Recombinant Nocardia farcinica UDP-N-acetylmuramoylalanine--D-glutamate ligase (murD)

What is Nocardia farcinica UDP-N-acetylmuramoylalanine--D-glutamate ligase (murD) and what role does it play in bacterial cell wall synthesis?

Nocardia farcinica UDP-N-acetylmuramoylalanine--D-glutamate ligase (murD) is a cytoplasmic enzyme crucial for peptidoglycan biosynthesis, which is essential for bacterial cell wall integrity. It specifically catalyzes the ATP-dependent addition of D-glutamate to UDP-N-acetylmuramoyl-L-alanine (UMA), forming UDP-N-acetylmuramoyl-L-alanine-D-glutamate (UMAG) . This reaction represents the second step in the sequential assembly of the peptide moiety of peptidoglycan, occurring after the addition of L-alanine catalyzed by MurC and before the addition of diaminopimelate or lysine (catalyzed by MurE) and D-alanyl-D-alanine (catalyzed by MurF) .

The reaction proceeds through a three-step mechanism:

• Phosphorylation of the C-terminal carboxylate of UDP-MurNAc-L-alanine by ATP's γ-phosphate to form an acyl phosphate intermediate

• Nucleophilic attack by the amide group of D-glutamate

• Formation of the final products: UDP-MurNAc-L-Ala-D-Glu, ADP, and inorganic phosphate

This enzyme is particularly significant because the L-Ala-D-Glu linkage it creates is universally present in the peptidoglycan of all eubacteria, making it an excellent target for broad-spectrum antibacterial drug development .

How does the structure of murD compare between Nocardia farcinica and other bacterial species?

While specific structural information for Nocardia farcinica murD is limited, comparative analysis with other bacterial murD enzymes, particularly the well-characterized Escherichia coli murD, reveals several conserved features across species:

The structural organization of murD typically comprises three domains with distinct functions:

• N-terminal domain: Exhibits a Rossmann fold structure involved in UDP-MurNAc-L-Ala binding

• Central domain: Contains a mononucleotide-binding fold similar to that found in the GTPase family, involved in ATP binding

• C-terminal domain: Also displays a Rossmann fold structure and participates in D-glutamate binding

Sequence identity analysis shows E. coli murD shares approximately 31% identity with Bacillus subtilis and 62% with Haemophilus influenzae murD . While specific sequence identity data for Nocardia farcinica murD is not provided in the search results, the sequence conservation pattern among Mur ligases generally ranges from 22-26% .

All murD enzymes contain a highly conserved ATP-binding site with a characteristic P-loop abundant in glycine residues, as well as conserved glutamic acid and histidine residues involved in regulating magnesium binding during catalysis .

What catalytic mechanism does Nocardia farcinica murD employ?

The catalytic mechanism of Nocardia farcinica murD follows the general mechanism determined for murD enzymes across bacteria:

• Initial step: ATP-dependent phosphorylation of the carboxylic acid of UDP-MurNAc-L-alanine, creating a reactive acyl-phosphate intermediate

• Middle step: Nucleophilic attack by the amino group of D-glutamate on this intermediate, forming a high-energy tetrahedral intermediate

• Final step: Collapse of the tetrahedral intermediate to yield the amide product (UDP-N-acetylmuramoyl-L-alanine-D-glutamate) and inorganic phosphate

This mechanism is supported by inhibition studies using phosphinate transition-state analogs, which have proven effective as murD inhibitors . The reaction follows an ordered substrate binding sequence, where the nucleotide substrate (UMA) binds first, followed by ATP and finally D-glutamate.

The reaction can be represented by the following equation:
UDP-MurNAc-L-Ala + D-Glu + ATP ⇔ UDP-MurNAc-L-Ala-D-Glu + ADP + Pi

What are the most effective techniques for expressing recombinant Nocardia farcinica murD?

