Recombinant Pseudomonas syringae pv. tomato FMN-dependent NADH-azoreductase 1 (azoR1)

What is the biochemical classification of Pseudomonas syringae pv. tomato FMN-dependent NADH-azoreductase 1?

Pseudomonas syringae pv. tomato FMN-dependent NADH-azoreductase 1 (azoR1) belongs to the azoreductase type 1 family of enzymes. It catalyzes the reductive cleavage of azo bonds in aromatic azo compounds to corresponding amines. The enzyme strictly requires NADH as an electron donor and cannot utilize NADPH for its catalytic activity . As observed in other characterized azoreductases, azoR1 contains Flavin Mononucleotide (FMN) as an essential prosthetic group that facilitates electron transfer during catalysis.

How does the structure of azoR1 relate to its catalytic function?

The azoR1 enzyme typically forms a homodimeric structure with each monomer adopting a short flavodoxin-like fold, similar to other characterized azoreductases . Crystal structure analysis of related azoreductases reveals a defined FMN binding cradle, which is a recurrent motif in many flavodoxin-like proteins . The active site contains binding pockets for both the FMN cofactor and the NADH substrate, positioned to facilitate efficient electron transfer. The substrate binding site accommodates aromatic azo compounds, with specific residues mediating substrate recognition and orientation for catalysis.

What is known about the NADH binding specificity of azoR1?

AzoR1 demonstrates strict specificity for NADH over NADPH as an electron donor . Structural studies of related azoreductases, such as PA2580, have provided insights into this cofactor preference by resolving the nicotinamide binding region interactions . The enzyme's NADH binding pocket likely contains specific residues that form hydrogen bonds with the 2'-hydroxyl group of NADH's ribose, which is absent in NADPH (containing a 2'-phosphate group instead), thereby explaining the strict NADH dependency.

Does azoR1 have a role in the pathogenicity of Pseudomonas syringae pv. tomato?

While the direct role of azoR1 in Pseudomonas syringae pv. tomato pathogenicity has not been definitively established, comparative genomic analyses suggest that metabolic enzymes can contribute to bacterial fitness during host infection . Unlike type III secretion effectors like HopM1, AvrE, or AvrPto that directly modulate plant defense responses , azoR1 likely plays an indirect role by potentially detoxifying host defense compounds or maintaining redox homeostasis during infection. The enzyme may contribute to bacterial survival by neutralizing antimicrobial compounds produced by host plants as part of their defense response.

What is the kinetic mechanism of azoR1's catalytic activity?

Based on stopped-flow kinetic analyses of similar azoreductases, azoR1 likely follows a sequential mechanism involving rapid hydride transfer from NADH to enzyme-bound FMN, followed by electron transfer from reduced FMN to the azo substrate . Key kinetic parameters include:

The rapid reduction of FMN by NADH (kred >900 s-1) suggests that the rate-limiting step in catalysis is likely the interaction with and reduction of the azo substrate rather than the reductive half-reaction .

How can researchers distinguish between different phases of the azoR1 reaction mechanism?

Researchers can employ rapid kinetics techniques to dissect the individual steps of the azoR1 reaction mechanism. Stopped-flow spectroscopy is particularly valuable, allowing the observation of:

• Flavin reduction phase by monitoring absorbance decrease at 370 and 461 nm

• Formation and decay of charge transfer complexes

• Substrate reduction kinetics

When applying these methods, researchers should ensure anaerobic conditions by using nitrogen-bubbled buffers containing glucose/glucose oxidase to scavenge residual oxygen . The distinct spectral properties of oxidized FMN, reduced FMN, and any intermediates facilitate the deconvolution of the reaction mechanism into discrete steps with associated rate constants.

What factors affect the substrate specificity of azoR1?

The substrate specificity of azoR1 is influenced by multiple factors that researchers should consider when designing experiments:

• Substrate structure: The enzyme typically shows preference for aromatic azo compounds with specific substitution patterns

• Active site architecture: Residues lining the substrate binding pocket determine steric constraints

• Electronic properties: Electron-withdrawing or -donating groups on the substrate affect reduction potential and reactivity

• pH and ionic strength: These factors influence substrate binding and catalytic efficiency

Differential scanning fluorimetry and thin-layer chromatography can be employed to assess substrate binding preferences and product formation, respectively . Additionally, enzymatic assays comparing various substrates can establish structure-activity relationships that define the substrate scope of azoR1.

