Recombinant Gloeobacter violaceus Leucine--tRNA ligase (leuS), partial

What is Gloeobacter violaceus leucyl-tRNA ligase and why is it significant for research?

Leucyl-tRNA ligase (LeuRS) in G. violaceus is an aminoacyl-tRNA synthetase responsible for attaching leucine to its cognate tRNA molecules, a critical step in protein synthesis. G. violaceus is of particular research significance because it represents one of the most primitive cyanobacterial lineages, having diverged before the evolution of thylakoid membranes and plant plastids. This organism's long and largely independent evolutionary history presents unique properties that reflect ancestral features of early oxygenic photoautotrophs, making its LeuRS enzyme an important subject for understanding the evolution of translation machinery . The study of G. violaceus LeuRS offers insights into primitive aminoacylation mechanisms and can serve as a model for investigating early protein synthesis systems.

What are the basic biochemical properties of recombinant G. violaceus LeuRS?

Recombinant G. violaceus LeuRS typically functions optimally under conditions that reflect its native environment. While specific data for G. violaceus LeuRS is limited in the provided search results, comparable aminoacyl-tRNA synthetases generally exhibit the following properties:

The enzyme catalyzes a two-step reaction: first activating leucine with ATP to form leucyl-AMP, then transferring the leucyl group to the appropriate tRNA molecule. The reaction specificity ensures accurate translation of the genetic code.

What are the recommended protocols for expressing and purifying recombinant G. violaceus LeuRS?

Based on established protocols for related tRNA synthetases, the following methodology is recommended for recombinant G. violaceus LeuRS:

• Expression System Selection: E. coli BL21(DE3) or Rosetta strains are preferred for heterologous expression of G. violaceus proteins due to their efficiency in expressing cyanobacterial proteins.

• Vector Design:

  • Incorporate a hexa-histidine or other affinity tag (preferably at the N-terminus)

  • Include a TEV protease cleavage site for tag removal

  • Optimize codon usage for E. coli expression

• Expression Protocol:

  • Culture cells at 30°C until OD600 reaches 0.6-0.8

  • Induce with 0.5-1.0 mM IPTG

  • Lower temperature to 18-20°C for overnight expression to improve protein folding

• Purification Workflow:

  • Initial capture with Ni-NTA chromatography (for His-tagged constructs)

  • Optional tag cleavage with TEV protease

  • Secondary purification via ion exchange chromatography using a salt gradient elution as employed for Gloeobacter membrane proteins

  • Final polishing step using size exclusion chromatography

• Storage Conditions:

  • Buffer composition: 20 mM HEPES/NaOH pH 7.5, 150 mM NaCl, 1 mM TCEP, and 10% glycerol

  • Flash-freeze in liquid nitrogen and store at -80°C in small aliquots

For maximum enzyme activity, it's critical to maintain reducing conditions throughout purification to protect active site cysteine residues.

How can the aminoacylation activity of G. violaceus LeuRS be reliably measured?

Several methodologies can be employed to assess LeuRS activity, each with specific advantages:

• Radioactive Aminoacylation Assay:

  • Incubate purified LeuRS with ³H or ¹⁴C-labeled leucine, ATP, and appropriate tRNA

  • At defined time points, precipitate aminoacylated tRNA with TCA

  • Filter precipitates and measure radioactivity via scintillation counting

  • Calculate aminoacylation rates from the slope of time course measurements

• HPLC-Based Assay:

  • Perform aminoacylation reactions with unlabeled components

  • Separate aminoacylated from non-aminoacylated tRNA by reverse-phase HPLC

  • Quantify peaks by UV absorbance at 260 nm

• Pyrophosphate Detection Assay:

  • Couple LeuRS activity to enzymatic detection of released pyrophosphate

  • Measure continuous kinetics using spectrophotometric methods

• Gel Shift Analysis:

  • Utilize acid urea PAGE to separate aminoacylated from non-aminoacylated tRNA

  • Quantify band intensities by densitometry, similar to methods described for tRNA ligation analysis

When establishing reaction conditions, it's crucial to include appropriate controls such as reactions without ATP or enzyme, and to benchmark activity against a well-characterized LeuRS enzyme.

What approaches can be used to generate and screen tRNA variants for G. violaceus LeuRS?

