Recombinant Yersinia pseudotuberculosis serotype O:3 Methylthioribose-1-phosphate isomerase (mtnA)

What is Methylthioribose-1-phosphate isomerase (mtnA) and its role in bacterial metabolism?

Methylthioribose-1-phosphate isomerase (mtnA) is a crucial enzyme involved in the universally conserved methionine salvage pathway (MSP). This enzyme catalyzes the conversion of 5-methylthioribose 1-phosphate (MTR-1-P) to 5-methylthioribulose 1-phosphate (MTRu-1-P) . The methionine salvage pathway is essential for recycling sulfur-containing metabolites and maintaining methionine homeostasis in bacteria. In pathogenic bacteria like Y. pseudotuberculosis, this pathway may play roles in bacterial survival during infection by helping the organism adapt to nutritional limitations within host environments.

The enzymatic reaction catalyzed by mtnA represents a critical isomerization step in the metabolic pathway that allows bacteria to recycle the methylthio group from methylthioadenosine, a byproduct of polyamine synthesis. This recycling mechanism is particularly important under conditions where de novo methionine synthesis is limited or when the organism faces sulfur starvation.

How does mtnA structurally compare to similar isomerases from other organisms?

The methylthioribose-1-phosphate isomerase from Y. pseudotuberculosis shares structural similarities with other related isomerases, while maintaining distinct features. Based on studies of similar enzymes, mtnA is likely to have a dimeric structure with specific N-terminal regions that contribute to its functional specificity . Unlike some related isomerases such as ribose-1,5-bisphosphate isomerase (R15Pi), mtnA likely contains unique structural attributes that create a hydrophobic microenvironment around the active site, which is favorable for its specific catalytic mechanism .

Crystallographic studies of related M1Pi enzymes have revealed that these proteins typically contain an active site with conserved catalytic residues that facilitate the isomerization reaction through a hydride transfer mechanism. The enzyme appears to operate through a cis-phosphoenolate intermediate formation during catalysis .

What are the sequence characteristics of mtnA in Y. pseudotuberculosis serotype O:3?

While the specific sequence of mtnA from Y. pseudotuberculosis serotype O:3 is not provided in the search results, we can infer some characteristics based on related proteins. The gene likely encodes a protein with distinct domains for substrate binding and catalysis. Similar to other bacterial isomerases, key catalytic residues would be conserved at the active site to facilitate the isomerization reaction.

The protein sequence would likely contain regions that distinguish it from other functionally related proteins such as ribose-1,5-bisphosphate isomerase (R15Pi) and the regulatory subunits of eukaryotic translation initiation factor 2B (eIF2B), despite sharing some structural similarities with these proteins .

What expression systems are most effective for producing recombinant Y. pseudotuberculosis mtnA?

For the expression of recombinant Y. pseudotuberculosis mtnA, several bacterial expression systems can be considered:

• E. coli-based expression systems: These represent the most commonly used approach for recombinant protein production due to:

  • Fast growth rates

  • High protein yields

  • Availability of various expression vectors and host strains

  • Compatibility with different affinity tags (His-tag, GST, MBP)

• Y. pseudotuberculosis homologous expression: For maintaining native post-translational modifications and folding characteristics, expressing mtnA in its native organism might provide advantages, though yields may be lower than heterologous systems.

• Cold-adapted expression systems: Given that Y. pseudotuberculosis can grow at lower temperatures, cold-adapted expression hosts might be beneficial for obtaining properly folded enzyme.

The choice between these systems should be guided by the specific requirements of downstream applications. For structural studies requiring large amounts of pure protein, E. coli systems with solubility-enhancing tags might be preferable, while for functional studies where native conformation is critical, homologous expression might be advantageous.

What purification strategies yield the highest activity for recombinant mtnA?

Optimal purification of enzymatically active recombinant mtnA typically involves a multi-step approach:

• Initial capture: Affinity chromatography using N-terminal or C-terminal tags (His6, GST)

• Intermediate purification: Ion exchange chromatography to separate charged variants

• Polishing step: Size exclusion chromatography to ensure monodispersity and remove aggregates

Throughout purification, it's critical to maintain a reducing environment (typically with 1-5 mM DTT or 2-10 mM β-mercaptoethanol) to prevent oxidation of cysteine residues that might affect enzyme activity. Additionally, including stabilizing agents such as glycerol (10-20%) can help maintain enzyme activity during storage.

