Recombinant Nitrosomonas europaea Succinyl-diaminopimelate desuccinylase (dapE)

What is Nitrosomonas europaea DapE and what is its biological function?

Nitrosomonas europaea DapE (N-succinyl-L,L-diaminopimelic acid desuccinylase) is an enzyme that catalyzes the conversion of N-succinyl-L,L-diaminopimelic acid to L,L-diaminopimelic acid and succinate. This reaction represents a critical step in the lysine biosynthetic pathway in bacteria.

N. europaea is a Gram-negative obligate chemolithoautotroph that derives all its energy from oxidizing ammonia to nitrite . It inhabits various environments including soil, sewage, freshwater, and the surfaces of buildings and monuments . As a key enzyme in the lysine biosynthetic pathway, DapE is essential for the production of both lysine and meso-diaminopimelic acid, which are critical components for bacterial protein synthesis and peptidoglycan cell wall remodeling .

The significance of DapE lies in its potential as an antimicrobial target, as the dap operon-encoded enzymes are absent in humans but essential for bacterial survival . The emergence of antibiotic-resistant bacterial strains underscores the importance of identifying new drug targets like DapE for developing novel antimicrobial compounds .

What is the structural composition of DapE and why is it important?

DapE belongs to the M20 peptidase family and has a distinct two-domain architecture:

• Catalytic domain: Contains the active site with metal-binding residues

• Dimerization domain: Essential for enzyme functionality

Structural studies have demonstrated that the dimerization domain is critical for enzymatic activity . When this domain is removed, the enzyme becomes inactive, even though the catalytic domain remains intact . Molecular dynamics simulations suggest that removal of the dimerization domain increases the flexibility of a conserved active site loop that may provide critical interactions with the substrate .

The active site of DapE typically contains a dinuclear zinc center. Based on structural comparisons with other M20 peptidases, DapE likely possesses a (μ-aquo)(μ-carboxylato)dizinc(II) core with one terminal carboxylate and one histidine residue at each metal site . Additionally, a putative bridging water/hydroxide molecule likely forms a hydrogen bond to an active site carboxylate group (Glu134), which functions as the general acid/base in the catalytic process .

What expression systems are recommended for recombinant N. europaea DapE?

For successful expression of recombinant N. europaea DapE, researchers should consider:

• Codon optimization: Sequences should be optimized for expression in E. coli, as noted in studies with other N. europaea proteins

• Expression vectors: pET-based systems with T7 promoters are commonly used

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

• Induction conditions: Typically 0.5-1.0 mM IPTG at mid-log phase

The following table outlines optimal expression parameters:

Why is DapE considered a potential antimicrobial target?

DapE represents an attractive antimicrobial target for several compelling reasons:

• Essential pathway: Lysine and meso-diaminopimelic acid are critical for bacterial protein synthesis and cell wall formation

• Absence in humans: The dap operon encodes enzymes specific to bacterial lysine biosynthesis with no human homologs

• Conservation: DapE is highly conserved across numerous bacterial species, suggesting broad-spectrum potential for inhibitors

• Structural characterization: The availability of structural data facilitates rational drug design approaches

• Antimicrobial resistance: The emergence of antibiotic-resistant bacterial strains necessitates identifying new drug targets

The pharmaceutical potential for DapE inhibitors is particularly significant as lysine is an essential amino acid that humans must obtain through diet, making the bacterial biosynthetic pathway an ideal target with minimal potential for off-target effects.

How does the lysine biosynthetic pathway function in bacteria?

The bacterial lysine biosynthetic pathway converts aspartic acid to lysine through multiple enzymatic steps. DapE catalyzes a critical step in this pathway:

• Aspartic acid undergoes several transformations to form N-succinyl-L,L-diaminopimelic acid

• DapE hydrolyzes N-succinyl-L,L-diaminopimelic acid to L,L-diaminopimelic acid and succinate

• L,L-diaminopimelic acid is converted to meso-diaminopimelic acid

• meso-Diaminopimelic acid serves two essential functions:

  • Direct incorporation into peptidoglycan cell wall structure

  • Conversion to lysine for protein synthesis

This pathway is absent in humans who obtain lysine through diet, making it an excellent target for antimicrobial development .