The directed evolution approach for optimizing tRNA functionality with LeuRS can be adapted from methods recently developed for leucyl-tRNA in mammalian systems :

• Library Construction Strategies:

  • Targeted mutagenesis of the acceptor stem, analogous to the approach used in mammalian cell evolution of tRNA^EcLeu

  • Randomization of key identity elements in the anticodon loop and variable region

  • Semi-rational design based on structural information from related LeuRS-tRNA complexes

• Selection/Screening Methods:

  • In vivo selection: Utilize amber suppression systems where cell growth depends on successful aminoacylation

  • In vitro selection: Employ SELEX-type approaches with immobilized LeuRS

  • Viral-assisted screening: Adapt the virus-assisted selection scheme successfully employed for tRNA optimization in mammalian cells

• Validation Protocol:

  • Purify candidate tRNA variants and assess aminoacylation efficiency using methods described in 2.2

  • Analyze structural determinants using chemical and enzymatic probing

  • Perform comparative cross-aminoacylation with LeuRS enzymes from related species

When evaluating tRNA variants, it's important to consider both aminoacylation efficiency and specificity to ensure that optimized tRNAs maintain fidelity in translation.

What are the key determinants for tRNA recognition by G. violaceus LeuRS?

Based on studies of related LeuRS systems, particularly in Streptomyces coelicolor, the following structural elements are likely critical for G. violaceus LeuRS recognition of its cognate tRNAs:

• Variable Loop Characteristics:

  • The length rather than the specific sequence of the variable loop appears to be a critical determinant for aminoacylation, as demonstrated in ScoLeuRS

  • G. violaceus LeuRS likely recognizes both type I (short variable loop) and type II (long variable loop) tRNAs, similar to ScoLeuRS

• Tertiary Structure Elements:

  • The D-loop and TΨC-loop interaction creates a tertiary structure important for recognition

  • The elbow region formed by these interactions provides a recognition platform for the synthetase

• Acceptor Stem Features:

  • Unlike many LeuRS systems, the discriminator base (position 73) may not be critical for recognition, based on findings in ScoLeuRS

  • The geometry of the acceptor stem rather than specific base pairs may be more important

• Anticodon Interaction:

  • The anticodon-binding domain of LeuRS likely makes specific contacts with the anticodon loop

  • These interactions contribute to specificity but may be secondary to acceptor stem and variable loop recognition

Understanding these recognition elements is crucial for engineering tRNAs with improved aminoacylation efficiency for biotechnological applications.

How does the leucine-specific domain (LSD) contribute to tRNA recognition in G. violaceus LeuRS?

The leucine-specific domain (LSD) plays a critical role in expanding the substrate scope of LeuRS enzymes. Based on research with ScoLeuRS:

• Dual tRNA Type Recognition:

  • The LSD appears essential for allowing LeuRS to aminoacylate both type I and type II tRNAs

  • The C-terminal region of the LSD contains amino acid residues crucial for type I tRNA recognition

• Structural Accommodation:

  • The LSD likely provides additional contact surfaces for interacting with structurally distinct tRNAs

  • It may function as an adaptable module that can conform to different tRNA architectures

• Evolutionary Significance:

  • The presence of an adaptable LSD in G. violaceus LeuRS would reflect an ancestral state where flexible tRNA recognition was advantageous

  • This flexibility may represent an early evolutionary stage before specialized tRNA synthetases emerged

To experimentally validate the role of LSD in G. violaceus LeuRS, domain swapping experiments with other LeuRS enzymes or targeted mutagenesis of conserved residues in the C-terminal region would be informative approaches.

What editing mechanisms exist in G. violaceus LeuRS to ensure translation fidelity?

Aminoacyl-tRNA synthetases typically possess editing mechanisms to prevent mischarging of tRNAs with incorrect amino acids. For G. violaceus LeuRS, the following editing mechanisms likely exist:

• Pre-transfer Editing:

  • Hydrolysis of misactivated aminoacyl-AMP intermediates before transfer to tRNA

  • Occurs within the synthetic active site through selective release of non-cognate amino acid adenylates

• Post-transfer Editing:

  • Hydrolysis of mischarged tRNAs through a separate editing domain

  • The CP1 (connective polypeptide 1) domain typically contains this activity in bacterial LeuRS enzymes

• Specificity Determinants:

  Editing Mechanism	Target Amino Acids	Structural Basis
  Pre-transfer	Isoleucine, Methionine	Active site discrimination
  Post-transfer	Isoleucine, Methionine, Valine	CP1 domain hydrolytic activity
  tRNA-dependent	Various near-cognate amino acids	Conformational changes induced by tRNA binding

• Evolutionary Conservation:

  • Given G. violaceus's ancient lineage, its editing mechanisms may represent ancestral forms of quality control in translation

  • Comparative analysis with other cyanobacterial LeuRS editing domains could reveal evolutionary patterns in fidelity mechanisms

Experimental approaches to study these mechanisms include site-directed mutagenesis of predicted editing site residues and misaminoacylation assays with near-cognate amino acids.

