Recombinant Gluconobacter oxydans Peptidyl-tRNA hydrolase (pth)

What is the fundamental role of peptidyl-tRNA hydrolase in bacterial systems?

Peptidyl-tRNA hydrolase (Pth) functions primarily to recycle tRNAs by cleaving off unfinished peptides from peptidyl-tRNAs that have prematurely dissociated from ribosomes during translation. This recycling is critical because the accumulation of peptidyl-tRNAs depletes the pool of available tRNAs for protein synthesis, inhibiting bacterial growth . The enzyme catalyzes the hydrolysis of the ester bond between the peptide and the 3' end of the tRNA molecule.

To study this function experimentally, researchers can employ:

• Northern blotting with specific tRNA probes to measure tRNA depletion

• Acid-PAGE separation combined with radioisotope labeling to monitor peptidyl-tRNA accumulation

• Cu-tRNAseq, a copper sulfate-based tRNA sequencing method, to quantitatively profile peptidyl-tRNA levels across all tRNA species simultaneously

How does Pth enzyme structure compare across bacterial species?

Pth enzymes across bacterial species share a conserved structural fold while exhibiting species-specific variations:

For G. oxydans Pth structural studies, researchers should consider:

• NMR spectroscopy with 15N/13C labeling to characterize protein dynamics

• X-ray crystallography to resolve high-resolution structures

• Molecular dynamics simulations to analyze substrate binding pocket flexibility

• Comparative structural analysis with homologs to identify unique features

Why is optimal Pth activity essential for bacterial survival?

Pth is essential in most bacteria because:

• In Pth-deficient cells, accumulation of peptidyl-tRNAs depletes the available pool of free tRNAs below the threshold required for protein synthesis

• Certain tRNA species are particularly sensitive to Pth deficiency - for example, in M. tuberculosis, peptidyl prolyl-tRNA was found to be especially dependent on Pth for recycling

• Pth deficiency can cause translation errors and growth arrest

To experimentally determine the essentiality of Pth in G. oxydans:

• Develop conditional knockdown systems using CRISPRi or degradation tags

• Monitor growth curves under Pth depletion conditions

• Quantify specific tRNA isoacceptor levels using Cu-tRNAseq

• Measure translation rates using pulse-chase experiments with radioactive amino acids

What expression systems are most effective for recombinant G. oxydans Pth?

For optimal expression of recombinant G. oxydans Pth, researchers should consider:

E. coli-based expression systems:

• BL21(DE3) or Rosetta(DE3) strains for high-level expression

• pET-based vectors with T7 promoter systems for controlled induction

• C-terminal or N-terminal His-tags for purification, ensuring tag placement doesn't interfere with active site residues

Expression optimization protocol:

• Transform expression plasmid into appropriate E. coli strain

• Grow cultures at 37°C until OD600 reaches 0.6-0.8

• Induce with 0.1-0.5 mM IPTG

• Lower temperature to 16-25°C post-induction

• Continue expression for 12-18 hours

• Harvest cells by centrifugation at 5000×g for 15 minutes

For NMR studies requiring isotope labeling, minimal M9 media supplemented with 15N-ammonium chloride and/or 13C-glucose would be necessary, as demonstrated with M. smegmatis Pth characterization .

What purification strategies yield the highest purity and activity of recombinant Pth?

A robust purification protocol for G. oxydans Pth should include:

Multi-step purification approach:

• Cell lysis: Sonication or high-pressure homogenization in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 5% glycerol, and protease inhibitors

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

• Intermediate purification: Ion exchange chromatography (IEX) - anion exchange using Q Sepharose

• Polishing step: Size exclusion chromatography using Superdex 75 or 200 columns

• Quality control: SDS-PAGE and Western blotting to confirm purity

Activity preservation considerations:

• Include 1-5 mM DTT or 2-5 mM β-mercaptoethanol to maintain reduced cysteines

• Add 5-10% glycerol to prevent aggregation

• Store purified enzyme at -80°C in small aliquots to avoid freeze-thaw cycles

• Validate activity using standard Pth assay substrates like diacetyl-Lys-tRNA

How can isotope labeling be optimized for NMR studies of G. oxydans Pth?

For high-quality NMR structural studies of G. oxydans Pth, optimize isotope labeling as follows:

Uniform 15N/13C labeling protocol:

• Transform expression plasmid into E. coli BL21(DE3)

• Grow initial culture in LB medium until OD600 = 0.8

• Harvest cells by centrifugation (5000×g, 15 min)

• Resuspend in M9 minimal medium containing:

  • 1 g/L 15N-ammonium chloride

  • 2-4 g/L 13C-glucose

  • Essential minerals (MgSO4, CaCl2, trace metals)

• Continue growth for 1-2 hours for adaptation

• Induce with 0.5 mM IPTG at 18°C

• Express for 16-20 hours

• Purify using standard protocol with deuterated buffers for final NMR samples

NMR sample preparation:

• Concentrate to 0.5-1.0 mM in 20 mM sodium phosphate buffer, pH 6.5

• Add 5-10% D2O for lock signal

• Include 0.02% NaN3 as preservative

• Filter through 0.22 μm filter to remove particulates

What are the most reliable methods to measure Pth activity in vitro?