How does the dimeric structure influence DapE catalytic activity?

Structural studies on DapE have revealed that the dimerization domain is not merely a structural feature but is essential for catalytic function. Research has demonstrated that:

• Deletion of the dimerization domain in DapE from Haemophilus influenzae and Vibrio cholerae resulted in complete loss of enzymatic activity

• Molecular dynamics simulations indicate that removal of the dimerization domain increases the flexibility of a conserved active site loop

• This active site loop appears to provide critical interactions with the substrate during catalysis

• Dimerization likely stabilizes the correct orientation of metal-coordinating residues in the active site

These findings suggest that the quaternary structure of DapE is fundamentally linked to its catalytic mechanism. The dimerization domain appears to play a crucial role in maintaining the proper conformation of the active site, particularly the positioning of the catalytic loop. Researchers investigating DapE must therefore consider the enzyme as a functional dimer rather than focusing solely on the catalytic domain .

What metal cofactors are essential for DapE activity?

DapE enzymes typically contain a dinuclear zinc center in the active site, though other metals can substitute with varying effects on catalytic efficiency:

For kinetic studies, it's recommended to determine parameters after incubating the enzyme with three equivalents of Zn(II) for one hour to ensure full metalation . EXAFS spectroscopic studies suggest a (μ-aquo)(μ-carboxylato)dizinc(II) core structure in the active site . The first metal ion is tightly bound and essential for catalysis, while the second metal ion may be more loosely associated and might play a regulatory role.

How can site-directed mutagenesis be used to study DapE function?

Site-directed mutagenesis represents a powerful approach to probe the structure-function relationships in DapE:

• Metal-binding residues: Mutation of histidine and aspartate residues that coordinate zinc can distinguish roles in metal binding versus catalysis. For example, studies with His67A and H349A altered DapE enzymes showed significantly altered kinetic parameters

• Catalytic residues: The putative general acid/base residue (Glu134) can be mutated to probe the catalytic mechanism

• Active site loop: Mutations in the conserved loop can assess substrate interactions and specificity

• Dimerization interface: Alterations can test the importance of specific residues in maintaining quaternary structure

• Experimental design recommendations:

  • Use conservative mutations first (His→Ala, Asp→Asn, Glu→Gln)

  • Purify mutants under identical conditions to wild-type

  • Verify structural integrity via circular dichroism or thermal shift assays

  • Assess metal content via ICP-MS or colorimetric assays

  • Perform detailed kinetic characterization

• Data analysis: Compare kcat/Km values to distinguish effects on substrate binding versus catalytic turnover

What experimental approaches are effective for screening potential DapE inhibitors?

Researchers investigating DapE inhibitors should consider these methodological approaches:

• High-throughput enzymatic assays:

  • Colorimetric detection of succinate or free amine formation

  • Coupled-enzyme assays tracking NADH consumption

  • Fluorescent substrate analogs for direct binding measurements

• Biophysical screening methods:

  • Thermal shift assays (Thermofluor) to identify stabilizing compounds

  • Surface plasmon resonance for binding kinetics

  • Isothermal titration calorimetry for thermodynamic parameters

• In silico approaches:

  • Structure-based virtual screening using DapE crystal structures

  • Pharmacophore modeling based on substrate recognition elements

  • Molecular docking with flexible active site residues

• Important controls:

  • Include metal chelator controls to distinguish specific inhibition from metal sequestration

  • Counter-screen against human metallopeptidases to assess selectivity

  • Test compounds against bacterial growth to confirm whole-cell activity

• Validation methods:

  • X-ray crystallography of enzyme-inhibitor complexes

  • Enzyme kinetics to determine inhibition mechanism (competitive, uncompetitive, etc.)

  • Mutagenesis of key binding residues to confirm interaction sites

How does environmental pH affect DapE activity and stability?