How can the enzymatic activity of recombinant mtnA be measured accurately?

The enzymatic activity of recombinant mtnA can be measured through several complementary approaches:

• Coupled enzyme assays: The production of 5-methylthioribulose 1-phosphate can be coupled to additional enzymatic reactions that generate a spectrophotometrically detectable product. For example:

  • Coupling with NADH-dependent reductases

  • Monitoring phosphate release using malachite green

• Direct product detection:

  • HPLC analysis of substrate consumption and product formation

  • Mass spectrometry to detect and quantify the product

• NMR spectroscopy:

  • Real-time monitoring of the isomerization reaction

  • Structural verification of reaction products

A standard activity assay protocol might include:

• Reaction buffer: 50 mM HEPES, pH 7.5, 5 mM MgCl₂

• Substrate: 0.1-1.0 mM 5-methylthioribose 1-phosphate

• Enzyme: 0.1-1.0 μg purified mtnA

• Temperature: 30-37°C (optimum for Y. pseudotuberculosis enzymes)

• Time: 10-15 minutes or until linear reaction rate is established

Results are typically expressed as specific activity (μmol product formed per minute per mg enzyme) or as kinetic parameters (Km, Vmax, kcat) determined through Michaelis-Menten analysis.

What is the proposed catalytic mechanism of mtnA and how can it be investigated?

The catalytic mechanism of mtnA likely involves a cis-phosphoenolate intermediate formation, similar to that observed in related isomerases . To investigate this mechanism, researchers can employ:

• Site-directed mutagenesis: Systematically altering predicted catalytic residues to determine their role in the reaction. Key targets would include:

  • Conserved acidic residues that may function as catalytic bases

  • Basic residues that stabilize transition states

  • Hydrophobic residues that create the necessary microenvironment

• Structural biology approaches:

  • X-ray crystallography with substrate analogs or transition state mimics

  • Cryo-EM to capture different conformational states during catalysis

• Computational methods:

  • Molecular dynamics simulations to model the reaction pathway

  • QM/MM calculations to determine energy barriers for proposed reaction steps

• Spectroscopic techniques:

  • NMR to detect intermediate formation

  • Infrared spectroscopy to monitor bond changes during catalysis

Based on studies of the related M1Pi from Pyrococcus horikoshii, the enzyme likely creates a hydrophobic microenvironment near the active site that facilitates the isomerization reaction . This environment, combined with precisely positioned catalytic residues, enables the efficient conversion of MTR-1-P to MTRu-1-P through the proposed cis-phosphoenolate intermediate.

How does the structure of mtnA contribute to its substrate specificity?

The substrate specificity of mtnA is likely determined by several structural features:

• Active site architecture: The enzyme contains a specific binding pocket that accommodates the methylthio group of MTR-1-P, distinguishing it from other sugar phosphates.

• N-terminal extension: Similar to related isomerases, mtnA likely contains an N-terminal extension that contributes to substrate recognition and binding specificity . This region may form part of the active site or influence enzyme dynamics during catalysis.

• Hydrophobic patch: The presence of a hydrophobic patch, absent in functionally related proteins like R15Pi, creates a microenvironment that favors binding of the methylthio moiety of the substrate .

• Domain movement: The enzyme likely undergoes specific domain movements upon substrate binding, characterized by shifts in loops covering the active site pocket rather than kink formations observed in related enzymes .

To experimentally investigate substrate specificity, researchers can:

• Test the enzyme with substrate analogs of varying structure

• Perform competitive inhibition studies with substrate-like molecules

• Use molecular docking and simulation to predict binding modes

• Conduct isothermal titration calorimetry to measure binding affinities for different substrates

What is the relationship between mtnA function and Y. pseudotuberculosis virulence?

