Recombinant Idiomarina loihiensis 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-succinyltransferase (dapD)

What is Idiomarina loihiensis and why is its dapD enzyme of research interest?

Idiomarina loihiensis is a deep-sea γ-proteobacterium isolated from a hydrothermal vent at 1,300-m depth on the Lōihi submarine volcano in Hawaii . The organism has evolved unique metabolic adaptations to survive in extreme conditions, including reliance on amino acid catabolism rather than sugar fermentation for carbon and energy acquisition . The dapD enzyme (2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-succinyltransferase) is part of the diaminopimelate (DAP)/lysine biosynthesis pathway, which is essential for bacterial cell wall formation.

What makes I. loihiensis particularly interesting is that its genome sequence revealed apparent contradictions in the DAP/lysine biosynthesis pathway. While some organisms in similar phylogenetic groups utilize the alternative DapL pathway and lack dapD genes, I. loihiensis shows genomic variations that warrant investigation regarding pathway selection and enzyme function . This makes recombinant I. loihiensis dapD a valuable subject for comparative biochemistry and evolutionary studies.

How does the genomic context of dapD in Idiomarina loihiensis differ from other organisms?

In many bacteria, dapD is found within operons containing other genes involved in lysine biosynthesis. In Idiomarina loihiensis, the genome comprises a single chromosome of 2,839,318 base pairs encoding 2,640 proteins . While some bacteria demonstrate a polycistronic structure where dapD is found in genomic contiguity with other dap genes, I. loihiensis shows a different genomic organization .

Research indicates that the presence or absence of dapD correlates with specific phylogenetic lineages. The occurrence of the alternative DapL pathway (using DapL transaminase) in a species generally correlates with the absence of genes for dapD and dapE . Analysis of the I. loihiensis genome provides insights into how different branches of the bacterial kingdom have evolved diverse solutions for similar biosynthetic challenges in amino acid metabolism.

What expression systems are recommended for recombinant production of Idiomarina loihiensis dapD?

When expressing recombinant I. loihiensis dapD, several factors must be considered due to the organism's unique adaptations to extreme environments. I. loihiensis can survive wide temperature ranges (4°C to 46°C) and high salinity conditions (0.5% to 20% NaCl) , which affects protein folding and stability.

For laboratory expression, E. coli systems are commonly used due to their well-established protocols, especially since functional complementation studies have shown that related enzymes can rescue E. coli dapD mutants . When designing expression systems, consider the following approach:

• Optimize codon usage for the host organism

• Include purification tags that minimize interference with enzyme activity

• Test expression at varying temperatures (20-37°C) to identify optimal conditions for soluble protein production

• Consider the addition of osmolytes in purification buffers to maintain enzyme stability

For challenging expressions, alternative hosts such as Pseudomonas or other halophilic systems might be more suitable than conventional E. coli strains.

How can I design experiments to determine if recombinant Idiomarina loihiensis dapD complements metabolic pathways in other organisms?

To design robust complementation experiments with recombinant I. loihiensis dapD, follow this methodological approach:

• Strain Selection:

  • Use E. coli dapD mutants (auxotrophic for DAP/lysine)

  • Include positive controls (native dapD gene) and negative controls (empty vector)

• Vector Design:

  • Clone the I. loihiensis dapD gene under both constitutive and inducible promoters

  • Include appropriate translation initiation signals for the host organism

• Complementation Assessment:

  • Perform growth assays on minimal media with and without DAP/lysine supplementation

  • Measure growth rates and final cell densities in different conditions

• Enzyme Activity Confirmation:

  • Extract proteins from complemented strains

  • Perform enzyme assays measuring the conversion of THDPA (tetrahydrodipicolinate) and succinyl-CoA to N-succinyl-THDPA

This approach builds on established methods for studying DapL function, where complementation of E. coli dapD and dapE mutants successfully demonstrated functional roles in DAP/lysine biosynthesis .

What are the recommended methods for assessing the enzymatic activity of recombinant Idiomarina loihiensis dapD?

