Recombinant Yersinia pseudotuberculosis serotype O:3 L-threonine 3-dehydrogenase (tdh)

What is L-threonine 3-dehydrogenase (tdh) and what is its role in Yersinia pseudotuberculosis metabolism?

L-threonine 3-dehydrogenase (tdh) is a key enzyme in the threonine metabolic pathway that catalyzes the NAD+-dependent oxidation of L-threonine to 2-amino-3-ketobutyrate, which can be further converted to glycine and acetyl-CoA. In Yersinia pseudotuberculosis, particularly serotype O:3, this enzyme plays a critical role in amino acid metabolism and energy production. The metabolism of threonine provides essential precursors for bacterial growth and may contribute to survival within host environments .

Methodological approach: To characterize the basic properties of tdh, researchers typically employ spectrophotometric assays measuring NAD+ reduction at 340 nm to determine enzyme kinetics (Km, Vmax, substrate specificity). Complementary approaches include isothermal titration calorimetry for binding studies and site-directed mutagenesis to identify catalytic residues.

How is recombinant Yersinia pseudotuberculosis tdh typically cloned and expressed?

The tdh gene from Y. pseudotuberculosis serotype O:3 is typically amplified using PCR with primers designed based on the published genome sequence. The cloning strategy generally follows these steps:

• PCR amplification of the tdh gene using high-fidelity polymerase

• Restriction digestion of the amplified product and selected expression vector

• Ligation and transformation into E. coli cloning strains

• Sequence verification of positive clones

• Transformation into expression hosts (typically E. coli BL21(DE3) or derivatives)

For expression, induction conditions (IPTG concentration, temperature, duration) must be optimized to maximize soluble protein yield. Similar to the approach used with Y. enterocolitica proteins, the tdh gene can be cloned into expression vectors with affinity tags (His6, GST) to facilitate purification .

What are the optimal conditions for purifying recombinant tdh protein?

The purification of recombinant tdh typically follows this methodological workflow:

For optimal stability, purified tdh should be stored with 1 mM DTT and 10% glycerol at -80°C. Protein purity should be verified by SDS-PAGE (>95%) and mass spectrometry for accurate mass determination.

How can researchers assess the enzymatic activity of recombinant tdh?

The enzymatic activity of recombinant tdh can be assessed using several complementary approaches:

• Spectrophotometric assay: Monitor NAD+ reduction at 340 nm in a reaction mixture containing L-threonine, NAD+, and buffer.

• Coupled enzyme assay: Link tdh activity to a secondary reaction that produces a colorimetric or fluorescent signal.

• Mass spectrometry: Detect and quantify reaction products directly.

A standard reaction mixture typically contains:

• 50 mM HEPES buffer (pH 8.0)

• 0.5 mM NAD+

• 10 mM L-threonine

• 0.1-1 μg purified tdh enzyme

• Total volume: 100 μL

Activity is calculated by measuring the initial velocity of NADH formation (ε340 = 6,220 M−1 cm−1) and expressed as μmol NADH formed per minute per mg protein.

What structural features distinguish Yersinia pseudotuberculosis tdh from homologs in other bacteria?

While specific structural data for Y. pseudotuberculosis tdh is limited in the provided search results, the enzyme typically belongs to the medium-chain dehydrogenase/reductase family. Key structural features likely include:

• NAD+ binding domain with Rossmann fold

• Substrate binding domain

• Catalytic zinc ion coordinated by conserved residues

• Dimeric or tetrameric quaternary structure

Methodological approach: Researchers can employ X-ray crystallography or cryo-EM to determine the three-dimensional structure. Comparative structural analysis with homologs can be conducted using tools like PyMOL or UCSF Chimera. Molecular dynamics simulations can provide insights into substrate binding and catalytic mechanisms.

How does recombinant tdh potentially contribute to the immunomodulatory effects of Yersinia pseudotuberculosis?

