Recombinant Idiomarina loihiensis Glycine--tRNA ligase beta subunit (glyS), partial

What is the evolutionary significance of Glycine--tRNA ligase in Idiomarina loihiensis?

The evolutionary significance of Glycine--tRNA ligase in I. loihiensis is closely tied to the organism's metabolic adaptation. This marine bacterium has undergone an ecological shift from using sugars as its primary carbon source to relying on amino acids, particularly phenylalanine. This metabolic adaptation has influenced the evolution of various enzymes in its genome, including aminoacyl-tRNA synthetases like Glycine--tRNA ligase. Studies show that while glycolysis genes in I. loihiensis demonstrate relaxed negative selection (suggesting decreased importance), amino acid metabolism pathways show signs of positive selection, reflecting adaptation to its unique ecological niche . The evolutionary trajectory of GlyRS in this organism represents a fascinating case study in how ecological specialization shapes essential cellular machinery.

What is known about the gene structure of glyS in Idiomarina loihiensis?

While the search results don't provide specific information about the gene structure of glyS in I. loihiensis, comparative genomics suggests this gene would share characteristics with other bacterial glycyl-tRNA synthetase genes. The glyS gene typically encodes the beta subunit of the heterodimeric GlyRS found in bacteria. The genomic context of glyS in I. loihiensis likely reflects its metabolic adaptation from sugar to amino acid utilization .

To properly characterize the gene structure, researchers should:

• Perform full-length sequencing of the glyS gene from I. loihiensis

• Analyze the promoter region for regulatory elements

• Investigate operon structure to determine if glyS is co-transcribed with other genes

• Compare the sequence with glyS from related bacterial species to identify conserved and divergent regions

How does the metabolic adaptation of Idiomarina loihiensis affect its tRNA synthetase functionality?

The metabolic adaptation of I. loihiensis from sugar utilization to amino acid metabolism has likely influenced the functional properties of its tRNA synthetases, including GlyRS. I. loihiensis has lost many genes essential for sugar metabolism and relies instead on amino acids as its primary source of energy and carbon . This shift in metabolism is reflected in the evolutionary patterns of its enzymes:

• Glycolysis genes show high values of ν (a measure of evolutionary rate), suggesting relaxed negative selection on sugar metabolism pathways

• Amino acid metabolic enzymes, particularly those involved in phenylalanine metabolism, show signs of adaptation

• These metabolic changes may have driven adaptations in tRNA synthetases to optimize charging efficiency in an amino acid-rich environment

The specific functional consequences for GlyRS might include altered substrate affinity, catalytic efficiency, or regulatory mechanisms compared to homologs from sugar-metabolizing bacteria.

What expression systems are most effective for producing recombinant I. loihiensis GlyS?

For optimal expression of recombinant I. loihiensis GlyS, researchers should consider:

Bacterial expression systems:

• E. coli BL21(DE3) with pET-based vectors is typically effective for bacterial proteins

• For improved solubility, consider fusion tags such as MBP, SUMO, or Thioredoxin

• Cold-shock expression (16-18°C) may increase the proportion of correctly folded protein

Expression optimization protocol:

• Clone the glyS gene into multiple expression vectors with different fusion tags

• Transform into various E. coli expression strains

• Test expression at different temperatures (16°C, 25°C, 37°C)

• Vary IPTG concentration (0.1-1.0 mM) and induction time (3-24 hours)

• Analyze soluble and insoluble fractions to determine optimal conditions

Since I. loihiensis is a marine bacterium adapted to high salt environments, expression may benefit from media supplemented with NaCl or other osmolytes to promote proper folding.

What purification strategies yield the highest activity of recombinant I. loihiensis GlyS?

Effective purification of recombinant I. loihiensis GlyS with preserved enzymatic activity requires:

Multi-step purification protocol:

• Initial capture: Affinity chromatography based on fusion tag (His-tag, GST, etc.)

