Recombinant Gloeobacter violaceus UDP-N-acetylenolpyruvoylglucosamine reductase (murB)

What is the function of UDP-N-acetylenolpyruvoylglucosamine reductase (murB) in bacterial cell wall synthesis?

Methodological Answer: UDP-N-acetylenolpyruvoylglucosamine reductase (MurB) catalyzes the NADPH-dependent reduction of UDP-N-acetylenolpyruvylglucosamine (EP-UNAG) to UDP-N-acetylmuramic acid (UDP-MurNAc), representing the second step in the cytoplasmic phase of peptidoglycan biosynthesis . This reaction is essential for bacterial cell wall formation across diverse bacterial species. To experimentally confirm MurB function, researchers should express and purify the recombinant enzyme, then conduct spectrophotometric assays monitoring NADPH oxidation at 340 nm. The activity can be further validated by LC-MS detection of UDP-MurNAc formation. G. violaceus, as an early-branching cyanobacterium lacking thylakoid membranes , presents a unique model for studying this enzyme in the context of primitive photosynthetic bacteria.

How can researchers overcome expression and purification challenges for recombinant G. violaceus murB?

Methodological Answer: Based on experience with other bacterial MurB enzymes, researchers should implement a systematic approach to optimize expression and purification:

• Expression system selection:

  • Use E. coli BL21(DE3) with pET vectors (pET28a+) as successfully employed for S. pneumoniae MurB

  • Consider the RSF1010-derived vector system (pKUT1121) that has been successfully used for G. violaceus transformation

• Solubility enhancement strategy:

  • Lower induction temperature (16-20°C) to slow folding

  • Reduce IPTG concentration (0.1-0.5 mM)

  • Add solubility enhancers to growth media (5-10% glycerol, 0.5M sorbitol)

  • Test multiple fusion tags (MBP, SUMO, Trx) as S. pneumoniae MurB showed significant insolubility issues

• Purification optimization:

  • Two-step purification approach as used for S. pneumoniae MurB

  • Implement on-column refolding if inclusion bodies persist

  • Verify flavoprotein status by monitoring characteristic absorption at ~460 nm

The success of these approaches should be monitored by SDS-PAGE and enzymatic activity assays at each purification step.

What are the optimal conditions for enzymatic assay of G. violaceus murB?

Methodological Answer: Based on characterization of other bacterial MurB enzymes, the following optimized assay conditions are recommended:

• Buffer composition:

  • 50 mM Tris-HCl, pH 7.5-8.0

  • 25-50 mM KCl (univalent cations activate MurB as observed with S. pneumoniae MurB)

  • 0.5 mM DTT (to maintain reduced cysteine residues)

• Substrate parameters:

  • NADPH: 50-200 μM

  • EP-UNAG: 10-500 μM (test for substrate inhibition effects, which have been documented for S. pneumoniae MurB)

• Reaction monitoring:

  • Primary assay: Spectrophotometric tracking of NADPH oxidation at 340 nm

  • Secondary validation: HPLC or LC-MS detection of UDP-MurNAc formation

• Control reactions:

  • Heat-inactivated enzyme

  • Substrate omission controls

  • Known MurB inhibitors (3,5-dioxopyrazolidines) as positive controls for inhibition

Monitor linearity and establish enzyme concentration ranges where reaction rate is proportional to enzyme concentration.

How should researchers approach sequence analysis and modeling of G. violaceus murB?

Methodological Answer: A comprehensive sequence analysis approach should include:

This approach will provide structural insights prior to experimental structure determination, guiding mutagenesis studies and inhibitor design.

How do 3,5-dioxopyrazolidines interact with murB enzymes, and what structural modifications might enhance specificity for G. violaceus murB?

