Recombinant Desulfovibrio vulgaris UDP-N-acetylenolpyruvoylglucosamine reductase (murB)

What is the biochemical function of murB in Desulfovibrio vulgaris?

MurB (UDP-N-acetylenolpyruvoylglucosamine reductase) in D. vulgaris catalyzes the second step in the cytoplasmic phase of peptidoglycan biosynthesis, a critical component of bacterial cell wall formation. Specifically, it reduces UDP-N-acetylenolpyruvoylglucosamine (UDP-GlcNAc-EP) to UDP-N-acetylmuramic acid (UDP-MurNAc) using NADPH as a cofactor . This reaction converts the enolpyruvyl moiety to a lactyl ether group, a crucial step in the UDP-N-acetylmuramoyl-pentapeptide biosynthesis pathway .

The enzyme contains a flavin adenine dinucleotide (FAD) binding domain characteristic of flavoproteins, which is essential for its catalytic function . As part of the Mur ligase family, murB represents a potential target for antibacterial compounds since the bacterial cell wall synthesis pathway is essential for bacterial survival but absent in humans and other animals .

What expression systems are recommended for recombinant D. vulgaris murB?

Based on research with D. vulgaris and related enzymes, the following expression systems have shown varying degrees of success:

For D. vulgaris murB specifically, protein expression has been tested but results have been challenging, as noted in DNA repository information (ProteinExpressed: "Tested_Not_Found") . Optimization strategies should include:

• Lowering induction temperature (16-25°C)

• Testing multiple E. coli strains optimized for proteins with different codon usage

• Using solubility-enhancing fusion tags beyond the standard His-tag

• Incorporating additives that maintain FAD association during expression

• Considering anaerobic expression conditions that mimic D. vulgaris's natural environment

What are the optimal conditions for purifying active recombinant D. vulgaris murB?

Purification of active D. vulgaris murB presents several challenges based on experiences with related MurB enzymes. The following protocol integrates insights from multiple studies:

• Lysis buffer optimization:

  • pH 7.5-8.0 phosphate or Tris buffer

  • 300-500 mM NaCl to enhance solubility

  • 10% glycerol as a stabilizing agent

  • 1-5 mM β-mercaptoethanol or DTT as reducing agent

  • Protease inhibitor cocktail

  • Consider adding FAD (10-100 μM) to stabilize the flavoprotein

• Purification strategy:

  • Initial capture: Nickel affinity chromatography (for His-tagged protein)

  • Intermediate purification: Ion exchange chromatography

  • Polishing step: Size exclusion chromatography

  • All steps should be performed at 4°C under reducing conditions

• Special considerations:

  • Maintain anaerobic conditions when possible (D. vulgaris is an anaerobe)

  • Include FAD in purification buffers to prevent cofactor loss

  • Monitor flavin absorption (~463 nm) to track active protein

  • Consider detergent addition (0.05-0.1% Triton X-100) if solubility remains an issue

Similar MurB purification approaches have yielded near-homogeneous protein from other bacterial species, though solubility issues required extensive optimization . For D. vulgaris murB specifically, expression and purification have been attempted but successful purification has not been reported ("Protein Purified: Tested_Not_Pur") .

How can researchers verify the enzymatic activity of recombinant D. vulgaris murB?

Multiple complementary approaches can be used to verify MurB activity:

• Spectrophotometric NADPH oxidation assay:

  • Monitor decrease in absorbance at 340 nm

  • Reaction mixture typically contains:

    • Purified recombinant MurB (0.1-1 μM)

    • UDP-GlcNAc-EP substrate (50-200 μM)

    • NADPH (100-250 μM)

    • Buffer (50 mM Tris-HCl, pH 7.5-8.0)

    • KCl (50-100 mM) for activation

  • Calculate activity based on NADPH consumption rate

• Coupled enzyme assay system:

  • Reconstruct sequential MurA-MurB reactions in vitro

  • MurA converts UDP-GlcNAc and PEP to UDP-GlcNAc-EP

  • MurB then reduces this product using NADPH

  • Monitor by NADPH consumption or product formation

• Product characterization methods:

  • HPLC analysis of UDP-MurNAc formation

  • Mass spectrometry confirmation of product identity

  • NMR analysis for structural verification

• FAD cofactor verification:

  • Absorption spectrum analysis for characteristic peak at ~463 nm

  • Fluorescence spectroscopy to confirm flavin incorporation

• Functional complementation:

  • Transform temperature-sensitive E. coli murB mutants with D. vulgaris murB

  • Test growth restoration at non-permissive temperatures

Control experiments should include enzyme-free reactions, substrate-free reactions, and inhibition with known MurB inhibitors to confirm specificity of the activity.

