Recombinant Gloeobacter violaceus D-alanine--D-alanine ligase (ddl)

What is Gloeobacter violaceus and why is it significant for evolutionary studies?

Gloeobacter violaceus represents one of the earliest branching lineages of cyanobacteria and exhibits several ancestral traits that make it particularly valuable for evolutionary studies. Most notably, G. violaceus lacks thylakoid membranes, which are present in virtually all other photosynthetic cyanobacteria . This unique characteristic suggests that G. violaceus diverged before the evolution of thylakoids in the cyanobacterial lineage.

Recent genomic studies have identified sister taxa to Gloeobacter, including Aurora vandensis, which together form a clade that is the closest cyanobacterial relative to the non-photosynthetic Vampirovibrionia (formerly Melainabacteria) . The unique phylogenetic position of Gloeobacter makes its proteins, including Ddl, particularly interesting for comparative biochemical studies aimed at understanding protein evolution in photosynthetic organisms.

What is D-alanine--D-alanine ligase (Ddl) and what is its function in bacterial cells?

D-alanine--D-alanine ligase (Ddl) is an ATP-dependent enzyme that catalyzes the formation of the D-alanyl-D-alanine dipeptide, which is an essential component of bacterial peptidoglycan . The peptidoglycan layer provides structural integrity to bacterial cell walls and is crucial for bacterial survival.

In the bacterial cell wall biosynthesis pathway, Ddl catalyzes the ATP-dependent ligation of two D-alanine molecules to form the terminal D-Ala-D-Ala dipeptide of the peptidoglycan precursor UDPMurNAc-pentapeptide . This reaction occurs in a sequential manner: first, ATP binds to the free enzyme, followed by the first D-alanine (D-Ala 1). The subsequent phosphorylation of the amino acid carboxylate by the γ-phosphate of ATP generates an acyl phosphate intermediate, which is then attacked by the amino group of the second D-alanine (D-Ala 2) to yield the D-Ala-D-Ala dipeptide . The formation of this dipeptide is a critical step in peptidoglycan synthesis, making Ddl an attractive target for antimicrobial drug development.

How does the structure of Ddl relate to its function?

The structure of D-alanine--D-alanine ligase directly supports its catalytic function through a specialized domain organization. Based on studies of Thermus thermophilus Ddl (TtDdl), which serves as a model system, Ddl consists of three primary domains: an N-terminal domain (Met 1–Gly 104), a central domain (Ala 105–Leu 192), and a C-terminal domain (Ser 193–Thr 319) . These domains work together to create the enzyme's active site.

Functionally, Ddl requires dimerization for enzymatic activity, with each subunit containing an ATP-binding site formed by the ATP-grasp fold, and two D-Ala-binding sites positioned adjacently at the center of the monomer . This arrangement facilitates the sequential binding of substrates and the formation of the dipeptide product.

The catalytic mechanism involves cumulative conformational changes induced by substrate binding. Studies have shown that the first D-Ala site demonstrates higher affinity for D-Ala than the second site , which aligns with the sequential nature of the reaction. This structural arrangement is critical for the proper positioning of substrates to facilitate the ATP-dependent formation of the D-alanyl-D-alanine dipeptide.

How does the Ddl enzyme from Gloeobacter violaceus differ from those of other bacterial species?

While the search results do not provide specific information about the unique features of Gloeobacter violaceus Ddl compared to other species, we can infer some distinctions based on the evolutionary position of Gloeobacter. As one of the earliest diverging lineages of cyanobacteria, G. violaceus likely possesses ancestral forms of many proteins, including Ddl .

Comparative studies with Ddl enzymes from other bacterial species, such as Thermus thermophilus, would be valuable for identifying specific structural and functional differences. These differences might include variations in the ATP-binding domain, substrate specificity, monovalent cation dependency, or regulatory mechanisms. The truncated Vipp homologue found in Gloeobacter violaceus, which lacks the approximately 30 amino-acid extension found in other oxygenic photosynthetic organisms , suggests that other proteins in G. violaceus, potentially including Ddl, might also exhibit unique structural features reflecting their evolutionary position.

What is the role of monovalent cations (particularly K+) in the activation of Ddl enzymes?

Monovalent cations, especially K+, play a crucial role in activating Ddl enzymes. Studies with Thermus thermophilus Ddl (TtDdl) have demonstrated that K+ significantly enhances the catalytic activity of the enzyme . This activation mechanism classifies Ddl as a type II activated enzyme, where the monovalent cation influences enzyme activity through conformational changes in the vicinity of the cation binding site.