What are the optimal conditions for recombinant expression of azoR1?

Successful expression of recombinant azoR1 typically involves the following optimized conditions:

• Expression system: Escherichia coli BL21(DE3) or similar strains designed for recombinant protein expression

• Vector selection: pET-based vectors with T7 promoter control for high-level expression

• Fusion tags: N-terminal SUMO or His-tag to facilitate purification and potentially enhance solubility

• Induction conditions: 0.1-0.5 mM IPTG at reduced temperature (16-25°C) for 16-20 hours

• Media supplementation: Addition of riboflavin (10-20 μM) to enhance FMN cofactor incorporation

• Growth media: Enriched media such as Terrific Broth to maximize protein yield

Researchers should monitor expression levels via SDS-PAGE analysis of whole cell lysates and ensure that the recombinant protein remains in the soluble fraction .

What purification strategy yields the highest quality recombinant azoR1?

A multi-step purification protocol typically yields the highest quality recombinant azoR1:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged constructs

• Tag removal: Site-specific protease treatment (e.g., TEV protease for His-tags or SUMO protease for SUMO fusion)

• Secondary purification: Ion exchange chromatography (IEX) to separate tag-free protein from uncleaved protein and protease

• Polishing step: Size exclusion chromatography (SEC) to achieve high purity and remove aggregates

• Quality control: Assessment of FMN content by measuring A450/A280 ratio (typically 0.1-0.2 for fully FMN-loaded enzyme)

Throughout purification, buffers should include stabilizing agents such as glycerol (10%) and potentially reducing agents like DTT or β-mercaptoethanol to maintain enzyme activity .

What analytical methods are most effective for characterizing azoR1 activity?

Several complementary analytical methods are recommended for comprehensive characterization of azoR1 activity:

• UV-Vis spectrophotometry: Monitoring absorbance changes at 340 nm (NADH oxidation) and wavelengths specific to azo substrate reduction

• HPLC analysis: Quantitative determination of substrate depletion and product formation

• Stopped-flow spectroscopy: Real-time measurement of rapid reaction kinetics and intermediate formation

• Differential scanning fluorimetry: Assessment of thermal stability and ligand binding

• Mass spectrometry: Identification of reaction products and potential protein modifications

For steady-state kinetic parameters, researchers should employ continuous assays under conditions where initial rates are measured (<10% substrate conversion) across a range of substrate concentrations (typically 0.2-5 × Km) .

How can researchers investigate the potential physiological roles of azoR1 in Pseudomonas syringae pv. tomato?

To investigate physiological roles of azoR1 in Pseudomonas syringae pv. tomato, researchers can employ a multi-faceted approach:

• Gene knockout studies: Generate azoR1 deletion mutants using homologous recombination or CRISPR-Cas technologies

• Complementation analysis: Reintroduce wild-type or mutant azoR1 versions to assess functional restoration

• Transcriptional profiling: Analyze azoR1 expression under various conditions using RNA-Seq or qRT-PCR

• Metabolomic screening: Identify potential physiological substrates by comparing metabolite profiles between wild-type and azoR1 mutant strains

• Plant infection assays: Assess the impact of azoR1 deletion on bacterial growth in planta and symptom development

Such approaches have successfully identified roles for other Pseudomonas syringae genes in pathogenicity and metabolic adaptation . For example, bacterial growth curves in minimal media containing plant-derived compounds could reveal substrates that require azoR1 for utilization or detoxification.

How does azoR1 interact with plant defense mechanisms during infection?

While direct evidence for azoR1 interaction with plant defense mechanisms is limited, researchers can explore this question using several approaches:

• Plant immune response assays: Compare defense gene expression and metabolite production in plants infected with wild-type versus azoR1 mutant bacteria

• Metabolite identification: Isolate and identify plant defense compounds that might serve as substrates for azoR1

• In vitro enzyme assays: Test azoR1 activity against purified plant defense compounds

• Apoplast fluid analysis: Extract and analyze apoplastic fluid composition during infection with wild-type versus azoR1 mutant bacteria

Studies have shown that other bacterial enzymes can modulate plant defense responses. For instance, 1-methyltryptophan (1-MT) produced by tomato plants in response to Pseudomonas syringae attack affects bacterial motility by reducing fliC gene expression . Similar mechanisms might involve azoR1 in detoxifying specific plant defense compounds.

