Recombinant Enterococcus faecalis Glycine--tRNA ligase alpha subunit (glyQ)

Basic Research Questions

• What is the structural organization of E. faecalis Glycine--tRNA ligase and how does it compare to other bacterial homologs?

The Glycine--tRNA ligase (GlyRS) in E. faecalis, like in other bacteria, is a heterotetrameric enzyme consisting of two α-subunits (encoded by glyQ) and two β-subunits (encoded by glyS). Structural studies reveal an X-shaped architecture, which differs significantly from the homodimeric GlyRS found in eukaryotes.

The α-subunits are primarily responsible for aminoacylation activity, while the β-subunits contribute to tRNA recognition and specificity. Based on homology to E. coli GlyRS, the α-subunit contains approximately 303 amino acid residues, with conserved motifs for ATP binding and aminoacylation .

Unlike single-chain synthetases, the X-shaped structure of bacterial GlyRS shows wide separation of abutting chain termini, suggesting evolutionary pressure against a single polypeptide format. This structural arrangement appears to be critical for enzyme function and explains why the α- and β-subunits are encoded by separate genes .

• What are the recommended expression systems and conditions for producing recombinant E. faecalis glyQ?

For successful expression of recombinant E. faecalis glyQ, a bacterial expression system using E. coli BL21(DE3) is typically employed. The gene can be amplified from E. faecalis genomic DNA using PCR with specific primers targeting the glyQ gene.

The optimized protocol involves:

• Insertion of the glyQ gene into an expression vector such as pET20b using appropriate restriction sites (e.g., NdeI and XhoI)

• Transformation into E. coli BL21(DE3) cells

• Growth of transformants in LB medium with appropriate antibiotics to OD600 = 0.6

• Induction of protein expression with IPTG

• Cell harvesting and protein purification using affinity chromatography

For co-expression of both α- and β-subunits, a dual plasmid system can be used, with the glyQ gene in one vector and glyS in another, using different antibiotic selection markers (e.g., ampicillin and kanamycin) .

• How can researchers verify the activity of recombinant E. faecalis glyQ?

Verification of recombinant E. faecalis glyQ activity involves multiple analytical approaches:

Aminoacylation Assay:
The primary function of GlyRS is to charge tRNA^Gly with glycine. This activity can be measured using:

• Radioactive assays with [³H]-glycine or [¹⁴C]-glycine

• Thin-layer chromatography to separate charged from uncharged tRNAs

• Spectrophotometric assays monitoring ATP consumption

Structural Verification:

• Circular dichroism to confirm proper folding

• Size-exclusion chromatography to verify tetrameric assembly

• Western blotting using antibodies specific to the α-subunit

Binding Studies:

• Surface plasmon resonance to measure interaction with tRNA^Gly

• Electrophoretic mobility shift assays to detect protein-tRNA complex formation

To generate in vitro transcribed tRNA^Gly for these assays, researchers can use templates produced by PCR with primers containing the T7 promoter sequence, similar to the methodology described for E. coli tRNA^Gly .

• What are the essential domains in E. faecalis glyQ and their functional significance?

The E. faecalis glyQ protein contains several functionally important domains that contribute to its aminoacylation activity:

The catalytic core within the Rossmann fold contains conserved HIGH and KMSKS motifs essential for ATP binding and aminoacylation. Mutations in these regions typically abolish enzymatic activity.

The dimerization interface is critical for forming the functional α₂β₂ tetramer, as isolated α-subunits show significantly reduced activity. This tetramerization is likely facilitated by a C-terminal helix, similar to what has been observed in E. coli GlyRS .

Advanced Research Questions

• How does the X-shaped architecture of bacterial GlyRS influence its catalytic mechanism in E. faecalis?

The X-shaped architecture of bacterial GlyRS, including E. faecalis GlyRS, has profound implications for its catalytic mechanism:

The spatial arrangement creates four active sites positioned at the extremities of the X-structure, allowing for independent aminoacylation reactions. This contrasts with the more constrained arrangement in homodimeric eukaryotic GlyRS.

The wide separation between chain termini observed in structural studies indicates strong evolutionary pressure against a single polypeptide format, suggesting that the flexible tetramer configuration is essential for proper tRNA binding and catalysis .

The architecture facilitates a communication network between subunits, where conformational changes in one subunit can influence the activity of others. This cooperativity likely enhances the enzyme's ability to respond to cellular conditions.

The β-subunit in E. faecalis GlyRS contains five domains, each playing distinct roles in:

• α-subunit binding

• ATP coordination

• tRNA recognition

• Stabilization of the aminoacylation transition state

This complex arrangement likely contributes to the enzyme's specificity and efficiency in aminoacylating tRNA^Gly, particularly under varying physiological conditions.

• What experimental approaches can be used to study the interaction between E. faecalis glyQ and tRNA^Gly?

