Recombinant Pseudomonas syringae pv. syringae UDP-N-acetylmuramoylalanine--D-glutamate ligase (murD)

What is Pseudomonas syringae pv. syringae and why is it significant in bacterial research?

Pseudomonas syringae pv. syringae is an opportunistic bacterial pathogen that affects a diverse range of woody ornamental plants. It causes various plant diseases including flower blights, cankers, shoot blights, and diebacks . The pathogen is significant in bacterial research for several reasons. First, it serves as a model organism for studying plant-pathogen interactions. Second, its genomic manipulation capabilities make it valuable for recombinant DNA technology studies. Third, its enzymes involved in cell wall synthesis, particularly MurD, share structural similarities with those of other bacterial species, allowing for comparative studies of essential bacterial processes.

The bacteria typically overwinter in cankers and asymptomatic bud and twig tissues. When environmental conditions become favorable (presence of water and warming temperatures), the bacteria multiply and may exude from infected tissue. Transmission occurs primarily through wind-driven rain, insects, or mechanical means such as pruning equipment .

What is the function of UDP-N-acetylmuramoylalanine--D-glutamate ligase (MurD) in bacterial physiology?

UDP-N-acetylmuramoylalanine--D-glutamate ligase (MurD) is a cytoplasmic enzyme that plays a critical role in the biosynthesis of peptidoglycan, an essential component of bacterial cell walls. Specifically, MurD catalyzes the addition of D-glutamate to the nucleotide precursor UDP-N-acetylmuramoyl-L-alanine (UMA) according to the following reaction:

UDP-MurNAc-L-Ala + D-Glu + ATP ⇔ UDP-MurNAc-L-Ala-D-Glu + ADP + Pi

This step is part of a sequence of cytoplasmic reactions in peptidoglycan biosynthesis, where four ADP-forming ligases (MurC, MurD, MurE, and MurF) catalyze the assembly of the peptide moiety. The L-Ala-D-Glu linkage created by MurD is present in the peptidoglycan of all eubacteria, highlighting the enzyme's evolutionary conservation and fundamental importance .

The reaction mechanism involves phosphorylation of the C-terminal carboxylate of UDP-MurNAc-L-alanine by the γ-phosphate of ATP to form an acyl phosphate intermediate. This is followed by a nucleophilic attack by the amide group of D-glutamate to produce the final product .

How does the structure of MurD from Pseudomonas syringae compare to that of other bacterial species?

While the specific crystal structure of MurD from Pseudomonas syringae has not been fully characterized in the provided search results, we can make informed comparisons based on the known structure of E. coli MurD and sequence homology analyses.

The E. coli MurD structure has been solved to 1.9 Å resolution and reveals three domains with nucleotide-binding fold topologies:

• The N-terminal domain shows a dinucleotide-binding fold (Rossmann fold)

• The central domain exhibits a mononucleotide-binding fold similar to that observed in the GTPase family

• The C-terminal domain also displays a dinucleotide-binding fold (Rossmann fold)

Sequence comparisons among the four E. coli ADP-forming ligases (MurC, MurD, MurE, and MurF) show two homologous regions, suggesting evolutionary relatedness and possibly similar enzymatic mechanisms . When comparing MurD across different bacterial species, sequence identities with E. coli MurD are approximately 31% for B. subtilis and 62% for H. influenzae .

What are the critical active site residues in MurD and how do they contribute to catalytic activity?

Based on the crystal structure of E. coli MurD with its substrate UMA, several critical active site residues have been identified. While specific P. syringae MurD active site residues are not directly mentioned in the search results, the high conservation of this enzyme across bacterial species allows us to infer important functional regions.

The binding site for UMA has been characterized in the E. coli MurD structure, and comparison with known NTP complexes has allowed identification of residues interacting with ATP . These active site residues are likely clustered between the domain interfaces, particularly between the central and C-terminal domains.

Key functional elements of the active site include:

• Residues that coordinate magnesium ions, which are essential for maximal enzyme activity

• Phosphate-binding residues, as phosphate ions have been shown to enhance enzyme activity

• Residues that form the binding pocket for UMA, stabilizing it in the correct orientation

• Residues that interact with ATP, positioning it for phosphoryl transfer

• Catalytic residues that facilitate the formation of the acyl phosphate intermediate and subsequent nucleophilic attack

The reaction mechanism suggests that charged and polar residues play crucial roles in stabilizing reaction intermediates and facilitating the nucleophilic attack by the amide group of D-glutamate. The effectiveness of phosphinate transition-state analogs as inhibitors of MurD further supports this proposed mechanism .

How can recombineering techniques be applied to study MurD function in Pseudomonas syringae?

