Recombinant Chromobacterium violaceum UDP-N-acetylmuramoylalanine--D-glutamate ligase (murD)

What is Chromobacterium violaceum MurD and what role does it play in bacterial cell wall synthesis?

MurD from Chromobacterium violaceum is a cytoplasmic enzyme involved in the biosynthesis of peptidoglycan, the essential structural component of bacterial cell walls. Specifically, MurD catalyzes the ATP-dependent addition of D-glutamate to UDP-N-acetylmuramoyl-L-alanine (UMA), which represents a critical step in the intracellular phase of bacterial cell wall formation .

The reaction catalyzed by MurD is part of a sequential pathway involving four Mur ligases (MurC, MurD, MurE, and MurF) that collectively build the peptide stem of bacterial peptidoglycan. In this pathway, MurD specifically catalyzes the second step, introducing D-glutamic acid to the growing peptidoglycan precursor . This addition occurs through a reaction mechanism involving acyl-phosphate and tetrahedral intermediates .

The peptidoglycan layer is essential for bacterial survival as it provides structural integrity and protection against osmotic pressure. Given that MurD catalyzes a reaction involving D-amino acids, which are rare in mammalian cells, it represents a selective target for antibacterial drug development .

What is the three-dimensional structure of MurD from Chromobacterium violaceum?

While the specific structure of Chromobacterium violaceum MurD has not been explicitly described in the provided search results, structural studies of MurD from other bacterial species provide valuable insights. The crystal structure of MurD in complex with its substrate UMA has been solved to 1.9 Å resolution .

The enzyme comprises three domains with topology reminiscent of nucleotide-binding folds:

• The N-terminal domain exhibits a dinucleotide-binding fold known as the Rossmann fold

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

• The C-terminal domain also features a Rossmann fold structure

This three-domain architecture creates a dynamic enzyme that undergoes significant conformational changes during catalysis. Molecular dynamics studies and targeted molecular dynamic (TMD) simulations have demonstrated that the C-terminal domain exhibits substantial motion during the enzymatic cycle, with the enzyme transitioning between open and closed conformations .

How does Chromobacterium violaceum MurD compare to MurD enzymes from other bacterial species?

Significant variations exist among MurD enzymes from different bacterial species, particularly in their amino acid sequences and the topologies of their active sites . These differences impact inhibitor efficacy, as compounds that effectively inhibit MurD from one bacterial species may show reduced activity against orthologs from other species.

In comparative studies, MurD inhibitors designed for Escherichia coli MurD showed variable efficacy against MurD enzymes from Staphylococcus aureus, Streptococcus pneumoniae, Borrelia burgdorferi, and Mycobacterium tuberculosis. Most E. coli MurD inhibitors demonstrated reduced effectiveness against these other bacterial orthologs .

This divergence in inhibitor efficacy can be directly attributed to the differences in amino acid sequences and active site topologies among MurD orthologs. These structural variations must be considered when developing broad-spectrum MurD inhibitors or species-specific antimicrobial agents .

What are the known substrates and cofactors required for Chromobacterium violaceum MurD activity?

Chromobacterium violaceum MurD requires several specific substrates and cofactors for its enzymatic activity:

• Primary substrates:

  • UDP-N-acetylmuramoyl-L-alanine (UMA) - The nucleotide precursor that serves as the acceptor substrate

  • D-glutamate - The amino acid added to UMA in the reaction catalyzed by MurD

• Essential cofactors:

  • ATP - Required for the activation of the carboxyl group of UMA through phosphorylation

  • Mg²⁺ - Necessary for ATP binding and catalysis

The binding site for UMA has been identified through structural studies, and comparison with known nucleoside triphosphate complexes has allowed researchers to identify residues that interact with ATP . This detailed knowledge of substrate and cofactor interactions provides valuable insights for the development of competitive inhibitors targeting the active site of MurD.

What approaches have proven successful for expressing and purifying recombinant Chromobacterium violaceum MurD?

While the search results don't specifically detail expression and purification methods for Chromobacterium violaceum MurD, successful approaches for recombinant MurD proteins from other bacterial species can be adapted:

Expression systems:

• E. coli expression systems using pET vectors under T7 promoter control have been effective for producing recombinant MurD enzymes

• Optimal expression typically occurs at lower induction temperatures (16-25°C) to enhance protein solubility

• Addition of chaperone co-expression plasmids may improve folding efficiency

Purification strategy:

• Cell lysis using sonication or pressure homogenization in buffer containing 50 mM Tris-HCl pH 7.5-8.0, 300 mM NaCl, and 10% glycerol

• Initial purification by immobilized metal affinity chromatography (IMAC) using an N-terminal or C-terminal His-tag

• Secondary purification by ion exchange chromatography

• Final polishing by size exclusion chromatography

• Storage in buffer containing 20-25 mM HEPES pH 7.5, 100-150 mM NaCl, and 10% glycerol at -80°C

For enzyme activity studies, ensuring the presence of appropriate cofactors (ATP and Mg²⁺) and substrates (UMA and D-glutamate) is essential for functional analysis .

