Recombinant Acinetobacter sp. 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-succinyltransferase (dapD)

What is the expression protocol for recombinant Acinetobacter sp. dapD in E. coli?

The expression of recombinant Acinetobacter sp. dapD follows a protocol similar to that used for related bacterial enzymes. The gene encoding dapD should be cloned into an expression vector containing an appropriate promoter (such as T7) and a purification tag (commonly His6). The protein can be expressed in E. coli using the following protocol:

• Transform the expression construct into a suitable E. coli strain (BL21(DE3) or derivatives)

• Culture transformed bacteria in appropriate media (LB or minimal media) supplemented with required antibiotics

• Grow culture at 37°C with shaking (210 rpm) until OD600 reaches 1.0-1.5

• Reduce temperature to 18°C and induce with IPTG (0.5 mM final concentration)

• Continue growth for 18 hours before harvesting cells

This protocol has been effective for related Acinetobacter enzymes, yielding sufficient protein for biochemical and structural studies . For crystallographic studies, selenomethionine-labeled protein can be produced by growing the bacteria in selenomethionine-supplemented minimal media .

What purification methods are recommended for recombinant dapD?

Purification of His-tagged recombinant dapD can be achieved through a combination of affinity chromatography and size exclusion techniques. Based on protocols for similar enzymes, the following purification steps are recommended:

• Resuspend cell pellet in lysis buffer (10 mM Tris-HCl pH 8.3, 500 mM NaCl, 5% glycerol, 20 mM imidazole, 5 mM TCEP) at a ratio of approximately 5 mL buffer per gram of cells

• Lyse cells by sonication (50% amplitude, 5s × 10s cycles for 20 min at 4°C)

• Clear lysate by centrifugation (18,000 × g for 30 minutes)

• Apply supernatant to a nickel affinity column pre-equilibrated with lysis buffer

• Wash column with 10 column volumes of lysis buffer

• Elute protein with buffer containing higher imidazole concentration (250-300 mM)

• Conduct size exclusion chromatography to remove aggregates and ensure homogeneity

This procedure typically yields protein with >95% purity suitable for enzymatic and structural studies .

How can I assess the enzymatic activity of purified dapD?

The enzymatic activity of dapD can be evaluated using a spectrophotometric assay similar to those developed for related enzymes in the lysine biosynthesis pathway. The following approach is recommended:

• Prepare reaction buffer containing appropriate substrate (2,3,4,5-tetrahydropyridine-2,6-dicarboxylate) and required cofactors

• Add purified enzyme at various concentrations

• Monitor product formation spectrophotometrically

• Analyze reaction kinetics using Michaelis-Menten kinetics

For product detection, a colorimetric ninhydrin-based assay can be employed, similar to the detection method used for N6-Me-L,L-DAP as described for DapE enzymes . The reaction product forms a complex with ninhydrin that can be measured by absorbance at 570 nm, which can be converted to reaction velocity using appropriate calibration curves.

Table 1: Representative enzymatic activity data for recombinant dapD (hypothetical values based on similar enzymes in the lysine biosynthesis pathway)

What are the optimal storage conditions for maintaining dapD stability?

To maintain enzyme stability, dapD should be stored under conditions that prevent denaturation and loss of activity. Based on protocols for related enzymes:

• Short-term storage (1-2 weeks): Store at 4°C in buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol, and 1 mM DTT

• Long-term storage: Flash-freeze aliquots in liquid nitrogen and store at -80°C

• Avoid repeated freeze-thaw cycles, which can lead to protein denaturation

• For crystallography work, fresh protein preparations are recommended

Activity assays should be performed before and after storage to assess stability. Typical half-life at 4°C is approximately 7-10 days for similar enzymes from Acinetobacter species .

What genetic manipulation techniques are most effective for studying dapD function in Acinetobacter sp.?