For successful expression of recombinant Nocardia farcinica murD, researchers should consider the following methodological approach:

Recommended Expression System:

• Escherichia coli BL21(DE3) or similar expression strain capable of high-level protein production

• A pET-based vector containing a T7 promoter for controlled induction

• Incorporation of an N-terminal or C-terminal His-tag for purification purposes

Expression Conditions:

• Culture growth in LB medium supplemented with appropriate antibiotic at 37°C until OD600 reaches 0.6-0.8

• Reduce temperature to 18-25°C prior to induction (helps maintain protein solubility)

• Induce expression with 0.5-1.0 mM IPTG

• Continue expression for 16-20 hours at reduced temperature

While the search results don't specifically detail the exact conditions for Nocardia farcinica murD expression, this approach is based on successful protocols for recombinant murD enzymes from other bacterial species . For E. coli murD, the recombinant protein has been successfully overproduced and purified to homogeneity, suggesting similar techniques could be adapted for Nocardia farcinica murD .

What purification strategies yield the highest purity and activity for Nocardia farcinica murD?

A multi-step purification process is recommended to obtain high-purity, active Nocardia farcinica murD:

Step 1: Initial Extraction

• Harvest cells by centrifugation and resuspend in a buffer containing:

  • 50 mM Tris-HCl (pH 8.0)

  • 300 mM NaCl

  • 10% glycerol

  • 1 mM DTT

  • Protease inhibitor cocktail

• Lyse cells using sonication or high-pressure homogenization

• Clarify lysate by centrifugation at 20,000 × g for 45 minutes at 4°C

Step 2: Affinity Chromatography

• Load clarified lysate onto a Ni-NTA column pre-equilibrated with binding buffer

• Wash extensively with binding buffer containing 20-40 mM imidazole

• Elute purified protein using an imidazole gradient (50-300 mM)

Step 3: Size Exclusion Chromatography

• Further purify the protein by gel filtration using a Superdex 200 column

• Use a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 1 mM DTT

Quality Control Checks:

• Assess purity by SDS-PAGE (≥95% purity)

• Verify enzyme activity using a coupled enzymatic assay measuring ADP production

• Confirm protein identity by mass spectrometry

The optimal buffer for storage of purified murD typically contains 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM DTT, and 10% glycerol. Storage at -80°C in small aliquots helps maintain enzyme activity.

What are the optimal conditions for assaying Nocardia farcinica murD activity in vitro?

For accurate and reproducible measurement of Nocardia farcinica murD enzyme activity, the following conditions are recommended:

Standard Reaction Mixture:

• 50 mM Tris-HCl buffer (pH 8.0)

• 10 mM MgCl₂ (essential cofactor)

• 5 mM ATP

• 0.5-1 mM UDP-MurNAc-L-Ala (substrate)

• 5-10 mM D-glutamate

• 10-20 nM purified murD enzyme

• Optional: 10 mM KCl and 0.1-1 mM DTT to enhance stability

Reaction Conditions:

• Temperature: 30-37°C (optimal temperature should be determined empirically)

• Reaction time: 10-30 minutes (within linear range)

• Total reaction volume: 50-100 μL

Detection Methods:

• Coupled Enzyme Assay: Monitoring ADP formation using pyruvate kinase and lactate dehydrogenase with spectrophotometric detection of NADH oxidation at 340 nm

• HPLC Analysis: Separating substrates and products using reverse-phase HPLC

• Radioactive Assay: Using ¹⁴C-labeled D-glutamate and measuring incorporation by scintillation counting

Based on existing research with murD enzymes, activity is maximal in the presence of magnesium and phosphate ions, which should be incorporated in the assay buffer . The reaction follows Michaelis-Menten kinetics, allowing the determination of kinetic parameters (KM, kcat) for each substrate.

How can site-directed mutagenesis be used to identify critical residues in Nocardia farcinica murD?

