Recombinant Buchnera aphidicola subsp. Baizongia pistaciae 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-succinyltransferase (dapD)

What is the taxonomic classification of Buchnera aphidicola subsp. Baizongia pistaciae?

Buchnera aphidicola subsp. Baizongia pistaciae belongs to the bacterial domain, specifically within the phylum Pseudomonadota (formerly Proteobacteria). Its complete taxonomic lineage is: Domain Bacteria, Phylum Pseudomonadota, Class Gammaproteobacteria, Order Enterobacterales, Family Erwiniaceae, Genus Buchnera, Species Buchnera aphidicola, and subspecies Baizongia pistaciae . This organism is the primary endosymbiont of aphids and has been extensively studied due to its highly reduced genome and obligate intracellular lifestyle . The classification reflects its evolutionary relationship to free-living Enterobacterales bacteria, such as Escherichia coli, though it has undergone substantial genome reduction during its evolutionary history as an endosymbiont.

Research methodologies for taxonomic verification typically involve 16S rRNA gene sequencing, which confirms the classification within the Erwiniaceae family. For comprehensive genomic studies, whole genome sequencing followed by comparative genomic analysis against related bacterial species provides deeper insights into its evolutionary adaptations and metabolic capabilities.

What genomic features characterize Buchnera aphidicola subsp. Baizongia pistaciae?

Buchnera aphidicola subsp. Baizongia pistaciae has a remarkably reduced genome of approximately 618,379 base pairs, consisting of a main chromosome (615,980 bp) and a small plasmid pBBp1 (2,399 bp) . The genome encodes a total of 560 genes, of which 520 are protein-coding genes, 38 are RNA genes, and 2 are pseudogenes . This represents one of the smallest genomes among non-viral organisms, reflecting its long-term co-evolution with its aphid host.

The genome contains 80 metabolic pathways, 511 enzymatic reactions, and 11 transport reactions . Despite its reduced gene set, B. aphidicola maintains essential metabolic functions, particularly those involved in amino acid biosynthesis pathways that are critical for the nutrition of its aphid host. The genomic data indicates the organism has a single 16S rRNA gene copy , which is consistent with its slow growth rate and specialized lifestyle.

Research methodology: Genome analysis typically involves next-generation sequencing technologies, followed by annotation using platforms such as the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) version 4.13 with GeneMarkS-2+ for gene prediction . Comparative genomics approaches are essential for understanding gene loss and retention patterns relative to free-living bacterial ancestors.

What is the biochemical function of 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-succinyltransferase (dapD)?

The enzyme 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-succinyltransferase (dapD) catalyzes a critical step in the diaminopimelate-lysine biosynthesis pathway. Specifically, dapD catalyzes the reaction between tetrahydrodipicolinate (THDP) and succinyl-CoA to form (S)-2-(3-carboxypropanamido)-6-oxoheptanedioic acid and coenzyme A . This reaction represents the third step in the pathway leading to the production of lysine, an essential amino acid for protein synthesis.

The diaminopimelate-lysine pathway produces two metabolites necessary for bacterial survival and growth: meso-diaminopimelate, which is required for cell wall peptidoglycan cross-linking in most bacteria, and lysine, an essential amino acid. Since humans lack this biosynthetic pathway and must obtain lysine from their diet, enzymes in this pathway, including dapD, represent potential targets for the development of antibiotics with minimal toxicity to human cells .

Methodologically, the enzyme activity can be assessed using both direct and coupled assays. Kinetic studies on dapD from related bacteria (e.g., Serratia marcescens) have shown that the enzyme follows a rapid equilibrium ordered bi bi kinetic mechanism, where the binding of substrates occurs in a specific order, and the conversion of the central enzyme complexes is the rate-limiting step in the reaction .

How does metal ion exposure affect dapD enzyme activity?