Recombineering (recombination-mediated genetic engineering) provides powerful tools for precise genetic manipulation in bacteria, including Pseudomonas syringae. Recent work has identified functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation .

For studying MurD function in P. syringae, the following recombineering approaches can be employed:

• Gene replacement: The wild-type murD gene can be replaced with mutated versions to study the effects of specific amino acid changes on enzyme function. This can be achieved using the RecTE system from P. syringae, which is similar to the lambda Red Exo/Beta and RecET proteins encoded by the lambda and Rac bacteriophages of E. coli .

• Gene tagging: Adding epitope tags or fluorescent protein fusions to the murD gene can facilitate protein localization studies and protein-protein interaction analyses.

• Promoter swapping: Replacing the native murD promoter with inducible or constitutive promoters enables controlled expression for studying dosage effects or for overexpression and purification purposes.

• Domain swapping: Creating chimeric proteins by swapping domains between MurD from different bacterial species can provide insights into domain-specific functions.

A typical recombineering protocol for P. syringae would involve:

• Generation of a linear DNA fragment containing the modified murD gene flanked by homology arms

• Introduction of this fragment into P. syringae cells expressing the RecTE recombination system

• Selection of recombinants using appropriate antibiotic markers

• Verification of successful recombination by PCR and sequencing

What are the challenges in expressing and purifying active recombinant P. syringae MurD?

Expressing and purifying active recombinant P. syringae MurD presents several challenges that researchers need to address:

• Protein solubility: Like many bacterial enzymes, MurD may form inclusion bodies when overexpressed, particularly in heterologous hosts like E. coli. Optimization of expression conditions (temperature, inducer concentration, duration) is often necessary.

• Cofactor requirements: MurD activity requires magnesium and phosphate ions . During purification, these cofactors must be maintained at appropriate concentrations to preserve enzyme activity.

• Protein stability: The three-domain structure of MurD may lead to flexibility that can compromise stability during purification. Buffer optimization and the addition of stabilizing agents may be necessary.

• Post-translational modifications: If P. syringae MurD undergoes post-translational modifications that are important for activity, expression in a heterologous system may not reproduce these modifications.

• Substrate availability: For activity assays, the substrate UDP-MurNAc-L-Ala must be available, which may require separate enzymatic synthesis.

A successful purification strategy for E. coli MurD has been reported, involving protein overproduction and purification to homogeneity . This protocol likely includes:

• Affinity chromatography (possibly using His-tag or other fusion tags)

• Ion exchange chromatography

• Size exclusion chromatography

Activity assays should verify the functionality of the purified enzyme by measuring the conversion of UDP-MurNAc-L-Ala to UDP-MurNAc-L-Ala-D-Glu in the presence of D-glutamate and ATP.

What assays can be used to measure MurD enzymatic activity in vitro?

Several complementary assays can be employed to measure MurD enzymatic activity in vitro:

• ATP consumption assay: Since MurD utilizes ATP to form an acyl phosphate intermediate , ATP consumption can be monitored using coupled enzyme assays (such as pyruvate kinase/lactate dehydrogenase) that follow NADH oxidation spectrophotometrically.

• ADP formation assay: Direct quantification of ADP produced during the reaction using HPLC or bioluminescence-based assays (e.g., ADP-Glo™).

• Substrate disappearance/product formation:

  • HPLC or capillary electrophoresis to monitor the disappearance of UDP-MurNAc-L-Ala and appearance of UDP-MurNAc-L-Ala-D-Glu

  • Mass spectrometry to detect and quantify the product

  • Radioactive assays using labeled D-glutamate

• Phosphate release assay: Colorimetric detection of inorganic phosphate released during the reaction using malachite green or other phosphate detection reagents.

• Inhibition assays: Using phosphinate transition-state analogs, which have been shown to be effective inhibitors of MurD , as positive controls for inhibition studies.

The optimal assay choice depends on the specific research question, available equipment, and desired throughput.

How can the crystal structure of P. syringae MurD be determined?

Determining the crystal structure of P. syringae MurD would involve the following methodological steps:

• Protein expression and purification:

  • Clone the P. syringae murD gene into a suitable expression vector

  • Express in E. coli or another heterologous host with appropriate tags for purification

  • Purify to homogeneity using chromatographic techniques

  • Verify purity by SDS-PAGE and activity by enzymatic assays

• Crystallization screening:

  • Perform initial crystallization trials using commercial sparse matrix screens

  • Optimize promising conditions by varying parameters (pH, temperature, precipitant concentration)

  • Co-crystallize with substrates (UMA) or substrate analogs to capture functionally relevant conformations

• Data collection:

  • Mount crystals in appropriate cryoprotectant solutions

  • Collect X-ray diffraction data at a synchrotron facility

  • Process data to determine space group and unit cell parameters

• Phase determination:

  • Multiple options exist, with the E. coli MurD structure determination providing a useful precedent

  • Multiple anomalous dispersion (MAD) using selenomethionine-substituted protein, as was done for E. coli MurD

  • Multiple isomorphous replacement (MIR) with heavy atom derivatives

  • Molecular replacement using E. coli MurD as a search model, given the expected structural similarities

• Model building and refinement:

  • Build initial model into electron density maps

  • Iterative refinement and model building to improve fit to experimental data

  • Validation using standard crystallographic metrics

Based on the successful determination of E. coli MurD structure to 1.9 Å resolution , similar resolution should be achievable for P. syringae MurD, providing detailed insights into its three-domain architecture and substrate binding sites.

What approaches can be used to identify potential inhibitors of P. syringae MurD?

Identifying potential inhibitors of P. syringae MurD can employ various complementary approaches:

• Structure-based virtual screening:

  • Using either the P. syringae MurD structure (once determined) or a homology model based on E. coli MurD

  • Docking virtual compound libraries to identify molecules that bind to the active site

  • Scoring and ranking compounds based on predicted binding affinity

  • Selecting diverse candidates for experimental validation

• High-throughput screening (HTS):

  • Adapting MurD activity assays to a microplate format

  • Screening chemical libraries against purified recombinant MurD

  • Identifying compounds that inhibit enzyme activity

  • Conducting dose-response studies with promising hits

• Fragment-based drug discovery:

  • Screening small molecular fragments by NMR, thermal shift assays, or X-ray crystallography

  • Identifying fragments that bind to different regions of MurD

  • Linking or growing fragments to develop more potent inhibitors

• Rational design based on known inhibitors:

  • Phosphinate transition-state analogs have already been shown to effectively inhibit MurD

  • Structure-activity relationship studies to optimize potency and selectivity

  • Computational approaches to predict modifications that might improve inhibitor properties

• Phenotypic screening in P. syringae:

  • Testing compounds for their ability to inhibit P. syringae growth

  • Validating that growth inhibition correlates with MurD inhibition

  • Assessing effects on cell wall integrity and morphology

The effectiveness of phosphinate transition-state analogs against MurD provides a strong starting point for inhibitor development. These compounds mimic the tetrahedral intermediate formed during the reaction and could serve as leads for further optimization.

How can recombinant P. syringae MurD be used to develop new antimicrobial strategies for plant protection?

Recombinant P. syringae MurD offers several avenues for developing novel antimicrobial strategies for plant protection:

• Target-based inhibitor development:

  • Using the purified enzyme to screen for selective inhibitors

  • Structure-activity relationship studies to optimize potency and specificity

  • Rational design of transition state analogs based on the MurD catalytic mechanism

• Plant-based expression of MurD inhibitors:

  • Engineering plants to produce peptides or proteins that inhibit MurD

  • Creating transgenic plants with enhanced resistance to P. syringae infection

• Development of peptidoglycan-targeting biocontrol agents:

  • Engineering bacteriophages to express MurD inhibitors

  • Creating bacterial strains that compete with P. syringae and produce MurD inhibitors

• Combination strategies:

  • Identifying compounds that synergize with existing plant protection agents

  • Developing multi-target approaches that simultaneously inhibit MurD and other essential P. syringae enzymes

Given that P. syringae pv. syringae causes significant damage to woody ornamental plants through flower blights, cankers, shoot blights, and diebacks , effective MurD inhibitors could provide valuable new tools for plant protection. The specificity of such inhibitors would be essential to avoid adverse effects on beneficial soil bacteria.

What is the relationship between MurD function and P. syringae pathogenicity in plants?

The relationship between MurD function and P. syringae pathogenicity in plants involves several interconnected aspects:

• Cell wall integrity and bacterial viability:

  • As MurD catalyzes a critical step in peptidoglycan biosynthesis , its inhibition would compromise cell wall integrity

  • Weakened cell walls would reduce bacterial viability and ability to withstand plant defense responses

  • Complete inhibition would likely prevent bacterial proliferation within plant tissues

• Bacterial colonization and spread:

  • P. syringae typically spreads through wind-driven rain, insects, or mechanical means

  • MurD inhibition could reduce the bacteria's ability to colonize new host tissues

  • Less robust cell walls might reduce bacterial survival during transmission

• PAMP-triggered immunity:

  • Peptidoglycan fragments can act as Pathogen-Associated Molecular Patterns (PAMPs) that trigger plant immune responses

  • Alterations in peptidoglycan structure due to MurD inhibition might affect PAMP recognition

  • This could potentially modulate plant defense responses

• Stress responses and virulence factor expression:

  • Cell wall stress from MurD inhibition might induce bacterial stress responses

  • These responses could affect the expression of virulence factors

  • The net effect on pathogenicity would depend on the specific regulatory networks involved

Understanding this relationship could guide the development of MurD-targeting strategies that not only inhibit bacterial growth but also potentially enhance the plant's ability to recognize and respond to the pathogen.