Site-directed mutagenesis represents a powerful approach to identify functionally important residues in Nocardia farcinica murD. A systematic mutagenesis strategy should target:

Priority Residues for Mutagenesis:

• Conserved residues in the P-loop (rich in glycine) involved in ATP binding

• Glutamic acid and histidine residues implicated in magnesium coordination

• Residues at the UMA binding site

• Residues at the D-glutamate binding site

• Residues at domain interfaces that may be involved in conformational changes

Recommended Mutagenesis Protocol:

• Design primers incorporating desired mutations using established primer design guidelines

• Perform PCR-based mutagenesis using a high-fidelity polymerase

• Verify mutations by DNA sequencing

• Express and purify mutant proteins using the same protocol as for wild-type enzyme

• Perform comparative kinetic analyses between wild-type and mutant enzymes

Analysis of Mutants:

• Determine kinetic parameters (KM, kcat) for each substrate

• Assess structural integrity using circular dichroism or thermal shift assays

• Evaluate binding affinities using isothermal titration calorimetry (ITC)

• For selected mutants, attempt X-ray crystallography to determine structural changes

By analyzing the effects of specific amino acid substitutions on enzyme activity, researchers can:

• Map the functional roles of specific residues

• Identify residues essential for catalysis versus substrate binding

• Understand the structural basis for substrate specificity

• Reveal potential sites for inhibitor design

What computational approaches are most effective for identifying potential inhibitors of Nocardia farcinica murD?

Computational approaches provide powerful tools for identifying potential inhibitors of Nocardia farcinica murD. A systematic multi-step computational workflow should include:

Step 1: Structural Preparation

• Obtain crystal structure or create a homology model of Nocardia farcinica murD

• Optimize the structure using molecular dynamics simulations

• Identify and characterize binding pockets using tools like SiteMap

Step 2: Virtual Screening

• Prepare a diverse compound library (e.g., ZINC database, FDA-approved drugs)

• Perform molecular docking using software like Glide, AutoDock, or GOLD

• Apply scoring functions to rank compounds based on binding affinity

• Select top-scoring compounds (typically top 10%) for further analysis

Step 3: Refinement and Selection

• Perform similarity analysis based on known inhibitors

• Evaluate drug-like properties using ADME prediction tools

• Calculate binding energies using more sophisticated methods (MM-GBSA or FEP)

• Select the most promising candidates (20-50 compounds) for experimental validation

Step 4: Molecular Dynamics Validation

• Perform extended molecular dynamics simulations (≥100 ns) of protein-ligand complexes

• Analyze stability of binding poses and key interactions

• Calculate binding free energy over the simulation trajectory

• Identify compounds maintaining stable conformations throughout the simulation

This approach has been successfully applied to identify potential inhibitors of Staphylococcus aureus murD, as described in search result . For example, after virtual screening and similarity analysis, compounds 46604 and 46608 were identified as promising candidates based on their favorable interactions with the binding pocket, good pharmacological properties, and stability during molecular dynamics simulations .

How can molecular dynamics simulations provide insights into the conformational changes of Nocardia farcinica murD?

Molecular dynamics (MD) simulations offer valuable insights into the conformational dynamics of Nocardia farcinica murD, particularly regarding substrate binding and catalytic mechanism:

Simulation Setup Protocol:

• Prepare the murD structure in various states:

  • Apo (unbound) state

  • Binary complex with UMA

  • Ternary complex with UMA and ATP

  • Complete complex with UMA, ATP, and D-glutamate

• Place protein in an explicit solvent box with physiological salt concentration

• Apply appropriate force field (e.g., AMBER ff14SB, CHARMM36)

• Perform energy minimization and equilibration

• Run production MD for at least 100-500 ns to capture relevant conformational changes

Analysis Approaches:

• Domain Motion Analysis:

  • Calculate root-mean-square deviation (RMSD) for individual domains

  • Analyze relative domain orientations and interdomain angles

  • Identify hinge regions mediating domain movements

• Binding Site Analysis:

  • Monitor key protein-ligand interactions throughout the simulation

  • Analyze water-mediated hydrogen bonds at the binding interface

  • Identify induced-fit conformational changes upon substrate binding

• Advanced Analyses:

  • Principal Component Analysis (PCA) to identify major conformational modes

  • Free energy landscapes to map conformational states

  • Markov State Models to characterize transition pathways between states

Expected Insights:

• Characterization of domain closure motion upon substrate binding

• Identification of key residues facilitating conformational changes

• Elucidation of the sequence of binding events and conformational changes

• Discovery of transient pockets that might be exploited for inhibitor design

MD simulations have successfully been used to verify the stability of inhibitor binding to murD proteins, with 100 ns simulations providing sufficient sampling to establish binding stability .

What strategies can be employed to develop selective inhibitors against Nocardia farcinica murD?

Developing selective inhibitors against Nocardia farcinica murD requires a multi-faceted approach combining structural insights, computational methods, and experimental validation:

Structure-Based Design Strategies:

• Transition State Mimics: Design compounds that mimic the tetrahedral intermediate or acyl-phosphate intermediate of the reaction

• ATP-Competitive Inhibitors: Target the ATP-binding site with analogs that compete with ATP binding

• Substrate Analogs: Develop modified versions of UMA or D-glutamate that bind but cannot undergo catalysis

• Allosteric Inhibitors: Identify allosteric sites that can modulate enzyme activity indirectly

Key Considerations for Selectivity:

• Exploiting Unique Features: Identify and target structural features or residues unique to Nocardia farcinica murD

• Comparative Analysis: Analyze differences in binding site architecture between murD from Nocardia farcinica and human pathogens

• Species-Specific Interactions: Design inhibitors that form interactions with non-conserved residues specific to Nocardia farcinica

Methodological Workflow:

• Perform detailed binding site analysis of Nocardia farcinica murD using SiteMap or similar tools

• Design focused compound libraries based on identified pharmacophore features

• Employ docking and scoring to prioritize compounds for synthesis

• Validate binding using biophysical methods (ITC, SPR, thermal shift assays)

• Assess inhibitory activity using enzymatic assays

• Evaluate selectivity against murD from other bacterial species and human enzymes

• Optimize lead compounds through iterative design-synthesis-testing cycles

Promising Scaffold Types:

• Phosphinate transition-state analogs (demonstrated effectiveness against murD)

• Nucleotide-based inhibitors targeting the UDP-binding region

• D-glutamate peptidomimetics targeting the D-glutamate binding pocket

• Fragment-based design starting with small molecules binding to sub-pockets

How can we address cell permeability challenges for Nocardia farcinica murD inhibitors?

Cell permeability represents a significant challenge in developing effective Nocardia farcinica murD inhibitors. The following strategies can help overcome this barrier:

Understanding Bacterial Cell Envelope:

• Nocardia species possess a complex cell envelope with mycolic acids, which creates a highly hydrophobic barrier

• Inhibitors must traverse both this hydrophobic layer and the peptidoglycan layer to reach cytoplasmic murD

Physicochemical Property Optimization:

• Lipophilicity Balancing: Maintain logP values between 1-3 for optimal membrane penetration

• Molecular Weight Consideration: Keep MW <600 Da to facilitate diffusion across membranes

• Hydrogen Bond Donors/Acceptors: Limit to ≤5 HBD and ≤10 HBA to enhance membrane permeability

• Charge State Management: Consider zwitterionic compounds to balance permeability with target binding

Formulation and Delivery Strategies:

• Prodrug Approach: Design prodrugs that are more permeable and are activated inside bacterial cells

• Nanoparticle Delivery: Encapsulate inhibitors in lipid or polymer nanoparticles to enhance delivery

• Siderophore Conjugation: Attach inhibitors to siderophores to exploit bacterial iron uptake systems

• Membrane Permeabilizers: Co-administer with sub-inhibitory concentrations of membrane-disrupting agents

Experimental Assessment:

• Implement bacterial accumulation assays to quantify intracellular concentration of inhibitors

• Use fluorescently labeled analogs to track cellular uptake and localization

• Establish correlation between in vitro enzyme inhibition and whole-cell activity

Recent research has highlighted that addressing cell permeability barriers is critical for effective drug development against murD and other intracellular targets in bacteria . Combining in silico methods with experimental work is recommended to overcome the catalytic machinery of murD enzyme and address permeability challenges .

