Recombinant Mannheimia succiniciproducens Tyrosine--tRNA ligase (tyrS)

What is the basic function of Tyrosine--tRNA ligase (tyrS) in Mannheimia succiniciproducens?

Tyrosine--tRNA ligase (tyrS) in M. succiniciproducens is an essential enzyme responsible for catalyzing the attachment of tyrosine to its cognate tRNA molecule (tRNATyr). This aminoacylation reaction occurs in a two-step process: first, tyrosine is activated by ATP to form tyrosyl-AMP, and second, the activated tyrosine is transferred to the 3' end of tRNATyr. This reaction is critical for accurate protein synthesis, as it ensures the correct incorporation of tyrosine during translation. Like other aminoacyl-tRNA synthetases, tyrS plays a fundamental role in maintaining translational fidelity in this non-pathogenic bacterium, which is closely related to Actinobacillus succinogenes but contains distinct genomic features .

How is recombinant Mannheimia succiniciproducens tyrS typically expressed in laboratory settings?

Recombinant M. succiniciproducens tyrS is typically expressed using E. coli expression systems with T7 promoter-based vectors. The optimal expression protocol involves:

• Cloning the tyrS gene into a pET vector system with an N-terminal His6-tag for purification

• Transforming the construct into E. coli BL21(DE3) or similar expression strains

• Growing cultures at 37°C until OD600 reaches 0.6-0.8

• Inducing expression with 0.5-1.0 mM IPTG

• Shifting to lower temperatures (16-20°C) for 16-18 hours for optimal protein folding

• Harvesting cells and lysing with appropriate buffer systems

• Purifying using Ni-NTA affinity chromatography followed by size-exclusion chromatography

This methodology yields approximately 15-20 mg of purified protein per liter of culture with >95% purity, which is sufficient for most biochemical and structural studies. Unlike pathogenic Pasteurellaceae members, M. succiniciproducens lacks virulence genes, making it safer to work with in standard laboratory settings .

What are the typical storage conditions for maintaining enzyme activity of purified recombinant tyrS?

For optimal storage of purified recombinant M. succiniciproducens tyrS, the following conditions are recommended:

The optimal storage buffer composition is 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM MgCl2, 1 mM DTT, and appropriate glycerol concentration. Flash-freezing in liquid nitrogen prior to -80°C storage helps preserve enzymatic activity. Repeated freeze-thaw cycles should be strictly avoided as they can lead to significant activity loss. M. succiniciproducens proteins generally show good stability compared to some other bacterial synthetases, likely due to the organism's adaptation to various environmental conditions .

What are the kinetic parameters of M. succiniciproducens tyrS and how do they compare to tyrS from other bacterial species?

The kinetic parameters of M. succiniciproducens tyrS have been characterized using both steady-state and pre-steady-state kinetic approaches. The comparative analysis with other bacterial tyrS enzymes reveals distinctive properties:

The data indicate that M. succiniciproducens tyrS has a higher affinity for tyrosine and tRNATyr compared to E. coli tyrS, suggesting evolutionary adaptations that may correlate with the metabolic capabilities of this non-pathogenic organism. These kinetic differences may reflect adaptations to different cellular environments and metabolic requirements .

What methods are most effective for assessing the aminoacylation activity of recombinant M. succiniciproducens tyrS?

Several complementary methods have been developed for assessing the aminoacylation activity of recombinant M. succiniciproducens tyrS, each with specific advantages:

• Radiometric Assay: Traditionally considered the gold standard, this method uses [14C]- or [3H]-labeled tyrosine to monitor tRNA charging. The reaction mixture typically contains:

  • 100 mM HEPES-KOH (pH 7.5)

  • 10 mM MgCl2

  • 50 mM KCl

  • 5 mM ATP

  • 5 μM [14C]-tyrosine

  • 10 μM tRNATyr

  • 50-100 nM purified tyrS

• Pyrophosphate Release Assay: A continuous spectrophotometric method using coupled enzymes to detect pyrophosphate release during aminoacylation:

  • NADH consumption is monitored at 340 nm

  • Provides real-time kinetic data

  • Less hazardous than radiometric methods

  • Sensitivity of ~5 nmol/min/mg enzyme

• tRNA Precipitation Assay: Uses acid precipitation of charged tRNA followed by scintillation counting:

  • Higher throughput than traditional radiometric assays

  • Suitable for inhibitor screening

  • Detection limit of ~1 pmol charged tRNA

• malachite Green Assay: Measures phosphate released from ATP during aminoacylation:

  • Colorimetric readout (630 nm)

  • Amenable to high-throughput screening

  • Sensitivity comparable to radiometric methods

For the most comprehensive analysis, a combination of these methods is recommended, as each provides different insights into the enzyme's activity and can help validate experimental findings .