Metal ion exposure, particularly Cu²⁺, has significant effects on dapD enzyme activity. Research on related bacterial dapD enzymes has shown that Cu²⁺ ions cause rapid and specific inactivation of the enzyme at low concentrations . This inactivation process involves:

• Binding of Cu²⁺ to histidine residues in the enzyme structure

• Reduction of Cu²⁺ to Cu⁺, as detected by EPR spectroscopy

• Oxidative inactivation of the enzyme

• Significant structural changes and protein denaturation

The binding of Cu²⁺ to dapD is characterized by a dissociation constant (KD) of approximately 2.7 μM, as determined by internal tryptophan fluorescence quenching studies . Importantly, the enzyme activity can be protected in reducing environments (e.g., in the presence of 1 mM dithiothreitol [DTT]), and the fluorescence quenching can be reversed by adding DTT or EDTA, which chelates the copper ions .

Research methodology: Metal ion effects on enzyme activity are typically studied using activity assays in the presence of varying concentrations of metal ions, spectroscopic techniques such as fluorescence spectroscopy to monitor structural changes, and electron paramagnetic resonance (EPR) to characterize metal binding properties.

What expression systems are most effective for producing recombinant Buchnera aphidicola dapD?

Based on studies with similar enzymes, heterologous expression of Buchnera aphidicola dapD is most effectively accomplished using E. coli expression systems. While there are no direct studies on recombinant expression of dapD from Buchnera aphidicola in the provided search results, successful expression of related enzymes provides valuable methodological insights.

For optimal expression, consider the following approach:

• Gene synthesis: Since Buchnera has different codon usage patterns than E. coli, codon optimization of the dapD gene sequence for E. coli expression is recommended. This involves adjusting the sequence to use codons preferred by E. coli while maintaining the amino acid sequence.

• Vector selection: pET-based expression vectors (particularly pET28a) with T7 promoter systems typically provide high-level expression of recombinant proteins. The inclusion of N-terminal or C-terminal His6-tags facilitates subsequent purification.

• Host strain selection: E. coli BL21(DE3) or derivatives like BL21(DE3)pLysS are commonly used for recombinant protein expression. For potentially toxic proteins, strains with tighter expression control such as BL21(DE3)pLysS or Rosetta(DE3) may be preferable.

• Induction conditions: Based on protocols for similar enzymes, induction with 0.5-1.0 mM IPTG at OD600 of 0.6-0.8, followed by expression at 18-25°C for 16-20 hours often provides optimal soluble protein yields .

• Culture media: While standard LB media can be used, enriched media such as Terrific Broth often provide higher cell densities and protein yields.

Experimental methodology: Optimization of expression conditions typically involves varying induction parameters (temperature, IPTG concentration, induction time), followed by SDS-PAGE and Western blot analysis to assess protein expression levels and solubility. Small-scale expression trials should precede large-scale production.

What purification strategies yield the highest purity and activity of recombinant dapD?

A multi-step purification strategy is recommended to obtain high-purity and enzymatically active dapD protein:

• Immobilized metal affinity chromatography (IMAC): For His-tagged dapD, Ni-NTA or Co-NTA affinity chromatography serves as an effective initial capture step. Use of a buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 20 mM imidazole for binding, followed by elution with an imidazole gradient (50-250 mM) typically yields 80-90% pure protein.

• Ion exchange chromatography: As a second purification step, anion exchange chromatography (e.g., Q-Sepharose) can remove remaining impurities. Buffer conditions should be adjusted based on the theoretical isoelectric point (pI) of dapD.

• Size exclusion chromatography: A final polishing step using gel filtration (e.g., Superdex 75 or 200) in a buffer containing 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 1 mM DTT typically yields >95% pure protein and separates aggregates from properly folded protein.

Throughout the purification process, it is essential to maintain reducing conditions (1-5 mM DTT or 0.5-2 mM TCEP) to prevent oxidation of cysteine residues, which could affect enzyme activity. Additionally, inclusion of 10% glycerol in storage buffers enhances protein stability during freeze-thaw cycles.

For activity assessments during purification, both direct and coupled assays can be employed. The direct assay measures the formation of CoA using DTNB (5,5'-dithiobis-(2-nitrobenzoic acid)), while the coupled assay monitors the reaction through subsequent enzymatic steps .

How is the kinetic mechanism of dapD characterized and what are its implications for inhibitor design?

The kinetic mechanism of dapD can be characterized using steady-state kinetic analyses with varying substrate concentrations. Based on studies of dapD from related organisms, the enzyme follows a rapid equilibrium ordered bi bi kinetic mechanism . In this mechanism:

• Substrates bind in a defined order: The first substrate (tetrahydrodipicolinate or an analog like 2-aminopimelate in assay systems) must bind before the second substrate (succinyl-CoA).

