Recombinant Pseudomonas syringae pv. syringae FMN-dependent NADH-azoreductase (azoR)

What is the structural organization of Pseudomonas syringae pv. syringae FMN-dependent NADH-azoreductase (azoR)?

Based on structural studies of related azoreductases, P. syringae pv. syringae azoR likely exists as a homodimeric enzyme containing FMN as a prosthetic group. Each monomer would be expected to have a flavodoxin-like structure with alternating alpha helices and beta sheets, similar to what has been observed in structurally related reductases. The tight binding of the FMN cofactor is typically mediated by interactions with numerous amino acid residues and water molecules .

Comparative analysis with other bacterial azoreductases indicates the presence of conserved residues involved in FMN binding, particularly in the region corresponding to Pro95-Gly148 in E. coli AzoR. Key residues like Tyr96, Asn97, Phe98, Gly141, and Gly142 are relatively conserved across various quinone reductases and play crucial roles in FMN binding .

How does the electron transfer mechanism work in azoreductases?

The electron transfer mechanism in azoreductases like azoR follows a ping-pong mechanism. NADH rapidly transfers a hydride to the enzyme-bound FMN, converting it to the two-electron-reduced state. This process can be observed spectroscopically as a decrease in absorbance at specific wavelengths (typically 370 and 461 nm) .

The hydride transfer is extremely efficient, with flavin reduction rates exceeding 900 s^-1 in some azoreductases. This rapid reduction means the catalytic rate is rarely limited by the hydride transfer process. During the reaction, a transient charge transfer complex forms between the oxidized enzyme and NADH, which can be observed as a decrease in absorbance around 600 nm .

What is the substrate specificity of bacterial azoreductases?

While originally characterized for their ability to reduce azo compounds, bacterial azoreductases like AzoR demonstrate broader substrate specificity. Research has shown that many azoreductases can efficiently reduce various quinone compounds, including benzo-, naphtho-, and anthraquinones, which often serve as better substrates than traditional azo compounds like methyl red .

This expanded substrate profile suggests azoreductases function physiologically as quinone reductases rather than being specialized for azo compound reduction. This broader specificity is consistent across azoreductases from different bacterial species, including E. coli and likely P. syringae .

What is the coenzyme preference of P. syringae pv. syringae azoR?

Based on studies of related azoreductases, P. syringae pv. syringae azoR likely exhibits a strict dependence on NADH as an electron donor. While some azoreductases show a preference for either NADH or NADPH, certain enzymes demonstrate absolute specificity. For example, when AzoA was tested with NADPH (50–200 μM) in the presence of indigo carmine (50 μM), no activity could be detected, confirming its strict preference for NADH .

This coenzyme specificity is a critical characteristic to consider when designing experimental assays for enzyme activity measurement.

What are the optimal conditions for measuring azoreductase activity in vitro?

For accurate measurement of azoreductase activity in vitro, researchers should consider the following methodological parameters:

• Anaerobic conditions: When studying the reduction of the enzyme by NADH, measurements should be performed under anaerobic conditions to prevent interference from oxygen .

• Enzyme concentration: Typically, low enzyme concentrations (approximately 50 nM) are sufficient for steady-state kinetic analyses .

• NADH concentration: For enzymes with strict NADH dependence, concentrations ranging from 50-200 μM are appropriate for initial activity assessments .

• Substrate selection: While azo compounds like methyl red can be used, quinone compounds may serve as better substrates for activity measurements .

• Spectrophotometric monitoring: Activity can be monitored by following the decrease in absorbance at wavelengths specific to the substrate or at 370 and 461 nm to track flavin reduction .

How can I determine the kinetic parameters of recombinant P. syringae pv. syringae azoR?

Determining kinetic parameters requires a combination of steady-state and pre-steady-state kinetic analyses:

• Steady-state kinetics: Measure initial reaction rates at varying substrate concentrations while keeping NADH concentration fixed. Plot the data using appropriate kinetic models (Michaelis-Menten, Lineweaver-Burk) to determine parameters such as K_m and k_cat .

• Stopped-flow kinetics: For more detailed mechanistic analysis, stopped-flow spectrophotometry allows measurement of rapid reactions. Mix the enzyme with NADH under anaerobic conditions and monitor absorbance changes at multiple wavelengths (e.g., 370, 461, and 600 nm) to track flavin reduction and potential formation of charge transfer complexes .

• Data analysis: Fit the kinetic traces to appropriate equations to extract parameters such as k_red(NADH) and K_d(NADH). Below is an example of pre-steady-state kinetic parameters determined for wild-type AzoA and its W60A variant:

This table demonstrates how mutations can significantly impact enzyme kinetics, with the W60A mutation causing 2-fold and 36-fold reductions in the two phases of NADH-mediated reduction .

What approaches can be used to investigate structure-function relationships in azoreductases?