Recombinant proteins from Yersinia species, including tdh, may contribute to immunomodulatory effects through several mechanisms. Similar to the lipoprotein described in Y. enterocolitica, tdh might affect immune response by:

• Stimulating cytokine production (particularly IL-6) from immune cells

• Acting as a B-cell mitogen

• Inducing expression of costimulatory molecules like B7.1 and B7.2

• Potentially generating cross-reactive antibodies with host proteins

These effects could influence bacterial survival and persistence within host tissues. Experimental approaches to study these effects include:

• Stimulation of immune cells (splenocytes, macrophages) with purified tdh and measurement of cytokine production

• Flow cytometry analysis of surface marker expression after tdh exposure

• Adoptive transfer studies to assess T-cell responses in vivo

What evidence exists for the role of tdh in bacterial survival within macrophages?

While specific information about tdh in the context of macrophage survival is not directly provided in the search results, Yersinia pseudotuberculosis employs various strategies to survive within phagocytes. The role of tdh in this process can be investigated through:

• Comparison of wild-type and tdh knockout strains in macrophage infection models

• Transcriptomic analysis of bacteria during macrophage infection to assess tdh expression

• Proteomics to determine if tdh is actively secreted or exposed to the host

Research has shown that Y. pseudotuberculosis proteins contribute to bacterial colonization of lymphoid organs through effects on immune cells, potentially polarizing macrophages toward an M2 phenotype that is less bactericidal . The role of tdh in this process requires further investigation using macrophage polarization assays and in vivo infection models.

How can researchers address contradictory findings regarding tdh expression levels in different Yersinia pseudotuberculosis strains?

Contradictory findings regarding tdh expression are common challenges in bacterial research. To address such contradictions, researchers should:

• Employ topological analysis of contradictions to systematically categorize discrepancies in reported findings

• Consider strain-specific differences that might affect gene expression

• Account for experimental conditions (growth medium, temperature, growth phase)

• Use standardized methodologies for quantification

A comprehensive approach involves:

This multi-omics approach allows for triangulation of findings to resolve apparent contradictions in the literature .

What methodological approaches can identify potential cross-reactivity between tdh and host proteins?

To identify potential cross-reactivity between tdh and host proteins, researchers can employ several methodological approaches:

• Generation of antibodies against purified recombinant tdh and testing for cross-reactivity with host tissue extracts using Western blot, as demonstrated with Y. enterocolitica lipoprotein

• Epitope mapping to identify shared sequences between tdh and host proteins

• Phage display technology to screen for cross-reactive peptides

• Protein array analysis to detect binding to host proteins

• In silico analysis using algorithms that predict antigenic determinants and structural similarities

For experimental validation, researchers should:

• Perform ELISAs with both recombinant tdh and candidate host proteins

• Conduct competition assays to confirm specificity

• Use surface plasmon resonance to measure binding kinetics

• Validate findings in animal models by detecting auto-antibodies after immunization with tdh

How can researchers design experiments to elucidate the role of tdh in Yersinia pseudotuberculosis virulence?

To elucidate the role of tdh in Y. pseudotuberculosis virulence, researchers should employ a comprehensive experimental design:

• Generate isogenic mutants:

  • Complete gene deletion (Δtdh)

  • Point mutations in catalytic residues (enzymatically inactive)

  • Complemented strains (restored expression)

• Conduct in vitro assays:

  • Growth curves in minimal media to assess metabolic requirements

  • Stress resistance tests (acid, oxidative, nutrient limitation)

  • Adhesion and invasion assays with epithelial cell lines

  • Survival in macrophages and neutrophils

• Perform in vivo experiments:

  • Animal infection models (typically mice)

  • Competitive index assays (wild-type vs. mutant)

  • Histopathological examination of infected tissues

  • Immune response characterization (cytokine profiles, cellular infiltration)

• Apply systems biology approaches:

  • Transcriptomics of both pathogen and host during infection

  • Metabolomics to trace threonine metabolism in vivo

  • Proteomics to identify interaction partners

This deductive qualitative analysis approach allows testing and refinement of theories about tdh's role in virulence through systematic examination of evidence .

What strategies can be employed to optimize crystallization of recombinant tdh for structural studies?

