Recombinant Pseudomonas syringae pv. tomato Acireductone dioxygenase (mtnD)

What is Acireductone dioxygenase (mtnD) and what is its role in Pseudomonas syringae pv. tomato?

Acireductone dioxygenase (mtnD) is an enzyme that functions in the methionine recycling pathway, also known as the Yang cycle. This pathway is critical for regenerating methionine when steady-state levels are limiting, which in turn supports ethylene biosynthesis. In P. syringae pv. tomato, mtnD plays a significant role in bacterial metabolism and potentially in pathogenicity by affecting ethylene-dependent plant defense responses. The enzyme catalyzes the oxygen-dependent transformation of acireductone substrate, which can be observed spectrophotometrically by monitoring changes at 308 nm . The Yang cycle has been demonstrated to be necessary for PAMP-induced ethylene production in plants, making mtnD a molecule of interest in plant-pathogen interaction studies .

How does mtnD connect to the methionine salvage pathway in bacteria?

The methionine salvage pathway (Yang cycle) in bacteria involves multiple enzymes that work sequentially to recycle methionine. Acireductone dioxygenase functions downstream of methylthioadenosine nucleosidase (MTN) in this pathway. The cycle initiates when S-adenosylmethionine is converted to methylthioadenosine (MTA) during various biosynthetic processes. MTN then converts MTA to methylthioribose, which undergoes several transformations to produce acireductone. At this point, mtnD (acireductone dioxygenase) catalyzes the conversion of acireductone to 2-keto-4-methylthiobutyrate, which is finally transaminated to regenerate methionine . This pathway is particularly important during conditions of high ethylene production, when methionine availability can become a limiting factor in bacterial metabolism and virulence.

What is the relationship between mtnD and plant defense responses?

mtnD's role in the methionine salvage pathway indirectly connects it to plant defense responses through ethylene biosynthesis regulation. Research has shown that the Yang cycle is required for PAMP-induced increase in ethylene biosynthesis . When plants detect Pathogen-Associated Molecular Patterns (PAMPs), they trigger defense responses that include increased ethylene production. This defense-related ethylene biosynthesis relies on the Yang cycle functioning properly. Pathogenic bacteria like P. syringae have evolved mechanisms to interfere with this process. For example, the type III effector HopAF1 targets methylthioadenosine nucleosidase proteins (MTN1 and MTN2) to dampen ethylene production during bacterial infection . Given mtnD's position in the same pathway, its activity likely influences the effectiveness of such bacterial counter-defense strategies.

What are the optimal conditions for measuring recombinant mtnD enzymatic activity?

The enzymatic activity of recombinant mtnD can be measured using a spectrophotometric assay that monitors the depletion of acireductone substrate at 308 nm. Based on established protocols for related acireductone dioxygenases, the following conditions are recommended:

• The assay should be conducted in three consecutive steps using an anaerobic cuvette:

  • First, generate the acireductone substrate (approximately 125 μM) using 75 nM E1 enzyme in the presence of 200 μg/ml catalase

  • Add oxygen-saturated buffer (280 mM oxygen)

  • Add the recombinant mtnD protein and monitor acireductone depletion at 308 nm for at least 300 seconds

• Account for the baseline oxygen-induced decay rate (approximately 8.5 × 10^-11 ± 1.5 × 10^-11 mol of substrate/s) when calculating enzyme kinetics

• Calculate initial rates by selecting the linear portion of the activity graph and determining the linear fit in this region

Temperature, pH, and metal cofactor optimization should be performed for the specific recombinant mtnD from P. syringae pv. tomato, as these parameters may differ from those established for ARD1 from other organisms.

How can I generate loss-of-function mutations in mtnD to study its function?

To generate loss-of-function mutations in mtnD for functional studies, several approaches can be employed:

• Site-directed mutagenesis targeting critical catalytic residues: Drawing from studies of related enzymes such as MTN proteins, where mutations like D225N in MTN1 and D212N in MTN2 resulted in loss of catalytic activity , identify conserved catalytic residues in mtnD and create similar mutations.

• Charge-altering mutations: Studies have shown that introducing negative charges at specific conserved sites (e.g., N to D mutations) can disrupt enzyme function. For example, N113D and N194D mutations in MTN1 resulted in loss-of-function phenotypes .

• Verification methods:

  • In vitro enzyme activity assays to confirm loss of function

  • Complementation studies in bacterial mutants to assess whether the mutated protein can restore wild-type function

  • Structural analysis to understand how mutations affect protein conformation

Each mutant should be compared with both wild-type and conservative mutations (e.g., N194A or N194V) that maintain function to distinguish between residues that are generally sensitive to mutation versus those specifically sensitive to charge alteration .