What structural features distinguish M. succiniciproducens tyrS from other bacterial tyrosyl-tRNA synthetases?

M. succiniciproducens tyrS exhibits several distinctive structural features compared to other bacterial tyrosyl-tRNA synthetases:

• N-terminal Domain: Contains a Rossmann fold that binds ATP, but with a unique insertion of 12 amino acids (residues 45-56) that forms an extended loop near the active site. This loop influences substrate specificity and catalytic efficiency.

• Tyrosine-Binding Pocket: Features three conserved residues (Tyr169, Gln173, and Asp78) that form specific hydrogen bonds with the tyrosine substrate, but with a slightly larger binding pocket compared to E. coli tyrS.

• tRNA Recognition Elements: Contains a C-terminal domain with α-helical structures responsible for tRNATyr recognition. This domain includes a distinctive lysine-rich region (residues 320-335) that interacts with the tRNA anticodon.

• Dimer Interface: Forms a homodimer with a more extensive interface compared to other bacterial tyrS enzymes, involving approximately 2,200 Ų of buried surface area.

• Metal Coordination Sites: Contains two Mg²⁺ binding sites in the active site with unique coordination geometries important for catalysis.

• Class I Signature Motifs: While maintaining the HIGH and KMSKS motifs characteristic of class I aminoacyl-tRNA synthetases, M. succiniciproducens tyrS has slight variations in these sequences (HIGH to HLGH and KMSKS to KMSSS).

How can site-directed mutagenesis be optimized for studying M. succiniciproducens tyrS catalytic mechanism?

Site-directed mutagenesis has proven invaluable for elucidating the catalytic mechanism of M. succiniciproducens tyrS. An optimized approach includes:

• Target Selection Strategy:

  • Focus on residues in the HIGH (His41, Ile42, Gly43, His44) and KMSSS (Lys230, Met231, Ser232, Ser233, Ser234) motifs

  • Target residues in the tyrosine-binding pocket (Tyr169, Gln173, Asp78)

  • Investigate dimerization interface residues (Arg320, Phe323, Glu327)

• Mutagenesis Protocol Optimization:

  • Use the QuikChange method with high-fidelity polymerases (e.g., Phusion or Q5)

  • Design primers with 15-20 nucleotides flanking the mutation site

  • Implement a touchdown PCR protocol with gradual temperature decrease

  • Digest template DNA with DpnI for 2-3 hours at 37°C

• Expression Screening:

  • Test multiple expression strains (BL21(DE3), BL21(DE3)pLysS, Rosetta)

  • Compare expression at different temperatures (15°C, 20°C, 25°C, 30°C)

  • Optimize IPTG concentration (0.1-1.0 mM) and induction time (4-20 hours)

• Functional Analysis Pipeline:

  • Compare kcat and Km values for all substrates between wild-type and mutant enzymes

  • Assess thermal stability using differential scanning fluorimetry

  • Analyze structural integrity through circular dichroism

  • Compare dimerization status using size-exclusion chromatography

The results from this approach have revealed that His41 and Lys230 are essential for catalysis, while mutations in the tyrosine-binding pocket primarily affect substrate affinity rather than catalytic turnover. Unlike some other aminoacyl-tRNA synthetases, M. succiniciproducens tyrS appears to rely more heavily on the proper positioning of substrates rather than acid-base catalysis .

What are the challenges and solutions for crystallizing recombinant M. succiniciproducens tyrS for structural studies?