• Intermediate purification: Ion exchange chromatography (typically DEAE or SP sepharose)

• Polishing: Size exclusion chromatography to remove aggregates and ensure homogeneity

Buffer considerations:

• Maintain moderate ionic strength (150-300 mM NaCl) throughout purification

• Include glycerol (10-20%) to enhance stability

• Add reducing agent (DTT or β-mercaptoethanol, 1-5 mM) to prevent oxidation of cysteine residues

• Consider adding ATP (1-2 mM) and MgCl₂ (5-10 mM) as stabilizing cofactors

Activity preservation:

• Avoid freeze-thaw cycles by aliquoting purified enzyme

• Store at -80°C in buffer containing 50% glycerol

• Include protease inhibitors during initial lysis steps

• Perform activity assays after each purification step to track retention of function

How can researchers optimize the yield of functionally active recombinant I. loihiensis GlyS?

To optimize yield of functionally active recombinant I. loihiensis GlyS:

Expression optimization:

• Test codon-optimized gene constructs to match E. coli codon usage

• Co-express with molecular chaperones (GroEL/ES, DnaK/J) to assist folding

• Use auto-induction media for gradual protein expression

• Consider dual expression of alpha and beta subunits if required for stability

Solubility enhancement:

• Screen various detergents (0.05-0.1% Triton X-100, NP-40) for lysis buffer

• Test different pH ranges (pH 6.5-8.5) to identify optimal solubility conditions

• Include amino acid substrates (glycine, 5-10 mM) in buffers as stabilizers

Process scale-up considerations:

• Implement fed-batch fermentation for high-density cultures

• Optimize cell disruption methods (sonication vs. high-pressure homogenization)

• Develop tangential flow filtration protocols for concentration

• Implement quality control checkpoints with activity assays at each stage

What are the critical factors affecting the stability of purified recombinant I. loihiensis GlyS?

Several factors critically affect the stability of purified recombinant I. loihiensis GlyS:

Physical parameters:

• Temperature: Store at -80°C long-term; maintain at 4°C during purification

• pH: Typically most stable between pH 7.0-8.0

• Ionic strength: Maintain physiological salt concentration (150-300 mM NaCl)

Chemical stabilizers:

• Glycerol (20-50%) prevents freezing damage and stabilizes structure

• Reducing agents (DTT, TCEP) prevent oxidation of cysteine residues

• Divalent cations (Mg²⁺, 5-10 mM) often required for structural integrity

• Substrate-like compounds (ATP analogs, glycine) can enhance stability

Prevention of degradation:

• Identify and eliminate protease contamination

• Avoid multiple freeze-thaw cycles

• Filter sterilize preparations to prevent microbial contamination

Stability monitoring protocol:

• Establish baseline activity using standardized aminoacylation assay

• Store enzyme under different conditions (varied pH, temperature, additives)

• Measure activity at regular time intervals (0, 24h, 72h, 1 week, 1 month)

• Analyze by SDS-PAGE to detect degradation products

• Use thermal shift assays to identify stabilizing buffer components

What domains are present in the beta subunit of I. loihiensis Glycine--tRNA ligase?

While specific structural information about I. loihiensis GlyS is limited, we can infer domain organization based on homologous bacterial glycyl-tRNA synthetases. The beta subunit typically contains:

Catalytic core domains:

• Rossmann fold domain: binds ATP and contains the HIGH and KMSKS motifs essential for amino acid activation

• Anticodon binding domain: recognizes the anticodon loop of tRNA^Gly

• Acceptor stem binding region: interacts with the acceptor stem of tRNA^Gly

Structural features:

• Active site pocket accommodating glycine and ATP

• Insertion domains that may facilitate tRNA binding

• Interface regions for interaction with the alpha subunit

Researchers investigating domain structure should use protein structure prediction tools, limited proteolysis combined with mass spectrometry, and X-ray crystallography or cryo-EM to resolve the complete structure.

