosynthesis and can potentially lead to novel antibacterial strategies targeting aminoacyl-tRNA synthetases .

What role might N. europaea ProRS play in the development of expanded genetic code systems?

While not directly discussed in the provided references for N. europaea ProRS, insights can be drawn from the expanding field of genetic code expansion:

• Engineering substrate specificity:

  • ProRS variants could potentially be engineered to incorporate proline analogs or non-canonical amino acids

  • Modification of the active site to accommodate non-natural substrates

  • Directed evolution approaches to select for variants with desired specificity

• Orthogonal translation systems:

  • Development of ProRS variants that specifically recognize engineered tRNAs

  • Creation of orthogonal ProRS/tRNA pairs that function independently of the endogenous translation machinery

  • This approach has been successfully demonstrated with pyrrolysyl-tRNA synthetase (PylRS) systems

• Applications in synthetic biology:

  • Incorporation of non-canonical amino acids with novel chemical properties

  • Expansion of the chemical diversity of proteins

  • Development of proteins with enhanced or novel functions

• Methodological approaches:

  • Application of selection strategies similar to those used for PylRS

  • Positive-negative selection schemes to identify variants that specifically incorporate desired non-canonical amino acids

  • High-throughput screening methods to evaluate aminoacylation activity and specificity

Drawing parallels from the extensive work on pyrrolysine systems , similar approaches could potentially be applied to engineer N. europaea ProRS for genetic code expansion applications.

What are the common issues encountered during expression and purification of recombinant ProRS, and how can they be addressed?

Researchers working with recombinant ProRS often face several challenges:

• Poor solubility and inclusion body formation:

  • Solution: Lower induction temperature (25-30°C), reduce IPTG concentration (25-50 μM), co-express with chaperones

  • Alternative: Use solubility-enhancing fusion tags (MBP, SUMO) or express in specialized E. coli strains (e.g., Arctic Express)

  • Last resort: Develop refolding protocols from inclusion bodies if active enzyme cannot be obtained in soluble form

• Low enzyme activity:

  • Check for proper folding: Use circular dichroism to assess secondary structure

  • Ensure cofactor presence: Include Mg2+ or Zn2+ in purification and assay buffers

  • Optimize storage conditions: Test stabilizing additives (glycerol, DTT, BSA)

  • Verify tRNA quality: Ensure tRNA is properly folded and lacks modifications that might interfere with aminoacylation

• Proteolytic degradation:

  • Include protease inhibitors throughout purification

  • Remove flexible regions through construct design

  • Optimize buffer conditions (pH, salt concentration) to minimize protease activity

• Inconsistent kinetic measurements:

  • Standardize enzyme preparation methods

  • Ensure consistent experimental conditions (temperature, pH, buffer composition)

  • Use internal controls to normalize between experiments

  • Account for potential product inhibition in kinetic analyses

These troubleshooting approaches are derived from experience with ProRS from various bacterial species and can be applied to work with N. europaea ProRS .

How can researchers differentiate between pre-transfer and post-transfer editing activities in N. europaea ProRS?

Differentiating between pre-transfer and post-transfer editing activities requires specialized experimental approaches:

• Rapid quench-flow experiments:

  • Monitor the formation and decay of aminoacyl-adenylate intermediates

  • Compare rates with cognate (proline) versus non-cognate amino acids

  • Fast kinetic measurements can capture transient intermediates

• tRNA-dependent versus tRNA-independent hydrolysis:

  • Pre-transfer editing can occur in the absence of tRNA

  • Compare AMP formation (indicator of adenylate hydrolysis) with and without tRNA

  • Higher rates in the presence of tRNA suggest post-transfer editing

• Use of modified substrates:

  • Non-hydrolyzable aminoacyl-adenylate analogs can help isolate post-transfer editing

  • 2'-deoxy-3'-end-modified tRNAs can block post-transfer editing

  • These tools allow separation of the two editing pathways

• Site-directed mutagenesis:

  • Target residues predicted to be involved specifically in either pre- or post-transfer editing

  • Mutations that selectively impair one pathway help quantify the contribution of each

• Experimental conditions that favor one pathway:

  • Temperature and pH can differentially affect pre- versus post-transfer editing

  • Systematic variation of conditions can help separate the contributions

The relative contribution of each editing pathway often varies with the non-cognate amino acid being tested, providing additional evidence for the operation of distinct mechanisms .