• Products are released in a specific order: CoA is released before the succinyltransferase product.

• The rate-limiting step is the chemical conversion of the central enzyme complexes rather than substrate binding or product release.

This mechanism has been determined through initial velocity pattern analysis using both direct and coupled enzyme assays . For dapD from Serratia marcescens, kinetic constants include a Kia (for 2-aminopimelate) of 1.9 ± 0.26 mM and a Kb (for succinyl-CoA) of 87 ± 15 μM .

The ordered binding mechanism has important implications for inhibitor design:

• Competitive inhibitors against the first substrate binding site can be effective regardless of second substrate concentration.

• Inhibitors designed to mimic the transition state of the reaction would likely be most effective.

• Bisubstrate analogs that simultaneously occupy both substrate binding sites could provide high-affinity inhibition.

• Metal-based inhibitors, particularly copper compounds, might be effective through oxidative inactivation and structural disruption of the enzyme.

Research methodology: Kinetic mechanisms are determined through initial velocity studies with varying concentrations of both substrates, product inhibition studies, and dead-end inhibition studies. Data are fitted to appropriate rate equations to determine kinetic constants and the most likely mechanism.

What structural features of dapD contribute to substrate specificity and catalytic activity?

While specific structural information for Buchnera aphidicola dapD is not provided in the search results, insights can be drawn from studies of dapD enzymes from related organisms. Key structural features that typically contribute to dapD substrate specificity and catalytic activity include:

• Active site architecture: The active site likely contains conserved residues that coordinate substrate binding and catalysis. In particular, histidine residues appear to be important, as suggested by the specific binding of Cu²⁺ to histidine residues in the enzyme .

• Substrate binding pockets: The enzyme must have distinct binding sites for its two substrates - tetrahydrodipicolinate (THDP) and succinyl-CoA. The ordered binding mechanism suggests these sites are structurally linked, with conformational changes likely occurring after binding of the first substrate.

• Conserved domains: As an acyltransferase, dapD likely contains nucleotide-binding domains characteristic of this enzyme family, which are essential for recognizing the CoA portion of succinyl-CoA.

• Oxidation-sensitive residues: The enzyme contains residues susceptible to oxidative inactivation, particularly in the presence of Cu²⁺ ions. These may include cysteine residues near the active site, which when oxidized could disrupt the catalytic mechanism .

• Conformational flexibility: Spectroscopic studies suggest significant structural changes upon metal binding, indicating conformational flexibility that may be important for catalysis .

Research methodology: Structural features are typically investigated through X-ray crystallography or cryo-electron microscopy of the purified enzyme, often in complex with substrates, products, or inhibitors. Site-directed mutagenesis of conserved residues followed by kinetic characterization helps identify key catalytic residues. Computational approaches such as homology modeling and molecular dynamics simulations can provide additional insights, especially when experimental structures are unavailable.

How has the dapD gene evolved in Buchnera aphidicola compared to free-living bacteria?

Despite this extreme genome reduction, Buchnera aphidicola has retained the dapD gene, suggesting its essential role in the bacteria's survival and in its symbiotic relationship with the aphid host. The retention of genes involved in amino acid biosynthesis, including the diaminopimelate-lysine pathway, is consistent with Buchnera's primary role in providing essential amino acids to its aphid host .

Several evolutionary patterns are likely to characterize the dapD gene in Buchnera:

• Accelerated sequence evolution: Like many genes in Buchnera, dapD likely shows higher rates of sequence evolution compared to free-living bacteria, due to relaxed selection pressure and genetic drift associated with small population sizes.

• Codon usage bias: The gene likely exhibits distinctive codon usage patterns reflective of the AT-rich genome of Buchnera.

• Gene order conservation: The genomic context of dapD may be conserved across different Buchnera strains, as suggested by the conserved gene order seen in other metabolic pathways like the leucine gene cluster .

• Loss of regulatory elements: Many regulatory elements present in free-living bacteria are likely to be absent in Buchnera's dapD, reflecting the more stable intracellular environment and reduced need for complex regulation.