How can structural biology approaches contribute to understanding azoR1 function?

Structural biology offers powerful tools for elucidating azoR1 function at the molecular level:

• X-ray crystallography: Determine three-dimensional structure of azoR1 alone and in complex with substrates or inhibitors

• Site-directed mutagenesis: Verify the importance of predicted catalytic residues through kinetic analysis of mutant variants

• Molecular dynamics simulations: Model enzyme dynamics and substrate interactions in solution

• Hydrogen-deuterium exchange mass spectrometry: Identify regions of conformational flexibility and solvent accessibility

• Cryo-electron microscopy: Visualize larger complexes involving azoR1 and potential interaction partners

For crystallization trials, researchers should consider screening conditions that have been successful for related azoreductases, typically including PEG precipitants (4,000-8,000 MW) at pH 6.0-8.0 with various salts . Co-crystallization with NADH and substrate analogs can provide valuable insights into the catalytic mechanism.

What genomic approaches can reveal the evolutionary history of azoR1 in plant pathogens?

To understand the evolutionary history of azoR1 in plant pathogens, researchers can employ several genomic approaches:

• Comparative genomics: Analyze azoR1 distribution and sequence conservation across different Pseudomonas species and pathovars

• Phylogenetic analysis: Construct evolutionary trees to trace the ancestry of azoR1 and identify potential horizontal gene transfer events

• Selection pressure analysis: Calculate dN/dS ratios to determine if azoR1 is under positive, negative, or neutral selection

• Synteny analysis: Examine the genomic context of azoR1 across different bacterial strains to identify conserved gene neighborhoods

• Population genomics: Survey azoR1 sequence variations within a species to identify recent evolutionary changes

Such approaches have revealed interesting evolutionary patterns in other Pseudomonas syringae genes. For example, the type III-secreted effector gene hopM1 shows evidence of selection for loss of function, contrary to its previously assumed role as a virulence factor .

How do mutations in the FMN binding site affect azoR1 catalytic efficiency?

Researchers investigating the role of the FMN binding site in azoR1 function should consider:

• Identification of key FMN binding residues through sequence alignment with characterized azoreductases

• Generation of point mutations targeting these residues

• Assessment of FMN binding affinity in mutant enzymes using spectroscopic methods

• Determination of kinetic parameters for mutant enzymes to quantify effects on catalytic efficiency

Studies on related azoreductases have identified a conserved FMN binding cradle motif . Mutations in this region typically lead to reduced FMN binding affinity and corresponding decreases in catalytic activity. For example, a W60A mutation in the AzoA enzyme resulted in 2-fold and 36-fold reductions in two phases of the FMN reduction rate . Similar structure-function relationships likely exist in azoR1 and can be systematically investigated through site-directed mutagenesis.

What high-throughput approaches can accelerate the discovery of azoR1 substrates?

Researchers can employ several high-throughput strategies to identify potential azoR1 substrates:

• Colorimetric microplate assays: Screen diverse azo compounds by monitoring absorbance changes upon reduction

• Fluorescence-based assays: Utilize fluorogenic azo substrates that become fluorescent upon reduction

• LC-MS metabolite profiling: Compare metabolite profiles of wild-type and azoR1 mutant bacteria when exposed to plant extracts

• Substrate docking simulations: Computationally screen compound libraries for potential binding to the azoR1 active site

• Activity-based protein profiling: Use chemical probes that react with active azoR1 to pull down bound substrates

These approaches can be combined with statistical analysis to identify substrate structural features that correlate with activity, thereby establishing structure-activity relationships for azoR1.

How can isothermal titration calorimetry enhance understanding of azoR1 cofactor binding?