Several genetic tools can be applied to study dapD function in Acinetobacter species, with selection of the appropriate technique depending on the specific research question:

Homologous Recombination for Gene Knockout

Homologous recombination represents an effective approach for dapD gene deletion to study its essentiality and function:

• Design a knockout cassette with an antibiotic resistance marker flanked by FRT sites and homologous regions upstream and downstream of dapD

• Clone the cassette into a suicide vector or maintain as a linear product

• Introduce into Acinetobacter via electroporation or natural transformation

• Select transformants on appropriate antibiotic media

• Optional: Remove the antibiotic marker using Flp recombinase for scarless deletion

For clinical isolates with high antibiotic resistance, alternative selection markers such as hygromycin or zeocin may be employed .

CRISPR-Cas9 Based Genome Editing

CRISPR-Cas9 provides precise genome editing capabilities for studying dapD:

• Construct a plasmid expressing Cas9 nuclease and appropriate sgRNA targeting dapD

• Include a repair template with desired modifications (point mutations, deletions, etc.)

• Introduce the system into Acinetobacter

• Select transformants and verify successful editing by sequencing

For Acinetobacter, an enhanced recombination efficiency can be achieved using RecAb from A. baumannii IS-123, which has demonstrated >10-fold higher efficiency compared to recombinases from other species .

How can site-directed mutagenesis be used to identify catalytic residues in dapD?

Site-directed mutagenesis provides valuable insights into the catalytic mechanism of dapD:

• Identify conserved residues through sequence alignment with related enzymes

• Design mutagenesis primers introducing specific amino acid substitutions

• Perform PCR-based site-directed mutagenesis

• Verify mutations by sequencing

• Express and purify mutant proteins

• Compare kinetic parameters of wild-type and mutant enzymes

Catalytic residues can be identified by evaluating the impact of mutations on key kinetic parameters:

Table 2: Hypothetical kinetic parameters for wild-type and mutant versions of dapD. Significant reductions in catalytic efficiency (kcat/Km) suggest involvement in catalysis or substrate binding.

Based on such analyses, residues showing dramatic reductions in catalytic efficiency when mutated (e.g., H67 and D103 in the hypothetical data) likely represent catalytic residues directly involved in the enzymatic mechanism.

What crystallization approaches are recommended for structural determination of dapD?

Crystallization of dapD for structural determination requires systematic screening and optimization:

• Concentrate purified protein to 10-15 mg/mL in crystallization buffer (typically 20 mM Tris-HCl pH 7.5, 150 mM NaCl)

• Screen various crystallization conditions using commercial sparse matrix screens

• Optimize promising conditions by varying:

  • Protein concentration

  • Precipitant concentration

  • pH

  • Temperature

  • Additives

• Collect high-resolution diffraction data at synchrotron sources

• Process data and solve structure using molecular replacement with related enzymes as search models

For selenomethionine-labeled protein, single-wavelength anomalous dispersion (SAD) or multi-wavelength anomalous dispersion (MAD) phasing methods can be employed .

Table 3: Representative crystallization conditions for dapD (hypothetical data based on crystallization of related enzymes)

Co-crystallization with substrates, products, or inhibitors can provide valuable insights into the enzyme's catalytic mechanism and binding mode .

How does dapD inhibition compare to related enzymes in the lysine biosynthesis pathway?

Inhibition studies of dapD can provide insights into potential antimicrobial development:

• Screen potential inhibitors at various concentrations

• Determine inhibition constants (Ki) using Michaelis-Menten kinetics

• Characterize inhibition mechanisms (competitive, non-competitive, uncompetitive)

• Compare inhibition profiles with related enzymes such as DapE

The evaluation of inhibitor efficacy follows methodologies established for related enzymes in the pathway . For enzyme inhibition studies, reactions are typically conducted with varying inhibitor concentrations while monitoring product formation spectrophotometrically.

Table 4: Hypothetical inhibition data comparing inhibitor effectiveness against dapD and DapE. Selectivity index represents the ratio of Ki values (DapE/dapD), with values >1 indicating greater selectivity for dapD.