Optimizing crystallization of recombinant tdh for structural studies requires a systematic approach:

• Protein preparation:

  • Ensure high purity (>95% by SDS-PAGE)

  • Verify monodispersity by dynamic light scattering

  • Test multiple constructs (full-length, truncations, surface mutations)

  • Consider co-purification with cofactors (NAD+)

• Initial screening:

  • Commercial sparse matrix screens (Hampton, Molecular Dimensions)

  • Varying protein concentrations (5-20 mg/mL)

  • Different temperatures (4°C, 18°C)

  • Addition of substrates or substrate analogs

• Optimization strategies:

  • Fine gradient screens around promising conditions

  • Additive screens (ions, polyols, detergents)

  • Seeding techniques (micro, streak, cross)

  • Surface entropy reduction mutations

• Alternative approaches if crystallization proves challenging:

  • Fusion with crystallization chaperones (T4 lysozyme, MBP)

  • In situ proteolysis to remove flexible regions

  • Nanobody co-crystallization

  • Cryo-EM as an alternative structure determination method

What are the most appropriate controls when studying recombinant tdh activity in vitro?

When studying recombinant tdh activity in vitro, researchers should include the following controls:

• Enzyme controls:

  • Heat-inactivated tdh (95°C for 10 minutes)

  • Catalytically inactive mutant (site-directed mutagenesis of active site residues)

  • Known concentration of commercial dehydrogenase with similar activity

• Substrate controls:

  • No substrate blank

  • Substrate analogs to assess specificity

  • Varying substrate concentrations for kinetic analysis

• Reaction condition controls:

  • Buffer-only control

  • No-cofactor control (NAD+ omitted)

  • pH series to determine optimum

• Inhibition controls:

  • Known dehydrogenase inhibitors

  • Reducing agents (DTT, β-mercaptoethanol)

  • Metal chelators (EDTA)

These controls help ensure specificity, reproducibility, and validity of enzyme activity measurements.

How can researchers apply Deductive Qualitative Analysis to improve experimental design when studying tdh?

Deductive Qualitative Analysis (DQA) can significantly improve experimental design for tdh research:

• Developing research questions and selecting guiding theory:

  • Review literature on tdh and related dehydrogenases

  • Formulate specific questions about tdh's role in metabolism or virulence

  • Select appropriate theoretical frameworks (e.g., enzyme kinetics, bacterial adaptation)

• Operationalizing theory:

  • Generate sensitizing constructs related to tdh function

  • Create working hypotheses about tdh's involvement in specific pathways

  • Define measurable outcomes that would support or refute hypotheses

• Data collection and analysis:

  • Design experiments that can provide all four types of evidence: supporting, contradicting, refining, and expanding

  • Employ negative case analysis to identify conditions where expected results don't occur

  • Alternate between deductive and inductive approaches during analysis

• Theory refinement:

  • Revise working hypotheses based on experimental results

  • Identify gaps in understanding that require additional experiments

  • Build an expanded model of tdh function and significance

What strategies can address low solubility of recombinant tdh during expression?

Low solubility of recombinant tdh is a common challenge that can be addressed through several strategies:

• Expression conditions optimization:

  • Lower induction temperature (16-20°C)

  • Reduce inducer concentration

  • Extended expression time (overnight)

  • Use richer media (TB, 2xYT)

• Construct modification:

  • Try different affinity tags (MBP, SUMO, TrxA)

  • Generate truncated constructs to remove hydrophobic regions

  • Codon optimization for expression host

  • Site-directed mutagenesis of aggregation-prone residues

• Solubility enhancement:

  • Co-expression with chaperones (GroEL/ES, DnaK)

  • Addition of osmolytes (glycerol, sorbitol) to growth media

  • Supplementation with enzyme cofactors during expression

• Alternative approaches if solubility issues persist:

  • Inclusion body isolation followed by denaturation and refolding

  • Cell-free protein expression systems

  • Expression in alternative hosts (yeast, insect cells)

How can researchers troubleshoot discrepancies in data when comparing tdh activity across different experimental setups?