How does murD contribute to the pathogenicity of Nocardia farcinica?

Nocardia farcinica murD plays a crucial role in pathogenicity through its essential function in cell wall biosynthesis:

Direct Contributions to Pathogenicity:

• Structural Integrity: By facilitating peptidoglycan synthesis, murD ensures bacterial cell wall integrity, which is essential for survival during infection

• Growth and Division: Functional murD is required for bacterial growth and division, enabling the expansion of bacterial populations during infection

• Antibiotic Resistance: The cell wall provides intrinsic resistance to various host defense mechanisms and certain antibiotics

Contextual Relevance to Nocardia farcinica Infections:

• Nocardia farcinica is an opportunistic pathogen causing various infections, particularly in immunocompromised patients

• Clinical manifestations include pulmonary, cutaneous, and disseminated infections

• Eight different Nocardia species have been identified in clinical settings, with Nocardia farcinica being one of the most prevalent (n=9 in the referenced study)

Relationship to Other Virulence Factors:
While murD itself is not classified as a virulence factor, it functions within a network of factors contributing to pathogenicity. Research has identified other proteins in Nocardia farcinica as potential virulence factors, such as:

• Nfa34810 protein: Located in the cell wall and facilitates bacterial invasion of host cells

• Nfa34810 promotes uptake and internalization into HeLa cells

• Deletion of the nfa34810 gene in N. farcinica affects invasion capabilities

• Nfa34810 promotes production of TNF-α in macrophages through activation of ERK, JNK, and NF-κB signaling pathways via TLR4

Understanding how murD interacts with or supports these virulence mechanisms requires further investigation, but its fundamental role in maintaining cell wall integrity underpins the ability of Nocardia farcinica to establish and maintain infection.

What are the future research directions for Nocardia farcinica murD as an antibiotic target?

Future research on Nocardia farcinica murD as an antibiotic target should focus on several promising directions:

Structural and Functional Characterization:

• Determine high-resolution crystal structures of Nocardia farcinica murD in different conformational states

• Elucidate species-specific features of the enzyme through comparative structural analysis

• Characterize the kinetic mechanism and substrate specificity in detail

• Identify potential allosteric sites that could be targeted for inhibition

Inhibitor Development and Optimization:

• Design and synthesize inhibitors specifically targeting Nocardia farcinica murD

• Develop transition state analogs based on the catalytic mechanism

• Explore combination approaches targeting multiple enzymes in the peptidoglycan biosynthesis pathway

• Address cell permeability challenges through innovative drug delivery systems

Validation in Infection Models:

• Establish appropriate in vitro and in vivo models for Nocardia farcinica infections

• Evaluate efficacy of murD inhibitors in these models

• Assess potential for resistance development and design counter-strategies

• Study pharmacokinetics and pharmacodynamics of promising inhibitors

Integration with Other Therapeutic Approaches:

• Investigate synergistic effects between murD inhibitors and existing antibiotics

• Explore potential for immunomodulatory approaches combined with murD inhibition

• Develop diagnostic tools based on murD-specific characteristics for early detection

Broader Impact:
The MurD enzyme represents one of the most appropriate targets for developing novel inhibitors against antibiotic-resistant bacterial pathogens . The rapid emergence of antibiotic resistance among various bacterial pathogens has been identified as a major concern for health organizations worldwide . Research on Nocardia farcinica murD could provide insights applicable to other bacterial pathogens, contributing to the broader effort to combat antimicrobial resistance.