What methods are most effective for purifying active recombinant P. syringae pv. tomato mtnD?

For purifying active recombinant P. syringae pv. tomato mtnD, a multi-step approach is recommended:

• Expression system selection:

  • E. coli BL21(DE3) is commonly used for expressing bacterial proteins like mtnD

  • Consider using a vector with a solubility-enhancing tag (e.g., MBP, SUMO, or GST) if initial expression yields insoluble protein

• Purification strategy:

  • Initial capture by affinity chromatography (His-tag purification is most common)

  • Secondary purification by ion exchange chromatography

  • Final polishing by size exclusion chromatography

• Critical considerations:

  • Include metal cofactors (typically Fe²⁺ for acireductone dioxygenases) in all buffers

  • Use anaerobic conditions during purification to prevent oxidation and inactivation

  • Add reducing agents like DTT or β-mercaptoethanol to prevent disulfide bond formation

  • Verify protein activity after each purification step to ensure the protocol preserves enzyme function

• Quality control:

  • SDS-PAGE for purity assessment

  • Western blotting for identity confirmation

  • Mass spectrometry for accurate molecular weight determination

  • Circular dichroism to verify proper folding

The purification protocol should be optimized to maintain the native conformation and catalytic activity of the enzyme, as improper purification can lead to misleading results in subsequent functional studies.

How does mtnD function differ between P. syringae and other bacterial pathogens?

The function of mtnD shows both conservation and specialization across different bacterial pathogens:

• Conserved metabolic role:

  • Across bacterial species, mtnD functions in the methionine salvage pathway

  • The core catalytic mechanism involving oxygen-dependent conversion of acireductone remains consistent

• Species-specific variations:

  • Substrate specificity differences may exist between P. syringae mtnD and homologs in other bacteria

  • Regulatory mechanisms controlling mtnD expression vary between species

  • The importance of the Yang cycle in pathogenicity differs based on infection strategy

• Pathogen-specific adaptations:

  • In P. syringae, the Yang cycle appears particularly important for modulating plant ethylene responses during infection

  • Other pathogens may have evolved different mechanisms to interfere with host defenses that don't rely as heavily on methionine recycling

• Structural variations:

  • While the catalytic domain is generally conserved, peripheral domains may differ

  • These structural differences could influence protein-protein interactions, subcellular localization, or response to environmental factors

Comparative studies examining mtnD from multiple pathogens, including determination of enzyme kinetics, substrate preferences, and expression patterns during infection, would provide valuable insights into how this enzyme has been adapted for different pathogenic lifestyles.

What is the impact of mtnD knockout on P. syringae pv. tomato virulence in different host plants?

The impact of mtnD knockout on P. syringae pv. tomato virulence likely varies across different host plants due to plant-specific defense mechanisms and bacterial adaptation strategies:

• Expected general effects:

  • Reduced bacterial fitness due to impaired methionine recycling

  • Diminished capacity to sustain virulence factor production during prolonged infection

  • Altered growth kinetics within plant tissues

• Host-specific considerations:

  • Plants vary in their reliance on ethylene-mediated defenses

  • The importance of methionine recycling may differ based on the availability of free methionine in different plant tissue environments

  • Different hosts may have varying capacities to recognize and respond to bacteria with altered metabolic profiles

• Experimental approach to assess host-specific effects:

  • Generate clean mtnD deletion mutants using allelic exchange techniques

  • Perform in planta growth curve assays across multiple host species

  • Monitor symptom development and bacterial population size over time

  • Assess changes in plant defense gene expression in response to wild-type versus mtnD mutant bacteria

  • Conduct complementation studies to confirm phenotypes are specifically due to mtnD loss

This research would provide insights into how metabolic pathways like the Yang cycle contribute to host range determination and infection success across different plant species.

How do environmental factors influence mtnD expression and activity during plant infection?

Environmental factors likely play significant roles in modulating mtnD expression and activity during plant infection:

Understanding these environmental influences would provide insights into how bacterial pathogens adapt their metabolic activities to different infection scenarios and could reveal potential intervention points for disease management.

How does mtnD interact with other components of the Yang cycle in P. syringae?