Research methodology: Comparative genomic analyses involving dapD sequences from multiple Buchnera strains and free-living relatives, calculation of dN/dS ratios to assess selection pressures, and analysis of genomic context conservation provide insights into the evolutionary trajectory of this gene.

What is the significance of dapD in the symbiotic relationship between Buchnera aphidicola and its aphid host?

The dapD enzyme, as part of the diaminopimelate-lysine biosynthesis pathway, plays a crucial role in the symbiotic relationship between Buchnera aphidicola and its aphid host. This significance stems from several factors:

• Essential amino acid provision: Lysine is an essential amino acid that aphids cannot synthesize themselves and must obtain from their diet or from their bacterial endosymbionts. By maintaining a functional diaminopimelate-lysine biosynthesis pathway, including dapD, Buchnera can synthesize lysine for its aphid host .

• Bacterial cell wall integrity: The diaminopimelate produced by this pathway is a critical component of peptidoglycan, which forms the bacterial cell wall. The maintenance of cell wall integrity is essential for Buchnera survival within specialized aphid cells called bacteriocytes.

• Metabolic integration: The retention of dapD and other enzymes in this pathway, despite massive genome reduction, reflects the metabolic integration between Buchnera and its host. The aphid likely provides metabolic precursors for this pathway, while Buchnera contributes enzymatic steps that the aphid genome lacks.

• Co-evolutionary implications: The retention and potential specialization of dapD in Buchnera represents an example of co-evolution between the endosymbiont and its host. The enzyme may have evolved specific properties adapted to the intracellular environment of aphid bacteriocytes.

• Potential vulnerability: As an essential enzyme for both bacterial survival and host nutrition, dapD represents a potential vulnerability in the symbiotic system. Disruption of dapD function could impact both Buchnera viability and aphid fitness.

Research methodology: The significance of dapD in the symbiotic relationship can be investigated through metabolic labeling experiments to track lysine synthesis and transfer, comparative genomics across different aphid-Buchnera associations, and experimental manipulation of the pathway using RNA interference or chemical inhibitors.

How can recombinant Buchnera aphidicola dapD be utilized in inhibitor screening and drug discovery?

Recombinant Buchnera aphidicola dapD can serve as a valuable tool for inhibitor screening and drug discovery, particularly in the development of novel insecticides that target the aphid-Buchnera symbiosis. Several methodological approaches can be employed:

• High-throughput screening (HTS): Purified recombinant dapD can be used in biochemical assays to screen chemical libraries for potential inhibitors. Both the direct assay (measuring CoA formation using DTNB) and coupled assays can be adapted to 96-well or 384-well format for HTS applications .

• Structure-based drug design: Once the three-dimensional structure of Buchnera dapD is determined through X-ray crystallography or modeled based on homologous proteins, virtual screening and structure-based design approaches can identify compounds predicted to bind at the active site or allosteric regulatory sites.

• Fragment-based drug discovery: This approach involves screening small chemical fragments that bind weakly to the enzyme, followed by fragment elaboration or linking to develop more potent inhibitors. Biophysical methods such as differential scanning fluorimetry, surface plasmon resonance, or NMR can detect such weak interactions.

• Natural product screening: Extracts from plants, particularly those resistant to aphids, can be screened for dapD inhibitory activity, potentially leading to the identification of novel scaffold structures for inhibitor development.

• Metal-based inhibitor development: Given the sensitivity of dapD to Cu²⁺-mediated inactivation , development of copper complexes or other metal-based compounds that specifically target dapD could be explored.

What are the most promising directions for future research on Buchnera aphidicola dapD?

Based on the available information and gaps in current knowledge, several promising research directions for Buchnera aphidicola dapD can be identified:

• Structural characterization: Determining the three-dimensional structure of Buchnera aphidicola dapD through X-ray crystallography or cryo-electron microscopy would provide valuable insights into its catalytic mechanism and facilitate structure-based inhibitor design. Comparing this structure with homologs from free-living bacteria could reveal adaptations specific to the endosymbiotic lifestyle.

• In vivo function analysis: Developing methods to study dapD function within intact Buchnera-aphid systems would enhance understanding of its role in the symbiosis. This could involve techniques such as metabolic labeling to track the flow of lysine from Buchnera to aphid tissues, or targeted inhibition of dapD in vivo.

