Recombinant Rhodopirellula baltica Methionine--tRNA ligase (metG), partial

What is Methionine-tRNA ligase (metG) and what is its fundamental role in Rhodopirellula baltica?

Methionine-tRNA ligase (metG), also known as methionyl-tRNA synthetase (MetRS), is an essential enzyme that catalyzes the attachment of methionine to its cognate tRNA. The reaction follows this biochemical pathway:

ATP + L-methionine + tRNAMet → AMP + diphosphate + L-methionyl-tRNAMet

In R. baltica, MetRS plays a critical role in protein synthesis, being responsible for aminoacylation of both initiator tRNA(Met) and elongator tRNA(Met) . This dual function makes it particularly important for both translation initiation and elongation processes. As a member of the ligase family (EC 6.1.1.10), it forms carbon-oxygen bonds in aminoacyl-tRNA compounds and participates in several metabolic pathways including methionine metabolism, selenoamino acid metabolism, and aminoacyl-tRNA biosynthesis .

What expression systems are recommended for producing recombinant R. baltica MetRS?

Multiple expression systems have been successfully employed for the production of recombinant R. baltica MetRS:

According to product specifications, expression typically includes affinity tags for purification, with final purities of ≥85% as determined by SDS-PAGE .

What purification protocols are most effective for isolating functional R. baltica MetRS?

Based on methodologies used for similar enzymes, an effective purification protocol would include:

• Expression with a His6-tag in an appropriate host system (E. coli BL21(DE3) RIL has been used for related MetRS proteins) .

• Cell lysis using buffer containing appropriate protease inhibitors.

• Initial purification via Immobilized Metal Affinity Chromatography (IMAC).

• Optional secondary purification via ion exchange chromatography or size exclusion chromatography.

• Determination of active enzyme concentration through active site titrations .

• Quality control via SDS-PAGE and activity assays including ATP/PPi exchange and aminoacylation kinetics.

The purification should yield protein with ≥85% purity as commonly specified for recombinant R. baltica proteins .

What methods are used to evaluate the enzymatic activity of recombinant R. baltica MetRS?

Several complementary approaches can be used to assess MetRS activity:

• ATP/PPi Exchange Assay: This measures amino acid activation by monitoring the exchange between 32P-PPi and ATP. Reactions are typically performed at 37°C in a buffer containing Na-HEPES (pH 7.2), KCl, MgCl2, NaF, ATP, 32P-PPi with varying concentrations of methionine (0-400 μM) .

• Aminoacylation Assay: This measures the charging of tRNA with 14C-labeled methionine. Reactions are performed in buffer containing Na-HEPES (pH 7.2), KCl, MgCl2, BSA, DTT, 14C-L-methionine with varying concentrations of tRNA(Met) .

• Band-Shift Analysis: This can be used to study the tRNA binding properties of MetRS and assess any non-specific tRNA binding activity contributed by additional domains .

What are the typical kinetic parameters of MetRS and how do they compare across species?

While specific kinetic parameters for R. baltica MetRS are not directly reported in the available literature, research on MetRS from other organisms provides comparative reference points:

Determining the specific kinetic parameters for R. baltica MetRS would require similar experimental approaches.

How does MetRS function in the context of R. baltica's unique cell biology?

R. baltica has several distinctive biological features that make its protein synthesis machinery of particular interest:

• Complex Life Cycle: R. baltica exhibits a complex life cycle with different cell morphologies, transitioning from flagellated swarmer cells to adult cells that form characteristic rosettes . MetRS activity likely varies across these different developmental stages.

• Unique Cell Compartmentalization: R. baltica, like other Planctomycetes, exhibits unusual cell compartmentalization . This may affect the spatial organization of translation machinery, including MetRS.

• Adaptation to Marine Environment: R. baltica has adapted to marine conditions and can tolerate various salt concentrations . Transcriptomic studies show that it regulates numerous genes, including those for translation, in response to environmental conditions .

Gene expression studies using microarrays have identified differential regulation patterns throughout R. baltica's growth curve, with up to 12% of genes showing regulation in late stationary phase compared to transition phase . The expression pattern of metG across this life cycle would provide valuable insights into its regulation and role.

What is the impact of metG mutations on bacterial physiology?

Studies on metG mutations in E. coli provide insights into potential effects in R. baltica:

• Translation Rate Effects: Mutations in metG can significantly reduce protein synthesis rates, as shown in E. coli where various mutations decreased rates from 16.2 to as low as 6.1 pmoles 35S-Met/min .

• Antibiotic Persistence: metG mutations have been associated with increased bacterial antibiotic persistence, where bacteria enter a low-metabolic state that allows survival during antibiotic exposure .

• Differential Effects on Translation Phases: Some metG mutations primarily affect translation initiation (~1.5-fold increase in initiation time), while others affect both initiation and elongation .

• Metabolic Interactions: metG mutations can affect homocysteine metabolism, with mutant strains showing hypersensitivity to homocysteine supplementation .

The table below summarizes the effects of various metG mutations observed in E. coli:

How can recombinant R. baltica MetRS be used for protein labeling and identification studies?