Methodological Answer: 3,5-dioxopyrazolidines represent a novel class of MurB inhibitors with demonstrated activity against E. coli and S. aureus MurB (IC50 values of 4.1-6.8 μM and 4.3-10.3 μM, respectively) . To investigate their interactions with G. violaceus MurB and develop improved inhibitors:

• Structural analysis of binding mode:

  • Obtain co-crystal structures of G. violaceus MurB with 3,5-dioxopyrazolidines

  • Compare with the 2.4 Å resolution crystal structure of E. coli MurB complexed with compound 4 (a C-4-unsubstituted 1,2-bis(3,4-dichlorophenyl)-3,5-dioxopyrazolidine)

  • Analyze specific interactions with active-site residues and the bound FAD cofactor

• Structure-activity relationship studies:

  • Synthesize analogs with modifications at positions showing species-specific interactions

  • Focus on 1,2-bis(4-chlorophenyl)-3,5-dioxopyrazolidine-4-carboxamide scaffold (compounds 1-3)

  • Evaluate impact of modifications on inhibition of G. violaceus MurB versus other bacterial MurB enzymes

• Binding affinity determination:

  • Use fluorescence-binding assays as employed for E. coli MurB (which gave a dissociation constant of 260 nM for compound 3)

  • Perform isothermal titration calorimetry to determine thermodynamic parameters

  • Monitor changes in FAD fluorescence upon inhibitor binding

This systematic approach will facilitate the development of selective inhibitors targeting G. violaceus MurB.

How can researchers address data discrepancies in kinetic characterization of G. violaceus murB?

Methodological Answer: When confronting inconsistent kinetic data for G. violaceus MurB, implement this methodological troubleshooting framework:

• Systematic analysis of experimental variables:

  • Enzyme preparation consistency: Standardize protein quantification methods, verify FAD content spectrophotometrically, and assess batch-to-batch variation in specific activity

  • Substrate quality: Analyze substrate purity by HPLC/LC-MS, prepare fresh NADPH solutions daily, and establish consistent substrate handling protocols

  • Assay conditions: Document complete buffer compositions, control temperature precisely (±0.5°C), and standardize order of reagent addition

• Advanced analytical techniques:

  • Global data fitting: Simultaneously fit multiple datasets using shared parameters

  • Statistical validation: Apply Akaike Information Criterion (AIC) to discriminate between kinetic models

  • Alternate assay formats: Validate primary assay results using orthogonal methods

• Data reconciliation strategy:

• Reporting recommendations:

  • Include raw data and detailed experimental conditions

  • Specify data fitting methods and software

  • Report complete parameter sets with confidence intervals

This systematic approach will help resolve discrepancies and establish reliable kinetic parameters for G. violaceus MurB.

What crystallization strategies are most effective for structure determination of G. violaceus murB?

Methodological Answer: Based on successful crystallization of other bacterial MurB enzymes, implement this comprehensive crystallization strategy:

• Protein sample optimization:

  • Achieve high purity (>95% by SDS-PAGE) through additional purification steps

  • Verify monodispersity by dynamic light scattering

  • Prepare protein with bound FAD cofactor (identifiable by characteristic absorption at ~460 nm)

  • Test both tag-free and His-tagged constructs

  • Concentrate to 5-15 mg/mL in optimized buffer

• Initial screening approach:

  • Deploy sparse matrix screens at multiple temperatures (4°C, 18°C, and 25°C)

  • Test vapor diffusion methods (sitting and hanging drop) with varying protein:precipitant ratios

  • Screen with ligands: substrate analogs, NADPH, 3,5-dioxopyrazolidines

  • Include additives known to promote crystallization of flavoproteins

• Optimization matrix for promising conditions:

• Advanced techniques for challenging cases:

  • Surface entropy reduction mutagenesis of surface lysine clusters

  • In situ proteolysis with trace amounts of trypsin or chymotrypsin

  • Crystallization chaperones (Fab fragments, nanobodies)

  • Lipidic cubic phase methods if membrane association is observed

• Crystal handling and data collection:

  • Optimize cryoprotectant conditions (glycerol, ethylene glycol, or sugars)

  • Test multiple crystals for diffraction quality

  • Consider micro-focus beamlines for small crystals

This systematic approach maximizes the probability of obtaining diffraction-quality crystals for structure determination.

