Recombinant Protochlamydia amoebophila Alanine--tRNA ligase (alaS), partial

What is Alanine--tRNA ligase (alaS) and what is its functional role in Protochlamydia amoebophila?

Alanine--tRNA ligase (alaS), also known as alanyl-tRNA synthetase (AlaRS), is an essential enzyme that catalyzes the attachment of alanine to its cognate tRNA(Ala) through a two-step reaction mechanism. First, alanine is activated by ATP to form alanyl-adenylate (Ala-AMP), followed by the transfer of the activated alanine to the 3' end of tRNA(Ala) . This aminoacylation process is critical for protein synthesis as it ensures the correct incorporation of alanine into growing polypeptide chains during translation.

In Protochlamydia amoebophila, an intracellular symbiont of amoebae, alaS plays a particularly significant role due to the organism's unique lifestyle. P. amoebophila elementary bodies (EBs) have been demonstrated to maintain metabolic activity outside their host cells, including protein synthesis capabilities . The presence of functional alaS would be essential for sustaining this protein synthesis activity during the infectious, extracellular stage of the chlamydial developmental cycle.

How does recombinant partial alaS differ from full-length native alaS in enzymatic activity?

Recombinant partial alaS proteins typically contain key catalytic domains but may lack certain regulatory regions present in the full-length enzyme. Studies with E. coli alanyl-tRNA synthetase have shown that the full-length enzyme (ARS875) differs from truncated versions (such as ARS461) in several functional aspects :

The partial recombinant alaS may retain aminoacylation activity but typically shows altered kinetic parameters and reduced editing function compared to the native enzyme . This difference is critical to consider when designing experiments or interpreting results using recombinant partial proteins.

What are the optimal storage and handling conditions for recombinant P. amoebophila alaS?

Based on recommendations for similar recombinant proteins, the following storage and handling conditions should be applied to maintain optimal activity of recombinant P. amoebophila alaS:

• Long-term storage: Store at -20°C to -80°C with 50% glycerol as a cryoprotectant. Lyophilized preparations generally have a shelf life of approximately 12 months at -20°C/-80°C, while liquid preparations typically remain stable for about 6 months .

• Working storage: Aliquot the protein and store working stocks at 4°C for up to one week. Avoid repeated freeze-thaw cycles as they can significantly reduce enzyme activity .

• Reconstitution: For lyophilized preparations, briefly centrifuge the vial before opening, then reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Adding glycerol to a final concentration of 5-50% is recommended for stability .

• Buffer considerations: Tris-based buffers are typically suitable, with pH optimized for alaS activity (generally pH 7.5-8.0) .

How does P. amoebophila alaS compare structurally and functionally to other bacterial alaS enzymes?

While specific structural data for P. amoebophila alaS is limited, insights can be gained by comparing it to well-characterized bacterial alaS proteins like that of E. coli:

Class II aminoacyl-tRNA synthetases, including alaS, typically function as dimers in solution, as confirmed by analytical ultracentrifugation studies . P. amoebophila alaS likely shares this dimeric structure, with each monomer containing distinct domains for aminoacylation and editing functions.

The enzyme contains multiple interaction sites with its tRNA substrate. In E. coli alaS, at least seven distinct sites have been identified through modification studies with periodate-oxidized tRNA(Ala), including:

• One site in the N-terminal adenylate synthesis domain (residue 74)

• Two sites at the opening of the editing domain (residues 526 and 585)

• Three sites on the posterior side of the editing domain (residues 637, 639, and 648)

Similar interaction patterns likely exist in P. amoebophila alaS, reflecting the conserved function of this essential enzyme across bacterial species.

What mechanisms ensure aminoacylation fidelity in alaS enzymes?

Alanyl-tRNA synthetase has evolved sophisticated mechanisms to maintain aminoacylation fidelity:

• Editing activity: AlaRS can misactivate glycine and serine at significant rates due to their structural similarity to alanine. To prevent misincorporation, the enzyme contains a dedicated editing domain that hydrolyzes misacylated Gly-tRNA(Ala) and Ser-tRNA(Ala) . This hydrolytic editing occurs at specific sites (residues 526 and 585 in E. coli AlaRS) and is crucial for translational accuracy.