The kinetic parameters of TtDdl have been determined in both low-MVC (monovalent cation) buffer and in the presence of varying concentrations of KCl (10, 50, and 100 mM) . The presence of K+ affects both the Km and kcat values for the enzyme, indicating that K+ influences both substrate binding and catalytic efficiency.

Crystallographic studies have captured TtDdl at different stages of the catalytic cycle to investigate K+-induced conformational changes. These structures include apo TtDdl (PDB code 6U1C), TtDdl in complex with ADP, Pi, and K+ (PDB code 6U1H), and TtDdl in complex with ADP, the phosphorylated form of d-cycloserine (DCSP), and K+ (PDB code 6U1I) . These structures provide valuable insights into how K+ influences the conformation and activity of Ddl during different stages of catalysis.

How do the kinetic parameters of Ddl enzymes vary with substrate concentration and environmental conditions?

The kinetic parameters of Ddl enzymes are significantly influenced by substrate concentration and environmental conditions, particularly the presence of monovalent cations. For Thermus thermophilus Ddl (TtDdl), the Km values for ATP and the first and second D-Ala substrates in a low-MVC reaction buffer have been determined to be 16.2 μM, 1250 μM, and 4020 μM, respectively .

When varying concentrations of KCl (10, 50, and 100 mM) were introduced, both Km and kcat values were affected, indicating that K+ influences both substrate binding affinity and catalytic rate . This relationship between monovalent cation concentration and enzyme kinetics is a critical consideration when designing experiments to study Ddl activity.

Table 1: Kinetic Parameters of T. thermophilus Ddl Under Various KCl Concentrations

*Exact kcat value not provided in the search results
†Values change in response to KCl but specific values not provided in the search results

What are the optimal conditions for expressing and purifying recombinant G. violaceus Ddl?

While the search results do not provide specific protocols for expressing and purifying recombinant Gloeobacter violaceus Ddl, general approaches for Ddl enzymes from other bacterial species can be adapted. Based on studies with Thermus thermophilus Ddl, which was used as a model system, the following considerations would be important:

• Expression System: A bacterial expression system such as E. coli with appropriate temperature control would be suitable. Given that G. violaceus is a cyanobacterium, optimization of codon usage may be necessary for efficient expression in E. coli.

• Purification Strategy: A multi-step purification process typically involving affinity chromatography (such as His-tag purification) followed by size exclusion chromatography would be appropriate. The dimeric nature of active Ddl should be considered during purification .

• Buffer Conditions: Buffer composition should account for the requirement of monovalent cations, particularly K+, for optimal enzyme activity. The buffer should also maintain pH stability and provide appropriate ionic strength .

• Stability Considerations: Addition of stabilizing agents such as glycerol or specific ions might be necessary to maintain enzyme stability during purification and storage.

• Quality Control: Assessing enzyme purity through SDS-PAGE and verifying activity through enzymatic assays would be essential steps in the purification process.

What assays are available for measuring Ddl enzyme activity in vitro?

Several assays are available for measuring Ddl enzyme activity in vitro, each with specific advantages depending on the research question:

• Colorimetric Antimony-Phosphomolybdate Assay: This method monitors the production of inorganic phosphate (Pi) from the enzymatic activity of Ddl. It has been used successfully with Thermus thermophilus Ddl to determine kinetic parameters such as kcat and Km for ATP and D-Ala substrates .

• Isotope Labeling Approaches: Using 2H-isotopically labeled alanine allows for tracking the flux of substrate through the enzyme reaction. This approach can be particularly useful for distinguishing between different steps in the reaction mechanism and for studying the effects of inhibitors .

• Coupled Enzyme Assays: These assays link Ddl activity to other enzymatic reactions that produce measurable products, providing a convenient way to monitor Ddl activity in real-time.

• ADP or ATP Detection Assays: Since Ddl consumes ATP and produces ADP during catalysis, assays that directly measure either ATP consumption or ADP production can be used to monitor enzyme activity.

• Mass Spectrometry-Based Assays: These can directly detect and quantify the D-Ala-D-Ala dipeptide product, providing a direct measure of enzyme activity without relying on coupled reactions or secondary detection methods.

How can X-ray crystallography be used to investigate the structural basis of Ddl catalysis?

X-ray crystallography has been instrumental in elucidating the structural basis of Ddl catalysis, particularly for understanding how the enzyme binds substrates and undergoes conformational changes during the catalytic cycle. The approach involves:

• Capturing Different Catalytic States: By crystallizing the enzyme in the presence of various ligands, different stages of the catalytic cycle can be captured. For Thermus thermophilus Ddl, structures have been solved representing at least four different stages: apo enzyme (PDB code 6U1C), enzyme-ADP-Pi-K+ complex (PDB code 6U1H), and enzyme-ADP-DCSP-K+ complex (PDB code 6U1I) .