For accurate assessment of recombinant I. loihiensis dapD enzymatic activity, three complementary assay approaches are recommended:

• Direct Product Formation Assay:

  • Measure the formation of N-succinyl-THDPA spectrophotometrically

  • Monitor decrease in succinyl-CoA at 232 nm (ε = 4.5 × 10³ M⁻¹ cm⁻¹)

  • Reaction conditions: 50 mM HEPES (pH 7.5), 0.5 mM THDPA, 0.1 mM succinyl-CoA, 1 mM MgCl₂

• Coupled Assay System:

  • Link dapD activity to a secondary reaction with a colorimetric/fluorescent output

  • Use DapE to convert N-succinyl-THDPA to succinate and THDPA

  • Measure THDPA by reaction with o-aminobenzaldehyde to form dihydroquinazolium derivative (λₘₐₓ = 440 nm)

• Comparative Kinetics Analysis:

  • Determine Kₘ values for both substrates under varying temperature and salt concentrations

  • Assess enzyme stability at different temperatures (4-46°C) and salt concentrations (0.5-20% NaCl)

For halophilic enzymes like those from I. loihiensis, activity should be measured across a range of salt concentrations to determine optimal conditions.

How does the biochemical function of Idiomarina loihiensis dapD compare with alternative lysine biosynthesis pathways?

The lysine biosynthesis pathway in bacteria shows remarkable diversity, with at least four known variants. Understanding how I. loihiensis dapD functions compared to alternative pathways requires systematic comparative analysis:

The I. loihiensis genome analysis reveals a complex picture regarding pathway utilization. While closely related Idiomarina species primarily utilize the DapL pathway, genomic evidence from I. loihiensis suggests potential pathway redundancy or specialization .

For experimental comparison:

• Express recombinant dapD from I. loihiensis alongside DapL from model organisms

• Compare enzyme kinetics under varying environmental conditions

• Assess relative efficiency in complementing DAP/lysine auxotrophs

• Perform growth competition experiments under different nutrient conditions

This approach will clarify whether I. loihiensis dapD represents an evolutionary adaptation to extreme environments or a conserved ancestral trait.

What experimental challenges arise when studying the structural biology of Idiomarina loihiensis dapD?

Structural studies of recombinant I. loihiensis dapD present several unique challenges:

• Protein Stability Issues:

  • As a halophilic organism surviving in 0.5-20% NaCl, I. loihiensis enzymes may require high salt concentrations for stability

  • Standard crystal growth conditions may destabilize the protein structure

• Post-translational Modifications:

  • Deep-sea adaptations may include unusual post-translational modifications

  • Expression in E. coli might yield protein lacking critical modifications

• Crystallization Challenges:

  • Optimize crystallization buffers to include appropriate salt concentrations

  • Test thermostability at various temperatures (4-46°C) given the organism's temperature range

  • Consider membrane mimetics if the protein has membrane associations

• Methodological Approach:

  • Begin with small-angle X-ray scattering (SAXS) to assess protein behavior in solution

  • Test multiple affinity tags and their positions to identify constructs amenable to crystallization

  • Consider cryo-EM as an alternative approach if crystallization proves challenging

  • Employ molecular dynamics simulations to understand flexibility under varying salt conditions

Approaches that have succeeded with other extremophile enzymes, such as incorporating stabilizing osmolytes in purification and crystallization buffers, should be considered.

How can site-directed mutagenesis be used to investigate the catalytic mechanism of Idiomarina loihiensis dapD?

To systematically investigate the catalytic mechanism of I. loihiensis dapD, a comprehensive site-directed mutagenesis approach should target key residues:

• Identification of Target Residues:

  • Perform multiple sequence alignment with well-characterized dapD enzymes

  • Use homology modeling based on existing dapD crystal structures

  • Identify conserved residues in the predicted active site

• Mutagenesis Strategy:

  • Generate alanine substitutions of conserved residues

  • Create conservative substitutions to probe specific interactions

  • Design mutations that alter substrate specificity

• Functional Assessment:

  • Measure enzyme kinetics (kcat, Km) for each mutant

  • Determine pH and temperature profiles compared to wild-type

  • Assess thermal stability using differential scanning fluorimetry

• Data Analysis Framework:

  • Plot relative activity of each mutant as percentage of wild-type

  • Create Michaelis-Menten curves to visualize kinetic parameter changes

  • Develop a structural model incorporating mutational data

This approach has been successfully applied to other enzymes in the DAP/lysine pathway, providing insights into catalytic mechanisms and evolutionary adaptations .