• Allosteric regulation: Binding of ATP and aminoacyl-adenylate analogues (such as ASAd and GSAd) alters the distribution of enzyme-tRNA interaction modes . This allosteric mechanism helps coordinate the synthetic and editing functions of the enzyme.

• Thermodynamic control: ATP binding to AlaRS is endothermic (ΔH = 3-4 kcal mol⁻¹), while binding of the correct aminoacyl-adenylate analogue (ASAd) is exothermic with a large negative heat capacity change (ΔCp = 0.48 kcal mol⁻¹ K⁻¹) . These thermodynamic properties contribute to the discrimination between correct and incorrect substrates.

What expression systems are optimal for producing functional recombinant P. amoebophila alaS?

Based on production approaches for similar proteins, the following expression systems are recommended:

E. coli expression systems are most commonly used for recombinant aminoacyl-tRNA synthetases, as evidenced by the successful production of recombinant AlaRS with good purity (>85% by SDS-PAGE) . When using E. coli, consider:

• Adding solubility-enhancing tags (His, GST, MBP) that can be cleaved post-purification

• Optimizing induction conditions (temperature, IPTG concentration, duration)

• Using specialized E. coli strains designed for expression of proteins with rare codons

• Co-expressing molecular chaperones if folding issues are encountered

How can I design assays to measure aminoacylation activity of recombinant P. amoebophila alaS?

Several approaches can be employed to assess the aminoacylation activity:

• ATP-PPi exchange assay: Measures the first step of the aminoacylation reaction (amino acid activation) by quantifying the exchange between ³²P-labeled PPi and ATP.

• Direct aminoacylation assay: Utilizes ¹⁴C or ³H-labeled alanine to monitor the formation of Ala-tRNA(Ala). The reaction mixture typically contains:

  • Purified recombinant alaS (10-100 nM)

  • tRNA(Ala) (1-10 μM)

  • Labeled alanine (10-50 μM)

  • ATP (2-5 mM)

  • Magnesium chloride (10 mM)

  • Buffer (typically Tris-HCl, pH 7.5)

• Pyrophosphate detection assay: A coupled enzymatic assay that measures PPi release during aminoacylation using pyrophosphatase and other coupling enzymes.

• Malachite green assay: Detects phosphate released from PPi during the reaction, allowing continuous monitoring of aminoacylation activity.

For kinetic analysis, varying substrate concentrations (alanine, tRNA, ATP) while maintaining constant enzyme concentration allows determination of Km and kcat values for each substrate.

What methods can be used to study the editing activity of P. amoebophila alaS?

The editing function of alaS can be studied using the following approaches:

• Misaminoacylation assays: Measure the rate of mischarging tRNA(Ala) with serine or glycine using radioactively labeled amino acids, followed by determination of the rate of hydrolysis of the misacylated tRNA.

• AMP formation assay: Monitor the hydrolysis of misactivated aminoacyl-adenylates by measuring AMP production using HPLC or coupled enzyme assays.

• Fluorescence-based assays: Utilize fluorescently labeled tRNAs to monitor both aminoacylation and deacylation in real-time.

• Site-directed mutagenesis: Introduce mutations in the predicted editing domain (based on homology with E. coli alaS) to identify residues critical for the editing function.

Experimental conditions should include appropriate controls:

• Reactions with canonical substrate (alanine) to establish baseline aminoacylation activity

• Reactions with non-cognate amino acids (glycine, serine) to assess misaminoacylation rates

• Reactions with pre-formed misacylated tRNAs to directly measure editing activity

How can P. amoebophila alaS be utilized to study the metabolic activity of elementary bodies?

Recent research has challenged the long-held view that chlamydial elementary bodies (EBs) are metabolically inert, demonstrating that P. amoebophila EBs maintain respiratory activity and can metabolize D-glucose in a host-free environment . To investigate the role of alaS in this context:

• Protein synthesis capability study: Use radiolabeled amino acids to track protein synthesis in purified EBs, with specific focus on the role of alaS through selective inhibition or depletion.

• Metabolic labeling: Employ stable isotope-labeled precursors combined with mass spectrometry analyses (IRMS, ICR/FT-MS, UPLC-MS) to trace metabolic activity, particularly examining how protein synthesis impacts central carbon metabolism in EBs .

• Nutrient deprivation experiments: Test how absence of specific nutrients affects EB viability and infectivity, potentially indicating essential metabolic pathways requiring functional alaS .

• Comparative analysis: Contrast the activity and regulation of alaS in P. amoebophila with that in pathogenic chlamydiae (e.g., C. trachomatis) to identify potential evolutionary adaptations related to different lifestyles.

What approaches can be used to identify potential inhibitors of P. amoebophila alaS?

Several strategies can be employed to identify inhibitors with potential antimicrobial applications:

• Structure-based design: Use homology modeling based on known alaS structures to design compounds targeting either the aminoacylation active site or the editing domain.

• High-throughput screening: Develop miniaturized assays suitable for screening compound libraries, focusing on:

  • ATP-PPi exchange inhibition

  • Aminoacylation inhibition

  • Disruption of tRNA binding

• Fragment-based approach: Screen small molecular fragments that bind to different regions of the enzyme, then link promising fragments to create more potent inhibitors.

• Exploitation of allosteric regulation: Target the allosteric sites identified through studies with aminoacyl-adenylate analogues (ASAd, GSAd) that have been shown to alter enzyme-tRNA interactions .

• Natural product screening: Test extracts from various sources, particularly focusing on compounds with known activity against related organisms.

For all inhibitor studies, it's essential to evaluate:

• Selectivity against bacterial versus human alaS enzymes

• Effect on whole-cell growth of P. amoebophila

• Mechanism of action (competitive, noncompetitive, uncompetitive)

• Structure-activity relationships to guide optimization

What factors might cause lower-than-expected activity in recombinant P. amoebophila alaS preparations?

Several factors can contribute to suboptimal enzyme activity:

Additionally, the partial nature of the recombinant protein might result in altered activity compared to the full-length enzyme. Studies with E. coli AlaRS have shown that N-terminal constructs (ARS461) exhibit different binding properties than the full-length enzyme (ARS875) .

How can I assess the quality and purity of recombinant P. amoebophila alaS preparations?

Multiple analytical techniques should be employed to evaluate protein quality:

• SDS-PAGE: Assess purity, with acceptable preparations typically showing >85% purity . Check for degradation products that might indicate proteolysis.

• Size exclusion chromatography: Evaluate the oligomeric state of the protein. AlaRS should primarily exist as a dimer in solution, as confirmed by analytical ultracentrifugation for other bacterial AlaRS enzymes .

• Mass spectrometry: Verify protein identity and detect any post-translational modifications or truncations.

• Thermal shift assays: Assess protein stability and the effect of different buffer conditions on the melting temperature.

• Activity assays: Compare specific activity (units/mg) to established benchmarks for similar enzymes to evaluate functional quality.

• Circular dichroism: Analyze secondary structure content to ensure proper folding.

What are the emerging research areas involving P. amoebophila alaS?

Several promising research directions exist for further investigation:

• Structural biology: Determining the three-dimensional structure of P. amoebophila alaS would provide valuable insights into its function and evolution, especially given its role in an organism with a unique intracellular lifestyle.

• Metabolic integration: Further studies on how alaS activity integrates with the central carbon metabolism in P. amoebophila EBs, particularly focusing on the pentose phosphate pathway and TCA cycle activities that have been observed in these supposedly dormant forms .

• Comparative enzymology: Systematic comparison of kinetic and thermodynamic properties of alaS from environmental chlamydiae (like P. amoebophila) versus pathogenic chlamydiae to identify adaptations related to different ecological niches.

• Antimicrobial development: Exploration of P. amoebophila alaS as a model for developing broad-spectrum inhibitors effective against related pathogens, potentially exploiting unique features of chlamydial AlaRS enzymes.

• Evolutionary studies: Investigation of horizontal gene transfer and evolutionary relationships among alaS enzymes from different domains of life, with particular focus on the unique evolutionary position of Protochlamydia as an ancestral relative of pathogenic chlamydiae.