To investigate structure-function relationships in azoreductases like P. syringae pv. syringae azoR, researchers can employ multiple complementary approaches:

• Site-directed mutagenesis: Target conserved residues, especially those involved in FMN binding or substrate interaction. For example, studies with AzoA showed that the W60A mutation significantly altered enzyme kinetics by affecting the interaction between tryptophan and the isoalloxazine ring of FMN .

• X-ray crystallography: Determine the three-dimensional structure of the enzyme in different states (oxidized, reduced, substrate-bound) to identify key structural features. Crystal structures can reveal unique elements like the extended loop in AzoA (residues 55-67) that contacts the binding site near the FMN cofactor .

• Comparative structural analysis: Compare the structure with other azoreductases to identify unique features that might explain differences in substrate specificity or catalytic efficiency .

• Molecular dynamics simulations: Use computational approaches to model enzyme flexibility and substrate binding dynamics, complementing static crystal structures.

What is the physiological role of azoR in bacterial species?

Research with E. coli AzoR has demonstrated that this enzyme plays a critical role in providing resistance to thiol-specific stress caused by electrophilic quinones. Several lines of evidence support this physiological function:

• Mutant susceptibility: The ΔazoR mutant displayed reduced viability when exposed to electrophilic quinones capable of depleting cellular reduced glutathione (GSH) .

• Rescue by GSH: Externally added GSH partially restored the impaired growth of the ΔazoR mutant caused by 2-methylhydroquinone, confirming the thiol-protective function .

• Transcriptional regulation: The transcription of azoR is induced by electrophiles, including 2-methylhydroquinone, catechol, menadione, and diamide, further supporting its role in stress response .

Given the conservation of azoreductases across bacterial species, P. syringae pv. syringae azoR likely serves a similar protective function against oxidative and electrophilic stresses, which could be particularly relevant during plant-pathogen interactions .

How does azoreductase activity correlate with pathogenicity in P. syringae pv. syringae?

While direct evidence linking azoR to pathogenicity in P. syringae is limited, integrating findings across studies suggests potential connections:

P. syringae pv. syringae isolates demonstrate differential pathogenicity phenotypes, with some strains causing cankers, leaf spots, and fruit lesions in the field, while others cause a more limited range of symptoms . These varying pathogenicity profiles correlate with specific genomic features, including the presence of certain virulence factors.

Given that plant defense responses often include oxidative bursts and production of antimicrobial compounds, including quinones, azoreductase activity might provide protection against host-derived defense molecules. This protective function could contribute to bacterial survival during infection and indirectly influence pathogenicity .

Future research should investigate whether:

• azoR expression levels correlate with virulence potential across different P. syringae isolates

• azoR mutants show attenuated virulence in plant infection models

• azoR activity is induced during plant-pathogen interactions

How does P. syringae pv. syringae azoR react with molecular oxygen?

Understanding the interaction between reduced azoreductases and molecular oxygen is crucial for both experimental design and physiological interpretations. Research with related azoreductases indicates extremely slow reaction rates with oxygen.

When reduced AzoA was mixed with various oxygen concentrations in stopped-flow experiments, an extremely slow increase in absorbance at 461 nm was observed (0.02–0.08 s^-1), consistent with very gradual enzyme reoxidation. This process was preceded by a lag period (10–20 s), potentially reflecting the buildup of initial reaction products such as flavin radical species and/or superoxide .

This negligible ability to use molecular oxygen as an electron acceptor distinguishes azoreductases from typical oxidases and has important implications for their physiological function and experimental handling. Researchers working with P. syringae pv. syringae azoR should ensure anaerobic conditions when studying the reduced enzyme to prevent slow background reoxidation .

What genomic context surrounds azoR in different Pseudomonas species?

Comparative genomic analysis of azoreductases across different Pseudomonas species can provide insights into evolutionary relationships and potential functional associations. The P. syringae species complex encompasses multiple genomospecies, including P. syringae pv. syringae, P. syringae, P. cerasi, P. viridiflava, and genomospecies A, each with distinct pathogenicity profiles .

While the search results don't provide specific information about the genomic context of azoR in these species, researchers investigating this question should examine:

• Whether azoR is found in conserved genomic neighborhoods across different Pseudomonas species

• If azoR co-occurs with specific gene clusters related to stress response or virulence

• Whether horizontal gene transfer has influenced azoR distribution

• If any regulatory elements upstream of azoR are conserved across species

Such analysis could provide valuable insights into the evolutionary history and functional significance of azoR in the Pseudomonas genus.

Why might I observe low activity with recombinant P. syringae pv. syringae azoR?

Several factors can contribute to lower-than-expected activity when working with recombinant azoreductases:

• FMN incorporation: Insufficient FMN incorporation during expression or purification can result in partially active enzyme. Consider supplementing growth media with riboflavin or adding FMN during purification steps .

• Oxidation state: Ensure the enzyme is in the correct oxidation state for your experiments. Pre-reduction with NADH may be necessary for certain assays .