• Comparative analysis across Buchnera strains: Analyzing dapD sequence, expression, and function across different Buchnera strains associated with diverse aphid hosts could reveal how this enzyme has evolved in different symbiotic contexts and identify conserved features essential for its function.

• Transcriptional and post-translational regulation: Investigating how dapD expression and activity are regulated in Buchnera could provide insights into the coordination of metabolic activities between the endosymbiont and its host. This might involve proteomics approaches to identify post-translational modifications or interacting proteins.

• Development of selective inhibitors: Designing inhibitors that specifically target Buchnera dapD without affecting related enzymes in other organisms could lead to novel aphid control strategies with minimal environmental impact. Exploring the Cu²⁺ sensitivity of dapD could provide one avenue for such selective targeting .

Research methodology: These directions require integration of multiple approaches, including molecular biology, biochemistry, structural biology, and systems biology. Collaboration between experts in bacterial metabolism, insect physiology, and structural biology would be particularly valuable for addressing these complex questions.

What are the key challenges in working with recombinant proteins from endosymbiotic bacteria like Buchnera aphidicola?

Working with recombinant proteins from endosymbiotic bacteria like Buchnera aphidicola presents several unique challenges that researchers must address:

• Codon usage bias: Buchnera aphidicola has a highly AT-rich genome with distinctive codon usage patterns that differ significantly from expression hosts like E. coli. This can lead to translation pauses, premature termination, or low expression levels. Codon optimization of the synthetic gene is typically necessary to overcome this challenge.

• Protein solubility and folding: Endosymbiont proteins have evolved to function within the specialized intracellular environment of host cells, which may differ in pH, ionic strength, and chaperone availability from typical expression systems. This can lead to solubility issues and misfolding when expressed recombinantly.

• Post-translational modifications: If the native protein undergoes specific post-translational modifications in Buchnera that are absent in the expression host, the recombinant protein may lack full activity or proper folding.

• Structural stability: The reduced selection pressure on endosymbiont proteins can lead to decreased structural stability when expressed outside their native context. Addition of stabilizing agents or optimization of buffer conditions may be necessary.

• Assay development: Establishing robust activity assays can be challenging when the natural substrates may be unknown or commercially unavailable, requiring development of suitable substrate analogs or coupled assay systems.

Methodological approaches to address these challenges include:

• Use of specialized E. coli strains (e.g., Rosetta strains that supply rare tRNAs)

• Expression as fusion proteins with solubility-enhancing tags (e.g., MBP, SUMO)

• Co-expression with chaperones from Buchnera or E. coli

• Extensive screening of expression conditions (temperature, media, induction parameters)

• Inclusion of stabilizing additives in purification buffers

How can researchers validate that recombinant dapD accurately represents the native enzyme's properties?

• Enzymatic characterization comparison: If possible, compare the kinetic parameters, substrate specificity, and inhibition profile of the recombinant enzyme with those determined for the native enzyme from Buchnera extracts. While challenging due to the difficulty of culturing Buchnera, this provides the most direct validation.

• Functional complementation: Express the Buchnera dapD gene in a bacterial strain with a dapD deletion mutation to test if it can restore growth in the absence of lysine supplementation. Successful complementation suggests the recombinant protein retains native functionality.

• Structural integrity assessment: Use circular dichroism spectroscopy, thermal shift assays, or limited proteolysis to assess if the recombinant protein has a stable, well-folded structure consistent with a functional enzyme.

• Mass spectrometry analysis: Compare the accurate mass and peptide mapping of the recombinant protein with theoretical predictions to confirm correct translation and processing.

• Comparative analysis with homologs: Compare the properties of recombinant Buchnera dapD with well-characterized homologs from related bacteria. Similar kinetic mechanisms, metal sensitivities, and substrate preferences would support the validity of the recombinant enzyme as a model.

• In silico validation: Use homology modeling and molecular dynamics simulations to assess if the recombinant protein's predicted structure and dynamics are consistent with a functional enzyme.

Research methodology: These validation approaches require integration of biochemical, biophysical, and computational techniques. Documentation of multiple lines of evidence supporting the recombinant protein's representation of native properties strengthens the reliability of subsequent findings.