Recombinant Mycoplasma pneumoniae Methionine--tRNA ligase (metG), partial

What is Methionine--tRNA ligase (MetG) and what is its function in Mycoplasma pneumoniae?

Methionine--tRNA ligase (MetG), also known as methionyl-tRNA synthetase, is an essential enzyme that catalyzes the attachment of methionine to its cognate tRNA. In Mycoplasma pneumoniae, MetG performs a critical two-step aminoacylation reaction:

• Activation of methionine using ATP to form methionyl-AMP

• Transfer of the methionyl group to the 3' end of tRNA^Met

This charged tRNA is essential for both initiation and elongation phases of protein synthesis. The initiator Met-tRNA^Met binds to the ribosomal P-site to begin translation, making MetG indispensable for bacterial viability and a potential antimicrobial target.

What are the conserved domains in Mycoplasma pneumoniae MetG?

Mycoplasma pneumoniae MetG contains several highly conserved domains that are characteristic of bacterial methionyl-tRNA synthetases:

• Catalytic domain: Located in the N-terminal region, containing the HIGH and KMSKS motifs essential for ATP binding and methionine activation

• Anticodon binding domain: Located in the C-terminal region, responsible for recognizing the anticodon loop of tRNA^Met

• Zinc-binding domain: A structural domain coordinating a zinc ion that contributes to protein stability

• Connecting peptide 1 (CP1): An insertion within the catalytic domain that contributes to amino acid specificity

Despite Mycoplasma pneumoniae's reduced genome compared to other bacteria, these domains remain highly conserved, highlighting their essential nature in protein synthesis.

How is recombinant MetG typically produced for research purposes?

Production of recombinant Mycoplasma pneumoniae MetG for research typically follows a systematic approach:

• Gene cloning: The metG gene (or a partial sequence of interest) is amplified from Mycoplasma pneumoniae genomic DNA and cloned into an expression vector with an appropriate promoter and affinity tag.

• Expression optimization:

  • Host selection: Usually E. coli BL21(DE3) or Rosetta strains to address codon bias

  • Temperature: Often lowered to 16-25°C to improve solubility

  • Induction conditions: IPTG concentration and induction time optimized for yield and solubility

• Purification protocol:

  • Affinity chromatography: Typically using His-tag for IMAC purification

  • Ion exchange chromatography: To remove contaminants with different charge properties

  • Size exclusion chromatography: For final polishing and buffer exchange

• Validation methods:

  • SDS-PAGE and Western blot: To confirm purity and identity

  • Activity assays: To verify enzymatic function through aminoacylation assays

  • Mass spectrometry: To confirm protein sequence and identify post-translational modifications

• Storage: Usually in buffer containing glycerol at -80°C to maintain activity

This systematic approach ensures production of functional enzyme suitable for structural and biochemical studies.

How do mutations in MetG affect antibiotic susceptibility in bacterial pathogens?

Mutations in MetG can significantly alter antibiotic susceptibility patterns in bacteria through mechanisms linked to the stringent response:

Research on Burkholderia thailandensis has demonstrated that MetG mutations confer tolerance to multiple antibiotic classes including β-lactams (meropenem, ampicillin, ceftazidime), quinolones (ciprofloxacin), and aminoglycosides (kanamycin) . This multi-drug tolerance is distinct from resistance mechanisms that typically affect specific antibiotic classes.

For Mycoplasma pneumoniae, which possesses both MetG and stringent response machinery, similar mutations could potentially lead to antibiotic tolerance, complicating treatment of respiratory infections.

How do MetG mutations specifically affect binding of methionine or tRNA?

MetG mutations can affect substrate binding in distinct but sometimes interconnected ways:

Methionine Binding Site Mutations:
Mutations like Pro27Ser in the methionine binding pocket directly alter the size, shape, or electrostatic properties of the binding cavity, typically affecting the Km for methionine . These changes can reduce enzymatic efficiency and increase the pool of uncharged tRNA^Met.

tRNA Binding Site Mutations:
Two distinct types of tRNA binding site mutations have been identified:

• Mutations in the catalytic domain (e.g., Leu216Pro, Phe316Ser) affecting tRNA binding near the active site

• Mutations in the anticodon recognition domain (e.g., Arg424Pro, Phe501Leu) that disrupt specific interactions with the tRNA anticodon

Allosteric Effects:
Surprisingly, molecular dynamics simulations have revealed that mutations in the tRNA-binding sites, particularly in the anticodon-binding domain, can increase the volume of the methionine-binding cavity despite their physical distance . This demonstrates how mutations in one functional domain can affect substrate binding in another domain through allosteric mechanisms.

