Recombinant Pseudomonas putida D-alanine--D-alanine ligase B (ddlB)

What is the primary function of D-alanine--D-alanine ligase B (ddlB) in Pseudomonas putida?

D-alanine--D-alanine ligase B (ddlB) in Pseudomonas putida catalyzes the ATP-dependent formation of the D-alanyl-D-alanine dipeptide, which is an essential precursor for peptidoglycan biosynthesis in bacterial cell walls. This enzyme belongs to the ATP-grasp superfamily and plays a critical role in cell wall integrity and bacterial survival. Like its homolog in P. aeruginosa, P. putida ddlB likely catalyzes the condensation of two D-alanine molecules using ATP as an energy source to form the peptide bond. The resulting D-alanyl-D-alanine dipeptide is then incorporated into the pentapeptide stem of the peptidoglycan precursor, which is essential for maintaining cell shape and protecting against osmotic pressure .

How does Pseudomonas putida ddlB differ structurally from homologous enzymes in other bacterial species?

What expression systems are most effective for producing recombinant P. putida ddlB?

For heterologous expression of P. putida proteins, including ddlB, several expression systems have proven effective:

• E. coli-based expression systems: Standard bacterial expression vectors containing T7 or tac promoters are commonly used, with BL21(DE3) or similar strains as hosts.

• Homologous expression in P. putida: Using P. putida itself as an expression host offers advantages for proper folding and potential post-translational modifications of native proteins.

• Promoter selection: For heterologous gene expression in P. putida, various promoters have been employed successfully:

For optimal recombinant ddlB production, an E. coli BL21(DE3) system with pET vectors often provides high yields, while expression in P. putida itself using the Ptac promoter may provide protein with native-like characteristics for functional studies .

How can I optimize enzymatic activity assays for recombinant P. putida ddlB?

Optimizing enzymatic activity assays for recombinant P. putida ddlB requires careful consideration of multiple factors:

• Assay methodology: The most common methods include:

  • ATP-consumption assays (coupled enzymatic assays monitoring ADP production)

  • Direct monitoring of D-alanyl-D-alanine formation using HPLC or LC-MS

  • Malachite green-based phosphate detection assays

• Buffer optimization:

  • Test pH range 7.0-8.5 (typically optimal around pH 7.8)

  • Include divalent cations (Mg²⁺ or Mn²⁺) at 5-10 mM

  • Test various ionic strengths (50-200 mM KCl or NaCl)

• Substrate concentrations:

  • D-alanine: 0.1-10 mM

  • ATP: 0.5-5 mM

• Controls:

  • Include negative controls (heat-inactivated enzyme)

  • Use D-cycloserine as a positive control inhibitor

  • Consider running parallel assays with commercially available E. coli or P. aeruginosa Ddl enzymes

Based on studies with P. aeruginosa DdlB, recombinant P. putida ddlB likely catalyzes D-alanyl-D-alanine production with comparable efficiency to its homologs. Activity can be effectively disrupted by D-cycloserine, which serves as a useful control in activity assays .

What strategies are effective for crystallizing P. putida ddlB for structural studies?

Based on successful crystallization of related D-alanine--D-alanine ligases, the following strategies are recommended for P. putida ddlB crystallization:

• Protein preparation:

  • Ensure high purity (>95% by SDS-PAGE)

  • Use size exclusion chromatography as a final purification step

  • Test both His-tagged and tag-cleaved versions

  • Concentrate to 10-15 mg/ml in a stabilizing buffer

• Crystallization conditions:

  • Screen with commercial sparse matrix kits

  • Focus on conditions containing:

    • PEG 3350-8000 (12-25%)

    • pH range 6.5-8.0

    • Various salts (ammonium sulfate, lithium sulfate)

• Co-crystallization approaches:

  • With ATP or non-hydrolyzable ATP analogs

  • With D-alanine or D-alanyl-D-alanine product

  • With inhibitors (e.g., D-cycloserine)

• Data collection considerations:

  • Cryoprotect crystals (typically 20-25% glycerol or ethylene glycol)

  • Consider using synchrotron radiation for high-resolution data

P. aeruginosa DdlA and DdlB were successfully co-crystallized with ATP and either D-alanyl-D-alanine or D-cycloserine, which allowed direct comparison of key structural features. Similar approaches would likely be effective for P. putida ddlB .

How do mutations in the active site of P. putida ddlB affect substrate specificity and catalytic efficiency?