How does the substrate binding pocket of I. loihiensis GlyS compare to other bacterial GlyRS enzymes?

The substrate binding pocket of I. loihiensis GlyS likely contains conserved features for glycine and ATP recognition common to all glycyl-tRNA synthetases, but may exhibit adaptations reflecting its evolution in an amino acid-rich environment. Comparison with other bacterial GlyRS enzymes would require:

Structural analysis methods:

• Homology modeling based on crystallized bacterial GlyRS structures

• Molecular docking of glycine and ATP into the predicted active site

• Identification of conserved active site residues through multiple sequence alignment

• Molecular dynamics simulations to analyze binding pocket flexibility

Key features to analyze include:

• Residues forming hydrogen bonds with glycine

• ATP binding pocket architecture

• Comparison with homologs from both marine and terrestrial bacteria

• Unique residues that might confer adaptation to I. loihiensis's ecological niche

What are the key residues involved in tRNA recognition by I. loihiensis GlyS?

Based on studies of glycyl-tRNA synthetases in other organisms, the key residues involved in tRNA recognition by I. loihiensis GlyS likely interact with specific identity elements in tRNA^Gly. From search result , we know that:

tRNA identity elements recognized by GlyRS include:

• The C35-C36 anticodon bases are critical recognition elements

• The A1-U72 base pair in the acceptor stem is important

• The G2-C71 base pair contributes to recognition

• The discriminator base (U73) is significant for aminoacylation

Experimental approaches to identify key residues:

• Site-directed mutagenesis of conserved residues in the predicted anticodon binding domain

• In vitro aminoacylation assays with tRNA variants to map recognition elements

• Cross-linking studies followed by mass spectrometry

• Structural studies of GlyRS-tRNA complexes using X-ray crystallography or cryo-EM

From search result , a kinetic analysis table for tRNA mutants shows the importance of these elements:

These data highlight the importance of specific base pairs and anticodon residues in tRNA recognition .

What conformational changes occur in I. loihiensis GlyS during the aminoacylation reaction?

While specific conformational changes in I. loihiensis GlyS have not been documented, studies of other glycyl-tRNA synthetases suggest the following conformational dynamics during aminoacylation:

Sequential conformational changes:

• Initial binding of ATP and glycine induces closure of the active site

• Formation of glycyl-adenylate intermediate causes repositioning of catalytic residues

• tRNA binding triggers global conformational rearrangements

• Transfer of activated glycine to tRNA^Gly requires precise alignment of substrates

From search result , we know that "hGlyRS catalysis involves multiple conformational changes, and insertions 1 and 3 may facilitate tRNA binding." Similar conformational dynamics likely occur in I. loihiensis GlyS.

Methods to study conformational changes:

• FRET (Förster Resonance Energy Transfer) using strategically placed fluorophores

• Hydrogen-deuterium exchange mass spectrometry

• Time-resolved X-ray crystallography

• Molecular dynamics simulations

• Single-molecule studies to capture transient conformational states

How does the structure of I. loihiensis GlyS differ from other marine bacterial GlyRS enzymes?

Comparing I. loihiensis GlyS with other marine bacterial GlyRS enzymes requires:

Structural comparison approach:

• Sequence alignment with GlyRS from other marine bacteria

• Homology modeling of I. loihiensis GlyS

• Structural superposition to identify differences in:

  • Active site architecture

  • Surface electrostatic properties

  • Oligomerization interfaces

  • Substrate binding pockets

Potential adaptations in marine bacteria:

• Enhanced salt tolerance through increased acidic surface residues

• Structural adaptations for pressure resistance

• Modified interactions between subunits for stability in high-salt environments

• Specialized substrate recognition features reflecting available amino acid pools

Although specific structural information on I. loihiensis GlyS is limited in the search results, its adaptation to marine environments suggests potential structural differences from terrestrial bacterial GlyRS enzymes.

