Recombinant Photorhabdus luminescens subsp. laumondii Glycine--tRNA ligase alpha subunit (glyQ)

How does P. luminescens GlyRS compare to GlyRS from other organisms?

P. luminescens GlyRS shares significant structural similarities with other bacterial GlyRS enzymes, particularly from the Enterobacteriaceae family. Comparative analysis reveals:

• Like Escherichia coli GlyRS, P. luminescens GlyRS forms an X-shaped tetrameric structure (α₂β₂) .

• While the residues involved in metal and ligand binding are relatively conserved between P. luminescens and other bacteria, distinct differences exist compared to Pseudomonas aeruginosa LecA (a related protein) .

• Unlike some eukaryotic GlyRS enzymes that function as dimers (α₂), bacterial GlyRS typically requires both α and β subunits for catalytic activity .

• Human GlyRS contains a large open reading frame (ORF) encoding 685 amino acids with approximately 60% identity to Bombyx mori GlyRS and 45% identity to Saccharomyces cerevisiae GlyRS .

What are the optimal conditions for expressing and purifying recombinant P. luminescens glyQ?

Based on established protocols for recombinant GlyRS production, the following methodological approach is recommended:

Expression System Selection:

• E. coli is the preferred heterologous expression system for bacterial proteins like glyQ .

• Mammalian cell expression systems can be utilized for applications requiring specific post-translational modifications .

Expression and Purification Protocol:

• Clone the glyQ gene into an appropriate expression vector (e.g., pBAD24 with L-arabinose induction system has been successful for other P. luminescens proteins) .

• Transform the construct into an E. coli expression strain.

• Induce protein expression with appropriate inducer (e.g., L-arabinose or IPTG depending on the vector).

• Lyse cells using sonication or French press in a buffer containing 50 mM Tris-HCl pH 7.5, 300 mM NaCl, and 5% glycerol.

• Purify using affinity chromatography (if a tag was added) followed by size-exclusion chromatography.

• Assess purity using SDS-PAGE (>85% purity is typically achievable) .

Storage Recommendations:

• For liquid formulations: Store at -20°C/-80°C with shelf life of approximately 6 months.

• For lyophilized formulations: Store at -20°C/-80°C with shelf life of approximately 12 months.

• Avoid repeated freeze-thaw cycles; store working aliquots at 4°C for up to one week.

• For long-term storage, add glycerol to a final concentration of 5-50% (50% is recommended) .

How can I assess the aminoacylation activity of recombinant P. luminescens GlyRS?

Standard Aminoacylation Assay Protocol:

• Reaction Components:

  • Purified recombinant GlyRS (both α and β subunits required for activity)

  • tRNA^Gly substrate (either purified native tRNA or in vitro transcribed)

  • [14C] or [3H]-labeled glycine

  • ATP

  • Appropriate buffer (typically containing Mg²⁺)

• Kinetic Parameter Measurement:
  Typical kinetic parameters for GlyRS enzymes that can be used as reference points include:

  Parameter	Substrate	Typical Values	Citations
  K_m	ATP	14.0-42.0 μM
  K_m	Glycine	30.0-160.0 μM
  K_m	tRNA^Gly	0.2-2.1 μM

• Data Analysis:
  Calculate the k_cat and k_cat/K_m values to determine the enzyme efficiency. For comparison, reference values from related GlyRS enzymes can be used. For example, from Lactobacillus plantarum (Lp) GlyRS studies:

  tRNA Variant	K_m (μM)	k_cat (×10⁻³/sec)	k_cat/K_m (relative)
  Lp tRNA^Gly(UCC) in vitro transcript	0.690 ± 0.087	123 ± 13	1
  mutant_(a)_A1U72	0.235 ± 0.004	3.6 ± 0.15	0.086
  mutant_(b)_G2C71	0.328 ± 0.063	23 ± 7	0.39
  mutant_(c)_G35	Nd	Nd	Nd
  mutant_(d)_A36	Nd	Nd	Nd

  Note: Nd = Not determined due to minimal activity

What are the key tRNA identity elements recognized by bacterial GlyRS enzymes?

Based on comparative studies of GlyRS from various bacterial species, several key identity elements in tRNA^Gly have been identified that are likely relevant to P. luminescens GlyRS:

How do mutations in tRNA^Gly affect recognition by bacterial GlyRS?

Studies with bacterial GlyRS enzymes have demonstrated the effects of various tRNA mutations on aminoacylation efficiency. These findings can guide experimental design when studying P. luminescens GlyRS specificity:

How can structural models of P. luminescens GlyRS-tRNA complexes inform enzyme engineering?