Mutations in the active site of P. putida ddlB can significantly alter its substrate specificity and catalytic properties. Based on structural and functional studies of related D-alanine--D-alanine ligases:

• ATP-binding pocket mutations:

  • Alterations to residues coordinating the adenine base can affect ATP binding affinity and orientation

  • Mutations in phosphate-binding regions may alter the rate of phosphoryl transfer

• D-alanine binding site mutations:

  • The D-alanine pocket is typically highly conserved

  • Even conservative substitutions can dramatically affect substrate specificity

  • Mutations may allow binding of non-canonical amino acids (e.g., D-serine, D-lactate)

• Omega-loop region:

  • Mutations in this flexible region can alter the coordination between ATP and D-alanine binding sites

  • May affect product release and enzyme turnover rates

While specific data for P. putida ddlB mutations is not directly provided in the search results, comparative analysis with P. aeruginosa DdlB suggests that both enzymes likely share similar catalytic mechanisms and sensitivity to active site perturbations. In P. aeruginosa, both DdlA and DdlB isoforms effectively catalyze D-alanine--D-alanine production with nearly identical efficiency, despite some differences in their ATP-binding pockets .

What are the optimal conditions for heterologous expression of P. putida ddlB?

Based on successful heterologous expression of other P. putida proteins, the following conditions are recommended for optimal recombinant ddlB production:

• Expression host selection:

  • E. coli BL21(DE3) or derivatives for high-yield expression

  • P. putida KT2440 for homologous expression with native characteristics

• Vector design considerations:

  • Include a strong, inducible promoter (T7, tac, or Pm/XylS)

  • Optimize codon usage if expressing in E. coli

  • Consider fusion tags for purification (His6, MBP, or GST)

• Culture conditions for E. coli expression:

  • LB or TB media supplemented with appropriate antibiotics

  • Growth at 37°C until OD600 = 0.6-0.8

  • Induction at lower temperatures (16-25°C) for enhanced solubility

  • IPTG concentration: 0.1-0.5 mM

  • Post-induction expression: 16-20 hours at 16°C or 4-6 hours at 25°C

• Culture conditions for P. putida expression:

  • M9 minimal media or nutrient-rich media as appropriate

  • Growth at 30°C to OD600 = 0.6-0.8

  • Induction with appropriate inducer based on promoter

  • Post-induction expression: 12-24 hours at 25-30°C

P. putida has emerged as an excellent platform for recombinant protein production, offering advantages such as versatile metabolism and xenobiotic tolerance. These characteristics make it particularly suitable for producing enzymes involved in its own metabolic pathways, including ddlB .

What purification strategy yields the highest activity of recombinant P. putida ddlB?

A multi-step purification strategy is recommended to obtain high-activity recombinant P. putida ddlB:

• Initial capture:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged protein

  • Buffer composition: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol

  • Imidazole gradient: 20-300 mM for elution

• Intermediate purification:

  • Ion exchange chromatography (typically Q-Sepharose)

  • Buffer: 20 mM Tris-HCl pH 7.5, 50 mM NaCl

  • Elution with 50-500 mM NaCl gradient

• Polishing step:

  • Size exclusion chromatography (Superdex 200)

  • Running buffer: 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM DTT

• Critical factors affecting enzyme activity:

  • Include 5-10 mM MgCl2 in all buffers to stabilize the active site

  • Add 0.5-1 mM DTT to prevent oxidation of cysteine residues

  • Consider adding 10% glycerol to enhance protein stability

  • Avoid freezing/thawing cycles; store aliquots at -80°C

Typically, this approach yields protein with >95% purity and specific activity comparable to native enzyme. Similar purification strategies have been successfully employed for other P. putida recombinant enzymes, and for homologous D-alanine--D-alanine ligases from related bacteria .

How can I develop selective inhibitors targeting P. putida ddlB?

Developing selective inhibitors against P. putida ddlB requires a multifaceted approach:

• Structure-based design strategy:

  • Identify unique structural features of P. putida ddlB compared to homologs

  • Target differences in the ATP-binding pocket

  • Focus on allosteric sites specific to P. putida ddlB

• High-throughput screening approach:

  • Develop a robust enzymatic assay suitable for HTS format

  • Screen diverse chemical libraries (natural products, synthetic compounds)

  • Use counter-screens against homologous enzymes to identify selective hits

• Rational modification of known inhibitors:

  • Start with D-cycloserine or phosphinate transition-state analogs

  • Modify to exploit unique features of P. putida ddlB

  • Optimize for selectivity over other D-alanine--D-alanine ligases

• Fragment-based approach:

  • Screen fragment libraries using biophysical methods (thermal shift, NMR)

  • Link or grow fragments that bind to different regions of the active site

  • Optimize for potency while maintaining selectivity

Based on studies with P. aeruginosa DdlA and DdlB, both isoforms possess the same structural architecture and share high conservation within the active site. While the D-alanine pocket is completely conserved, the ATP-binding pocket shows several amino acid substitutions resulting in a different chemical environment around the ATP adenine base. These differences could be exploited for developing selective inhibitors against specific ddlB variants .

How can I address solubility issues when expressing recombinant P. putida ddlB?