The table below summarizes the effects of specific MetG mutations identified in B. thailandensis:

These structure-function relationships provide valuable insights for understanding how MetG mutations might affect enzyme function in Mycoplasma pneumoniae.

How can 3D modeling be used to predict the effects of mutations in Mycoplasma pneumoniae MetG?

3D modeling provides crucial insights into the structural consequences of mutations in Mycoplasma pneumoniae MetG through a systematic approach:

Modeling Methodology:

• Template selection: Identify crystallized MetG structures from related organisms with high sequence identity (E. coli MetG serves as an excellent template with 54% identity)

• Sequence alignment: Align Mycoplasma pneumoniae MetG sequence with the template, focusing on conserved motifs (HIGH, KMSKS)

• Model building: Generate the 3D structure using homology modeling software (Modeller, SWISS-MODEL)

• Energy minimization: Refine the model using molecular dynamics simulations

• Validation: Assess model quality using Ramachandran plots and other validation tools

Mutation Analysis Protocol:

• Map functional sites: Identify residues involved in methionine, ATP, and tRNA binding

• In silico mutagenesis: Introduce mutations of interest and analyze structural changes

• Molecular dynamics simulations: Perform extended simulations (>1 ns) to observe dynamic effects

• Binding pocket analysis: Measure changes in pocket volume and electrostatic properties

• Binding energy calculations: Estimate changes in substrate affinity

Practical Application Example:
The study on B. thailandensis MetG demonstrated how mutations in the tRNA-binding sites (Arg424Pro, Phe501Leu) unexpectedly increased the volume of the methionine-binding cavity despite being located in a different domain . This structural insight explained how mutations in the anticodon-binding domain could affect substrate binding in the catalytic domain through allosteric effects.

For Mycoplasma pneumoniae MetG, similar modeling approaches could help predict:

• How specific mutations might affect enzyme function

• Potential sites for rational drug design

• Structural mechanisms of antibiotic tolerance

• Evolutionary constraints on the enzyme

What is the relationship between MetG mutations and the stringent response in bacteria?

The relationship between MetG mutations and the stringent response involves a sophisticated cellular adaptation mechanism with significant implications for antibiotic tolerance:

Mechanistic Pathway:

• Altered tRNA charging: Specific mutations in MetG reduce the enzyme's efficiency in charging tRNA^Met with methionine

• Increased uncharged tRNA pool: The inefficient aminoacylation leads to accumulation of uncharged tRNA^Met

• Ribosome sensing: Uncharged tRNA^Met binds to the ribosomal A-site, which is recognized by RelA

• (p)ppGpp synthesis: RelA synthesizes (p)ppGpp, the alarmone of the stringent response

• Global transcriptional changes: (p)ppGpp alters gene expression patterns, downregulating growth-related processes and upregulating stress responses

Experimental Evidence:

• Growth phenotypes: MetG mutants exhibit significantly reduced growth rates compared to wild-type strains

• RelA dependency: Inactivation of relA in MetG mutants significantly lowers MICs of various antibiotics

• Multi-drug tolerance: MetG mutations confer tolerance to multiple classes of antibiotics

• Killing kinetics: Extended survival times (MDK99 values) in lethal antibiotic concentrations

Implications for Mycoplasma pneumoniae:
While the specific details of the stringent response in Mycoplasma pneumoniae may differ from those in other bacteria due to its reduced genome, the conservation of MetG and RelA suggests that similar mechanisms could operate in this pathogen. This connection between MetG function and the stringent response represents a potential mechanism for antibiotic tolerance that could complicate treatment of Mycoplasma pneumoniae infections.

What methodologies are most effective for studying the kinetics of recombinant Mycoplasma pneumoniae MetG?

