Recombinant Desulfovibrio vulgaris D-alanine--D-alanine ligase (ddl)

What is D-alanine--D-alanine ligase (Ddl) and what is its role in bacterial peptidoglycan synthesis?

D-alanine--D-alanine ligase (Ddl) catalyzes the ATP-dependent formation of the D-alanyl-D-alanine dipeptide, which is essential for bacterial cell wall biosynthesis. This enzyme belongs to the ATP-grasp superfamily, characterized by an atypical nucleotide-binding site known as the ATP-grasp fold. The dipeptide product serves as a critical building block for peptidoglycan synthesis, contributing to bacterial cell wall integrity and structure .

The catalytic mechanism involves ATP activation, followed by the sequential binding of two D-alanine molecules to form the dipeptide. This process is accompanied by conformational changes in the enzyme structure that facilitate substrate binding and product formation . In many bacteria, this pathway represents a viable target for antimicrobial agents, as disruption of peptidoglycan synthesis compromises cell wall integrity.

What structural features characterize Ddl enzymes?

D-alanine--D-alanine ligase typically exists as a dimer, with each monomer consisting of three distinct domains: an N-terminal domain (approximately residues 1-104), a central domain (residues 105-192), and a C-terminal domain (residues 193-319) . Each subunit contains a single ATP-binding site formed by the ATP-grasp fold and two D-alanine binding sites strategically positioned to facilitate dipeptide formation .

The first D-alanine binding site typically exhibits higher affinity for the substrate compared to the second site, which influences the sequential binding mechanism. The enzyme undergoes significant conformational changes during catalysis, which are essential for proper substrate positioning and reaction progression .

How does Ddl function relate to vancomycin sensitivity in bacteria?

The relationship between Ddl function and vancomycin sensitivity is determined by the type of peptidoglycan termini generated by the enzyme. Dipeptide ligase activity produces D-alanyl-D-alanine (D-Ala-D-Ala) termini in peptidoglycan, which serve as high-affinity binding sites for vancomycin, resulting in bacterial sensitivity to this antibiotic . In contrast, depsipeptide ligase activity generates D-alanyl-D-lactate (D-Ala-D-Lac) termini, which have low-affinity binding for vancomycin, conferring resistance .

Importantly, a single amino acid in the Ddl active site can determine whether the enzyme functions as a dipeptide or depsipeptide ligase. For instance, phenylalanine at this position typically confers depsipeptide ligase activity, while tyrosine promotes dipeptide ligase activity . This structural distinction has significant implications for predicting and potentially modifying vancomycin sensitivity in various bacterial species.

What approaches are effective for expressing recombinant Desulfovibrio vulgaris Ddl?

For successful expression of recombinant Desulfovibrio vulgaris Ddl, researchers should consider several methodological approaches:

• Expression system selection: While E. coli remains the most common heterologous expression host, the anaerobic nature of Desulfovibrio vulgaris may necessitate special considerations regarding protein folding and post-translational modifications. Systems such as BL21(DE3) with additional plasmids encoding rare codons may improve expression levels.

• Fusion tag optimization: Affinity tags such as His6, GST, or MBP can facilitate purification while potentially enhancing solubility. C-terminal His-tagging often preserves enzymatic activity better than N-terminal tagging for many ATP-grasp fold enzymes.

• Culture conditions: Since Ddl requires proper folding for activity, expression at lower temperatures (16-20°C) following induction may improve the yield of active enzyme. Additionally, considering the anaerobic native environment of Desulfovibrio vulgaris, oxygen-limited growth conditions might improve proper folding.

• Solubility enhancement: Co-expression with chaperone proteins or addition of osmolytes like sorbitol or betaine to the culture medium can improve solubility of recombinant Ddl.

For purification, a combination of affinity chromatography followed by size exclusion chromatography typically yields purified enzyme suitable for structural and functional studies.

How can site-directed mutagenesis be used to modify Ddl substrate specificity?

Site-directed mutagenesis represents a powerful approach for investigating and modifying the substrate specificity of Desulfovibrio vulgaris Ddl. Based on studies of Ddl from other organisms, researchers should consider the following strategic approach:

• Target active site residues known to influence specificity, particularly focusing on residues that interact with the substrate. The phenylalanine/tyrosine switch position (analogous to F261 in Leuconostoc mesenteroides or F258 in Lactobacillus reuteri) represents a primary target, as this residue determines dipeptide versus depsipeptide ligase activity .

• Implement a PCR-based mutagenesis protocol using complementary primers containing the desired mutation. For challenging GC-rich regions often found in Desulfovibrio species, the addition of DMSO or specialized polymerases may improve amplification efficiency.