Researchers frequently encounter solubility challenges with recombinant P. putida ddlB. Here are evidence-based strategies to overcome these issues:

• Expression optimization:

  • Lower induction temperature (16-18°C)

  • Reduce inducer concentration (0.1 mM IPTG)

  • Use slower growing media (M9 minimal media with glucose)

  • Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

• Fusion partners to enhance solubility:

  • MBP (maltose-binding protein)

  • SUMO

  • Thioredoxin

  • GST (glutathione S-transferase)

• Buffer optimization during lysis and purification:

  • Include stabilizing agents: 10% glycerol, 0.1% Triton X-100

  • Add osmolytes: 0.5 M sorbitol, 0.5-1 M proline

  • Test various salt concentrations (150-500 mM NaCl)

  • Include cofactors: 5 mM MgCl2, 1 mM ATP

• Refolding strategy if inclusion bodies form:

  • Solubilize inclusion bodies with 8 M urea or 6 M guanidine HCl

  • Remove denaturant by gradual dialysis or rapid dilution

  • Include an oxidation/reduction system (GSH/GSSG) if disulfide bonds are present

How do I troubleshoot inconsistent activity in recombinant P. putida ddlB preparations?

Inconsistent enzymatic activity is a common challenge when working with recombinant P. putida ddlB. A systematic troubleshooting approach includes:

• Protein quality assessment:

  • Verify protein purity by SDS-PAGE (>95% purity recommended)

  • Confirm identity by mass spectrometry or western blotting

  • Assess protein homogeneity by size exclusion chromatography

  • Check for proper folding using circular dichroism

• Storage condition optimization:

  • Test stability at different storage temperatures (4°C, -20°C, -80°C)

  • Compare stability in different buffer compositions

  • Evaluate the impact of additives (glycerol, DTT, metal ions)

  • Determine optimal protein concentration for storage

• Enzymatic assay variables:

  • Ensure consistent buffer composition and pH

  • Verify quality and concentration of substrates

  • Control reaction temperature precisely

  • Standardize enzyme addition and mixing protocols

• Batch-to-batch variation sources:

  • Expression conditions (media composition, induction timing)

  • Purification protocol consistency

  • Buffer preparation accuracy

  • Equipment calibration status

Based on studies with P. aeruginosa DdlA and DdlB, both isoforms effectively catalyze D-alanine--D-alanine production with near identical efficiency, and activity is effectively disrupted by D-cycloserine. Similar behavior would be expected for P. putida ddlB, and D-cycloserine could be used as a control to validate assay performance across different enzyme preparations .

What approaches can resolve conflicting kinetic data for P. putida ddlB with different substrates?

Researchers often encounter contradictory kinetic data when characterizing P. putida ddlB with various substrates. To resolve such conflicts, consider these methodological approaches:

• Standardize experimental conditions:

  • Use identical buffer systems across experiments

  • Maintain consistent temperature and pH

  • Employ the same enzyme preparation or standardize between batches

  • Define a standard assay protocol and adhere to it rigorously

• Employ multiple, complementary assay methods:

  • ATP consumption (luciferase-based assay)

  • Phosphate release (malachite green assay)

  • Product formation (HPLC or LC-MS)

  • Calorimetric methods (isothermal titration calorimetry)

  Assay Method	Advantages	Limitations	Best For
  Coupled enzyme (ADP detection)	Continuous, high-throughput	Potential coupling enzyme interference	Initial rate determination
  HPLC product detection	Direct product measurement	Discontinuous, labor-intensive	Definitive product verification
  Malachite green	Simple, cost-effective	Discontinuous, phosphate contamination sensitive	High-throughput screening
  ITC	Label-free, direct	Requires specialized equipment, high protein amounts	Thermodynamic parameters

• Comprehensive kinetic analysis:

  • Determine full kinetic parameters (kcat, Km) for each substrate

  • Analyze reaction mechanisms (ordered vs. random)

  • Assess product inhibition effects

  • Evaluate cofactor dependencies

• Statistical validation:

  • Perform experiments in triplicate at minimum

  • Apply appropriate statistical tests

  • Consider Bayesian analysis for complex datasets

  • Validate with independent enzyme preparations

D-alanine--D-alanine ligases typically follow an ordered kinetic mechanism where ATP binds first, followed by the first D-alanine, then the second D-alanine. Deviations from this mechanism or substrate preference variations between enzyme preparations could explain contradictory results and should be systematically investigated .

How can I distinguish between the roles of ddlA and ddlB in Pseudomonas putida?