How can G. violaceus LeuRS be engineered for genetic code expansion applications?

Engineering G. violaceus LeuRS for genetic code expansion involves several strategic approaches:

• Active Site Engineering:

  • Identify and mutate key residues in the amino acid binding pocket to accommodate non-canonical amino acids (ncAAs)

  • Focus on residues that interact with the side chain of leucine to alter substrate specificity

• Directed Evolution Strategies:

  • Adapt the virus-assisted selection scheme used for leucyl-tRNA evolution in mammalian cells

  • Implement selection systems that link cell survival to successful incorporation of ncAAs

  • Screen libraries focusing on both the enzyme and its cognate tRNA

• tRNA Optimization:

  • Engineer the acceptor stem of tRNA^Leu to improve aminoacylation efficiency, following approaches that have yielded remarkably improved variants for incorporating ncAAs

  • Focus particularly on positions identified as critical in the directed evolution of bacterial leucyl tRNA in mammalian cells

• Application Development:

  • Generate stable cell lines expressing the optimized G. violaceus LeuRS/tRNA pair for consistent ncAA incorporation

  • Develop viral vectors incorporating this machinery to facilitate genetic code expansion in difficult-to-transfect cells

The evolutionary distance of G. violaceus from model organisms provides an orthogonality advantage for genetic code expansion systems, potentially allowing the development of highly specific synthetase/tRNA pairs with minimal cross-reactivity with host machinery.

What can G. violaceus LeuRS reveal about the evolution of aminoacyl-tRNA synthetases?

G. violaceus LeuRS represents a valuable evolutionary model due to the organism's primitive lineage:

The genomic comparison between G. violaceus PCC 7421 and G. kilaueensis JS1 reveals substantial divergence despite sharing orthologous genes , suggesting that studying LeuRS across these species could provide insights into enzyme evolution within this ancient cyanobacterial lineage.

What methodological challenges exist in studying the kinetics of G. violaceus LeuRS, and how can they be addressed?

Several methodological challenges must be overcome for rigorous kinetic analysis of G. violaceus LeuRS:

• Protein Stability Issues:

  • Challenge: G. violaceus proteins may exhibit instability under standard laboratory conditions

  • Solution: Optimize buffer conditions by screening various additives (glycerol, reducing agents, salt concentrations) to improve enzyme stability; consider fusion protein approaches to enhance solubility

• tRNA Substrate Preparation:

  • Challenge: Obtaining properly folded, homogeneous tRNA substrates for kinetic studies

  • Solution: Implement in vitro transcription protocols with careful refolding procedures, similar to those used for HAC1 RNA studies ; alternatively, consider chemical synthesis of defined tRNA fragments for binding studies

• Product Separation and Detection:

  • Challenge: Distinguishing charged from uncharged tRNAs in real-time kinetic assays

  • Solution: Adapt fluorescence-based assays using labeled tRNA substrates similar to those employed for RNA ligation kinetics ; implement FRET-based approaches to monitor the aminoacylation reaction in real-time

• Data Analysis Complexities:

  • Challenge: Complex kinetic models involving multiple steps and potentially cooperative effects

  • Solution: Utilize global fitting approaches to simultaneously analyze multiple datasets; apply model discrimination methods to identify the most appropriate kinetic model

• Recommended Analytical Framework:

  Kinetic Parameter	Experimental Approach	Data Analysis Method
  kcat/KM for tRNA	Varying tRNA concentrations at fixed enzyme and amino acid levels	Initial velocity analysis
  KM for leucine	Varying leucine concentrations with saturating tRNA and ATP	Michaelis-Menten fitting
  Pre-steady state kinetics	Rapid quench-flow techniques	Burst phase analysis
  Inhibition constants	Competitive inhibitor studies	Dixon plots and global fitting

Addressing these challenges requires combining traditional enzyme kinetics approaches with modern biophysical techniques to fully characterize the aminoacylation mechanism of G. violaceus LeuRS.