Studying the kinetics of recombinant Mycoplasma pneumoniae MetG requires sophisticated biochemical techniques to dissect the two-step aminoacylation reaction:

Steady-State Kinetic Methods:

Pre-Steady-State Kinetics:

Data Analysis Approaches:

• Michaelis-Menten analysis: To determine Km and kcat values for each substrate

• Linear and non-linear regression: To fit experimental data to kinetic models

• Global fitting: To analyze data across multiple substrate concentrations

• Inhibition studies: To elucidate reaction mechanism through product inhibition patterns

Through these complementary approaches, researchers can construct a comprehensive kinetic model of Mycoplasma pneumoniae MetG function, identify rate-limiting steps, and evaluate the effects of mutations or potential inhibitors on enzyme activity.

What are the implications of MetG as a potential antimicrobial target in Mycoplasma pneumoniae?

MetG presents compelling advantages as an antimicrobial target in Mycoplasma pneumoniae, with several important considerations:

Target Validation Criteria:

• Essentiality: MetG is absolutely required for protein synthesis and bacterial survival

• Conservation: The enzyme is highly conserved across bacterial species

• Structural uniqueness: Bacterial MetGs differ from human cytoplasmic and mitochondrial methionyl-tRNA synthetases

• Druggability: Contains well-defined pockets suitable for small molecule binding

Drug Development Strategies:

Challenges and Considerations:

• Resistance development: Mutations in MetG could confer resistance, though such mutations might compromise fitness

• Stringent response activation: Sub-inhibitory concentrations might trigger the stringent response, potentially leading to tolerance to other antibiotics

• Delivery challenges: Mycoplasma pneumoniae lacks a cell wall, potentially affecting compound penetration

• Cross-reactivity: Ensuring selectivity over human methionyl-tRNA synthetases

MetG inhibitors could address difficult-to-treat Mycoplasma pneumoniae infections, particularly those showing resistance to macrolides and other conventional antibiotics. The connection between MetG and the stringent response also suggests that combination therapies targeting both mechanisms might be effective in preventing the development of antibiotic tolerance.

How can site-directed mutagenesis be used to study the function of specific domains in Mycoplasma pneumoniae MetG?

Site-directed mutagenesis offers a powerful approach to dissect the structure-function relationships in Mycoplasma pneumoniae MetG:

Strategic Target Selection:

• Conserved motifs: HIGH and KMSKS motifs involved in ATP binding and catalysis

• Methionine binding pocket: Residues that interact directly with the methionine substrate

• tRNA binding interface: Residues in both the catalytic domain and anticodon-binding domain

• Interdomain communication: Residues that mediate allosteric effects between domains

Experimental Workflow:

• Primer design and mutagenesis:

  • Design mutagenic primers to introduce specific amino acid substitutions

  • Use PCR-based mutagenesis techniques to generate mutant constructs

  • Verify mutations by DNA sequencing

• Protein expression and purification:

  • Express mutant proteins under optimized conditions

  • Purify using affinity chromatography and additional purification steps

  • Verify protein folding using circular dichroism or thermal shift assays

• Functional characterization:

  • Assess aminoacylation activity using standard assays

  • Determine kinetic parameters for each substrate

  • Measure binding affinities using biophysical techniques

Strategic Mutations Based on Related Research:

This systematic mutational analysis can provide insights into the specific roles of various domains and residues in Mycoplasma pneumoniae MetG function, with implications for understanding bacterial persistence and developing targeted antimicrobials.

What are the key research priorities for studying Mycoplasma pneumoniae MetG?

Future research on Mycoplasma pneumoniae MetG should focus on several priority areas to advance both basic understanding and therapeutic applications:

• Structural characterization:

  • Determine the crystal structure of Mycoplasma pneumoniae MetG

  • Compare with structures from other bacterial species to identify unique features

  • Analyze complex structures with substrates and potential inhibitors

• Antibiotic tolerance mechanisms:

  • Investigate the relationship between MetG mutations and the stringent response in Mycoplasma pneumoniae

  • Characterize the transcriptional and metabolic changes associated with MetG dysfunction

  • Develop strategies to overcome tolerance mechanisms

• Inhibitor development:

  • Design and screen selective inhibitors targeting Mycoplasma pneumoniae MetG

  • Optimize lead compounds for antimicrobial activity and selectivity

  • Evaluate combination therapies targeting both MetG and the stringent response

• Evolutionary considerations:

  • Analyze the evolutionary constraints on MetG in the context of Mycoplasma's reduced genome

  • Investigate the co-evolution of MetG with other components of the translation machinery

  • Assess the fitness costs of resistance-conferring mutations