Advanced structural analyses of GlyRS-tRNA complexes provide valuable insights for rational enzyme engineering:

• Docking Model Insights:

  • The docking model of Lactobacillus plantarum GlyRS with tRNA^Gly (which can serve as a model for P. luminescens GlyRS) shows that the acceptor stem and anticodon region of L-shaped tRNA are recognized by the enzyme .

  • The T-stem region may interact with the B1 domain of the β subunit .

  • While the D-loop and T-loop do not directly interact with GlyRS in the model, they may interact with the enzyme through the flexible B2 domain of the β subunit .

• Specific Residue Interactions:

  • In the anticodon-binding domain, specific residues interact with C35 and C36 through hydrogen bonds. For example, in E. coli GlyRS, C35 forms hydrogen bonds with the main chain O or NH of Phe652, Val655, Val657, and Met658, while C36 hydrogen bonds to Arg585 .

  • Many of these residues are conserved in P. luminescens GlyRS, suggesting similar recognition mechanisms .

• Discriminator Base Recognition:

  • The discriminator base U73 is recognized differently among GlyRS enzymes. P. luminescens GlyRS may recognize U73 more strictly than E. coli GlyRS .

  • In E. coli GlyRS, both the base and the ribose/phosphate moiety of U73 are recognized, with Arg482 forming hydrogen bonds with the phosphate oxygen and Gln270 with the 2'-OH of the ribose .

This structural information can guide the design of mutations to alter substrate specificity or improve catalytic efficiency in engineered GlyRS variants.

What are the potential applications of recombinant P. luminescens GlyRS in synthetic biology?

Recombinant GlyRS enzymes, including P. luminescens GlyRS, have several potential applications in synthetic biology:

• Expanding the Genetic Code:

  • Engineered GlyRS variants can be used to incorporate non-canonical amino acids at glycine codons, expanding the genetic code for novel protein functions.

  • By altering the amino acid binding pocket while maintaining tRNA recognition, GlyRS can be engineered to aminoacylate tRNA^Gly with non-natural amino acids.

• Orthogonal Translation Systems:

  • P. luminescens GlyRS could be engineered to function as part of an orthogonal translation system in heterologous hosts, allowing site-specific incorporation of non-canonical amino acids without interfering with the host's translation machinery.

• Cell-Free Protein Synthesis:

  • Recombinant GlyRS can be used in cell-free protein synthesis systems to produce proteins with specific modifications at glycine positions.

What are common challenges in expressing functionally active P. luminescens GlyRS?

Several technical challenges may arise when working with recombinant P. luminescens GlyRS:

• Subunit Coordination:

  • GlyRS requires both α (glyQ) and β (glyS) subunits for activity. Expressing only the α subunit will not yield a functional enzyme .

  • Ensure stoichiometric expression of both subunits or consider creating a fusion protein of the α and β subunits, which has been shown to maintain catalytic activity in some systems .

• Protein Solubility:

  • If encountering solubility issues, consider:

    • Lowering the expression temperature (16-18°C)

    • Using solubility-enhancing fusion tags (e.g., MBP, SUMO)

    • Optimizing buffer conditions (pH, salt concentration, additives)

• Assessing Activity:

  • When testing aminoacylation activity, use both native tRNA^Gly and in vitro transcribed tRNA to rule out potential post-transcriptional modification requirements.

  • Include positive controls with well-characterized GlyRS enzymes (e.g., E. coli GlyRS) when establishing new activity assays.

How can I differentiate between structural and catalytic defects in mutant GlyRS variants?

When analyzing mutant GlyRS variants with altered activity, it's important to distinguish between structural defects and specific catalytic effects:

• Structural Analysis Approaches:

  • Circular dichroism (CD) spectroscopy to assess secondary structure integrity

  • Size-exclusion chromatography to verify proper oligomeric state (α₂β₂ tetramer)

  • Thermal shift assays to evaluate protein stability

• Functional Dissection Methods:

  • ATP-PP_i exchange assays to isolate the amino acid activation step from tRNA charging

  • tRNA binding assays (e.g., electrophoretic mobility shift assay) to assess tRNA binding independently of catalysis

  • Active site titration to determine the fraction of active enzyme

• Comprehensive Kinetic Analysis:

  • Determine k_cat and K_m values for all substrates (ATP, glycine, tRNA^Gly)

  • Calculate the effect of mutations on binding (K_m) versus catalysis (k_cat)

  • Use different tRNA variants to probe specific recognition elements