What are the kinetic parameters of D. vulgaris murB compared to other bacterial species?

While specific kinetic parameters for D. vulgaris murB are not directly reported in literature, comparative data from related bacterial MurB enzymes provides context for experimental design:

For characterizing D. vulgaris murB, researchers should:

• Determine Km and Vmax for both substrates under varying pH, temperature, and ionic conditions

• Analyze potential substrate inhibition patterns similar to those observed in other bacterial MurB enzymes

• Investigate metal ion effects, particularly univalent cations like potassium

• Compare catalytic efficiency (kcat/Km) with other bacterial MurB enzymes to identify unique characteristics

Such comparative kinetic analysis would provide insights into potential adaptations of D. vulgaris murB to its anaerobic, sulfate-reducing lifestyle.

What computational approaches best predict D. vulgaris murB structure and function?

A multi-faceted computational approach is recommended for predicting D. vulgaris murB structure and function:

• Homology modeling:

  • Use well-characterized MurB structures as templates (E. coli, S. aureus)

  • Employ multiple modeling algorithms (SWISS-MODEL, I-TASSER, Rosetta)

  • Validate models through Ramachandran plots, QMEAN scores, and ProSA z-scores

  • This approach has been successfully applied to Mycobacterium tuberculosis MurB

• Molecular dynamics simulations:

  • Refine homology models through extensive MD simulations (100+ ns)

  • Use OPLS_2005 or AMBER force fields with explicit solvent

  • Analyze protein stability, flexibility, and conformational changes

  • Identify binding pocket dynamics critical for substrate and inhibitor interactions

• Active site identification and characterization:

  • Identify catalytic and substrate-binding residues through sequence alignment with characterized MurB enzymes

  • Focus on residues equivalent to those identified in other MurB enzymes (e.g., Tyr155, Arg156, Ser237, Asn241, His304 in M. tuberculosis MurB)

  • Analyze FAD-binding motif and interaction patterns

• Virtual screening for potential inhibitors:

  • Prepare computational libraries from multiple repositories (ChemSpider, DrugBank, ZINC)

  • Use AutoDock Vina or similar tools for docking simulations

  • Set binding affinity thresholds (e.g., -9.0 Kcal/mol) for hit identification

  • Further assess promising hits through MM-GBSA binding free energy calculations

These computational approaches provide a robust foundation for experimental validation and can significantly accelerate structure-function studies and inhibitor development targeting D. vulgaris murB.

How do structural differences in D. vulgaris murB affect its sensitivity to known inhibitors?

Understanding structure-activity relationships for D. vulgaris murB requires analysis of binding site architecture and inhibitor interactions:

• Comparative binding site analysis:
  While specific structural information for D. vulgaris murB is limited, insights from other bacterial MurB enzymes highlight key structural determinants of inhibitor sensitivity:

  • Hydrophobic pockets accommodating bulky side chains significantly impact inhibitor binding

  • The meta position of terminal phenyl rings in inhibitors is critical for activity, particularly with electron-withdrawing groups (-NO₂ > -F)

  • Electron-donating substituents at ortho positions enhance activity (-OMe > -OH)

  • Binding free energies range from -8.19 to -11.56 kcal/mol for effective inhibitors

• Binding interactions to consider:

  • Hydrogen bonding networks with key residues

  • π-π stacking interactions with aromatic residues

  • Hydrophobic interactions in binding pockets

  • Electrostatic complementarity between inhibitor and binding site

• Structure-based inhibitor optimization strategy:

  Structural Feature	Impact on Activity	Design Considerations for D. vulgaris murB
  Electron-withdrawing groups at meta positions	Increased activity	Incorporate -NO₂, -F, or -Br at meta positions of terminal rings
  Electron-donating groups at ortho positions	Enhanced binding	Include -OMe or -OH groups at ortho positions
  Bulky side chains	Essential for filling hydrophobic pockets	Design inhibitors with appropriate space-filling groups
  Di-substitution patterns	Can cause steric hindrance	Avoid overcrowding in certain positions (e.g., 2,5-dimethoxy)

For D. vulgaris murB specifically, researchers should:

• Identify unique residues in the binding site through sequence analysis and homology modeling

• Consider the anaerobic environment of D. vulgaris and potential redox sensitivity of the enzyme

• Evaluate inhibitors with varying physicochemical properties to establish structure-activity relationships specific to D. vulgaris murB

• Test inhibitors with demonstrated activity against other bacterial MurB enzymes as starting points

How can site-directed mutagenesis enhance our understanding of D. vulgaris murB?