How do structural dynamics influence the enzymatic activity of Chromobacterium violaceum MurD?

Structural dynamics play a crucial role in MurD enzymatic activity. Based on computational studies of MurD enzymes:

Large-scale conformational changes occur during the catalytic cycle, particularly involving the C-terminal domain. These conformational changes have been investigated using Targeted Molecular Dynamic (TMD) simulation and the Off-Path Simulation (OPS) technique .

Key findings regarding MurD structural dynamics include:

• The N-terminal and central MurD domains are energetically most favorable and more accessible to the enzyme .

• The Off-Path Simulation (OPS) 1EEH–2UAG analysis suggests that all conformational states on the closing pathway are readily accessible to the MurD enzyme, indicating an equilibrium of available protein structures between the free unbound and closed conformation .

• The closed conformation of the MurD enzyme corresponds to the lowest energy conformation on the investigated OPS energy landscape .

The energy required for conformational changes varies between different replica systems. The 1E0D–2UAG replica system requires minimal energy for large-scale conformational change. These findings suggest that MurD exists in a dynamic equilibrium between open and closed states, with the energy barriers between these states influencing catalytic efficiency .

What current strategies exist for designing inhibitors targeting Chromobacterium violaceum MurD?

Current strategies for designing MurD inhibitors employ multiple approaches:

Structure-based design strategies:

• Multiple protein conformation analysis: Using various MurD protein conformations selected based on computational data to account for structural flexibility. This approach combines structure-based pharmacophores with molecular docking calculations .

• Hot spot identification: Using GRID map calculations to identify energetically favorable binding regions within the protein structure .

• ATP-binding site targeting: Many inhibitor design efforts focus on the ATP-binding site of the MurD enzyme. This approach has yielded compounds like aminothiazole (compound 3), which has been discovered to act as a dual MurC/MurD inhibitor in the micromolar range .

Computational approaches:

• Virtual screening: Two-stage virtual screening approaches combining derived structure-based pharmacophores with molecular docking calculations .

• Molecular dynamics simulations: Performing MD simulations of potential inhibitors in complex with selected MurD conformations to understand binding stability and provide rationale for experimental results .

Kinetic analysis:
Performing steady-state kinetic studies on the MurD enzyme to provide information about the mechanistic aspects of inhibition, which guides further inhibitor optimization .

One notable challenge in MurD inhibitor design is the structural differences between enzymes from different bacterial species. Inhibitors designed for E. coli MurD often show reduced effectiveness against other bacterial orthologs, highlighting the need for species-specific design considerations .

How does violacein production in Chromobacterium violaceum influence MurD activity and cell wall synthesis?

The relationship between violacein production and MurD activity in Chromobacterium violaceum involves complex regulatory networks:

Violacein is a purple pigment expressed by C. violaceum that has created significant research interest due to its virulence properties and antibiotic-inhibiting characteristics . The regulatory mechanisms controlling violacein production and cell wall synthesis in C. violaceum include:

• Quorum sensing regulation: Violacein expression is under control of the CviI/CviR quorum sensing system and is negatively regulated by VioS, an otherwise uncharacterized regulator . This system allows population-dependent regulation of violacein production.

• VioS repressor function: VioS acts as a novel repressor protein that negatively controls violacein biosynthesis. The violacein operon is regulated negatively by VioS and positively by the CviI/R system .

• Antibiotic-induced response: Sublethal doses of translation-inhibiting antibiotics induce violacein production in C. violaceum ATCC31532. This response involves the antibiotic-induced response (air) two-component regulatory system .

While direct interactions between violacein production and MurD activity are not explicitly described in the search results, both processes are linked through bacterial cell wall metabolism. As a cytoplasmic enzyme involved in peptidoglycan biosynthesis, MurD contributes to cell wall integrity, while violacein production influences antibiotic resistance properties.

The regulatory networks controlling these processes may allow C. violaceum to coordinate cell wall synthesis and secondary metabolite production in response to environmental stressors, including the presence of antibiotics .

What assay methods are most reliable for measuring Chromobacterium violaceum MurD activity?

Several reliable assay methods can be employed to measure MurD enzymatic activity:

1. Coupled enzyme assays:

• Coupling MurD activity to ADP production using pyruvate kinase and lactate dehydrogenase

• Monitoring NADH oxidation at 340 nm as a readout of ATP consumption

• This method provides continuous monitoring of enzyme activity

2. HPLC-based assays:

• Separation and quantification of substrate (UDP-MurNAc-L-Ala) and product (UDP-MurNAc-L-Ala-D-Glu)

• Detection at 268 nm (absorption maximum for uridine)

• Allows direct product quantification but requires sample processing

3. Radiometric assays:

• Using ¹⁴C-labeled D-glutamate to measure incorporation into the peptidoglycan precursor

• Separation of labeled product from unreacted substrate by paper chromatography or ion-exchange techniques

• High sensitivity but requires radioactive materials handling

4. Malachite green-based assays:

• Measuring inorganic phosphate release from ATP hydrolysis

• Colorimetric detection at 650 nm

• Simple endpoint assay but may be subject to interference

For inhibitor testing, steady-state kinetic studies have been performed on MurD enzymes to provide information about the mechanistic aspects of inhibition . These studies typically involve varying substrate concentrations in the presence of fixed inhibitor concentrations to determine inhibition mechanisms (competitive, uncompetitive, or noncompetitive) and kinetic parameters.