• Following mutagenesis, express and purify the variant enzymes using standardized protocols, then characterize substrate specificity through:

  • Kinetic analysis comparing utilization of D-alanine versus D-lactate

  • Structural analysis using X-ray crystallography or cryo-EM to visualize substrate binding

  • Product analysis using mass spectrometry to confirm dipeptide or depsipeptide formation

Previous studies with Lactobacillus reuteri demonstrated that a single F258Y substitution was sufficient to convert the enzyme from a depsipeptide to a dipeptide ligase, fundamentally altering its function and the resulting vancomycin sensitivity of the bacterium .

How can metabolomics approaches be used to study Ddl function in vivo?

Metabolomics offers powerful tools for investigating Ddl function in living bacterial cells. The following methodological approach can be implemented:

• Employ validated filter-based techniques coupled to high-resolution mass spectrometry to monitor changes in intracellular metabolite pools related to the D-alanine pathway in response to Ddl inhibition or gene manipulation .

• Utilize isotope labeling to track metabolic flux through the Ddl pathway. For example, cells can be challenged with D-alanine containing stable isotopes (such as D-Ala-13C-2H), allowing researchers to monitor the appearance of labeled dipeptide products and related metabolites over time .

• Compare differential effects on metabolite pools between specific Ddl inhibitors and inhibitors of other enzymes in the pathway to distinguish primary and secondary effects. This comparative approach enabled researchers to determine that D-cycloserine primarily targets Ddl rather than alanine racemase in some bacterial species .

• Implement time-course experiments to capture the dynamic response of the metabolome to Ddl inhibition, revealing both immediate effects and compensatory mechanisms that may develop over time.

This approach successfully demonstrated that D-cycloserine treatment leads to rapid and dose-dependent depletion of the D-Ala:D-Ala dipeptide pool, supporting its mechanism of action through inhibition of Ddl rather than other potential targets .

How can Ddl be utilized as a counterselection marker in genetic engineering?

D-alanine--D-alanine ligase can be strategically employed as a counterselection marker for genetic engineering of bacteria with intrinsic vancomycin resistance. The methodology involves exploiting the relationship between Ddl activity and vancomycin sensitivity:

• Construct a suicide vector containing a dipeptide ligase gene (such as ddlF258Y) that produces D-Ala-D-Ala termini in peptidoglycan, increasing sensitivity to vancomycin in otherwise resistant bacteria .

• Introduce this vector into the target bacterium, where it will integrate into the chromosome through homologous recombination, simultaneously introducing the dipeptide ligase gene.

• The integrated vector will express the dipeptide ligase, rendering the bacterium sensitive to vancomycin. When grown in the presence of vancomycin, only cells that have lost the vector through a second recombination event (potentially resulting in the desired genetic modification) will survive .

• Implement a liquid-based approach to identify recombinants, which can reduce the time required to identify successful transformants to approximately 5 days, roughly half the time required by conventional approaches .

This system has been demonstrated to be effective in vancomycin-resistant lactobacilli and potentially has broad applications across bacterial species with similar resistance profiles. Importantly, this represents a "plug and play" counterselection system that does not require prior genome editing or synthetic medium preparation .

How does Ddl from different bacterial species compare structurally and functionally?

The structural and functional characteristics of D-alanine--D-alanine ligase vary across bacterial species, with important implications for antibiotic sensitivity and potential applications in genetic engineering.

This comparative analysis highlights the critical role of a single amino acid residue in determining enzyme function and subsequent antibiotic sensitivity. Phylogenetic analysis suggests that approximately 81% of Lactobacillus species are intrinsically resistant to vancomycin, indicating they likely possess a Ddl enzyme with depsipeptide ligase activity .

What insights does structural biology provide about Ddl catalytic mechanism?

Structural biology studies reveal that D-alanine--D-alanine ligase undergoes significant conformational changes during catalysis. The enzyme typically forms a dimer, with each monomer containing three domains and a single ATP-binding site formed by the ATP-grasp fold .

The ATP and two D-alanine substrates bind in adjacent sites at the center of the Ddl monomer to facilitate dipeptide formation. The first D-alanine binding site typically exhibits higher affinity for the substrate than the second site, influencing the sequential binding mechanism .

The catalytic mechanism involves:

• ATP binding and activation

• Binding of the first D-alanine substrate at the high-affinity site

• Formation of an acylphosphate intermediate

• Binding of the second D-alanine at the lower-affinity site

• Nucleophilic attack by the amino group of the second D-alanine on the acylphosphate intermediate

• Formation of the dipeptide product and release

Each step is accompanied by conformational changes that optimize substrate positioning and facilitate the reaction progression .