How should researchers analyze evolutionary relationships between G. violaceus murB and other bacterial homologs?

Methodological Answer: G. violaceus represents an early-branching cyanobacterium that lacks thylakoid membranes , making its MurB enzyme particularly valuable for evolutionary studies. Implement this comprehensive evolutionary analysis framework:

• Sequence-based phylogenetic analysis:

  • Curate a diverse dataset of bacterial MurB sequences, including representatives across major bacterial phyla

  • Generate multiple sequence alignments using structure-aware methods (PROMALS3D)

  • Construct phylogenetic trees using maximum likelihood (IQ-TREE) and Bayesian inference (MrBayes)

  • Assess node support through ultrafast bootstrap approximation and posterior probabilities

• Structure-based evolutionary analysis:

  • Perform structural alignment of available MurB crystal structures

  • Map sequence conservation onto structural models

  • Identify lineage-specific structural features

  • Analyze domain architecture and insertion/deletion patterns

• Functional divergence investigation:

  • Identify Type I (rate shift) and Type II (property shift) functional divergence using DIVERGE software

  • Correlate divergent sites with substrate binding, catalysis, or regulation

  • Map potentially adaptive sites onto the structural model

• Selection pressure analysis:

  • Apply codon-based likelihood methods (PAML, HyPhy) to detect positive selection

  • Identify episodic selection using branch-site models

  • Correlate selection patterns with ecological niches and cellular architecture

This integrated approach will provide insights into the evolution of peptidoglycan biosynthesis enzymes in relation to G. violaceus's unique evolutionary position among photosynthetic organisms.

What are the recommended protocols for site-directed mutagenesis of conserved residues in G. violaceus murB?

Methodological Answer: Based on structural and functional studies of bacterial MurB enzymes, implement this comprehensive mutagenesis strategy:

• Target residue selection rationale:

  • Catalytic residues corresponding to E. coli MurB Arg159 and Glu325 (involved in transition state stabilization)

  • Diphosphate-binding residues analogous to E. coli Tyr190, Lys217, Asn233, and Glu288

  • FAD-binding motif residues identified through sequence alignment with S. pneumoniae MurB

  • Interface residues potentially involved in oligomerization

• Mutagenesis protocol optimization:

  • QuikChange site-directed mutagenesis for single mutations:

    • Design primers with 25-45 nucleotides with mutation centered

    • Use high-fidelity polymerase (Q5 or Pfu Ultra)

    • Optimize annealing temperature based on primer Tm

    • Perform 16-18 cycles followed by DpnI digestion

  • Gibson Assembly for multiple mutations:

    • Design overlapping fragments incorporating desired mutations

    • Assemble using isothermal assembly mixture

    • Transform into high-efficiency competent cells

• Mutant verification strategy:

  • Complete sequencing of the G. violaceus murB gene

  • Expression testing using SDS-PAGE analysis

  • Spectroscopic verification of FAD incorporation

  • Thermal stability assessment via differential scanning fluorimetry

• Functional characterization framework:

  • Steady-state kinetics (Km, kcat for NADPH and EP-UNAG)

  • Substrate specificity alterations

  • Inhibitor sensitivity profiling using 3,5-dioxopyrazolidines

  • Structural studies when possible (X-ray crystallography)

This systematic approach will elucidate structure-function relationships in G. violaceus MurB and provide insights into species-specific features.

How can researchers optimize activity assays for high-throughput screening of G. violaceus murB inhibitors?