• Investigating Monovalent Cation Effects: Crystallography has been used to determine how K+ influences Ddl conformation and activity. By solving structures in the presence and absence of K+, the conformational changes induced by this monovalent cation can be visualized .

• Substrate Binding Analysis: Crystal structures with bound substrates or substrate analogues reveal the precise interactions that determine substrate specificity and positioning within the active site. These structures can show how the first and second D-Ala binding sites differ in affinity and arrangement .

• Inhibitor Binding Studies: Structures with bound inhibitors, such as D-cycloserine or phosphinate/phosphonate transition state mimetics, provide insights into inhibition mechanisms and guide the design of new antimicrobial agents .

• Mutation Analysis: Combining site-directed mutagenesis with crystallography allows for the identification of key residues involved in catalysis, substrate binding, or structural integrity. For instance, a single amino acid in the Ddl active site (phenylalanine or tyrosine) determines whether the enzyme produces a depsipeptide or dipeptide .

How might the unique evolutionary position of Gloeobacter violaceus influence the properties of its Ddl enzyme?

The unique evolutionary position of Gloeobacter violaceus as one of the earliest diverging cyanobacterial lineages likely has profound implications for the properties of its Ddl enzyme. G. violaceus exhibits several ancestral traits, most notably the absence of thylakoid membranes, which are present in virtually all other photosynthetic cyanobacteria . This suggests that G. violaceus Ddl might retain ancestral features that could provide insights into the evolutionary trajectory of this enzyme family.

Comparative genomic analyses have positioned Gloeobacter plus candidatus Aurora vandensis as the closest cyanobacterial relatives to the non-photosynthetic Vampirovibrionia (formerly Melainabacteria) . This phylogenetic relationship suggests that studying G. violaceus Ddl could provide insights into how essential metabolic enzymes evolved during the transition to photosynthetic lifestyles.

The observation that Gloeobacter violaceus possesses a truncated Vipp homologue lacking the approximately 30 amino-acid extension found in other oxygenic photosynthetic organisms raises the possibility that other proteins, including Ddl, might also exhibit unique structural features reflecting their evolutionary position. These features could include differences in substrate specificity, catalytic efficiency, or regulatory mechanisms compared to Ddl enzymes from more recently diverged bacterial lineages.

What are the potential implications of G. violaceus Ddl structure for the design of novel antimicrobial agents?

The unique structural features of Gloeobacter violaceus Ddl could have significant implications for the design of novel antimicrobial agents. As an ATP-dependent enzyme essential for bacterial cell wall biosynthesis, Ddl represents an attractive target for antibacterial drug development . Understanding the specific structural and functional characteristics of G. violaceus Ddl could lead to the development of inhibitors with unique properties or mechanisms of action.

Mixed dipeptide analogues, phosphinate and phosphonate dipeptides (as transition-state mimetics), and inhibitor scaffolds identified through structure-based drug design and virtual screening represent promising avenues for developing new Ddl inhibitors . The unique evolutionary position of G. violaceus might make its Ddl particularly valuable for comparative studies aimed at identifying conserved features that could be targeted for broad-spectrum activity, or unique features that could enable selective targeting.

How does the molecular mechanism of Ddl catalysis compare between G. violaceus and other bacterial species?

While the search results do not provide specific information comparing the molecular mechanism of Ddl catalysis between G. violaceus and other bacterial species, general insights can be inferred from studies of Ddl enzymes from other organisms.

The catalytic mechanism of Ddl involves formation of a dimer, with each monomer containing distinct domains that work together to create the enzyme's active site. In Thermus thermophilus Ddl (TtDdl), the enzyme consists of an N-terminal domain, a central domain, and a C-terminal domain . ATP and two D-Ala substrates bind in adjacent sites at the center of the Ddl monomer to facilitate formation of the D-alanyl-D-alanine dipeptide .

The reaction proceeds through an acylphosphate intermediate formed by phosphorylation of the first D-alanine's carboxylate group by ATP . This intermediate is then attacked by the amino group of the second D-alanine to form the dipeptide product . This mechanism involves cumulative conformational changes induced by substrate binding, and the first D-Ala binding site has been shown to have higher affinity for D-Ala than the second site .

The activation of Ddl by monovalent cations, particularly K+, represents another important aspect of the catalytic mechanism. In TtDdl, K+ has been shown to influence both substrate binding (Km) and catalytic rate (kcat) . Whether G. violaceus Ddl exhibits similar dependencies on monovalent cations would be an interesting area for comparative studies.