While the direct relationship between mtnA and Y. pseudotuberculosis virulence is not explicitly described in the search results, we can formulate hypotheses based on known bacterial pathogenesis mechanisms:

• Metabolic adaptation: The methionine salvage pathway, in which mtnA plays a crucial role, may enable Y. pseudotuberculosis to adapt to nutrient-limited environments encountered during infection, particularly within lymph nodes where the bacterium establishes infection .

• Potential interaction with virulence factors: Y. pseudotuberculosis virulence is known to involve various factors encoded on the pYV virulence plasmid, including adhesins and type III secretion systems . Metabolic enzymes like mtnA could indirectly support these virulence mechanisms by maintaining cellular homeostasis during infection.

• Stress response: The methionine salvage pathway may contribute to bacterial survival under host-induced stress conditions, such as oxidative stress encountered during interaction with neutrophils and inflammatory monocytes in infected tissues .

Research approaches to investigate potential links between mtnA and virulence could include:

• Construction and characterization of mtnA deletion mutants

• Virulence testing of mtnA mutants in animal infection models

• Transcriptomic analysis to determine if mtnA expression changes during infection

• Metabolomic profiling to assess the impact of mtnA deletion on methionine metabolism during infection

How can structural biology approaches enhance our understanding of mtnA?

Structural biology approaches offer powerful tools for elucidating the molecular basis of mtnA function:

By integrating these approaches, researchers can develop a comprehensive understanding of how mtnA structure relates to its function in the methionine salvage pathway. This information can also guide rational design of inhibitors or engineering of the enzyme for biotechnological applications.

What bioinformatic tools are most useful for analyzing evolutionary conservation of mtnA across Yersinia species?

For comprehensive evolutionary analysis of mtnA across Yersinia species, several bioinformatic approaches are particularly valuable:

• Sequence alignment and phylogenetic analysis:

  • MUSCLE or T-Coffee for accurate multiple sequence alignment

  • RAxML or MrBayes for phylogenetic tree construction

  • PAML for detection of selection pressures on specific codons

• Structural conservation mapping:

  • ConSurf for mapping sequence conservation onto three-dimensional structures

  • FTMap for predicting functionally important surface regions

  • ProBiS for structural alignment and binding site comparison

• Comparative genomics:

  • MicrobesOnline for analyzing genomic context and operon structures

  • STRING for predicting functional protein associations

  • IslandViewer for identifying potential horizontally transferred regions

• Coevolutionary analysis:

  • PSICOV or DCA for detecting coevolving residues

  • EVcouplings for predicting structural contacts from sequence data

When applying these tools to mtnA analysis, researchers should pay special attention to:

• Conservation patterns in putative catalytic residues

• Coevolution between residues forming the active site

• Structural features unique to pathogenic Yersinia species

• Genomic context of the mtnA gene across different Yersinia strains

How can isothermal titration calorimetry be applied to study mtnA-substrate interactions?

Isothermal Titration Calorimetry (ITC) provides valuable thermodynamic information about mtnA-substrate interactions:

• Experimental design considerations:

  • Protein concentration: 20-50 μM mtnA in the cell

  • Ligand concentration: 200-500 μM 5-methylthioribose 1-phosphate in the syringe

  • Buffer conditions: 20 mM HEPES pH 7.5, 150 mM NaCl (matched precisely between protein and ligand solutions)

  • Temperature: 25°C (standard) or multiple temperatures for entropy-enthalpy compensation analysis

  • Control experiments: Buffer-into-buffer, buffer-into-protein, ligand-into-buffer

• Parameters that can be determined:

  • Binding affinity (Kd)

  • Binding stoichiometry (n)

  • Enthalpy change (ΔH)

  • Entropy change (ΔS)

  • Gibbs free energy change (ΔG)

• Advanced applications:

  • Testing substrate analogs to develop structure-activity relationships

  • Measuring binding at multiple temperatures to determine heat capacity changes

  • Comparing wild-type and mutant enzymes to identify key binding residues

  • Investigating the role of divalent cations in substrate binding

By comparing these parameters across different experimental conditions, researchers can gain insights into the energetic basis of substrate recognition and the role of specific residues in the binding process.