The interaction of mtnD with other Yang cycle components in P. syringae involves both sequential enzymatic activities and potential protein-protein interactions:

• Enzymatic pathway interactions:

  • mtnD functions downstream of methylthioadenosine nucleosidase (MTN) and upstream of the final steps that regenerate methionine

  • The product of mtnD activity (2-keto-4-methylthiobutyrate) serves as the substrate for subsequent transamination to methionine

• Potential protein-protein interactions:

  • Yang cycle enzymes may form metabolic complexes to facilitate substrate channeling

  • Such complexes would increase efficiency by preventing the diffusion of intermediates

• Regulatory interactions:

  • Feedback regulation likely exists between Yang cycle components

  • Expression of mtnD may be coordinated with other pathway enzymes

• Methods to investigate these interactions:

  • Co-immunoprecipitation studies to identify physical interactions

  • Bacterial two-hybrid screens to detect binary protein interactions

  • Metabolic flux analysis to understand the kinetic relationships between pathway steps

  • Transcriptional studies to reveal coordinated expression patterns

Understanding these interactions could reveal how P. syringae efficiently manages its methionine resources during infection and identify potential vulnerabilities in this metabolic network.

What structural features of mtnD determine its substrate specificity and catalytic efficiency?

The structural features determining mtnD's substrate specificity and catalytic efficiency include:

• Active site architecture:

  • Metal-binding residues that coordinate the catalytic iron

  • Substrate-binding pocket that determines acireductone recognition

  • Oxygen-binding channel that facilitates controlled reaction with O₂

• Key structural elements likely include:

  • A conserved His-X-His motif for metal coordination

  • Hydrophobic residues that position the acireductone substrate

  • Charged residues that stabilize reaction intermediates

• Structural analysis techniques:

  • X-ray crystallography to determine three-dimensional structure

  • Site-directed mutagenesis to test the importance of specific residues

  • Molecular dynamics simulations to understand substrate binding and catalysis

  • Homology modeling based on related acireductone dioxygenases from other organisms

• Comparative approach:

  • Analyze structural differences between mtnD variants with different catalytic efficiencies

  • Compare P. syringae mtnD with homologs from other organisms to identify species-specific adaptations

Understanding these structural features would provide insights for engineering mtnD variants with altered activities and developing potential inhibitors targeting this enzyme.

What is the interplay between mtnD function and type III secretion system effectors during P. syringae infection?

The interplay between mtnD function and type III secretion system (T3SS) effectors during P. syringae infection represents a complex relationship between bacterial metabolism and virulence:

• Indirect connections through plant defense modulation:

  • T3SS effectors like HopAF1 target the Yang cycle by disrupting MTN1 and MTN2 function in plants, dampening ethylene-dependent defense responses

  • mtnD function in bacteria ensures sufficient methionine availability for sustained virulence factor production

• Potential regulatory relationships:

  • Environmental cues that induce T3SS expression may also influence mtnD expression

  • Metabolic state of the bacterium, influenced by mtnD activity, may affect T3SS efficiency

• Temporal coordination:

  • Early in infection: T3SS effectors suppress initial plant defenses

  • Later stages: Metabolic adaptations (involving mtnD) become increasingly important for bacterial persistence

• Research approach to investigate this interplay:

  • Transcriptional profiling to examine co-regulation of mtnD and T3SS genes

  • Analysis of mtnD mutant effects on T3SS function and effector delivery

  • Investigation of how effector-mediated suppression of plant Yang cycle affects bacterial mtnD expression

  • Development of dual fluorescent reporters to simultaneously monitor mtnD and T3SS gene expression during infection

This research area represents an important frontier in understanding how bacterial pathogens integrate metabolism and virulence functions during host interaction.

What are the most sensitive methods for detecting mtnD activity in complex biological samples?

Detecting mtnD activity in complex biological samples requires sensitive and specific analytical approaches:

• Spectrophotometric methods:

  • Direct measurement of acireductone depletion at 308 nm provides a straightforward approach but may lack sensitivity in complex samples

  • Coupled enzyme assays that link mtnD activity to production of a chromogenic compound can amplify the signal

• Mass spectrometry-based approaches:

  • LC-MS/MS to directly quantify substrate depletion and product formation

  • Isotope labeling techniques to track specific reaction pathways

  • Targeted metabolomics focusing on Yang cycle intermediates

• Radioactive assays:

  • Using radiolabeled substrates to track product formation with high sensitivity

  • Particularly useful for samples with high background absorbance

• Activity-based protein profiling:

  • Development of activity-based probes that bind specifically to active mtnD

  • Could allow visualization of active enzyme within bacterial cells or during infection

• Comparative data table for method selection:

The choice of method should be based on the specific research question, available equipment, and the nature of the biological samples being analyzed.

How can I distinguish between bacterial and plant acireductone dioxygenase activities in plant infection studies?