Studying the interaction between E. faecalis glyQ and tRNA^Gly requires sophisticated experimental approaches:

In vitro Transcription of tRNA^Gly:
Researchers can generate E. faecalis tRNA^Gly using T7 RNA polymerase and DNA templates created through overlapping PCR. A typical protocol involves:

• Design of primers containing the T7 promoter (5′-TAATACGACTCACTATA-3′) followed by the tRNA^Gly gene sequence

• PCR amplification to generate the DNA template

• In vitro transcription using T7 RNA polymerase

• Purification of transcribed tRNA by gel electrophoresis

Binding Assays:

• Electrophoretic mobility shift assays (EMSA) to visualize complex formation

• Filter binding assays to determine binding constants

• Surface plasmon resonance for real-time analysis of association/dissociation kinetics

• Isothermal titration calorimetry to measure thermodynamic parameters

Structural Studies:

• X-ray crystallography of GlyRS-tRNA^Gly complexes

• Cryo-electron microscopy for visualizing the complex in different conformational states

• Hydrogen-deuterium exchange mass spectrometry to identify interaction interfaces

Functional Assays:

• Aminoacylation kinetics using purified components

• Competition assays with tRNA variants to assess specificity determinants

• Mutagenesis of specific glyQ residues to identify critical interaction points

• How can researchers address the challenges of distinguishing between GlyRS-mediated canonical and non-canonical functions in E. faecalis?

Distinguishing between canonical (aminoacylation) and non-canonical functions of GlyRS in E. faecalis requires multiple complementary approaches:

Separation-of-Function Mutants:
Create specific mutations that disrupt aminoacylation activity while preserving other potential functions. This typically involves:

• Site-directed mutagenesis of catalytic residues

• Expression of these mutants in E. faecalis

• Assessment of phenotypic effects independent of translation

Protein-Protein Interaction Studies:

• Immunoprecipitation followed by mass spectrometry (IP-MS) to identify interaction partners

• Bacterial two-hybrid assays to confirm specific interactions

• Proximity labeling techniques to capture transient interactions

Transcriptome and Proteome Analysis:

• RNA-Seq to identify genes affected by GlyRS dysfunction beyond translation effects

• Ribosome profiling to distinguish translational from non-translational effects

• Proteomics to identify proteins affected by GlyRS alterations

In vivo Localization:

• Fluorescence microscopy with tagged GlyRS to track subcellular localization

• Fractionation studies to identify GlyRS in unexpected cellular compartments

• ChIP-Seq if DNA binding activity is suspected

Recent studies with similar aminoacyl-tRNA synthetases have revealed unexpected roles in stress responses, gene regulation, and bacterial pathogenicity . Similar non-canonical functions may exist for E. faecalis GlyRS, particularly under stress conditions.

• What role might E. faecalis glyQ play in bacterial stress responses and virulence?

E. faecalis glyQ likely contributes to stress responses and virulence through multiple mechanisms:

Integration with Stringent Response:
GlyRS function is linked to the bacterial stringent response through interactions with RelA and (p)ppGpp metabolism. Under amino acid starvation:

• Uncharged tRNAs accumulate, potentially altering GlyRS activity

• RelA produces (p)ppGpp, which may modulate GlyRS function

• This coordination helps bacteria adapt to nutrient limitation

Stress Response Regulation:
Transcriptome studies of E. faecalis under alkaline stress (pH 10) have revealed differential expression of genes involved in:

• Carbohydrate metabolism

• Amino acid biosynthesis

• Nucleotide transport and metabolism

While specific data on glyQ regulation under stress is limited, aminoacyl-tRNA synthetases often show altered expression or activity during stress adaptation in bacteria.

Biofilm Formation:
E. faecalis can form biofilms in hostile environments, including alkaline conditions (pH 10) . Proper protein synthesis mediated by functional GlyRS is likely essential for:

• Production of adhesins and extracellular matrix components

• Stress adaptation proteins needed for biofilm persistence

• Coordination of metabolic shifts during biofilm formation

Virulence Connection:
E. faecalis is known for its robust ability to survive outside the host and has high intrinsic antimicrobial resistance . The core genome, which includes essential genes like glyQ, serves as:

• A genetic scaffold for evolution in healthcare settings

• A source of potential vaccine and drug targets

• A contributor to survival during infection

• How can recombinant E. faecalis glyQ be utilized for structural studies aimed at developing novel antibiotics?

Recombinant E. faecalis glyQ presents a valuable target for structural studies with antibiotic development potential:

Structural Determination Approaches:

• X-ray crystallography of the isolated α-subunit

• Cryo-EM of the complete α₂β₂ tetramer

• NMR studies of specific domains

For successful crystallization, researchers typically:

• Express the glyQ fragment with a cleavable His-tag

• Purify using nickel affinity chromatography

• Remove the tag with site-specific proteases

• Further purify by size-exclusion chromatography

• Screen multiple crystallization conditions

Structure-Based Drug Design:
Once high-resolution structures are obtained, researchers can:

• Identify unique pockets in E. faecalis GlyRS not present in human homologs

• Perform in silico screening of compound libraries

• Design competitive inhibitors that block ATP or glycine binding

• Develop allosteric inhibitors that disrupt tetramer formation

Inhibitor Validation Pipeline:

• Biochemical assays measuring aminoacylation inhibition

• Determination of minimum inhibitory concentrations (MICs)

• Cytotoxicity testing against mammalian cells

• Pharmacokinetic and pharmacodynamic studies

• Animal infection models

The unique X-shaped architecture of bacterial GlyRS provides distinct targeting opportunities compared to other bacterial aminoacyl-tRNA synthetases . The differences between bacterial and human GlyRS make this enzyme an attractive antibiotic target.