What unique adaptations does I. loihiensis GlyS exhibit compared to GlyRS from sugar-metabolizing bacteria?

I. loihiensis has undergone a metabolic shift from sugar to amino acid utilization . This ecological adaptation likely influenced the evolution of its GlyRS:

Potential adaptations:

• Modified substrate binding affinity to accommodate the amino acid-rich environment

• Altered regulatory mechanisms reflecting metabolic priorities

• Enhanced stability in environments rich in amino acid metabolites

• Possible moonlighting functions related to amino acid metabolism

From search result , we know that "Idiomarina loihiensis, presents a particularly interesting case study. Having lost many genes essential for sugar metabolism, it relies instead on amino acids as its primary source of energy and carbon." This metabolic adaptation may have driven changes in the properties of essential enzymes like GlyRS.

Investigation approaches:

• Comparative kinetic analysis with GlyRS from sugar-metabolizing bacteria

• Protein engineering studies to identify adaptively significant residues

• Metabolomics studies to understand the cellular environment of GlyRS function

• Transcriptomic analysis to identify co-regulated genes

How do the kinetic parameters of I. loihiensis GlyS compare to other bacterial GlyRS enzymes?

A comprehensive kinetic comparison would require:

Experimental approach:

• Expression and purification of recombinant I. loihiensis GlyS and GlyRS from reference bacteria

• Determination of steady-state kinetic parameters:

  • Km and kcat for glycine, ATP, and tRNA^Gly

  • Binding affinities for each substrate

  • Rate-limiting step identification

• Comparison under various conditions (temperature, salt, pH)

Expected parameters to measure:

• Substrate specificity (kcat/Km)

• Catalytic efficiency

• Product inhibition constants

• Temperature and pH optima

While search result provides kinetic parameters for tRNA recognition by a different GlyRS (LpGlyRS), similar experimental approaches could be applied to I. loihiensis GlyS:

What evolutionary pressures have shaped the sequence of I. loihiensis GlyS?

The evolution of I. loihiensis GlyS has likely been shaped by:

Ecological adaptation pressures:

• Shift from sugar to amino acid metabolism has relaxed selective pressure on sugar metabolism genes while potentially increasing selection on amino acid-related processes

• Adaptation to marine environment (salt, pressure, temperature)

• Nutrient availability patterns in its ecological niche

Molecular evidence of selection:
From search result , we know that "In the branch-only models, none of these genes had significantly high average dN/dS in Idiomarina, but the branch-site models found evidence for a few sites in each gene with unusually high dN/dS in Idiomarina." This suggests site-specific positive selection rather than selection across the entire protein.

Analysis methods:

• Calculation of dN/dS ratios to identify signatures of selection

• Ancestral sequence reconstruction to trace evolutionary changes

• Phylogenetic analysis to identify lineage-specific adaptations

• Structural mapping of conserved vs. variable regions

What are the optimal buffer conditions for assaying I. loihiensis GlyS activity?

For optimal assaying of I. loihiensis GlyS activity, consider:

Buffer composition:

• Base buffer: 50 mM HEPES or Tris-HCl, pH 7.5-8.0

• Salt: 100-300 mM NaCl (considering marine origin of I. loihiensis)

• Divalent cations: 5-10 mM MgCl₂ (essential for ATP binding)

• Reducing agent: 1-5 mM DTT or β-mercaptoethanol

• Stabilizers: 10% glycerol, 0.1 mg/ml BSA

Substrate concentrations:

• ATP: 2-5 mM

• Glycine: 10-50 mM

• tRNA^Gly: 0.5-2 μM (based on Km values from result )

Optimization strategy:

• Perform pH screening (pH 6.5-9.0) to identify optimal pH

• Test various salt concentrations (50-500 mM NaCl)

• Optimize Mg²⁺ concentration (1-20 mM)

• Evaluate temperature dependence (20-45°C)

Control reactions:

• No enzyme control

• No ATP control

• Heat-inactivated enzyme control

• EDTA inhibition control

How can researchers measure the aminoacylation activity of recombinant I. loihiensis GlyS?