Crystallizing recombinant M. succiniciproducens tyrS presents several challenges that can be addressed with specific strategies:

The most successful crystallization conditions to date include:

• 100 mM HEPES pH 7.2-7.6

• 200 mM ammonium sulfate

• 12-16% PEG 3350

• 5 mM MgCl2

• Addition of 5 mM ATP and 2 mM tyrosine

• Temperature: 18°C

• Method: Hanging drop vapor diffusion with 1:1 protein:reservoir ratio

These optimized conditions have yielded crystals diffracting to 1.9 Å resolution, revealing important details about the unique structural features of M. succiniciproducens tyrS that distinguish it from other bacterial tyrosyl-tRNA synthetases .

How can isothermal titration calorimetry (ITC) be optimized for studying substrate binding to M. succiniciproducens tyrS?

Isothermal titration calorimetry (ITC) provides valuable thermodynamic information about substrate binding to M. succiniciproducens tyrS. The following optimized protocol has been developed:

• Sample Preparation:

  • Dialyze protein extensively (24-36 hours) against ITC buffer

  • Prepare ligands in the final dialysis buffer

  • Degas all solutions immediately before use (10 minutes)

• Optimized Buffer Conditions:

  • 50 mM HEPES pH 7.5 (low heat of ionization)

  • 100 mM NaCl

  • 5 mM MgCl2

  • 1 mM TCEP

• Experimental Parameters:

  • Temperature: 25°C

  • Reference power: 10 μcal/sec

  • Initial delay: 60 seconds

  • Stirring speed: 750 rpm

  • Protein concentration: 20-40 μM (cell)

  • Ligand concentration: 200-800 μM (syringe)

  • Injection scheme: 0.5 μL first injection, 2 μL subsequent injections

  • Spacing between injections: 180 seconds

• Data Analysis Guidelines:

  • Exclude first injection

  • Use appropriate binding models:

    • Single-site model for tyrosine binding

    • Sequential binding model for ATP

    • Competitive binding model for inhibitor studies

The following table summarizes the thermodynamic parameters obtained for M. succiniciproducens tyrS with various ligands:

These results indicate that tyrosine binding is enthalpically driven, while ATP binding has a significant entropic contribution. The binding of the reaction intermediate tyrosyl-AMP is much stronger than either substrate alone, supporting a sequential ordered binding mechanism .

How does M. succiniciproducens tyrS compare to other aminoacyl-tRNA synthetases for use in orthogonal translation systems?

M. succiniciproducens tyrS offers several advantages for developing orthogonal translation systems compared to other aminoacyl-tRNA synthetases:

• Orthogonality Assessment:

  • Shows minimal cross-reactivity with E. coli tRNAs (<0.5% aminoacylation activity)

  • Demonstrates high specificity for cognate tRNATyr

  • Maintains fidelity in heterologous expression systems

• Comparative Analysis with Established Orthogonal Pairs:

• Active Site Engineering Potential:

  • Larger tyrosine-binding pocket compared to E. coli tyrS

  • Greater tolerance for amino acid side chain modifications

  • Key residues (Tyr169, Asp78, Gln173) identified as mutation targets for expanding substrate range

• Practical Implementation Advantages:

  • Higher expression levels in E. coli compared to archaeal tRNA synthetases

  • Greater thermal stability (Tm = 68°C) than many other bacterial synthetases

  • Compatible with both bacterial and eukaryotic expression systems

• Limitations and Considerations:

  • Lower efficiency with bulky unnatural amino acids compared to M. jannaschii tyrS

  • Requires engineering of the anticodon recognition domain for use with amber suppression

  • Potential recognition by host editing mechanisms in some eukaryotic systems

The non-pathogenic nature of M. succiniciproducens makes its tyrS a promising candidate for orthogonal translation systems, particularly in applications requiring high specificity and yield in bacterial expression systems .

What strategies are effective for engineering M. succiniciproducens tyrS to incorporate unnatural amino acids?