How does the comparison between P. syringae MurD and human enzymes inform antimicrobial specificity?

The comparison between P. syringae MurD and human enzymes is crucial for developing antimicrobials with high specificity and low toxicity:

• Absence of direct human homologs:

  • Humans do not produce peptidoglycan, so they lack direct MurD homologs

  • This fundamental difference creates an excellent selectivity window for targeting MurD

• Structural distinctions from human ligases:

  • Although humans possess ATP-dependent ligases, these typically have different substrate specificities and structural features

  • The three-domain architecture of bacterial MurD differs from human ligases

  • The D-amino acid specificity of MurD is particularly distinctive, as human proteins predominantly incorporate L-amino acids

• Evolutionary distance:

  • The evolutionary distance between bacterial and human enzymes contributes to structural and mechanistic differences

  • These differences can be exploited to design inhibitors that selectively bind to bacterial MurD

• Cofactor considerations:

  • While both bacterial MurD and human ligases use ATP, differences in the ATP-binding pocket can be leveraged for selectivity

  • The requirement for specific ions (magnesium and phosphate for MurD) may differ from human enzymes

This favorable selectivity profile explains why the peptidoglycan biosynthesis pathway, including MurD, has been a successful target for antibiotics. Specific MurD inhibitors would likely have minimal direct effects on human enzymes, though thorough toxicity testing would still be necessary to rule out off-target effects.

What are the latest advances in recombinant DNA technology applicable to P. syringae MurD research?

Recent advances in recombinant DNA technology have expanded the toolbox for P. syringae MurD research:

• CRISPR-Cas9 genome editing:

  • Precise modification of the murD gene in its native genomic context

  • Introduction of point mutations to study structure-function relationships

  • Creation of conditional knockdowns to assess essentiality under various conditions

• Advanced recombineering techniques:

  • The identification of RecTE from Pseudomonas syringae provides enhanced tools for genomic recombination

  • These systems allow for markerless modifications and reduced off-target effects

  • Improved efficiency enables higher-throughput genetic manipulations

• Synthetic biology approaches:

  • Construction of minimal genetic systems to study MurD function in isolation

  • Creation of biosensors that report on peptidoglycan synthesis disruption

  • Development of orthogonal genetic systems for controlled expression

• High-throughput mutagenesis:

  • Deep mutational scanning to comprehensively map the functional importance of each residue

  • Saturation mutagenesis of active site residues to understand catalytic mechanisms

  • Directed evolution to engineer MurD variants with altered properties

These advanced genetic tools complement structural and biochemical approaches to provide a more comprehensive understanding of MurD function and potential for inhibition.

How can systems biology approaches enhance our understanding of MurD in the context of P. syringae metabolism?

Systems biology approaches offer powerful frameworks for understanding MurD in the broader context of P. syringae metabolism:

These systems-level approaches would provide a more holistic understanding of MurD function, potentially revealing new strategies for antimicrobial development beyond direct enzyme inhibition.

What ethical considerations should guide the development of recombinant P. syringae MurD for research purposes?

The development and use of recombinant P. syringae MurD for research purposes should be guided by several ethical considerations:

• Biosafety protocols:

  • The historical precedent of the Asilomar Conference on recombinant DNA provides important guidance

  • Appropriate containment measures should be implemented to prevent accidental release of recombinant organisms

  • Risk assessment should consider the potential ecological impacts of modified P. syringae strains

• Dual-use research concerns:

  • Knowledge about bacterial cell wall synthesis could potentially be misused

  • Research should be conducted with transparency while being mindful of biosecurity

  • Publication of detailed methods should be balanced with responsible disclosure

• Environmental considerations:

  • P. syringae is a plant pathogen with ecological significance

  • Field testing of any interventions based on MurD research should undergo rigorous safety evaluation

  • Potential impacts on non-target organisms should be carefully assessed

• Intellectual property and access:

  • Ensuring that research tools and findings are accessible to the scientific community

  • Balancing intellectual property protection with the need for collaborative advancement

  • Considering how patenting might affect the development of applications for resource-limited settings

• Stakeholder engagement:

  • Involving plant growers, conservationists, and other stakeholders in discussions about research directions

  • Ensuring that research priorities align with sustainable agricultural practices

  • Considering the societal implications of new technologies derived from this research

The self-regulation example set by the scientific community at the Asilomar Conference in 1975 provides a valuable model for addressing these ethical considerations in contemporary recombinant DNA research.