What makes Ddl an attractive target for antimicrobial drug development?

D-alanine--D-alanine ligase (Ddl) represents an attractive target for antimicrobial drug development for several compelling reasons:

• Essential Role in Cell Wall Biosynthesis: Ddl catalyzes the formation of the D-alanyl-D-alanine dipeptide, which is an essential component of bacterial peptidoglycan . Inhibition of Ddl prevents bacterial growth, making it a viable target for antibacterial drugs .

• Absence in Mammalian Cells: D-amino acids and the enzymes that process them, including Ddl, are generally absent in mammalian cells, providing a basis for selective toxicity against bacteria without affecting host cells .

• Evolutionary Conservation: The essential nature of Ddl across bacterial species makes it a potential broad-spectrum target, although structural variations between species could also enable the development of narrow-spectrum agents targeting specific bacterial pathogens.

• Established Proof of Concept: D-cycloserine, a structural analogue of D-alanine, is a known inhibitor of Ddl that has been used clinically, primarily for the treatment of tuberculosis . While its use has been limited due to side effects and high MIC values, it provides proof of concept that Ddl inhibition can be achieved in a clinical setting.

• Diverse Inhibition Strategies: Multiple approaches to Ddl inhibition have been identified, including substrate analogues, product analogues, transition state mimetics, and novel scaffolds identified through screening or modeling . This diversity of potential inhibition mechanisms expands the opportunities for drug discovery.

What are the major classes of Ddl inhibitors and their mechanisms of action?

Four main categories of Ddl inhibitors have been described, each with distinct mechanisms of action:

• Analogues of D-Alanine: These compounds compete with the natural substrate for binding to the enzyme. The most important example is D-cycloserine (D-4-amino-3-isoxazolidone), which is a structural analogue of D-alanine with a Ki of 27 μM . D-cycloserine is the only Ddl inhibitor that has been used clinically, primarily for tuberculosis treatment, although its use has been limited due to neurological side effects and high MIC values .

• Analogues of Product (D-Ala-D-Ala): Since Ddl is strongly inhibited by its reaction product D-Ala-D-Ala, various mixed dipeptide analogues have been developed as inhibitors. Several of these have proven to be slightly more effective inhibitors than the natural reaction product .

• Transition State Analogues: Phosphinate and phosphonate dipeptides have been described as transition-state mimetics that inhibit Ddl by mimicking the acylphosphate intermediate formed during catalysis. Despite potent activity against isolated enzymes, these compounds have generally failed to show significant antibacterial activity, likely due to poor transport into bacterial cells .

• Novel Inhibitor Scaffolds: In recent years, several new inhibitor scaffolds that show no structural similarity with the substrate, product, or reaction intermediate have been identified through de novo structure-based drug design and virtual screening approaches . These compounds offer potential advantages in terms of pharmacokinetic properties and resistance profiles.

How can metabolomics approaches be used to validate Ddl as a drug target and assess inhibitor specificity?

Metabolomics approaches offer powerful tools for validating Ddl as a drug target and assessing inhibitor specificity in complex biological systems. These approaches can:

• Track Substrate-Product Relationships: Using isotopically labeled substrates, the flow of metabolites through the Ddl-catalyzed reaction can be monitored. For instance, by using 2H-isotopically labeled alanine, researchers can track the disappearance of the α-2H isotopologue over time as alanine is processed by alanine racemase (Alr) and subsequently by Ddl .

• Identify On-Target Effects: By measuring pool sizes of key metabolites such as D-Ala, L-Ala, and D-Ala-D-Ala in response to potential Ddl inhibitors, researchers can confirm that observed antibacterial effects are indeed due to Ddl inhibition rather than off-target effects .

• Distinguish Between Multiple Targets: Some compounds, such as D-cycloserine (DCS), can inhibit multiple enzymes in the same pathway. Metabolomics can help distinguish between inhibition of Ddl and other targets such as alanine racemase. In one study, the addition of DCS at 1× MIC corresponded to a complete absence of newly synthesized D-Ala-D-Ala, while alanine racemase retained some activity under the same conditions, providing evidence for a key role of Ddl inhibition in the antibiotic action of DCS .

• Assess Temporal Dynamics: Metabolomics can track changes in metabolite pools over time, providing insights into the kinetics of inhibition and potential compensatory mechanisms that might affect drug efficacy.

• Evaluate System-Wide Effects: Beyond the immediate pathway involving Ddl, metabolomics can reveal broader metabolic consequences of Ddl inhibition, potentially identifying synergistic drug targets or explaining observed side effects.