• Anaerobic conditions: When measuring reduction reactions, ensure strict anaerobic conditions are maintained, as even trace amounts of oxygen can affect results .

• Coenzyme specificity: Verify you are using the correct electron donor. If the enzyme is strictly NADH-dependent like many azoreductases, NADPH will not function as a substitute .

• Protein folding: Improper folding during recombinant expression can affect activity. Consider optimizing expression conditions (temperature, induction time) or using different fusion tags .

• Critical residues: Mutations in key residues can dramatically affect activity, as demonstrated by the W60A variant of AzoA showing significantly reduced reduction rates .

How can I distinguish between direct and indirect effects when studying azoR knockout phenotypes?

When investigating phenotypes associated with azoR deletion, several approaches can help distinguish direct from indirect effects:

• Complementation studies: Reintroduce the wild-type azoR gene into the knockout strain. Restoration of the wild-type phenotype confirms direct causality .

• Enzyme activity assays: Perform in vitro assays with purified recombinant azoR to determine if the enzyme can directly act on substrates of interest .

• Metabolite profiling: Compare metabolite profiles of wild-type and ΔazoR strains to identify specific biochemical pathways affected by the deletion.

• Thiol status measurement: Since azoR protects against thiol-specific stress, measure GSH levels in wild-type and mutant strains under various conditions to confirm the mechanism of action .

• Transcriptional profiling: Identify genes differentially expressed in response to azoR deletion, which can reveal compensatory mechanisms or regulatory networks.

These approaches collectively provide a more comprehensive understanding of azoR function than phenotypic observations alone.

How do environmental conditions affect azoR expression and activity?

Understanding how environmental factors influence azoR expression and activity is crucial for both experimental design and physiological interpretation:

• Electrophilic inducers: In E. coli, azoR transcription is induced by various electrophiles, including 2-methylhydroquinone, catechol, menadione, and diamide. Similar compounds likely induce expression in P. syringae .

• Oxidative stress conditions: While azoR shows negligible reaction with molecular oxygen, oxidative stress conditions may indirectly regulate its expression through redox-sensitive transcription factors .

• Growth phase: Expression levels may vary depending on bacterial growth phase, with potential upregulation during stationary phase when stress responses are typically enhanced.

• Host environment: For pathogenic species like P. syringae pv. syringae, host-derived signals and defense compounds encountered during infection may influence azoR expression .

When designing experiments, researchers should consider standardizing growth conditions and potentially pre-inducing azoR expression with appropriate compounds to ensure consistent enzyme levels.

What controls should I include when investigating azoR's role in pathogenicity?

When studying azoR's potential role in P. syringae pv. syringae pathogenicity, comprehensive controls should include:

• Genetic controls:

  • Wild-type strain

  • Clean azoR deletion mutant

  • Complemented mutant strain expressing wild-type azoR

  • Site-directed mutants with catalytically inactive azoR

• Pathogenicity assays:

  • Multiple host tissues (leaves, fruits, stems) as P. syringae pv. syringae can cause various symptoms including cankers, leaf spots, and fruit lesions

  • Different infection time points to capture dynamic responses

  • Various inoculation methods to assess different aspects of the infection process

• Biochemical controls:

  • Measurement of in planta quinone/electrophile levels

  • Assessment of bacterial thiol status during infection

  • Quantification of reactive oxygen species in host tissues

• Expression analysis:

  • azoR expression levels under different infection conditions

  • Correlation between expression and virulence factor production

  • Comparison with other stress response genes

Such comprehensive controls will help establish whether any observed effects on pathogenicity are directly attributable to azoR function or represent secondary consequences of altered bacterial physiology.

How can structural insights inform the development of specific inhibitors for P. syringae pv. syringae azoR?

Understanding the unique structural features of P. syringae pv. syringae azoR could enable the development of specific inhibitors with potential applications in plant protection. The unique loop structure observed in AzoA (residues 55-67) that contacts the binding site near the FMN cofactor represents one potential target for selective inhibition .

Future research should focus on:

• Obtaining high-resolution crystal structures of P. syringae pv. syringae azoR

• Computational modeling of potential inhibitor binding sites

• Structure-guided design of molecules that selectively target P. syringae azoR without affecting beneficial microorganisms

What role might azoR play in antibiotic resistance in Pseudomonas syringae?

Studies with P. syringae have identified a relatively high level of resistance to copper among the population of P. syringae pv. syringae (47.5%), suggesting copper cannot be effectively used for control . Given azoR's role in protecting against electrophilic stress, it could potentially contribute to resistance against other antimicrobial compounds.

Future research should investigate:

• Whether azoR expression correlates with resistance to specific antibiotics

• If azoR can directly reduce antibiotic compounds

• Whether azoR induction occurs in response to antibiotic exposure

• If azoR knockout increases susceptibility to certain classes of antibiotics

Such studies could provide valuable insights into bacterial defense mechanisms and inform more effective antimicrobial strategies.