What are optimal conditions for assaying Ddl activity?

For accurate assessment of D-alanine--D-alanine ligase activity from Desulfovibrio vulgaris, researchers should implement the following methodological approach:

• Enzyme preparation: Purified recombinant enzyme should be maintained in a buffer containing:

  • 50 mM Tris-HCl (pH 7.5-8.0)

  • 10 mM MgCl₂ (essential for ATP binding)

  • 1-5 mM DTT or β-mercaptoethanol (to maintain reduced cysteines)

  • 10% glycerol (for stability)

• Reaction conditions: Standard assay conditions should include:

  • 50 mM Tris-HCl (pH 7.5-8.0)

  • 10 mM MgCl₂

  • 10 mM KCl (as a monovalent cation activator)

  • 5 mM ATP

  • 10-50 mM D-alanine

  • Purified enzyme (0.1-1 μg)

  • Incubation at 30-37°C (temperature optimization may be necessary)

• Activity detection methods:

  • Coupled enzyme assay monitoring ADP formation using pyruvate kinase and lactate dehydrogenase with spectrophotometric detection of NADH oxidation

  • Direct detection of dipeptide formation using HPLC or mass spectrometry

  • Radioactive assay using ¹⁴C-labeled D-alanine

• Kinetic analysis:

  • Determine Km values for both D-alanine binding sites

  • Measure kcat and catalytic efficiency

  • Assess the effects of potential inhibitors on enzyme activity

When studying Ddl from anaerobic organisms like Desulfovibrio vulgaris, consider performing assays under anaerobic conditions or including reducing agents to maintain enzyme activity.

How can isotope labeling track D-alanine metabolism and Ddl activity?

Isotope labeling provides powerful insights into D-alanine metabolism and Ddl activity in living bacterial cells. The following methodological approach can be implemented:

• Stable isotope preparation: Use commercially available or synthetically prepared isotope-labeled D-alanine, such as:

  • D-Ala-¹³C for carbon tracing

  • D-Ala-²H for tracking racemization through proton exchange

  • D-Ala-¹⁵N for nitrogen incorporation studies

• Experimental design: Challenge bacterial cells with labeled D-alanine under various conditions:

  • Add 1 mM labeled D-alanine to bacterial cultures

  • Include potential inhibitors or genetic modifications to assess their impact

  • Collect samples at various time points for metabolite extraction

• Sample preparation and analysis:

  • Extract intracellular metabolites using a validated filter-based technique

  • Analyze using high-resolution mass spectrometry to detect isotope-labeled compounds

  • Track the appearance of labeled D-Ala:D-Ala dipeptide and related metabolites

• Data interpretation:

  • Measure the rate of incorporation of labeled alanine into the dipeptide pool

  • Assess the impact of inhibitors on dipeptide formation

  • Track racemization by observing disappearance of α-²H isotopologue over time

This approach has been successfully employed to demonstrate that D-cycloserine inhibits Ddl at lower concentrations than it inhibits alanine racemase, by showing that while some racemization of L-Ala-¹³C-²H to D-Ala continued in the presence of D-cycloserine at 1× MIC, there was a complete absence of newly formed labeled D-Ala:D-Ala dipeptide under the same conditions .

How can discrepancies between in vitro and in vivo Ddl inhibition studies be reconciled?

Reconciling discrepancies between in vitro and in vivo inhibition studies of D-alanine--D-alanine ligase requires a multifaceted approach addressing several potential sources of variation:

• Implement metabolomics approaches to track the effects of inhibitors on intracellular metabolite pools, particularly focusing on D-alanine and D-Ala:D-Ala levels. This can reveal whether observed inhibition in vitro translates to expected metabolic changes in vivo .

• Consider the impact of cellular permeability, efflux mechanisms, and potential metabolic conversion of inhibitors, which may affect effective inhibitor concentrations at the target site in vivo but not in isolated enzyme studies.

• Account for potential compensatory mechanisms in living cells, such as upregulation of Ddl expression or activation of alternative pathways that may partially overcome inhibition.

• Validate the primary target of inhibitors using genetic approaches, such as creating resistant mutants and identifying the genetic basis of resistance, which can confirm whether observed effects are primarily due to Ddl inhibition.

• Design time-course experiments to capture the dynamic response to inhibition, as the immediate effects may differ from long-term adaptations in living cells.

This integrated approach successfully resolved discrepancies in the mechanism of action of D-cycloserine, demonstrating through metabolite pool analysis that while this compound inhibits both alanine racemase and D-alanine--D-alanine ligase, the primary lethal effect at clinically relevant concentrations is through Ddl inhibition .