What is the optimal protocol for purifying active recombinant Idiomarina loihiensis dapD?

For purification of active recombinant I. loihiensis dapD, the following optimized protocol incorporates considerations for the halophilic nature of the source organism:

• Expression System:

  • Construct: pET28a vector with N-terminal His6-tag

  • Host: E. coli BL21(DE3) or Rosetta(DE3) for rare codon optimization

  • Induction: 0.5 mM IPTG at OD600 = 0.6-0.8, 25°C for 16-18 hours

• Cell Lysis Buffer:

  • 50 mM Tris-HCl (pH 8.0)

  • 300 mM NaCl (initial purification)

  • 10% glycerol

  • 1 mM DTT

  • 1 mM PMSF

  • 5 mM imidazole

• Purification Steps:

  • Ni-NTA affinity chromatography (wash with 20 mM imidazole, elute with 250 mM imidazole)

  • Size exclusion chromatography (Superdex 200)

  • Salt gradient testing (100 mM to 2 M NaCl) to determine optimal stability conditions

• Storage Conditions:

  • 50 mM Tris-HCl (pH 7.5)

  • Optimized NaCl concentration (typically 300-500 mM)

  • 20% glycerol

  • Store at -80°C in small aliquots

• Quality Control:

  • SDS-PAGE for purity assessment

  • Dynamic light scattering for aggregation analysis

  • Activity assay to confirm functional enzyme

This protocol takes into account I. loihiensis' adaptation to high salinity environments (up to 20% NaCl) and should be further optimized based on empirical stability and activity data.

How can the temperature and salt concentration dependencies of Idiomarina loihiensis dapD be systematically characterized?

For systematic characterization of temperature and salt dependencies of I. loihiensis dapD, implement this experimental design approach:

• Temperature Dependence Assessment:

  • Measure enzyme activity at 5°C intervals from 5°C to 50°C

  • Determine temperature optimum and compare to the growth temperature range of I. loihiensis (4-46°C)

  • Calculate activation energy (Ea) using Arrhenius plot

• Salt Concentration Experiments:

  • Test NaCl concentrations from 0-3 M in 0.25 M increments

  • Examine effects of different salt types (KCl, LiCl, MgCl2) at equivalent ionic strengths

  • Determine if salt effects are specific or follow Hofmeister series patterns

• Combined Temperature-Salt Matrix:

  • Create a 5×5 experimental matrix of temperature and salt combinations

  • Measure enzyme activity, stability, and kinetic parameters across the matrix

  • Develop a 3D response surface model of activity

• Data Analysis:

  • Calculate half-life at different temperature-salt combinations

  • Determine kinetic parameters (Km, Vmax) under varying conditions

  • Model the relationship between environmental conditions and enzyme function

This approach addresses I. loihiensis' remarkable adaptability to both temperature variations and high salinity (up to 20% NaCl) , providing insights into structural adaptations that maintain enzyme function in extreme environments.

What control experiments are essential when comparing wild-type and mutant versions of Idiomarina loihiensis dapD?

• Protein Quality Controls:

  • Circular dichroism spectroscopy to confirm similar secondary structure

  • Thermal shift assays to detect potential stability differences

  • Size exclusion chromatography to verify oligomeric state

  • SDS-PAGE and western blot to confirm protein purity and concentration

• Activity Baseline Controls:

  • Include enzyme-free reaction controls for each assay condition

  • Perform time-course measurements to ensure linear reaction rates

  • Test multiple substrate concentrations to identify potential substrate inhibition

  • Include known dapD inhibitors as positive controls for activity reduction

• Environmental Variation Controls:

  • Test multiple buffer systems to identify potential buffer-specific effects

  • Include reducing agent controls (±DTT, ±β-mercaptoethanol)

  • Measure activity with standardized substrate batches

• Complementation Controls:

  • Include vector-only control in all complementation experiments

  • Use well-characterized dapD mutants as references

  • Perform complementation with varying expression levels

This experimental design approach ensures that observed differences between wild-type and mutant enzymes reflect genuine biochemical changes rather than experimental artifacts or protein quality issues.