Several complementary approaches can be employed to accurately measure G. oxydans Pth activity:

Radioactive assay:

• Prepare diacetyl-[14C]Lys-tRNA as a substrate

• Incubate with purified Pth enzyme

• Stop reaction at different time points with trichloroacetic acid

• Separate products by thin-layer chromatography

• Quantify using PhosphorImager or scintillation counting

• Calculate initial rates for enzyme kinetics

Spectrophotometric assay:

• Use synthetic p-nitrophenyl ester derivatives of amino acids as substrates

• Monitor release of p-nitrophenol at 400 nm

• Determine kinetic parameters (KM, kcat) from initial rates

• Compare with natural substrate kinetics for validation

Northern blot validation:

• Perform enzyme reaction with natural peptidyl-tRNA substrates

• Isolate RNA at various time points

• Separate using acid-PAGE (6.5% urea gel, 7M urea, 100 mM sodium acetate, pH 5.0)

• Transfer to nitrocellulose membrane

• Probe with radiolabeled oligonucleotides specific to tRNA of interest

• Visualize using PhosphorImager

How can Cu-tRNAseq be adapted to study G. oxydans Pth specificity?

Cu-tRNAseq is a powerful method to profile peptidyl-tRNAs and can be adapted for G. oxydans Pth research:

Cu-tRNAseq protocol adaptation:

• Extract total RNA from G. oxydans (wild-type and Pth-depleted)

• Divide samples into three treatment groups:

  • Untreated (control for total tRNA)

  • NaIO4-treated (oxidizes 3' ends of uncharged tRNAs)

  • CuSO4-treated (deacylates aminoacyl-tRNAs but not peptidyl-tRNAs)

• Perform tRNA-specific library preparation:

  • Dephosphorylate tRNA 3' ends

  • Ligate 3' adapter

  • Reverse transcribe

  • PCR amplify with indexed primers

• Sequence on Illumina platform

• Analyze data to calculate:

  • Peptidyl fraction = (CuSO4-treated - Untreated)/(NaIO4-treated - Untreated)

  • Compare peptidyl fractions across tRNA isoacceptors

This method allows comprehensive profiling of all tRNA species affected by Pth deficiency and can reveal substrate preferences of G. oxydans Pth.

What are appropriate substrates for kinetic characterization of G. oxydans Pth?

For comprehensive kinetic characterization of G. oxydans Pth:

Natural substrates:

• Bulk peptidyl-tRNAs isolated from G. oxydans strains

• Specific peptidyl-tRNA species synthesized in vitro

• Focus on prolyl-tRNA species (shown to be particularly dependent on Pth in mycobacteria)

Synthetic substrates:

• Diacetyl-Lys-tRNA (standard Pth substrate)

• N-acetylated aminoacyl-tRNAs

• Fluorescently labeled peptidyl-tRNA analogs for high-throughput assays

Substrate preparation protocol:

• Generate peptidyl-tRNAs by in vitro translation of truncated mRNAs

• Purify by size exclusion chromatography

• Verify integrity by acid-PAGE

• Determine concentration by UV absorbance and/or radioactive counting

• Use in enzyme assays at concentrations ranging from 0.1-10× KM

How can NMR spectroscopy be used to characterize the dynamics of G. oxydans Pth?

NMR spectroscopy provides valuable insights into protein dynamics and can be applied to G. oxydans Pth following the approach used for M. smegmatis Pth:

NMR experimental design:

• Prepare uniformly 15N/13C-labeled Pth samples

• Collect standard triple-resonance experiments for backbone assignment:

  • HNCA, HN(CO)CA, HNCACB, CBCA(CO)NH

• Measure 15N-relaxation parameters:

  • R1 (longitudinal relaxation rate)

  • R2 (transverse relaxation rate)

  • {1H}-15N heteronuclear NOE

• Calculate order parameters (S2) to quantify flexibility

• Perform CPMG relaxation dispersion experiments to detect slow timescale motions

• Map dynamics data onto structural model to identify:

  • Rigid regions (gate loop)

  • Regions with slow motions (base loop)

  • Highly flexible regions (lid loop)

Expected outcomes:

• Identification of dynamic regions involved in substrate recognition

• Correlation between flexibility and catalytic activity

• Comparison with dynamics of Pth enzymes from other species

What crystallization conditions are suitable for G. oxydans Pth?