Site-directed mutagenesis provides powerful insights into structure-function relationships. A systematic approach should include:

• Strategic selection of mutation targets:

  Residue Type	Purpose	Expected Outcome
  Catalytic residues	Verify enzymatic mechanism	Substantial reduction in catalytic activity
  Substrate-binding residues	Probe substrate specificity	Altered Km values; substrate preference changes
  FAD-binding motif residues	Examine cofactor interactions	Reduced FAD binding; spectral changes
  Interface residues	Investigate protein-protein interactions	Modified interaction with other enzymes in the pathway
  D. vulgaris-specific residues	Identify unique functional adaptations	Potentially altered kinetics or substrate specificity

• Comprehensive functional analysis framework:

  • Express and purify mutant proteins using optimized protocols for wild-type

  • Conduct spectroscopic analysis to confirm proper folding and FAD incorporation

  • Perform enzyme kinetic studies to determine Km, kcat, and catalytic efficiency changes

  • Test substrate analogs to identify specificity alterations

  • Evaluate thermal and chemical stability of mutants compared to wild-type

• Structural consequences assessment:

  • Use circular dichroism to detect secondary structure perturbations

  • Apply differential scanning fluorimetry to analyze thermal stability changes

  • Perform computational modeling of mutations to predict structural impacts

  • When possible, obtain crystal structures of key mutants to confirm structural changes

This systematic mutagenesis approach has proven valuable for other bacterial MurB enzymes, with computational analysis guiding the selection of key residues (e.g., Tyr155, Arg156, Ser237, Asn241, His304 in M. tuberculosis MurB) .

What methodologies are most effective for identifying novel inhibitors against D. vulgaris murB?

An integrated approach combining computational and experimental methodologies offers the most efficient path to novel inhibitor discovery:

• Virtual screening cascade:

  • Generate homology models and identify binding pockets

  • Screen large compound libraries against the target using AutoDock Vina

  • Apply binding affinity filters (e.g., -9.0 Kcal/mol threshold)

  • Perform molecular dynamics simulations (100+ ns) on top candidates to assess binding stability

  • Calculate binding free energies using MM-PBSA or MM-GBSA methods

  • Select diverse compounds with favorable predicted properties for experimental testing

• Biochemical screening methodology:

  • Establish reliable enzymatic assays for D. vulgaris murB (NADPH oxidation monitoring)

  • Screen selected compounds at multiple concentrations (typically 1-100 μM)

  • Determine IC₅₀ values for promising inhibitors

  • Evaluate inhibition mechanisms (competitive, noncompetitive, uncompetitive)

  • Assess selectivity against human enzymes and other bacterial MurB variants

• Structure-activity relationship development:

  • Group effective inhibitors by chemical scaffolds

  • Systematically modify substituents to improve potency

  • Test the impact of specific structural features:

    • Electron-withdrawing groups at meta positions (-NO₂ > -F)

    • Electron-donating groups at ortho positions (-OMe > -OH)

    • Bulky side chains to fill hydrophobic pockets

  • Avoid di-substitution patterns that may cause steric hindrance

• Cellular evaluation of promising candidates:

  • Assess antibacterial activity against D. vulgaris and other bacterial species

  • Determine minimum inhibitory concentrations (MICs)

  • Evaluate cytotoxicity against mammalian cells

  • Assess membrane permeability and potential efflux pump susceptibility

This integrated approach has successfully identified potent inhibitors against other bacterial MurB enzymes, with binding affinities ranging from -9.70 to -13.0 Kcal/mol for top candidates .

How might D. vulgaris murB contribute to antimicrobial resistance mechanisms?

Although direct evidence on D. vulgaris murB's role in antimicrobial resistance is limited, several potential mechanisms can be inferred from our understanding of bacterial cell wall biosynthesis and resistance:

• Structural adaptations affecting inhibitor binding:

  • Mutations in key binding site residues could reduce inhibitor affinity while maintaining catalytic function

  • Altered protein dynamics might affect inhibitor access to binding sites

  • Research focus: Compare murB sequences from resistant and susceptible strains

• Enzymatic modifications influencing substrate processing:

  • Kinetic adaptations allowing continued function at lower efficiency despite inhibition

  • Altered substrate specificity providing alternative pathways for cell wall synthesis

  • Research focus: Characterize kinetic parameters of murB from resistant strains

• Regulatory adaptations:

  • Overexpression of murB to overcome competitive inhibition

  • Altered regulation of the entire peptidoglycan synthesis pathway

  • Research focus: Quantify murB expression levels in response to antimicrobial pressure

• Unique aspects for D. vulgaris:

  • As a sulfate-reducing anaerobe found in inflammatory conditions like ulcerative colitis , D. vulgaris may experience distinctive selective pressures

  • The connection between D. vulgaris and inflammation could influence antimicrobial exposure patterns and resistance development

  • D. vulgaris biofilm formation, which has been demonstrated in rat colon models , may contribute to antimicrobial tolerance

• Horizontal gene transfer considerations:

  • Acquisition of variant murB genes with inherent resistance properties

  • Exchange of resistance determinants within microbial communities

  • Research focus: Phylogenetic analysis of murB variations across Desulfovibrio species

Understanding these potential resistance mechanisms would enable the development of strategies to overcome resistance, such as dual-targeting inhibitors or combination therapies that address multiple steps in the peptidoglycan synthesis pathway.