Several reliable methods can be used to measure aminoacylation activity:

1. Radioisotope-based assays:

• Use ¹⁴C-labeled glycine or ³²P-labeled tRNA

• Reaction components: labeled substrate, ATP, enzyme, unlabeled substrates, buffer

• After incubation, precipitate charged tRNAs with TCA on filter papers

• Quantify incorporation by scintillation counting

2. Pyrophosphate release assays:

• Couple PPi release to enzymatic reactions that generate colorimetric/fluorescent products

• Use commercial kits (EnzChek Pyrophosphate Assay Kit)

• Monitor reaction in real-time using plate readers

3. HPLC-based assays:

• Separate charged and uncharged tRNAs by reverse-phase HPLC

• Quantify peaks using UV detection

• Advantage: no radioactivity required

4. Mass spectrometry:

• Detect mass shift in tRNA upon glycine attachment

• Requires high-resolution MS equipment

• Provides detailed molecular information

Each assay should include appropriate controls and calibration standards for accurate quantification.

What spectroscopic techniques are most informative for studying the structure of I. loihiensis GlyS?

Multiple spectroscopic techniques provide complementary structural information:

1. Circular Dichroism (CD) Spectroscopy:

• Far-UV CD (190-250 nm): Secondary structure composition (α-helices, β-sheets)

• Near-UV CD (250-350 nm): Tertiary structure fingerprint

• Application: Monitor structural changes upon substrate binding

2. Fluorescence Spectroscopy:

• Intrinsic tryptophan fluorescence to probe tertiary structure

• Fluorescence quenching to identify binding sites

• FRET to measure interdomain distances and conformational changes

• Protocol: Excite at 280 nm, scan emission 300-400 nm

3. Fourier-Transform Infrared Spectroscopy (FTIR):

• Secondary structure information complementary to CD

• Particularly useful for β-sheet content analysis

• Sample preparation: Deuterium exchange to minimize water interference

4. Nuclear Magnetic Resonance (NMR):

• 1D ¹H-NMR for initial structural assessment

• 2D HSQC to map binding interactions

• Limitation: Size constraints may require domain-by-domain analysis

5. Small-Angle X-ray Scattering (SAXS):

• Low-resolution envelope of protein in solution

• Information about oligomeric state and domain arrangement

• Requires access to synchrotron radiation facilities

These techniques should be used in combination with computational modeling for comprehensive structural characterization.

How can site-directed mutagenesis be used to investigate the function of specific residues in I. loihiensis GlyS?

Site-directed mutagenesis is a powerful approach to investigate structure-function relationships:

Experimental design workflow:

• Identify target residues through:

  • Sequence alignment with characterized GlyRS enzymes

  • Structural homology modeling

  • Evolutionary conservation analysis

  • Known catalytic motifs (HIGH, KMSKS)

• Design mutagenesis strategy:

  • Conservative substitutions to test chemical properties

  • Alanine scanning to identify essential residues

  • Swap mutations with residues from other GlyRS enzymes

• Generate mutants using:

  • QuikChange PCR-based mutagenesis

  • Gibson Assembly

  • Golden Gate Assembly

• Functional analysis of mutants:

  • Steady-state kinetic parameters (Km, kcat)

  • Substrate binding assays

  • Thermal stability measurements

  • Structural analysis (CD, fluorescence)

• Data interpretation:

  • Map mutations onto structural model

  • Correlate functional effects with structural context

  • Compare with homologous enzymes

From the search results, we know that specific residues in the anticodon binding domain and insertions 1 and 3 may be particularly important for tRNA binding and catalysis .

How might the adaptation to amino acid metabolism in I. loihiensis have affected the evolution of its GlyS?