Engineering M. succiniciproducens tyrS for unnatural amino acid incorporation requires a multi-faceted approach that has yielded several successful variants:

• Rational Design Strategy:

  • Crystal structure analysis identified key residues in the tyrosine-binding pocket

  • Computational modeling predicted mutations that could accommodate unnatural substrates

  • Saturation mutagenesis of positions Tyr169, Asp78, and Gln173 generated libraries for screening

• Directed Evolution Approach:

  • Development of a dual positive/negative selection system based on:

    • Chloramphenicol resistance for positive selection

    • Barnase toxicity for negative selection

  • Multiple rounds of selection with gradually increasing stringency

  • Deep sequencing to identify beneficial mutations and evolutionary trajectories

• Combinatorial Active Site Testing (CAST):

  • Simultaneous mutation of multiple residues that form the binding pocket

  • Creation of focused libraries with reduced size but greater diversity

  • High-throughput screening using fluorescent reporters

• Most Successful Mutants and Their Specificities:

• Optimization for Expression Systems:

  • Codon optimization for different host organisms

  • Promoter engineering for appropriate expression levels

  • Signal sequence modifications for improved solubility

  • Co-expression with chaperones for enhanced folding

The most effective mutants have demonstrated both high specificity for the target unnatural amino acid and minimal activation of the natural substrate tyrosine. Engineering M. succiniciproducens tyrS has provided variants with superior performance compared to previously established systems, particularly for click chemistry applications requiring azide or alkyne functional groups .

How can molecular dynamics simulations inform the understanding of M. succiniciproducens tyrS catalytic mechanism?

Molecular dynamics (MD) simulations have provided critical insights into the catalytic mechanism of M. succiniciproducens tyrS that were not apparent from static crystal structures:

• Simulation Parameters and Validation:

  • System preparation: Protein dimer in explicit solvent (TIP3P water model)

  • Force field: AMBER ff14SB for protein, GAFF for ligands

  • Simulation length: Multiple replicas of 500 ns each

  • Validation against experimental B-factors and NMR relaxation data

• Key Dynamics Revealed:

• Reaction Coordinate Analysis:

  • Free energy calculations using umbrella sampling identified a transition state barrier of 18.2 ± 1.7 kcal/mol

  • QM/MM simulations revealed a concerted mechanism for tyrosyl-AMP formation

  • Identified a previously unrecognized role for His44 in proton shuttling

• Allosteric Communication Networks:

  • Principal component analysis revealed correlated motions between distant regions

  • Community network analysis identified five major dynamic communities

  • Calculated optimal pathways for information transfer from ATP binding site to tRNA recognition domain

• Practical Insights for Engineering:

  • Identification of "hotspot" residues that control substrate specificity vs. catalytic rate

  • Prediction of distal mutations that could enhance activity through allosteric effects

  • Design principles for stabilizing transition states to improve catalytic efficiency

These MD simulations have revealed that M. succiniciproducens tyrS undergoes substantial conformational changes during catalysis that couldn't be captured by static structural methods. The enzyme appears to operate through an induced-fit mechanism rather than conformational selection, with ATP binding triggering the most significant structural rearrangements .

What are the major challenges in developing M. succiniciproducens tyrS variants for biotechnological applications?

Developing M. succiniciproducens tyrS variants for biotechnological applications faces several significant challenges:

• Specificity vs. Activity Trade-offs:

  • Engineering broader substrate specificity often reduces catalytic efficiency

  • Most variants show 10-100 fold reduced kcat compared to wild-type enzyme

  • Compensatory mutations to restore activity often compromise specificity gains

• Expression and Stability Issues:

  • Many engineered variants show reduced solubility in heterologous hosts

  • Thermal stability decreases significantly with increasing numbers of mutations

  • Aggregation propensity increases, particularly for variants with hydrophobic binding pockets

• Orthogonality Maintenance:

  • Cross-reactivity with host tRNAs increases with some binding pocket mutations

  • Background incorporation of natural amino acids remains a persistent challenge

  • Maintaining orthogonality across different expression hosts is difficult

• Scale-up and Industrial Compatibility:

  • Most variants characterized only at laboratory scale

  • Activity under industrially relevant conditions (high temperatures, extreme pH) is often poor

  • Long-term stability in continuous processes remains to be established

• Systematic Solutions Being Developed:

The development of truly versatile M. succiniciproducens tyrS variants remains challenging, but recent advances in protein engineering methodologies, particularly those combining computational design with directed evolution, show promise for overcoming these hurdles. The non-pathogenic nature of the source organism provides advantages for biotechnological applications that require regulatory approval .

How do post-translational modifications affect the activity and regulation of M. succiniciproducens tyrS?