Isothermal titration calorimetry (ITC) provides thermodynamic parameters for interactions between azoR1 and its cofactors or substrates:

• Binding affinity (Ka or Kd): Determine the strength of FMN and NADH binding to azoR1

• Binding stoichiometry (n): Confirm the number of binding sites per enzyme molecule

• Enthalpy change (ΔH): Measure the heat released or absorbed during binding

• Entropy change (ΔS): Calculate from ΔG and ΔH to understand the role of disorder in binding

• Temperature dependence: Assess how binding parameters change with temperature to determine heat capacity changes (ΔCp)

These thermodynamic parameters can help elucidate the molecular basis for azoR1's strict preference for NADH over NADPH and its specific FMN binding characteristics. Researchers should perform ITC experiments under carefully controlled buffer conditions to minimize interference from buffer ionization enthalpy.

What considerations are important when designing site-directed mutagenesis studies of azoR1?

When designing site-directed mutagenesis studies of azoR1, researchers should consider:

• Selection of target residues based on:

  • Sequence conservation across azoreductase family members

  • Structural information from homologous proteins

  • Computational prediction of functionally important residues

  • Proximity to cofactor or substrate binding sites

• Choice of amino acid substitutions:

  • Conservative substitutions to probe subtle effects

  • Charge reversal to test electrostatic interactions

  • Size changes to investigate spatial requirements

  • Removal of functional groups to identify specific chemical contributions

• Experimental validation through:

  • Thermal stability analysis to ensure proper folding

  • FMN binding assessment to confirm cofactor incorporation

  • Comprehensive kinetic characterization to quantify functional effects

  • Structural analysis to confirm predicted conformational changes

Comparison of mutational effects across multiple substrates can reveal substrate-specific interactions and help define the catalytic mechanism of azoR1.

What are the major challenges in working with recombinant azoR1 and how can they be addressed?

Researchers face several challenges when working with recombinant azoR1:

• Protein solubility issues:

  • Solution: Optimize expression conditions (temperature, induction time)

  • Use solubility-enhancing fusion tags (SUMO, MBP, TrxA)

  • Screen buffer compositions for stabilizing additives

• Cofactor incorporation:

  • Solution: Supplement expression media with riboflavin

  • Add FMN during purification or reconstitute apo-enzyme with FMN post-purification

  • Quantify FMN content spectrophotometrically and ensure consistent loading

• Enzyme stability:

  • Solution: Include glycerol and reducing agents in storage buffers

  • Determine optimal pH and ionic strength conditions

  • Consider flash-freezing aliquots in liquid nitrogen for long-term storage

• Activity assays:

  • Solution: Ensure anaerobic conditions for accurate activity measurements

  • Use appropriate substrate concentrations to avoid solubility issues

  • Control for non-enzymatic reduction of azo compounds

By systematically addressing these challenges, researchers can establish robust protocols for working with recombinant azoR1.

How might azoR1 be exploited for biotechnological applications?

While commercial applications are outside the scope of this academic research-focused FAQ, potential biotechnological applications of azoR1 that might be explored in research settings include:

• Bioremediation research: Investigating azoR1's ability to degrade azo dyes in environmental samples

• Biosensor development: Using azoR1 activity to detect specific compounds in research samples

• Biocatalysis studies: Exploring azoR1's potential for stereoselective reduction reactions

• Structure-based inhibitor design: Using azoR1 structural information to develop specific inhibitors for research purposes

• Protein engineering: Modifying azoR1 to alter substrate specificity or improve stability for research applications

These research directions could provide valuable insights into enzyme function while potentially leading to applications that might eventually address real-world problems.

What are promising future directions for understanding azoR1's role in bacterial physiology?

Future research into azoR1's physiological roles could explore:

• Systems biology approaches: Integrate transcriptomic, proteomic, and metabolomic data to place azoR1 within cellular networks

• In vivo substrate identification: Develop chemical biology approaches to trap and identify physiological substrates

• Regulation studies: Characterize mechanisms controlling azoR1 expression and activity in response to environmental conditions

• Protein-protein interaction networks: Identify potential interaction partners that might influence azoR1 function

• Subcellular localization: Determine if azoR1 is associated with specific cellular compartments or structures

Understanding azoR1's physiological role could provide insights into bacterial adaptation to host environments and potentially reveal new targets for controlling bacterial plant pathogens.