Analysis of inhibitor binding can be further enhanced through structural studies of enzyme-inhibitor complexes, revealing specific interactions within the active site .

What approaches can be used to study dapD in the context of multi-drug resistant clinical isolates?

Studying dapD in multi-drug resistant (MDR) clinical isolates presents unique challenges that require specialized approaches:

• Genetic manipulation requires alternative selection markers beyond commonly used antibiotics

• Single-step homologous recombination techniques can create scarless deletions, allowing for the creation of multiple gene deletions using the same selection marker

• Evaluating essentiality of dapD across diverse clinical isolates provides insight into its potential as an antimicrobial target

For genetic manipulations in MDR strains, non-clinical antibiotic markers like hygromycin, zeocin, or apramycin can be used . Alternatively, non-antibiotic selection methods can be employed to circumvent resistance issues.

Table 5: Hypothetical data showing dapD essentiality across clinical isolates with varying resistance profiles. CAR: carbapenems, FLQ: fluoroquinolones, AMG: aminoglycosides, POL: polymyxins. MDR: multi-drug resistant, XDR: extensively drug resistant.

The uniform essentiality of dapD across diverse clinical isolates would support its potential as a broad-spectrum antimicrobial target.

How can I troubleshoot low expression yields of recombinant dapD?

Low expression yields of recombinant dapD can be addressed through systematic optimization:

• Codon optimization: Adapt the dapD gene sequence for optimal expression in E. coli

• Expression strain selection: Screen multiple E. coli strains (BL21(DE3), C41(DE3), Rosetta, etc.)

• Expression temperature optimization: Test induction at various temperatures (15°C, 18°C, 25°C, 30°C)

• IPTG concentration: Optimize inducer concentration (0.1-1.0 mM)

• Media composition: Compare rich media (LB, TB) versus minimal media

• Expression time: Evaluate different post-induction incubation periods

Systematic optimization can significantly improve protein yields:

Table 6: Hypothetical optimization data for recombinant dapD expression under various conditions

What strategies can address protein solubility issues with dapD?

Solubility issues with recombinant dapD can be addressed through various strategies:

• Fusion tags: Incorporate solubility-enhancing tags (MBP, SUMO, GST)

• Buffer optimization: Screen buffers with varying pH, salt concentration, and additives

• Co-expression with chaperones: Express dapD alongside molecular chaperones (GroEL/ES, DnaK/J)

• Truncation constructs: Design and express stable domains of dapD if the full-length protein is insoluble

• Detergents: Include mild detergents in lysis and purification buffers for proteins with hydrophobic regions

Comparing solubility enhancement strategies:

Table 7: Hypothetical data comparing various strategies for improving dapD solubility

How can structural analysis of dapD inform drug discovery efforts?

Structural analysis of dapD can significantly accelerate drug discovery through:

• Identification of catalytic residues and binding pocket characteristics

• Structure-based virtual screening to identify potential inhibitors

• Fragment-based drug design targeting specific regions of the active site

• Analysis of conformational changes during catalysis to identify allosteric sites

• Comparison with human enzymes to ensure selectivity

Structure-guided design has proven successful for related bacterial enzymes, with structural information providing insights into inhibitor specificity and potency . The potential of dapD as an antimicrobial target can be further evaluated through structure-activity relationship studies of identified inhibitors.

What are the current challenges in developing dapD as an antimicrobial target?

Development of dapD inhibitors as antimicrobials faces several challenges:

• Selectivity: Ensuring inhibitors target bacterial dapD without affecting human enzymes

• Cell penetration: Designing molecules that can penetrate the bacterial cell envelope

• Efflux resistance: Avoiding recognition by bacterial efflux pumps

• Metabolic stability: Ensuring sufficient stability for in vivo efficacy

• Target validation: Confirming essentiality across diverse clinical isolates

Addressing these challenges requires multidisciplinary approaches combining structural biology, medicinal chemistry, and microbiology. Comparative analysis of dapD with related enzymes like DapE can provide insights into designing selective inhibitors .