When troubleshooting discrepancies in tdh activity data across different experimental setups, researchers should systematically:

• Standardize measurement conditions:

  • Use consistent buffer systems, pH, and ionic strength

  • Control temperature precisely (±0.5°C)

  • Standardize enzyme concentrations using Bradford or BCA assays

  • Prepare fresh substrate and cofactor solutions

• Instrument calibration:

  • Calibrate spectrophotometers using NADH standards

  • Verify linear range of detection

  • Use the same pathlength cuvettes or microplates

• Data analysis consistency:

  • Apply uniform methods for calculating initial velocities

  • Use standardized software for enzyme kinetics (e.g., GraphPad Prism)

  • Apply appropriate statistical tests

• Systematic investigation of variables:

  • Test effects of protein batch variations

  • Examine storage conditions impact

  • Assess reagent lot-to-lot variation

  • Compare results between different laboratory members

When contradictions persist, apply topological analysis of contradictions to categorize and resolve discrepancies, as described in search result . This approach helps identify whether contradictions arise from methodological differences, biological variations, or other factors.

How can CRISPR-Cas9 genome editing advance studies of tdh function in Yersinia pseudotuberculosis?

CRISPR-Cas9 genome editing offers several advantages for studying tdh function in Y. pseudotuberculosis:

• Precise genetic manipulation:

  • Generation of clean deletions without antibiotic resistance markers

  • Introduction of point mutations to study specific residues

  • Creation of reporter fusions at native loci

  • Simultaneous editing of multiple genes

• Methodological approach:

  • Design sgRNAs targeting tdh with minimal off-target effects

  • Clone sgRNAs and repair templates into Cas9-expressing vectors

  • Transform into Y. pseudotuberculosis using electroporation

  • Screen transformants using PCR and sequencing

  • Confirm phenotypes with complementation

• Advanced applications:

  • CRISPRi for tunable gene repression without gene deletion

  • CRISPRa for enhanced expression under native regulation

  • Base editing for introducing specific mutations without double-strand breaks

  • CRISPR screening to identify genetic interactions with tdh

This technology enables more sophisticated genetic analysis than traditional methods, allowing researchers to dissect tdh function with unprecedented precision.

What potential exists for using computational approaches to predict novel functions or interactions of tdh?

Computational approaches offer powerful tools for predicting novel functions or interactions of tdh:

• Structural bioinformatics:

  • Homology modeling based on related dehydrogenases

  • Molecular docking to identify potential substrates beyond threonine

  • Molecular dynamics simulations to study conformational changes

  • Virtual screening for potential inhibitors

• Systems biology approaches:

  • Metabolic network analysis to predict pathway interactions

  • Protein-protein interaction networks to identify functional partners

  • Gene co-expression analysis across conditions

  • Flux balance analysis to predict metabolic consequences of tdh alterations

• Machine learning applications:

  • Prediction of post-translational modifications

  • Identification of regulatory motifs in the tdh gene region

  • Classification of tdh variants based on virulence potential

  • Deep learning for function prediction from sequence

• Integrative approaches:

  • Multi-omics data integration to place tdh in biological context

  • Evolutionary analysis to identify conserved features

  • Host-pathogen interaction modeling

These computational methods generate testable hypotheses that can guide experimental design and reveal unexpected aspects of tdh biology.

How might understanding tdh function contribute to novel therapeutic approaches for Yersinia pseudotuberculosis infections?

Understanding tdh function could contribute to novel therapeutic approaches through several mechanisms:

• Target-based drug development:

  • Structure-based design of specific inhibitors

  • Fragment-based screening for binding molecules

  • Allosteric modulators that affect enzyme regulation

  • Covalent inhibitors targeting catalytic residues

• Metabolic vulnerability exploitation:

  • Identification of metabolic bottlenecks created by tdh inhibition

  • Combination approaches targeting related pathways

  • Nutrient restriction strategies that enhance tdh dependency

• Immunomodulatory approaches:

  • Attenuated vaccine strains with modified tdh

  • Targeting tdh-mediated immune evasion mechanisms

  • Development of antibodies that neutralize extracellular tdh

• Diagnostic applications:

  • tdh-based biomarkers for infection detection

  • Strain typing based on tdh variants

  • Monitoring treatment efficacy through tdh activity measurement

These approaches could address the growing concern of antibiotic resistance by providing alternative treatment strategies for Y. pseudotuberculosis infections, including Far East scarlet-like fever which often involves liver pathology .