The pH environment significantly impacts both the catalytic activity and stability of DapE enzymes:

These pH effects can be explained by several factors:

• Metal binding: Extreme pH conditions can affect zinc coordination, with acidic conditions potentially causing metal loss

• Catalytic residues: The protonation state of key catalytic residues (histidine, glutamate) is pH-dependent

• Substrate interactions: Ionic interactions between enzyme and substrate vary with pH

• Protein stability: Extreme pH can cause partial unfolding or aggregation

N. europaea, which tolerates pH 6.0-9.0 with optimal growth in slightly basic conditions , may have a DapE adapted to function optimally at higher pH values than DapE enzymes from neutrophilic bacteria.

What are the challenges in crystallizing recombinant N. europaea DapE?

Researchers face several technical challenges when attempting to crystallize N. europaea DapE:

• Protein production issues:

  • Slow growth rate of N. europaea makes native purification impractical

  • Recombinant expression requires codon optimization for E. coli

  • Ensuring consistent metalation state is critical

• Protein stability concerns:

  • Metal loss during purification

  • Oxidation of metal-coordinating cysteine or histidine residues

  • Conformational heterogeneity of loop regions

• Crystallization strategies:

  • Screening with and without substrate analogs or inhibitors

  • Surface entropy reduction mutations to promote crystal contacts

  • Exploration of both full-length protein and catalytic domain constructs

  • Metal ion screening (Zn2+, Co2+) to identify optimal conditions

  • Anaerobic crystallization if the protein is oxygen-sensitive

• Data collection considerations:

  • Radiation damage can alter metal coordination

  • Low-temperature data collection to minimize radiation effects

  • Multiple wavelength anomalous dispersion (MAD) using zinc anomalous signal

How do N. europaea DapE properties compare to those from other bacteria?

Comparative analysis reveals both similarities and differences between N. europaea DapE and homologs from other bacterial species:

• Sequence conservation:

  • Metal-binding residues (histidines, aspartates) are strictly conserved across species

  • The catalytic glutamate residue is conserved across DapE enzymes

  • Active site architecture appears largely conserved based on sequence analysis

• Environmental adaptations:

  • N. europaea, being an obligate chemolithoautotroph , may have evolved unique adaptations

  • As N. europaea grows optimally in slightly basic conditions , its DapE may have a higher pH optimum

  • The slow growth rate of N. europaea suggests its metabolic enzymes may have evolved for efficiency over speed

• Inhibition susceptibility:

  • Conservation of active site suggests similar inhibition profiles across species

  • Subtle differences in substrate binding loops may enable species-selective inhibitors

  • N. europaea's unique environmental niche may have driven evolutionary adaptations in its DapE

• Regulatory mechanisms:

  • N. europaea harbors numerous toxin-antitoxin systems that may regulate DapE expression

  • The presence of MazF endoribonuclease in N. europaea may modulate translation profiles

  • Environmental stresses trigger regulatory responses that may differ between species

How might N. europaea's lifestyle influence DapE function?

N. europaea's unique physiological characteristics likely influence DapE function:

• Chemolithoautotrophic metabolism:

  • N. europaea derives energy from ammonia oxidation and carbon from CO2

  • This unique metabolism may affect amino acid biosynthetic pathways including lysine synthesis

  • DapE may be regulated differently than in heterotrophic bacteria

• Environmental sensitivity:

  • N. europaea is susceptible to various environmental factors including temperature, pH, nitrite and ammonia concentrations, heavy metals, and organic/inorganic compounds

  • These sensitivities may be reflected in DapE adaptations

• Stress response mechanisms:

  • N. europaea harbors more than 50 type II toxin-antitoxin pairs

  • The MazF endoribonuclease system may regulate cellular activities during stress

  • Environmental stresses trigger specific responses that could affect DapE expression and activity

• Growth characteristics:

  • Slow division rate due to high ammonia consumption

  • Adaptation to alkaline environments (optimal pH slightly basic)

  • These factors may be reflected in kinetic properties of DapE

• Nitrogen cycle involvement:

  • N. europaea plays important roles in industrial, agricultural, and environmental nitrogen cycles

  • This ecological niche may have driven unique adaptations in nitrogen-containing amino acid pathways