What are the current knowledge gaps in understanding mtnA function in Y. pseudotuberculosis?

Despite advances in understanding methylthioribose-1-phosphate isomerases, several knowledge gaps remain specifically for mtnA in Y. pseudotuberculosis:

• Structural characterization: The three-dimensional structure of Y. pseudotuberculosis mtnA has not been determined, limiting our understanding of its specific catalytic mechanism.

• Regulation of expression: The factors controlling mtnA expression during different growth phases and infection stages remain poorly understood.

• Metabolic integration: How the methionine salvage pathway interfaces with other metabolic pathways during infection, particularly in nutrient-limited environments within host tissues.

• Host-pathogen interactions: Whether mtnA or its metabolic products influence host immune responses or contribute to bacterial survival within phagocytes.

• Potential as a drug target: The druggability of mtnA and whether specific inhibitors could attenuate Y. pseudotuberculosis virulence without affecting commensal microbiota.

Addressing these knowledge gaps requires integrated approaches combining structural biology, biochemistry, molecular microbiology, and infection models to provide a comprehensive understanding of mtnA's role in Y. pseudotuberculosis physiology and pathogenesis.

How might inhibitors of mtnA be designed and what experimental approaches would validate their efficacy?

The design of effective mtnA inhibitors would follow a rational, structure-based approach:

• Inhibitor design strategies:

  • Substrate analogs that compete for the active site

  • Transition state mimics targeting the proposed cis-phosphoenolate intermediate

  • Allosteric inhibitors targeting enzyme conformational changes

  • Fragment-based approaches to identify novel chemical scaffolds

• In vitro validation approaches:

  • Enzyme inhibition assays (IC50, Ki determination)

  • Binding affinity measurements (ITC, SPR, MST)

  • Co-crystallization or soaking experiments to confirm binding modes

  • Thermal shift assays to evaluate stabilization effects

• Cellular validation approaches:

  • Growth inhibition assays under conditions requiring methionine salvage

  • Metabolomic profiling to confirm pathway disruption

  • Combination studies with existing antibiotics

  • Resistance development assessment

• Infection model validation:

  • Cell culture infection models

  • Animal infection models focusing on lymph node colonization

  • Pharmacokinetic and pharmacodynamic studies

  • Assessment of effects on normal microbiota

When developing inhibitors for bacterial metabolic enzymes like mtnA, researchers must navigate challenges including specificity, permeability across bacterial membranes, and potential for resistance development. The distinct structural features of bacterial mtnA compared to mammalian enzymes provide opportunities for selective targeting.

What experimental approaches could reveal the role of mtnA in Y. pseudotuberculosis infection of lymph nodes?

Investigating the role of mtnA during lymph node infection by Y. pseudotuberculosis requires specialized approaches:

• Genetic approaches:

  • Construction of clean mtnA deletion mutants

  • Complementation studies with wild-type and catalytically inactive variants

  • Conditional expression systems for temporal control

  • Fluorescent protein fusions for localization studies

• Animal infection models:

  • Oral infection of mice to recapitulate natural infection route

  • Tracking bacterial colonization of Peyer's patches and mesenteric lymph nodes (MLNs)

  • Comparison of wild-type and mtnA mutant strains for MLN colonization

  • Analysis of pyogranuloma formation, a characteristic feature of Y. pseudotuberculosis infection

• Ex vivo systems:

  • Precision-cut lymph node slices for controlled infection studies

  • Organoid models incorporating lymphoid tissues

  • Primary lymphoid cell cultures for host-pathogen interaction analysis

• 'Omics approaches:

  • Transcriptomics to assess mtnA expression during different infection stages

  • Proteomics to identify mtnA interaction partners during infection

  • Metabolomics to quantify methionine pathway metabolites in infected tissues

  • Dual RNA-seq to simultaneously profile host and bacterial responses

These approaches would help determine whether mtnA contributes to the ability of Y. pseudotuberculosis to survive and replicate within lymph nodes, potentially influencing the formation of characteristic pyogranulomas and the development of mesenteric lymphadenitis .