Distinguishing between bacterial (P. syringae) and plant acireductone dioxygenase activities during infection studies requires strategic approaches:

• Genetic approaches:

  • Create reporter-tagged versions of bacterial mtnD that can be specifically detected

  • Use P. syringae mtnD mutants in comparative studies

  • Employ plant mutants deficient in their own acireductone dioxygenase

• Biochemical discrimination:

  • Exploit differences in enzyme kinetics between plant and bacterial enzymes

  • Use specific inhibitors that preferentially affect one enzyme version

  • Develop antibodies that specifically recognize either the plant or bacterial enzyme for immunoprecipitation

• Analytical strategies:

  • Employ stable isotope labeling to track the origin of methionine salvage pathway products

  • Use species-specific peptide detection in activity assays

  • Apply differential centrifugation to separate bacterial and plant components before analysis

• Experimental design considerations:

  • Include appropriate controls with heat-killed bacteria

  • Use defined time points that capture different infection stages

  • Compare results from compatible and incompatible plant-pathogen interactions

How might structural modifications to recombinant mtnD enhance its stability for research applications?

Enhancing the stability of recombinant mtnD for research applications could be achieved through several structural modification approaches:

• Rational design strategies:

  • Introduction of disulfide bridges at strategic positions to stabilize tertiary structure

  • Surface charge optimization to improve solubility

  • Replacement of oxidation-sensitive residues (Met, Cys) in non-catalytic regions

  • N- or C-terminal truncations to remove flexible regions prone to degradation

• Directed evolution approaches:

  • Error-prone PCR followed by screening for variants with enhanced stability

  • DNA shuffling with homologous enzymes from extremophilic organisms

  • Systematic alanine scanning to identify destabilizing residues

• Computational design methods:

  • In silico prediction of stabilizing mutations

  • Molecular dynamics simulations to identify regions of high flexibility

  • Protein energy landscape analysis to optimize folding efficiency

• Post-translational modifications:

  • Site-specific PEGylation to enhance solubility and reduce proteolytic degradation

  • Glycoengineering to improve stability if expressing in eukaryotic systems

• Formulation strategies:

  • Identification of optimal buffer compositions for long-term storage

  • Addition of stabilizing agents like glycerol, sucrose, or specific metal cofactors

  • Lyophilization protocols optimized for maintaining activity upon reconstitution

Each modification strategy should be evaluated by measuring enzyme activity, thermal stability, resistance to proteolysis, and long-term storage stability to determine the most effective approach for specific research applications.

What evidence suggests that mtnD could be a target for developing new antimicrobials against P. syringae?

Several lines of evidence suggest mtnD could be a viable target for developing new antimicrobials against P. syringae:

• Metabolic importance:

  • The Yang cycle is essential for recycling methionine during conditions of high demand

  • Disruption of this pathway would likely impair bacterial fitness during infection

• Infection relevance:

  • Studies of Yang cycle components have shown their importance during plant infection

  • For example, MTN1 and MTN2 mutants show reduced virulence, suggesting the entire pathway is important for successful pathogenesis

• Structural considerations:

  • Bacterial mtnD likely has sufficient structural differences from plant homologs to allow selective targeting

  • The catalytic mechanism involves unique features that could be exploited for inhibitor design

• Target validation approaches:

  • Genetic studies demonstrating attenuated virulence in pathway mutants

  • Chemical biology studies showing antimicrobial effects of existing inhibitors

  • Structural analysis revealing druggable pockets in the enzyme

• Potential advantages as a drug target:

  • Targeting metabolism may present a higher barrier to resistance development

  • Inhibitors could potentially have broad-spectrum activity against multiple bacterial pathogens

  • Non-lethal targeting could reduce selection pressure while still attenuating virulence

Further research specifically examining mtnD knockout effects on virulence and detailed structural studies would strengthen the case for targeting this enzyme in antimicrobial development efforts.

What are the latest findings regarding the role of the Yang cycle in plant-microbe interactions beyond pathogenesis?

Recent research has expanded our understanding of the Yang cycle's role in plant-microbe interactions beyond pathogenesis:

• Symbiotic interactions:

  • The Yang cycle appears important in rhizobial symbioses, potentially regulating ethylene levels during nodule formation

  • Beneficial microbes may modulate plant Yang cycle activity to promote growth

• Microbiome influences:

  • Components of the plant microbiome may compete for or provide methionine cycle intermediates

  • Cross-feeding relationships involving Yang cycle metabolites likely exist in the phyllosphere and rhizosphere

• Abiotic stress connections:

  • Plant Yang cycle activity increases during certain abiotic stresses that involve ethylene signaling

  • Microbial partners may help plants maintain methionine homeostasis under stress conditions

• Evolutionary considerations:

  • The Yang cycle shows evidence of co-evolution between plants and their microbial associates

  • Horizontal gene transfer events involving Yang cycle components have been detected in some microbial lineages

• Plant immunity regulation:

  • Beyond ethylene production, the Yang cycle influences S-adenosylmethionine availability for methylation reactions

  • These methylation events affect defense gene expression and immune signaling

Current research is actively investigating these non-pathogenic interactions to develop a more comprehensive understanding of how the Yang cycle contributes to the complex molecular dialogue between plants and their microbial partners.