Methodological Answer: To develop a robust high-throughput screening assay for G. violaceus MurB inhibitors, implement this optimization strategy:

• Assay format selection and validation:

  • Primary assay: NADPH fluorescence detection (excitation 340 nm, emission 460 nm)

  • Alternative format: Coupled enzyme assay with diaphorase and resazurin (higher sensitivity)

  • Counter-screen: Direct binding assay using fluorescence polarization with labeled ligands

• Assay optimization parameters:

• Validation compound sets:

  • Positive controls: 3,5-dioxopyrazolidines with known IC50 values (4.1-35 μM against E. coli MurB)

  • Negative controls: Structurally related inactive compounds

  • Reference compounds: Thiazolidinones and imidazolidinones reported as MurB inhibitors

• Data analysis pipeline:

  • Calculate % inhibition relative to controls on each plate

  • Apply plate pattern correction algorithms

  • Set initial hit threshold at >50% inhibition

  • Confirm hits with dose-response curves (8-point, 3-fold dilutions)

  • Counter-screen against related flavoenzymes for selectivity

This optimized approach will enable efficient identification of selective G. violaceus MurB inhibitors while minimizing false positives and negatives.

What methods should be employed to study the effects of pH and temperature on G. violaceus murB activity?

Methodological Answer: To comprehensively characterize the pH and temperature dependence of G. violaceus MurB, implement this systematic approach:

• pH-dependence protocol:

  • Prepare a consistent buffer system across the pH range:

    • Use overlapping buffers: MES (pH 5.5-6.5), MOPS (pH 6.5-7.5), HEPES (pH 7.0-8.0), Tris (pH 7.5-9.0)

    • Maintain consistent ionic strength with NaCl adjustments

    • Include 0.5 mM DTT and univalent cations (K+) which activate MurB

  • Determine full kinetic parameters at each pH:

    • Measure Km and kcat for both NADPH and EP-UNAG

    • Plot log(parameter) vs. pH to identify ionizable groups

    • Fit data to appropriate ionization models to determine pKa values

• Temperature dependence analysis:

  • Kinetic measurements:

    • Determine initial rates at temperatures from 10-45°C

    • Create Arrhenius plots to calculate activation energy

    • Analyze temperature effects on Km and kcat separately

  • Stability assessment:

    • Monitor activity retention after pre-incubation at various temperatures

    • Determine half-life of enzyme at elevated temperatures

    • Correlate with thermal unfolding data from differential scanning fluorimetry

• Combined pH-temperature matrix study:

  • Generate 3D activity profiles across pH-temperature combinations

  • Identify optimal conditions for maximum activity

  • Determine conditions that maximize stability

  • Create stability maps to guide long-term storage conditions

• Comparative analysis with other bacterial MurB enzymes:

  • Compare G. violaceus MurB pH-temperature profiles with E. coli and S. pneumoniae MurB

  • Correlate differences with the ecological niche of G. violaceus

  • Analyze potential adaptations related to the lack of thylakoid membranes

This comprehensive approach will provide insights into the biochemical adaptations of MurB in this evolutionarily distinct cyanobacterium.

How should researchers interpret binding interactions between G. violaceus murB and inhibitors using computational methods?

Methodological Answer: To accurately model and interpret inhibitor binding to G. violaceus MurB, implement this integrated computational workflow:

• Homology model preparation and validation:

  • Generate models based on E. coli MurB crystal structures, particularly those with bound inhibitors

  • Validate model quality through:

    • Ramachandran analysis

    • DOPE score assessment

    • Stereochemical validation

    • Cavity analysis compared to template structures

• Molecular docking protocol:

  • Prepare 3,5-dioxopyrazolidine inhibitor structures :

    • Generate 3D structures with appropriate tautomeric and ionization states

    • Perform conformational analysis to identify low-energy conformers

  • Configure docking settings:

    • Define binding site based on known interactions with FAD cofactor

    • Include key water molecules observed in crystal structures

    • Enable flexibility for binding site residues

    • Use extra precision (XP) scoring functions for final pose evaluation

• Molecular dynamics simulation analysis:

  • System preparation:

    • Embed protein-inhibitor complex in explicit solvent

    • Add physiological ion concentration

    • Apply AMBER or CHARMM force fields

  • Simulation protocol:

    • Equilibration (50-100 ns)

    • Production runs (200-500 ns minimum)

    • Replicate simulations for statistical validity

  • Analysis metrics:

    • Ligand RMSD and binding pose stability

    • Protein-ligand contact persistence

    • Hydrogen bond network analysis

    • Water-mediated interaction patterns

    • Binding free energy calculations (MM-PBSA/MM-GBSA)

• Integration with experimental data:

  • Correlate computational binding energies with experimental IC50 values

  • Guide site-directed mutagenesis based on predicted key interactions

  • Design modified inhibitors targeting unique features of G. violaceus MurB

This systematic computational approach will provide atomic-level insights into inhibitor binding mechanisms and guide rational optimization of selective inhibitors.