• What are the current methods for analyzing interactions between E. faecalis glyQ and small molecule regulators?

Several sophisticated methods can be employed to study interactions between E. faecalis glyQ and small molecule regulators:

Biophysical Interaction Analysis:

• Differential scanning fluorimetry (DSF) to monitor thermal stability changes upon ligand binding

• Microscale thermophoresis (MST) to measure binding affinities in solution

• Surface plasmon resonance (SPR) for real-time binding kinetics

• Isothermal titration calorimetry (ITC) for complete thermodynamic profiles

Structural Analysis:

• X-ray crystallography of glyQ-ligand complexes

• NMR for mapping binding interfaces in solution

• Hydrogen-deuterium exchange mass spectrometry to identify conformational changes

Functional Assays:

• Enzyme inhibition/activation assays measuring aminoacylation rates in the presence of regulators

• ATP consumption monitoring using coupled enzymatic assays

• tRNA charging efficiency using gel-based methods

A particularly intriguing finding from related aminoacyl-tRNA synthetases is the discovery that the small alarmone synthetase RelQ from E. faecalis (which produces the alarmone nucleotides pppGpp and ppGpp) is regulated by a sophisticated mechanism involving RNA binding and allosteric regulation :

Similar complex regulatory mechanisms may exist for E. faecalis glyQ, particularly in response to stress conditions where both RNA availability and small molecule regulators might fluctuate significantly .

• How does E. faecalis glyQ activity correlate with antibiotic resistance mechanisms?

The relationship between E. faecalis glyQ activity and antibiotic resistance involves several interconnected mechanisms:

Translation Quality Control:
GlyQ is essential for accurate protein synthesis. Modulation of its activity affects:

• Translational fidelity and error rates

• Stress response protein production

• Cell wall synthesis enzyme accuracy

Stress Response Integration:
The bacterial stringent response, mediated in part by aminoacyl-tRNA synthetases like GlyQ, is connected to antibiotic tolerance through:

• (p)ppGpp production influencing persister cell formation

• Metabolic adaptation to antibiotic stress

• Transcriptional reprogramming affecting antibiotic targets

Cross-resistance Phenomena:
Alterations in aminoacyl-tRNA synthetase activity can contribute to:

• Adaptive responses to multiple antibiotic classes

• Changes in cell envelope properties

• Altered metabolic states that reduce antibiotic efficacy

E. faecalis is notorious for its high intrinsic and acquired antimicrobial resistance, complicating treatment of hospital-acquired enterococcal infections. The core genome, which includes essential genes like glyQ, serves as a genetic scaffold for evolution in healthcare settings .

Researchers investigating these connections typically employ:

• Comparative transcriptomics of resistant vs. sensitive strains

• Mutagenesis studies of glyQ and phenotypic analysis

• Time-kill experiments under various antibiotic pressures

• Fitness studies in the presence of sublethal antibiotic concentrations

• What glycan analysis techniques can be integrated with E. faecalis glyQ studies to understand broader cellular processes?

Integrating glycan analysis with E. faecalis glyQ studies provides insights into the relationship between protein synthesis and glycosylation processes:

Glycan Analysis Platforms:
The Gly-Q Glycan Analysis System represents a comprehensive approach that can be applied to E. faecalis studies:

• Electropherogram-based analysis of glycan profiles

• Use of glucose unit (GU) ladders for standardization

• Migration standards (MS) for alignment purposes

• Integration parameters for peak detection and analysis

Integrated Research Approaches:

• Glycan Profile Changes During Stress:

  • Monitor glycan profiles when glyQ is inhibited or mutated

  • Analyze impacts on cell wall glycan structures

  • Track changes in secreted glycoproteins

• Metabolic Connections:

  • Investigate how amino acid and sugar metabolism are coordinated

  • Examine how translation quality affects glycosylation machinery

  • Study shared metabolic precursors between protein and glycan synthesis

• Glycan Utilization During Nutrient Limitation:
  E. faecalis can utilize glycans as carbon sources through specific hydrolases:

  • GH18 family glycosyl hydrolases (EfEndo18A) primarily deglycosylate high-mannose glycoproteins

  • The enzyme EndoE targets complex-type glycoproteins like IgG

  • These activities are regulated by carbon catabolite repression via CcpA

• Technical Integration:

  • Glycomic quintavariate-informed quantification (GlyQ-IQ) enables identification of N-glycans in LC-MS data

  • This approach uses a priori glycan information for improved sensitivity

  • Biological targeting approach offers key advantages over traditional processing methods

This integrated approach is particularly relevant as E. faecalis can metabolize glucosamine, and colonic glucosamine levels increase during experimental colitis, potentially exacerbating inflammation .