How does G. violaceus LeuRS function within the context of the organism's unique cellular architecture?

G. violaceus lacks thylakoid membranes, a distinctive feature among cyanobacteria, which creates a unique cellular context for translation machinery:

• Spatial Organization Effects:

  • Without the compartmentalization provided by thylakoids, translation machinery including LeuRS likely operates in a more directly interfaced environment with photosynthetic components

  • This proximity may impose unique requirements on protein-protein interactions and localization signals

• Membrane Association:

  • LeuRS may associate with the cytoplasmic membrane where photosynthetic complexes are embedded in G. violaceus

  • This association could influence enzyme activity and regulation compared to thylakoid-containing cyanobacteria

• Metabolic Integration:

  • The connection between photosynthesis, carbon fixation, and protein synthesis pathways is likely more direct in G. violaceus

  • LeuRS activity may be more immediately responsive to changes in photosynthetic output and cellular energy status

• Structural Adaptations:

  • G. violaceus exhibits unusual protein structures in its photosynthetic machinery, with Photosystem I containing approximately 150 chlorophylls per P700 instead of the usual 90

  • Similarly, LeuRS may possess structural adaptations that reflect this primitive cellular organization

Understanding these unique contexts requires integrating structural studies of LeuRS with cellular imaging techniques to visualize its distribution and interactions within the distinctive architecture of G. violaceus cells.

What insights can comparative analysis of G. violaceus and G. kilaueensis LeuRS provide?

The discovery of G. kilaueensis enables comparative analysis of LeuRS between Gloeobacter species:

• Sequence Divergence Assessment:

  • Despite G. violaceus and G. kilaueensis sharing 2842 orthologous genes, they show limited gene synteny

  • Comparing the sequence conservation in LeuRS between these species can identify both critical conserved regions and species-specific adaptations

• Adaptive Evolution Signatures:

  • Areas of high sequence divergence may indicate regions under selective pressure for adaptation to different environmental conditions

  • G. kilaueensis was isolated from a lava cave in Hawai'i , representing a distinct habitat from G. violaceus

• Functional Conservation Testing:

  • Experimental comparison of aminoacylation efficiency and specificity between the two LeuRS enzymes

  • Cross-species complementation studies to test functional equivalence

• Evolutionary Rate Comparison:

  Domain	Expected Conservation Level	Functional Implication
  Catalytic Core	High	Essential aminoacylation chemistry
  tRNA Recognition Elements	Moderate	Adaptation to species-specific tRNAs
  Editing Domain	Variable	Possible habitat-specific optimization
  LSD	Potentially divergent	Adaptation to different tRNA populations

This comparative approach can provide insights into how aminoacyl-tRNA synthetases adapt during speciation while maintaining essential functions.

How can systems biology approaches integrate G. violaceus LeuRS function with broader cellular processes?

Systems biology offers powerful frameworks for understanding LeuRS function in the context of global cellular regulation:

• Translation Efficiency Networks:

  • Construct models integrating LeuRS activity with tRNA abundance, codon usage patterns, and protein expression levels

  • Identify potential bottlenecks in the translation machinery specific to G. violaceus

• Stress Response Integration:

  • Investigate how LeuRS activity responds to environmental stressors like nutrient limitation, light stress, or temperature changes

  • Map interactions between LeuRS and stress response regulators that might directly or indirectly modulate translation

• Metabolic Flux Analysis:

  • Trace the connection between amino acid metabolism, particularly leucine biosynthesis and degradation, and LeuRS activity

  • Develop models predicting how changes in metabolic flux affect translation fidelity

• Experimental System Biology Approaches:

  Approach	Application to LeuRS Research	Expected Outcome
  Transcriptomics	Monitor leuS expression under various conditions	Regulatory network identification
  Proteomics	Identify LeuRS interaction partners	Protein complex formation insights
  Metabolomics	Measure amino acid pools during translation stress	Metabolic adaptation mechanisms
  Network Modeling	Integrate multi-omics data	Predictive models of translation efficiency

These systems approaches can reveal how this ancient enzyme functions within the broader context of one of the most primitive photosynthetic organisms, providing insights into the co-evolution of translation and metabolism during the early history of life.