What computational approaches can be used to identify potential inhibitors of Chromobacterium violaceum MurD?

Multiple computational approaches have proven effective for identifying potential MurD inhibitors:

1. Structure-based virtual screening:

• Using multiple protein conformations to account for enzyme structural flexibility

• Combining structure-based pharmacophores with molecular docking calculations

• This two-stage approach has been utilized successfully to identify novel MurD ligase inhibitors

2. Molecular dynamics (MD) simulations:

• Simulating potential inhibitors in complex with selected MurD conformations

• Using the CHARMM molecular modeling suite and CHARMM-GUI environment for protein manipulation and system construction

• Performing production phase MD simulations (typically 10 ns or longer) to observe dynamical behavior of protein-ligand systems

3. Targeted Molecular Dynamic (TMD) and Off-Path Simulation (OPS):

• Investigating the C-terminal domain motion of the MurD ligase

• Selecting MurD protein structures based on TMD/OPS data as initial criteria for inhibitor design

• Using energy landscape analysis to identify accessible conformational states

4. GRID map calculations:

• Identifying hot spots (energetically favorable binding regions) within the protein structure

• Focusing particularly on the ATP-binding site of the MurD enzyme

These computational approaches can be combined in a comprehensive workflow:

• Select multiple MurD conformations based on dynamics studies

• Identify hot spots using GRID calculations

• Develop structure-based pharmacophores

• Perform virtual screening with molecular docking

• Validate promising compounds through MD simulations

• Test selected compounds in enzyme binding assays

This integrated computational strategy has successfully identified compounds like aminothiazole (compound 3), which acts as a dual MurC/MurD inhibitor .

What expression systems yield the highest activity for recombinant Chromobacterium violaceum MurD?

Based on general practices for producing recombinant bacterial enzymes, the following expression systems are recommended for obtaining high-activity Chromobacterium violaceum MurD:

Bacterial expression systems:

• E. coli BL21(DE3) or BL21(DE3)pLysS strains with T7 RNA polymerase-based vectors (pET series)

• Arctic Express or Rosetta-gami strains to enhance proper folding of proteins with complex structures

• Auto-induction media to achieve high cell density without monitoring growth and manual IPTG addition

Expression conditions optimization:

• Lower induction temperatures (16-20°C) to minimize inclusion body formation

• Extended expression times (18-24 hours) at reduced temperatures

• Reduced IPTG concentration (0.1-0.5 mM) to slow expression rate

Fusion tag selection:

• N-terminal His₆-tag for convenient purification

• MBP (maltose-binding protein) fusion for enhanced solubility

• SUMO fusion system for native N-terminus after tag removal

Buffer composition:

• Addition of glycerol (5-10%) to stabilize protein structure

• Inclusion of reducing agents (DTT or β-mercaptoethanol) to prevent oxidation

• Appropriate cofactors (Mg²⁺) to maintain enzyme in active conformation

When expressing recombinant MurD, it's important to consider that the activity may be influenced by the folding environment. The enzyme structure comprises three domains with nucleotide-binding folds , which may require specific conditions for proper folding and domain arrangement.

How can site-directed mutagenesis be used to explore the functional domains of Chromobacterium violaceum MurD?

Site-directed mutagenesis provides a powerful approach to investigate structure-function relationships in MurD:

Key residues for targeted mutagenesis:

• ATP-binding site residues - Identified through structural studies and comparison with known NTP complexes

• UMA-binding site residues - Determined through co-crystallization studies with the substrate UMA

• Catalytic residues - Involved in phosphoryl transfer and substrate activation

• Domain interface residues - Mediating interdomain communications during conformational changes

Mutagenesis strategy:

• Alanine scanning - Systematic replacement of key residues with alanine to assess their contribution to catalysis or binding

• Conservative substitutions - Replacing residues with chemically similar amino acids to probe specific interactions

• Non-conservative substitutions - Introducing charged or bulky residues to disrupt specific interactions

• Domain swapping - Exchanging domains between MurD enzymes from different species to investigate specificity determinants

Functional analysis:

• Enzymatic activity assays comparing wild-type and mutant enzymes

• Substrate binding studies using isothermal titration calorimetry or surface plasmon resonance

• Thermal stability analysis using differential scanning fluorimetry

• Crystallization of mutant proteins to determine structural changes

Expected insights:

• Identification of residues critical for catalysis versus substrate binding

• Understanding of domain motion requirements for enzymatic activity

• Elucidation of species-specific differences in substrate recognition

• Identification of allosteric sites that could be targeted for inhibitor design