How can missing data be appropriately handled in structural and functional studies of D. vulgaris murB?

Research on D. vulgaris murB often confronts missing data challenges that require methodologically sound approaches:

How does D. vulgaris murB compare to fusion enzymes like MurB/C found in other bacteria?

The discovery of fusion enzymes in the peptidoglycan synthesis pathway presents interesting evolutionary questions relevant to understanding D. vulgaris murB:

• Structural and functional comparison:

  Feature	D. vulgaris murB	MurB/C Fusion Enzymes
  Genetic organization	Single gene encoding murB function	Fusion ORF encoding both murB and murC activities
  Enzyme architecture	Single functional domain with FAD-binding motif	Two distinct functional domains with separate active sites
  Evolutionary distribution	Common across diverse bacterial phyla	Identified in specific lineages (e.g., Verrucomicrobia)
  Activity verification	Challenging to verify in recombinant form	MurC activity demonstrated in vitro; murB activity verified through complementation

• Evolutionary significance:

  The fusion of murB and murC genes in certain bacterial lineages suggests potential advantages:

  • Coordinated expression of sequential enzymes in the pathway

  • Enhanced substrate channeling between reactions

  • Potential regulatory advantages in specific environmental niches

  • Evolutionary adaptation to streamline the genome

• Functional implications for D. vulgaris:

  • D. vulgaris maintains separate murB and murC genes, similar to most bacterial species

  • The distinct regulation of these genes may be advantageous in the sulfate-reducing, anaerobic lifestyle of D. vulgaris

  • Comparative analysis between D. vulgaris murB and the murB domain of fusion proteins could reveal functional adaptations to different ecological niches

• Research opportunities:

  • Investigating whether D. vulgaris murB interacts physically with murC despite being encoded by separate genes

  • Exploring the possibility of creating artificial fusion proteins with D. vulgaris enzymes to study substrate channeling

  • Examining the expression correlation between murB and other peptidoglycan synthesis genes in D. vulgaris under various conditions

This comparative analysis provides evolutionary context for understanding D. vulgaris murB and may suggest novel approaches for studying its function and regulation.

How can knowledge of D. vulgaris murB be applied to understanding its role in microbial communities and disease states?

D. vulgaris's prevalence in certain disease states provides context for understanding the broader significance of murB research:

• D. vulgaris in inflammatory conditions:

  • Increased abundance of Desulfovibrio has been observed in ulcerative colitis patients

  • D. vulgaris can stably colonize the rat colon for extended periods (3.5+ months)

  • Higher levels of hydrogen sulfide (H₂S) production correlate with higher tumor burden in some models

  • Biofilm formation by D. vulgaris may contribute to persistent colonization

• Potential applications of murB research:

  Research Area	Application to Health/Disease	Methodological Approach
  Biofilm inhibition	Targeting murB could disrupt D. vulgaris biofilm formation	Screen for murB inhibitors that reduce biofilm formation without causing resistance
  Microbiome modulation	Selective inhibition of D. vulgaris without disrupting beneficial bacteria	Design species-specific murB inhibitors based on structural differences
  Inflammatory disease	Reducing D. vulgaris colonization in ulcerative colitis	Explore combination therapies targeting murB and inflammatory pathways
  Diagnostic development	Using murB as a biomarker for D. vulgaris presence	Develop molecular or serological tests targeting unique aspects of D. vulgaris murB

• Research methodology for clinical applications:

  • In vitro colonization models to test murB inhibitors against D. vulgaris biofilms

  • Animal models of inflammatory bowel disease to assess the impact of targeting D. vulgaris murB

  • Metagenomic analysis to correlate murB variants with disease severity

  • Combination therapy approaches that target multiple aspects of D. vulgaris pathobiology

• Ecological considerations:

  • Impact of targeting D. vulgaris murB on broader microbial communities

  • Potential for horizontal gene transfer of resistance determinants

  • Ecological role of D. vulgaris in healthy versus diseased states

  • Relationship between D. vulgaris and host factors like mucin production

This research direction connects basic enzymatic studies of D. vulgaris murB to potential clinical applications, particularly in inflammatory conditions where Desulfovibrio species have been implicated.