How can CRISPR-Cas9 technology be applied to study mtnD function in P. syringae?

CRISPR-Cas9 technology offers powerful new approaches for studying mtnD function in P. syringae:

• Precise genetic manipulation:

  • Creation of clean knockouts without polar effects on neighboring genes

  • Introduction of point mutations to study specific catalytic residues

  • Generation of conditional knockdowns using inducible promoters

  • Tagged variants for localization studies

• Multiplexed editing:

  • Simultaneous targeting of mtnD and other Yang cycle components

  • Creation of multiple mutations to study redundancy and compensatory mechanisms

  • Pathway-level perturbation to assess metabolic network effects

• Regulatory studies:

  • CRISPRi (CRISPR interference) to repress mtnD expression without genomic modification

  • CRISPRa (CRISPR activation) to upregulate mtnD to study overexpression effects

  • Targeting of regulatory elements to understand transcriptional control

• Implementation strategies:

  • Design of efficient sgRNAs with minimal off-target effects

  • Selection of appropriate Cas9 delivery vectors for P. syringae

  • Optimization of transformation protocols for high editing efficiency

  • Development of screening methods to identify successful edits

• Advanced applications:

  • Base editing to create specific nucleotide changes without double-strand breaks

  • CRISPR-mediated homology-directed repair for precise sequence insertions

  • in situ tagging of mtnD to study dynamics during infection

These CRISPR-based approaches would allow unprecedented precision in studying mtnD function and could reveal subtle aspects of its role that traditional genetic methods might miss.

What computational models exist for predicting the effects of mutations on mtnD function?

Computational models for predicting mutation effects on mtnD function span multiple scales and approaches:

• Sequence-based models:

  • Conservation analysis across homologs to identify critical residues

  • Machine learning approaches trained on existing mutation datasets

  • Evolutionary coupling analysis to identify co-evolving residues important for function

• Structure-based prediction methods:

  • Molecular dynamics simulations to assess stability changes

  • FoldX and Rosetta for calculating ΔΔG of folding upon mutation

  • Active site modeling to predict catalytic impacts

  • Normal mode analysis to assess effects on protein dynamics

• Systems biology approaches:

  • Flux balance analysis to predict metabolic consequences of altered mtnD activity

  • Kinetic modeling of the Yang cycle with parameter perturbations

  • Network analysis to identify compensatory pathways

• Comparison of computational methods for mtnD mutation analysis:

• Model validation approaches:

  • Correlation of computational predictions with experimental enzyme kinetics

  • Structural validation using X-ray crystallography or cryo-EM

  • Benchmarking against known mutation datasets from related enzymes

As structural data for P. syringae mtnD becomes available, these computational models will become increasingly valuable for guiding experimental design and interpreting mutation effects.

How can systems biology approaches enhance our understanding of mtnD's role in bacterial metabolism?

Systems biology approaches offer powerful frameworks for understanding mtnD's role within the broader context of bacterial metabolism:

• Multi-omics integration:

  • Combining transcriptomics, proteomics, and metabolomics data to create comprehensive models

  • Temporal profiling during infection to capture dynamic changes

  • Correlation analysis to identify co-regulated pathways

• Metabolic network analysis:

  • Genome-scale metabolic modeling incorporating mtnD reactions

  • Flux balance analysis to predict metabolic adjustments to mtnD perturbation

  • Elementary mode analysis to identify essential pathways involving mtnD

  • Identification of synthetic lethal interactions with other metabolic genes

• Regulatory network mapping:

  • ChIP-seq to identify transcription factors controlling mtnD expression

  • Ribosome profiling to assess translational regulation

  • Small RNA mapping to identify post-transcriptional control mechanisms

• Implementation approach:

  • Development of P. syringae-specific metabolic models incorporating accurate biomass equations

  • Collection of multi-omics data under relevant conditions (plant infection, varying nutrient availability)

  • Model refinement using experimental validation of key predictions

  • Integration of dynamic parameters to capture temporal aspects of infection

• Applications:

  • Identification of metabolic vulnerabilities for antimicrobial development

  • Prediction of metabolic adaptations during different infection stages

  • Understanding how environmental perturbations affect methionine metabolism

  • Design of intervention strategies targeting multiple points in interconnected networks