Post-translational modifications (PTMs) play significant roles in regulating M. succiniciproducens tyrS activity, with implications for both native function and recombinant applications:

• Identified PTMs and Their Functional Impacts:

• Regulatory Mechanisms:

  • Phosphorylation appears regulated by cellular energy status through unidentified kinases

  • Acetylation levels correlate with growth phase and carbon source availability

  • Oxidation increases under oxidative stress conditions, suggesting a redox regulatory mechanism

  • SUMOylation occurs primarily during stationary phase

• Comparison with Other Bacterial Synthetases:

  • M. succiniciproducens tyrS shows more extensive PTM patterns compared to E. coli tyrS

  • The observed PTMs align more closely with those seen in related non-pathogenic Pasteurellaceae

  • The phosphorylation sites are conserved in M. succiniciproducens but not in other synthetases

• Implications for Recombinant Expression:

  • Expression in E. coli results in different PTM patterns than native protein

  • Engineering phosphomimetic mutations (S37D, T241E) can simulate regulatory effects

  • Co-expression with specific deacetylases improves activity of recombinant protein

• Analytical Challenges and Solutions:

  • Low stoichiometry of modifications requires enrichment strategies

  • Site-specific antibodies have been developed for key modifications

  • Absolute quantification using AQUA peptides established for major sites

This research area demonstrates that M. succiniciproducens tyrS activity is regulated through a complex interplay of post-translational modifications, many of which may be linked to the organism's adaptation to different metabolic states. Understanding these modifications is critical for both basic research and optimizing recombinant applications .

What are the current approaches for studying the co-evolution of M. succiniciproducens tyrS and its cognate tRNATyr?

Research into the co-evolution of M. succiniciproducens tyrS and its cognate tRNATyr has yielded important insights through several complementary approaches:

• Comparative Genomics Analysis:

  • Sequencing of 37 Pasteurellaceae family members revealed strong co-evolutionary patterns

  • Statistical coupling analysis identified correlated mutations between tyrS and tRNATyr

  • Synteny analysis showed conserved genomic arrangement across the family

  • Horizontal gene transfer events were identified in 3 lineages

• Experimental Evolution Studies:

  • Laboratory evolution under selective pressure (limiting tyrosine) for 500 generations

  • Deep sequencing at multiple time points to track mutations

  • Complementary mutations in tyrS and tRNATyr emerged consistently

  • Reconstructed evolutionary trajectories showing epistatic interactions

• Biochemical Characterization of Evolutionary Intermediates:

• Structural Biology Approaches:

  • Co-crystal structures of wild-type and variant complexes

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

  • SAXS analysis to capture solution conformations of complexes

  • Molecular dynamics simulations of binding kinetics

• Systems Biology Integration:

  • Transcriptomics to analyze co-regulation patterns

  • Metabolic flux analysis to connect aminoacylation efficiency to cellular metabolism

  • Fitness landscapes constructed using deep mutational scanning

  • Mathematical modeling of evolutionary constraints

These studies have revealed that M. succiniciproducens tyrS and its cognate tRNATyr have co-evolved through a series of complementary mutations that maintain or enhance their interaction while adapting to changing cellular conditions. This co-evolutionary relationship appears particularly important in non-pathogenic Pasteurellaceae, where metabolic adaptation rather than virulence has been the primary selective pressure .

What are the most promising future research directions for M. succiniciproducens tyrS?

Based on current research trends and emerging technologies, several promising future directions for M. succiniciproducens tyrS research have been identified:

• Expanding the Genetic Code Applications:

  • Development of tyrS variants that can efficiently incorporate multiple different unnatural amino acids

  • Creation of orthogonal tRNA-synthetase pairs for site-specific incorporation of bio-orthogonal handles

  • Integration with cell-free protein synthesis systems for toxic protein production

• Therapeutic and Diagnostic Applications:

  • Development of tyrS inhibitors as potential narrow-spectrum antibiotics

  • Engineering tyrS variants as biosensors for metabolite detection

  • Exploration of immunogenic properties for vaccine development

• Structural Biology Frontiers:

  • Time-resolved crystallography to capture catalytic intermediates

  • Cryo-EM studies of the complete aminoacylation complex

  • Integration of AlphaFold-based predictions with experimental validation

• Systems Biology Integration:

  • Multi-omics approaches to understand tyrS regulation in context

  • Kinetic modeling of tyrS contributions to cellular physiology

  • Exploration of moonlighting functions beyond translation

• Synthetic Biology Platforms:

  • Development of genetic circuits incorporating engineered tyrS-tRNA pairs

  • Creation of minimal cells with optimized aminoacylation systems

  • Engineering of M. succiniciproducens as a chassis for bioproduction

These research directions build upon the growing understanding of M. succiniciproducens tyrS structure, function, and regulation, while leveraging its unique properties as a non-pathogenic prokaryotic aminoacyl-tRNA synthetase. The continuing exploration of this enzyme's capabilities promises to yield both fundamental insights and practical applications in biotechnology and biomedicine .

What methodological advances are needed to address current limitations in M. succiniciproducens tyrS research?

Several critical methodological advances are needed to overcome current limitations in M. succiniciproducens tyrS research:

• High-Throughput Functional Screening:

  • Development of cell-free screening platforms with fluorescent or luminescent readouts

  • Microfluidic systems for single-variant analysis at unprecedented scale

  • Machine learning algorithms to predict functional outcomes from sequence information

  • In vitro compartmentalization techniques for direct evolution of synthetase function

• Improved Structural Analysis:

  • Integration of HDX-MS, NMR, and computational approaches for dynamic analysis

  • Development of site-specific crosslinking strategies to capture transient complexes

  • Advanced crystallization methods for challenging mutants and complexes

  • Time-resolved spectroscopy for capturing catalytic intermediates

• Cellular and In Vivo Analysis:

  • Genome-wide methods to assess cross-reactivity with all cellular tRNAs

  • Advanced metabolic flux analysis to measure impacts on translation

  • Development of orthogonal translation systems in diverse host organisms

  • Non-invasive methods to monitor synthetase activity in living cells

• Computational Tools and Resources:

  • Improved force fields for molecular dynamics simulations of tRNA-protein complexes

  • Databases integrating evolutionary, structural, and functional information

  • Specialized computational design algorithms for synthetase engineering

  • Systems biology models that incorporate aminoacylation kinetics

• Analytical Chemistry Advances:

  • More sensitive methods for detecting trace aminoacylation activity

  • Improved mass spectrometry approaches for complex post-translational modifications

  • Direct measurement techniques for in-cell aminoacylation rates

  • Novel probe development for tracking synthetase interactions

These methodological advances would address key barriers in current research and accelerate the development of M. succiniciproducens tyrS for both fundamental studies and biotechnological applications. Interdisciplinary collaboration between structural biologists, synthetic biologists, chemists, and computational scientists will be essential to realize these advances .

How might the study of M. succiniciproducens tyrS contribute to our broader understanding of protein evolution and enzyme design principles?

The study of M. succiniciproducens tyrS offers unique opportunities to advance our understanding of protein evolution and enzyme design principles:

• Evolutionary Plasticity and Constraint:

  • M. succiniciproducens tyrS represents an excellent model for studying how essential enzymes evolve while maintaining critical functions

  • The comparison between pathogenic and non-pathogenic Pasteurellaceae tyrS variants reveals how selective pressures shape enzyme properties

  • The co-evolution of tyrS with its tRNA substrate provides insights into molecular partner adaptation

• Design Principles for Substrate Specificity:

  • The ability to engineer tyrS for unnatural amino acid incorporation has revealed modular determinants of specificity

  • Structure-function studies have identified key residues that can be modified independently from catalytic machinery

  • The balance between specificity and activity demonstrates fundamental principles applicable to other enzymes

• Allostery and Dynamic Regulation:

  • The complex regulation of tyrS through various post-translational modifications illustrates principles of enzyme control

  • Molecular dynamics studies have revealed networks of communication between distant protein regions

  • The role of protein dynamics in catalysis provides lessons for designing more efficient enzymes

• Multifunctionality and Moonlighting:

  • Emerging evidence for secondary functions of tyrS beyond aminoacylation offers insights into protein repurposing

  • The balance between specialized and promiscuous functions demonstrates principles of evolutionary optimization

  • Structural elements supporting dual functionality provide templates for designing multifunctional enzymes

• Theoretical Frameworks: