Recombinant Acinetobacter sp. Aspartate--tRNA ligase (aspS), partial

What is Aspartate-tRNA ligase (AspRS) and what is its role in bacterial protein synthesis?

Aspartate-tRNA ligase (EC 6.1.1.12), also known as Aspartyl-tRNA synthetase (AspRS), is an essential enzyme responsible for charging tRNA^Asp with aspartate during protein synthesis. This enzyme catalyzes a critical step in translation by ensuring the correct amino acid is attached to its cognate tRNA, maintaining the fidelity of the genetic code. In Acinetobacter species, AspRS is encoded by the aspS gene and functions within the complex machinery of protein synthesis .

How do researchers classify different types of AspRS enzymes?

AspRS enzymes are classified into two main functional categories: discriminating and non-discriminating forms. Discriminating AspRS specifically charges tRNA^Asp with aspartate but cannot acylate tRNA^Asn. In contrast, non-discriminating AspRS can misacylate tRNA^Asn with aspartate, creating Asp-tRNA^Asn as an intermediate. This misacylated tRNA is later corrected by amidotransferases to form Asn-tRNA^Asn. While it was previously thought that bacterial AspRS enzymes were exclusively discriminating and archaeal ones non-discriminating, research has shown that both forms exist in archaea, highlighting the evolutionary complexity of these enzymes .

What are the structural domains of Acinetobacter AspRS and their functions?

Acinetobacter AspRS exhibits a multi-domain architecture typical of class II aminoacyl-tRNA synthetases:

What are the optimal conditions for reconstitution and storage of recombinant Acinetobacter AspRS?

Based on product specifications, researchers should follow these guidelines for optimal handling:

Proper reconstitution and storage are essential for maintaining enzymatic activity and ensuring experimental reproducibility .

What methods can be used to assess the activity of recombinant Acinetobacter AspRS?

Several complementary approaches allow researchers to evaluate different aspects of AspRS function:

• ATP-PPi exchange assay: Measures the first step of aminoacylation (amino acid activation) independent of tRNA charging.

• tRNA aminoacylation assay: Quantifies the rate of aspartate attachment to tRNA^Asp using radioisotope-labeled aspartate or ATP.

• Thin-layer chromatography (TLC): Can be used to separate and visualize charged from uncharged tRNAs, as demonstrated with tRNA modifications in Acinetobacter studies .

• LC-MS analysis: Provides precise quantification of aminoacylated tRNAs and can detect modifications, similar to techniques used to analyze m^7G modifications in Acinetobacter tRNAs .

• Gel mobility shift assays: Assess the binding affinity between AspRS and its tRNA substrates through electrophoretic mobility differences.

How can researchers generate AspRS mutants for functional studies?

Based on methodologies applied to related tRNA-modifying enzymes in Acinetobacter, researchers can follow this procedure:

• Design primers flanking the aspS gene or target region with appropriate overlaps for subsequent cloning steps.

• Use overlap extension PCR to fuse fragments with an antibiotic resistance cassette (e.g., apramycin resistance).

• Electroporate the purified linear DNA product into Acinetobacter strains containing a recombinase-expressing plasmid (such as pAT04 with IPTG-inducible RecAB).

• Select transformants using appropriate antibiotics and confirm gene replacement through whole-genome sequencing.

• For marker removal, introduce a plasmid encoding FLP recombinase (such as PAT03) to excise the antibiotic cassette if FRT sites were included.

• For complementation studies, use mini-Tn7 systems (such as pUCT18T-miniTn7-Zeo) to reintroduce the wild-type gene at a neutral site in the chromosome .

How does AspRS relate to bacterial stress responses and pathogenesis?

While AspRS itself has not been directly studied in Acinetobacter stress responses, research on other tRNA-modifying enzymes provides valuable insights into potential parallels. The tRNA methyltransferase TrmB in Acinetobacter baumannii has been shown to be critical for responses to oxidative stress, low pH, and iron deprivation - all conditions encountered during infection. Loss of TrmB dramatically attenuates virulence in murine pneumonia models .

By extension, as a key enzyme in translation, AspRS likely plays a role in:

• Maintaining translational fidelity under stress conditions

• Supporting adaptation to hostile host environments

• Enabling appropriate expression of virulence factors

• Contributing to antibiotic tolerance mechanisms

The essential nature of AspRS makes it a potential target for antimicrobial development, particularly given the rise of multidrug-resistant Acinetobacter strains .

What is the significance of recombinant partial AspRS in drug discovery research?

Recombinant partial AspRS proteins offer several advantages for drug discovery efforts:

• They provide accessible molecular targets for high-throughput screening of potential inhibitors.

• The partial protein may contain key catalytic domains while being easier to express and manipulate than the full-length enzyme.

• Structural studies of partial AspRS can reveal binding pockets and interaction sites for rational drug design.

• Comparison between bacterial AspRS and human homologs can identify bacterial-specific features for selective targeting.

• Partial AspRS constructs can serve as tools for validating hit compounds before progressing to whole-cell assays.

Recent efforts to develop inhibitors against another tRNA methyltransferase, TrmD, highlight the therapeutic potential of targeting tRNA-modifying enzymes to combat multidrug-resistant A. baumannii .

How does the genomic context of aspS in Acinetobacter inform its functional role?

The genomic neighborhood of aspS provides important insights into its evolutionary and functional relationships:

• The presence or absence of genes encoding asparaginyl-tRNA synthetase (AsnRS) and Asp-tRNA^Asn amidotransferase in the genome correlates with whether AspRS is discriminating or non-discriminating.

• Species with non-discriminating AspRS typically lack AsnRS but possess amidotransferase genes, supporting the indirect pathway for Asn-tRNA^Asn formation.

• Conversely, species with discriminating AspRS generally possess AsnRS but lack amidotransferase genes, utilizing the direct aminoacylation pathway .

This genomic context analysis helps researchers predict the functional characteristics of AspRS in different Acinetobacter strains and understand the evolutionary pressures shaping aminoacyl-tRNA synthetase systems.

How can researchers investigate the role of AspRS in coordinating translation with stress responses?

To explore the relationship between AspRS and stress adaptation, researchers can implement these sophisticated approaches:

• Conditional depletion systems: Utilize inducible promoters or degradation tags to modulate AspRS levels and assess the impact on stress response pathways.

• Ribosome profiling: Compare translational profiles between wild-type and AspRS-depleted cells under various stress conditions to identify differentially translated mRNAs.

• Proteomics analysis: Similar to studies with TrmB mutants, examine how altered AspRS activity affects protein expression during stress responses, particularly focusing on stress-response regulons and virulence factors .

• Post-transcriptional regulation analysis: Investigate whether AspRS is subject to post-transcriptional control during stress, similar to the regulation observed for acinetobactin cluster proteins in response to oxidative stress .

• In vivo infection models: Assess how AspRS mutations or inhibition affects bacterial survival during host infection, immune clearance, and disease progression.

What approaches can identify specific inhibitors of Acinetobacter AspRS?

Developing selective inhibitors requires sophisticated screening and validation strategies:

• Structure-based virtual screening: Use computational methods to identify compounds that bind to unique pockets in Acinetobacter AspRS.

• Fragment-based drug discovery: Screen small molecular fragments for binding to AspRS and optimize hits through medicinal chemistry.

• High-throughput biochemical assays: Develop miniaturized assays to screen compound libraries for inhibition of AspRS activity.

• Selectivity profiling: Test promising compounds against human DARS (cytosolic) and DARS2 (mitochondrial) aspartyl-tRNA synthetases to ensure bacterial selectivity.

• Resistance development studies: Assess the potential for resistance development by selecting for mutations that confer resistance to AspRS inhibitors.

• Combinatorial approaches: Explore synergistic inhibition of multiple tRNA-processing enzymes, such as combining AspRS inhibitors with those targeting TrmB or other tRNA methyltransferases .

How do evolutionary changes in AspRS contribute to Acinetobacter pathoadaptation?

Evolutionary analysis of AspRS can provide insights into bacterial adaptation processes:

• Sequence conservation analysis: Comparative genomics across Acinetobacter species can reveal conserved regions essential for function versus variable regions that may contribute to species-specific adaptations.

• Positive selection detection: Statistical methods can identify amino acid positions under positive selection pressure, potentially indicating adaptation to host environments.

• Horizontal gene transfer assessment: Analysis of codon usage, GC content, and phylogenetic incongruence can detect horizontal acquisition of aspS genes or domains.

• Host-pathogen co-evolution: Comparison of AspRS sequences from isolates adapted to different hosts may reveal host-specific adaptations.

• Mutational pathway reconstruction: Experimental evolution studies combined with structural analysis can elucidate how mutations in AspRS affect enzyme function and bacterial fitness.

The high sequence identity (up to 60%) observed between discriminating and non-discriminating archaeal AspRS enzymes suggests that relatively few mutations may be required to alter tRNA discrimination properties, highlighting the adaptability of these essential enzymes .

What are common challenges in working with recombinant Acinetobacter AspRS?

Researchers frequently encounter several technical challenges:

Implementing rigorous quality control procedures and standardized handling protocols can minimize these challenges and ensure consistent experimental results.

How can researchers verify the specificity of Acinetobacter AspRS activity?

To ensure accurate characterization of enzyme specificity:

• Substrate panel testing: Assess activity with various amino acids and tRNAs to confirm aspartate and tRNA^Asp specificity.

• Mischarging detection: Test whether the AspRS can charge tRNA^Asn with aspartate to determine if it is discriminating or non-discriminating.

• Amidotransferase coupling assay: If available, use Asp-tRNA^Asn amidotransferase to verify formation of Asp-tRNA^Asn, as demonstrated in studies with M. thermautotrophicus AspRS .

• Competition assays: Examine the ability of non-cognate amino acids or tRNAs to inhibit the canonical reaction.

• Kinetic parameter determination: Compare kinetic constants (K_m, k_cat) for cognate versus non-cognate substrates to quantify specificity differences.

These approaches provide a comprehensive assessment of AspRS specificity, which is essential for understanding its biological role and potential as a drug target.