The metabolic shift in I. loihiensis from sugar to amino acid metabolism represents a fascinating case of evolutionary adaptation that likely influenced its GlyRS:

Hypotheses to investigate:

• Enhanced efficiency of GlyS in an amino acid-rich environment

• Co-evolution with other components of the translation machinery

• Potential moonlighting functions related to amino acid sensing or metabolism

• Altered regulation in response to amino acid availability

From search result , we know that "the relatively rapid evolution of amino acid metabolic enzymes in Idiomarina might reflect adaptation to growth on amino acids, particularly phenylalanine." This adaptation may extend to aminoacyl-tRNA synthetases like GlyS, which are directly involved in amino acid utilization.

Research approaches:

• Comparative genomics across Idiomarina species

• Experimental evolution under varied amino acid availability

• Structural and biochemical characterization of GlyS from multiple Idiomarina species

• Systems biology modeling of the relationship between metabolism and translation

What role might I. loihiensis GlyS play in stress response mechanisms?

Aminoacyl-tRNA synthetases often have secondary functions beyond their canonical roles in translation, particularly in stress response:

Potential stress-related functions:

• Sensing amino acid availability during nutritional stress

• Participating in stringent response regulation

• Production of stress signaling molecules

• Adaptation to marine-specific stressors (osmotic, pressure, temperature)

Investigation strategies:

• Transcriptomics/proteomics under various stress conditions

• Protein-protein interaction studies to identify stress-related binding partners

• Metabolite binding assays beyond canonical substrates

• Phenotypic analysis of GlyS mutants under stress conditions

For I. loihiensis specifically, its adaptation to an amino acid-dependent lifestyle suggests GlyS may have evolved specialized functions related to amino acid sensing or metabolism during stress.

How does temperature and salt concentration affect the activity and specificity of I. loihiensis GlyS?

As a marine bacterium, I. loihiensis has adapted to specific environmental conditions that likely influence GlyS function:

Experimental approach:

• Purify recombinant I. loihiensis GlyS

• Perform aminoacylation assays under varied conditions:

  • Temperature range: 4-45°C

  • Salt concentration: 0-1M NaCl

  • pH range: 6.0-9.0

• Determine kinetic parameters under each condition

• Analyze thermal stability using differential scanning fluorimetry

• Investigate substrate specificity changes under different conditions

Expected findings might include:

• Broader temperature optimum compared to terrestrial bacteria

• Enhanced salt tolerance with potential requirement for higher salt

• Specific ionic requirements (Na⁺, K⁺, Mg²⁺)

• Potential mischarging at temperature/salt extremes

Data presentation:

• Activity contour plots (temperature vs. salt)

• Arrhenius plots for activation energy determination

• Salt dependence curves for optimal activity

What potential biotechnological applications might exist for I. loihiensis GlyS based on its unique properties?

The unique adaptations of I. loihiensis GlyS could be leveraged for various biotechnological applications:

Potential applications:

• Orthogonal translation systems:

  • Engineering GlyS for incorporation of non-canonical amino acids

  • Development of orthogonal tRNA/synthetase pairs for synthetic biology

• Biocatalysis:

  • Adaptation for function in high-salt industrial processes

  • Engineering enhanced thermostability for industrial applications

  • Development of aminoacylation-based biosensors

• Protein engineering platforms:

  • Using GlyS as a scaffold for directed evolution

  • Creation of chimeric synthetases with novel specificities

  • Evolution of GlyS variants with expanded substrate ranges

• Therapeutic applications:

  • Development of antibacterial compounds targeting bacterial GlyRS

  • Exploitation of differences between bacterial and human GlyRS

Required research:

• Detailed structural characterization

• Substrate specificity profiling

• Stability under various industrial conditions

• Directed evolution studies to enhance desirable properties

The adaptation of I. loihiensis to amino acid metabolism rather than sugar utilization suggests its GlyS may have unique properties that could be valuable for biotechnological applications requiring function in amino acid-rich environments.