How can isothermal titration calorimetry (ITC) be optimized for studying substrate binding to Idiomarina loihiensis dapD?

Optimizing ITC for studying substrate binding to I. loihiensis dapD requires specific considerations for this halophilic enzyme:

• Buffer Optimization:

  • Match experimental buffer to physiological conditions of I. loihiensis

  • Include salt concentrations reflecting the organism's natural environment (0.5-20% NaCl)

  • Test multiple buffer systems to minimize heat of ionization effects

• Experimental Design:

  • Perform initial experiments at 25°C, then optimize based on enzyme stability

  • Use protein concentrations of 10-20 μM in the cell

  • Titrate substrate at 10-20× protein concentration in the syringe

  • Set injection volumes to 2-3 μL with 3-5 minute intervals

• Control Titrations:

  • Buffer-into-buffer control to establish baseline

  • Substrate-into-buffer to measure dilution heats

  • Buffer-into-protein to detect potential dilution effects

• Data Analysis Approach:

  • Fit data to multiple binding models (one-site, two-site, sequential binding)

  • Report all thermodynamic parameters (ΔH, ΔS, ΔG, n, Ka)

  • Perform experiments at multiple temperatures to calculate ΔCp

This methodological approach addresses the challenges of working with enzymes from extremophiles, accounting for potential salt and temperature dependencies that might influence binding thermodynamics.

What approaches can resolve contradictory results when studying Idiomarina loihiensis dapD in different experimental systems?

When faced with contradictory results studying I. loihiensis dapD across different experimental systems, implement this systematic troubleshooting approach:

• Identify Source of Discrepancies:

  • Compare protein production methods (expression system, purification protocol)

  • Examine buffer compositions (salt concentration, pH, additives)

  • Review assay conditions (temperature, substrate concentrations, detection method)

• Cross-validation Experiments:

  • Transfer protocols between laboratories with standardized reagents

  • Perform parallel experiments with identical protein preparations

  • Use orthogonal assay methods to confirm observations

• Systematic Variation Analysis:

  • Create a matrix of experimental conditions to identify key variables

  • Test native I. loihiensis cell extracts as reference points

  • Compare recombinant protein from multiple expression systems

• Reconciliation Framework:

  • Determine if contradictions reflect context-dependent enzyme behavior

  • Develop unified model incorporating environmental dependencies

  • Identify experimental artifacts through statistical analysis

This approach recognizes that enzymes from extremophiles like I. loihiensis, which thrive in unique environments (hydrothermal vents, high salinity, wide temperature range) , may exhibit context-dependent behaviors that manifest as apparent contradictions across different experimental systems.

How can omics approaches be integrated with biochemical studies of Idiomarina loihiensis dapD?

Integrating omics approaches with biochemical studies of I. loihiensis dapD creates a comprehensive understanding of its biological context:

• Transcriptomics Integration:

  • Analyze dapD expression patterns under varying nutrient conditions

  • Compare expression with other lysine biosynthesis genes

  • Identify potential co-regulated genes suggesting functional relationships

• Proteomics Approaches:

  • Quantify dapD protein levels in different growth conditions

  • Identify post-translational modifications using mass spectrometry

  • Perform protein-protein interaction studies to detect functional complexes

• Metabolomics Analysis:

  • Track lysine pathway intermediates using LC-MS

  • Compare metabolite profiles between wild-type and dapD mutants

  • Measure flux through competing pathways

• Integrated Experimental Design:

  • Culture I. loihiensis under conditions mimicking its natural hydrothermal vent habitat

  • Sample for simultaneous transcriptomic, proteomic, and metabolomic analysis

  • Correlate biochemical enzyme properties with in vivo activity

This multi-omics approach provides context for understanding how I. loihiensis dapD functions within the organism's unique deep-sea adaptation strategy, which relies primarily on amino acid catabolism rather than sugar fermentation .