A powerful application of MetRS in research is cell-type-specific protein labeling:

• Bioorthogonal Labeling: Mutant MetRS (MetRS*) can incorporate non-canonical amino acids like azidonorleucine (ANL) into newly synthesized proteins .

• Click Chemistry: The incorporated ANL contains an azide group that can be linked to alkyne-containing tags via click chemistry, allowing for selective labeling of proteins .

• Selective Purification: The tagged proteins can be isolated using affinity purification methods and subsequently identified by mass spectrometry .

This methodology allows for:

• Cell-type-specific proteome analysis

• Temporal tracking of protein synthesis

• Study of protein degradation kinetics

• Investigation of protein-protein interactions

While this approach has been demonstrated using other MetRS systems, adapting it to use R. baltica MetRS could potentially offer advantages for specific research applications.

What are the prospects for using R. baltica MetRS inhibitors in antibiotic development?

Research on MetRS inhibitors has shown promising results as potential antibiotics:

• Selective Inhibition: MetRS inhibitors can be designed to selectively target bacterial MetRS without affecting the mammalian counterpart, providing a basis for antibiotic specificity .

• Demonstrated Efficacy: Some MetRS inhibitors have shown high activity against Gram-positive bacteria such as Staphylococcus, Enterococcus, and Streptococcus with MICs of ≤1.3 μg/ml .

• In Vivo Efficacy: Certain compounds have demonstrated efficacy in animal models, with 3-4 log decreases in bacterial load in a Staphylococcus aureus murine thigh infection model, comparable to established antibiotics like vancomycin and linezolid .

While R. baltica is not a pathogen, studies of its MetRS could contribute to understanding the evolution and diversity of these enzymes, potentially informing broader antibiotic development efforts.

How does the EMAPII-like domain in MetRS contribute to enzyme function?

The EMAPII-like domain found in some MetRS enzymes has significant functional implications:

• Non-specific tRNA Binding: This domain provides MetRS with non-specific tRNA binding properties, as demonstrated in rice MetRS .

• Enhanced Catalytic Efficiency: The presence of this domain results in a 10-fold decrease in Km for tRNA in the aminoacylation reaction, improving catalytic efficiency at physiological tRNA concentrations .

• Binding to tRNA Minihelix: The domain specifically binds to the acceptor minihelix of tRNA(Met) and facilitates its aminoacylation .

• Evolutionary Significance: This domain is hypothesized to be a relic of an ancient tRNA binding domain that was incorporated into primordial synthetases for aminoacylation of RNA minihelices, the proposed ancestors of modern tRNA .

Determining whether the R. baltica MetRS contains this domain would require sequence analysis and structural studies.

What can R. baltica MetRS teach us about the evolution of aminoacyl-tRNA synthetases?

R. baltica belongs to the phylum Planctomycetes, which occupies an interesting position in bacterial evolution:

• Ancient Lineage: Planctomycetes represent an ancient bacterial lineage with unique cellular features, including compartmentalized cells .

• Genomic Adaptations: R. baltica's genome reveals adaptations to marine environments and distinctive metabolic capabilities, including unusual pathways for compatibility solute biosynthesis .

• Evolutionary Context: Studies of R. baltica MetRS could provide insights into the evolution of translation machinery in this distinctive bacterial lineage.

• Domain Architecture: Analysis of domain architecture in R. baltica MetRS could inform our understanding of how aminoacyl-tRNA synthetases evolved and diversified across different bacterial phyla.

Comparative studies with other bacterial and archaeal MetRS enzymes could help establish the evolutionary relationships and functional adaptations of these essential enzymes.

What are the major challenges in working with R. baltica and its proteins?

Research with R. baltica presents several technical challenges:

• Slow Growth: R. baltica has a relatively slow growth rate compared to model organisms like E. coli, making cultivation time-consuming .

• Genetic Manipulation: Traditional genetic tools are still being developed for R. baltica. Recent work has focused on developing DNA transformation methods, but these remain challenging .

• Complex Life Cycle: R. baltica's complex life cycle with different cell morphologies complicates studies that require synchronized cultures .

• Cell Wall Composition: Despite being Gram-negative, R. baltica has an unusual cell wall composition that requires specialized protocols for protoplast formation .

• Limited Commercial Tools: Few specific tools and reagents are commercially available for R. baltica research compared to model organisms.

What methodological approaches can overcome these challenges?

Several strategies can address the challenges of working with R. baltica:

• Heterologous Expression: Express R. baltica proteins in well-established systems like E. coli for biochemical and structural studies .

• Adapted Protocols: Develop specialized protocols for R. baltica, such as the protoplast formation and regeneration method described in the literature .

• Transcriptome Amplification: Whole transcriptome amplification by adaptor-ligation PCR can be used when RNA quantity is limiting .

• Chemical Transformation: Methods for chemical transformation with homologous chromosomal DNA have been developed specifically for R. baltica .

• Comparative Approaches: When direct studies are challenging, comparative genomics and proteomics can provide insights by analyzing related species or homologous proteins.

These methodological adaptations can facilitate research on R. baltica and its proteins despite the technical challenges presented by this unique organism.