What analytical strategies should be employed to compare substrate specificity between G. violaceus murB and other bacterial homologs?

Methodological Answer: To comprehensively characterize and compare substrate specificity profiles, implement this integrated analytical framework:

• Kinetic parameter determination:

  • Measure complete kinetic parameters for natural substrates:

    • Determine Km, kcat, and kcat/Km for EP-UNAG and NADPH

    • Analyze substrate inhibition patterns observed with other MurB enzymes

    • Quantify effects of univalent cations on activity

  • Establish comparative kinetic profiles:

*Values to be determined experimentally

• Substrate analog profiling:

  • Synthesize and test substrate analogs with systematic modifications:

    • EP-UNAG analogs with modified sugar moieties

    • Nucleotide modifications in the UDP portion

    • Alternative nicotinamide cofactors (NADH vs. NADPH)

  • Analyze structure-activity relationships

  • Generate specificity fingerprints for each enzyme variant

• Pre-steady state kinetic analysis:

  • Employ stopped-flow techniques to resolve:

    • Order of substrate binding

    • Rate-limiting steps

    • Formation of intermediates

  • Compare transient kinetic parameters between bacterial homologs

• Integration with structural information:

  • Map specificity-determining residues onto structural models

  • Correlate specificity differences with active site architecture

  • Design chimeric enzymes to test hypotheses about specificity determinants

This comprehensive analytical approach will provide mechanistic insights into the substrate recognition properties of G. violaceus MurB and identify evolutionary adaptations in substrate utilization.

How can researchers analyze and interpret potential cofactor modifications in G. violaceus murB?

Methodological Answer: To systematically investigate FAD cofactor modifications and their functional implications in G. violaceus MurB, implement this analytical workflow:

• Spectroscopic characterization of FAD status:

  • UV-visible spectroscopy:

    • Record complete spectra (300-700 nm) of purified enzyme

    • Compare with characteristic flavoprotein absorption patterns (peaks at ~370 and ~450 nm)

    • Analyze peak ratios (A274/A450) to assess FAD occupancy

    • Monitor spectral changes upon reduction with NADPH

  • Fluorescence spectroscopy:

    • Measure FAD fluorescence (excitation ~450 nm, emission ~520 nm)

    • Assess quenching upon substrate binding

    • Compare quantum yield with free FAD

• Chemical analysis of FAD modifications:

  • Cofactor extraction protocol:

    • Heat treatment (95°C, 5 min) or acid precipitation

    • Ultrafiltration to separate protein and FAD

    • HPLC analysis of extracted cofactor

  • Mass spectrometry characterization:

    • LC-MS/MS analysis to detect covalent modifications

    • Compare observed mass with theoretical FAD mass

    • Fragmentation analysis to locate modification sites

• Redox properties assessment:

  • Determine redox potential:

    • Use xanthine/xanthine oxidase system with redox indicators

    • Calculate midpoint potentials for FAD/FADH2 couple

    • Compare with standard FAD and other bacterial MurB enzymes

  • Analyze reaction with oxygen:

    • Monitor oxygen consumption rates

    • Quantify hydrogen peroxide formation

    • Assess stability of reduced enzyme

• Functional implications analysis:

  • Correlate cofactor properties with catalytic parameters

  • Compare with FAD properties in E. coli and S. pneumoniae MurB

  • Analyze potential adaptations related to G. violaceus's unique cellular architecture lacking thylakoid membranes

This comprehensive analytical approach will provide insights into potential unique features of the FAD cofactor in G. violaceus MurB and their functional significance.