Based on successful crystallization of Pth from other bacterial species, the following strategy is recommended:

Initial screening:

• Concentrate purified G. oxydans Pth to 10-15 mg/mL

• Screen using commercial sparse matrix kits:

  • Hampton Crystal Screen 1 & 2

  • Molecular Dimensions JCSG+

  • Qiagen PACT Premier

• Set up sitting-drop vapor diffusion plates:

  • 1 μL protein + 1 μL reservoir solution

  • 500 μL reservoir volume

  • Incubate at 16-20°C

Optimization parameters:

• pH range: 6.0-8.5

• Precipitants: PEG 3350 (10-25%), ammonium sulfate (1.0-2.5 M)

• Salt additives: 0.1-0.3 M NaCl, MgCl2, or Li2SO4

• Additives: glycerol, MPD, or low concentrations of detergents

• Seeding techniques for improved crystal quality

Co-crystallization with substrates:

• Include tRNA fragments or peptidyl-tRNA analogs

• Try both soaking and co-crystallization approaches

• Optimize ligand:protein molar ratios (3:1 to 10:1)

How has Pth evolved across different bacterial species including G. oxydans?

Comparative evolutionary analysis of Pth enzymes reveals:

Evolutionary patterns:

• Pth is universally conserved across bacteria, reflecting its essential function

• Sequence conservation is highest in the catalytic core and substrate binding regions

• Greater sequence divergence occurs in peripheral regions

• Acetic acid bacteria like G. oxydans have Pth adaptations related to their acidophilic lifestyle

Methodological approach for evolutionary analysis:

• Collect Pth sequences from diverse bacterial phyla

• Perform multiple sequence alignment using MUSCLE or MAFFT

• Generate phylogenetic trees using Maximum Likelihood methods

• Calculate conservation scores for each residue

• Map conservation onto structural models

• Correlate conservation with:

  • Catalytic residues

  • Substrate binding regions

  • Species-specific adaptations

Can G. oxydans Pth be targeted for antibiotic development?

Targeting Pth for antibiotic development is promising based on findings from M. tuberculosis research:

Rationale for Pth as antimicrobial target:

• Pth is essential in most bacteria but absent in eukaryotes

• Inhibition leads to accumulation of toxic peptidyl-tRNAs

• Pth depletion increases susceptibility to certain antibiotics

Drug development strategy:

• Perform high-throughput screening against purified G. oxydans Pth

• Design structure-based inhibitors targeting the active site

• Validate hits in cellular assays using G. oxydans

• Assess selectivity against human enzymes

• Optimize pharmacokinetic properties

Potential synergistic applications:

• Combine Pth inhibitors with macrolides (rendered more effective against M. tuberculosis when Pth was depleted)

• Pair with tRNA synthetase inhibitors for enhanced activity

• Use in combination with existing acetic acid bacteria control agents

How can Pth inhibition affect tRNA pools in G. oxydans?

Pth inhibition has profound effects on tRNA pools that can be studied using:

Cu-tRNAseq analysis protocol:

• Treat G. oxydans with sub-lethal doses of Pth inhibitors or use conditional knockdown

• Extract total RNA at various time points

• Perform Cu-tRNAseq as described previously

• Analyze changes in:

  • Total charged tRNA fractions

  • Peptidyl-tRNA accumulation profiles

  • Specific tRNA isoacceptor depletion

Expected outcomes based on M. tuberculosis studies:

• Initial accumulation of peptidyl-tRNAs across most isoacceptors

• Progressive depletion of free tRNA pools

• Potential selective effects on specific tRNAs (e.g., prolyl-tRNA in M. tuberculosis)

• Correlation between tRNA depletion patterns and growth inhibition

How can CRISPR interference be applied to study Pth function in G. oxydans?

CRISPR interference (CRISPRi) offers a powerful approach for conditional knockdown:

CRISPRi system development:

• Design a two-plasmid system:

  • First plasmid: dCas9 (catalytically dead Cas9) under inducible promoter

  • Second plasmid: sgRNA targeting the pth gene promoter region

• Transform both plasmids into G. oxydans

• Induce dCas9 expression with appropriate inducer

• Validate knockdown efficiency by RT-qPCR and Western blot

• Monitor growth phenotypes under various conditions

Experimental applications:

• Titrate dCas9 expression to achieve varying levels of Pth depletion

• Combine with Cu-tRNAseq to correlate Pth levels with tRNA profiles

• Test antibiotic susceptibility under Pth depletion conditions

• Perform complementation studies with Pth variants

• Create a chemical-genetic profile by screening drug libraries

What computational approaches can predict substrate specificity of G. oxydans Pth?

Computational methods can provide insights into substrate specificity:

Structural bioinformatics approach:

• Generate homology model of G. oxydans Pth using M. smegmatis Pth (PDB: 2NAF) as template

• Perform molecular dynamics simulations to assess binding pocket flexibility

• Use docking studies with various peptidyl-tRNA substrates

• Calculate binding energies and identify key interaction residues

• Predict substrate preferences based on:

  • Electrostatic complementarity

  • Hydrogen bonding networks

  • Hydrophobic interactions

Machine learning strategy:

• Compile dataset of known Pth enzyme-substrate pairs with activity data

• Extract sequence and structural features

• Train machine learning models to predict:

  • Substrate binding affinity

  • Catalytic efficiency

  • Specificity determinants

• Validate predictions with experimental assays

• Apply to design improved Pth variants or inhibitors