Distinguishing between the biological roles of the two D-alanine--D-alanine ligase isoforms (ddlA and ddlB) in P. putida requires multiple experimental approaches:

• Gene expression analysis:

  • qRT-PCR to measure transcript levels under various conditions

  • RNA-seq to determine expression patterns in different growth phases

  • Reporter gene fusions to monitor promoter activity in situ

• Genetic manipulation strategies:

  • Generate single knockout mutants (ΔddlA and ΔddlB)

  • Create double knockout with complementation plasmids

  • Employ conditional expression systems for essential genes

  • Use RecET-based markerless gene deletion protocols for cleaner genetic backgrounds

• Biochemical characterization:

  • Compare enzyme kinetics of purified recombinant ddlA and ddlB

  • Assess substrate specificity differences

  • Evaluate inhibitor sensitivity profiles

  • Determine temperature and pH optima for each isoform

• Physiological role assessment:

  • Growth rate comparisons of mutants under various conditions

  • Cell morphology analysis

  • Peptidoglycan composition analysis

  • Antibiotic susceptibility testing

  • Stress response evaluation

Based on studies in P. aeruginosa, both DdlA and DdlB isoforms effectively catalyze D-alanine--D-alanine production with nearly identical efficiency, but may have different expression patterns or regulatory mechanisms. While the D-alanine binding pocket is completely conserved between isoforms, differences in the ATP-binding pocket create a different chemical environment, which may be relevant to their specific cellular roles .

What potential exists for engineering P. putida ddlB for biotechnological applications?

Engineering P. putida ddlB holds significant promise for various biotechnological applications:

• Expanding substrate specificity:

  • Engineer ddlB to accept non-canonical amino acids for novel peptide production

  • Modify binding pockets to incorporate D-hydroxy acids for depsipeptide synthesis

  • Create chimeric enzymes with regions from other ATP-grasp ligases

• Biocatalytic applications:

  • Develop ddlB variants for industrial peptide synthesis

  • Engineer thermostable or solvent-tolerant variants

  • Create immobilized enzyme systems for continuous production

• Platform for drug discovery:

  • Use engineered ddlB variants to screen for novel antimicrobials

  • Develop high-throughput screening systems based on modified ddlB

  • Create biosensors for detecting antimicrobial compounds

• Integration with synthetic biology approaches:

  • Incorporate engineered ddlB into synthetic peptidoglycan biosynthesis pathways

  • Develop cell-free systems for peptide synthesis using modified ddlB

  • Create orthogonal cell wall biosynthesis pathways

P. putida has emerged as an excellent platform for synthetic biology applications due to its versatile metabolism and tolerance to xenobiotics. These characteristics make it particularly suitable for housing engineered enzymes like modified ddlB for various biotechnological processes. The recombinant biosynthesis capabilities of P. putida have been demonstrated for various valuable natural products, suggesting that engineered ddlB could similarly be exploited for novel applications .

How can structural data on P. putida ddlB inform new antimicrobial development?

Structural insights into P. putida ddlB can drive innovative antimicrobial discovery through several approaches:

• Structure-based inhibitor design:

  • Identify unique structural features of bacterial ddlB enzymes

  • Design transition-state analogs based on the catalytic mechanism

  • Develop allosteric inhibitors targeting non-conserved regions

  • Create covalent inhibitors targeting specific active site residues

• Comparative structural analysis:

  • Align structures of ddlB from various bacterial pathogens

  • Identify conserved pockets for broad-spectrum inhibitor design

  • Map species-specific features for selective targeting

  • Analyze differences between bacterial and mammalian ATP-utilizing enzymes

• Dynamics-based approaches:

  • Use molecular dynamics simulations to identify transient binding pockets

  • Design inhibitors targeting enzyme conformational changes

  • Identify cooperative motions essential for catalysis

  • Develop compounds that disrupt protein-protein interactions

• Crystallographic fragment screening:

  • Identify fragment binding sites across the protein structure

  • Link fragments binding to adjacent pockets

  • Optimize fragment hits based on structural data

  • Develop fragment-derived lead compounds

Structural studies of P. aeruginosa DdlA and DdlB have revealed that both isoforms possess the same structural architecture with high conservation in the active site. The ATP-binding pocket shows several amino acid substitutions between isoforms, creating different chemical environments around the ATP adenine base. These findings support that the discovery of dual-acting competitive inhibitors targeting both ddlA and ddlB is a viable approach for developing new antibiotics .

What is the potential for using P. putida ddlB inhibitors in combination therapy against resistant pathogens?

The strategic use of P. putida ddlB inhibitors in combination therapy offers promising approaches against resistant pathogens:

• Synergistic combinations with existing antibiotics:

  • Pair ddlB inhibitors with β-lactams to enhance cell wall targeting

  • Combine with efflux pump inhibitors to increase intracellular concentrations

  • Use alongside membrane-disrupting agents for enhanced penetration

  • Couple with quorum sensing inhibitors to reduce virulence

• Resistance mitigation strategies:

  • Target multiple steps in peptidoglycan biosynthesis simultaneously

  • Develop dual-target inhibitors affecting both ddlA and ddlB

  • Create hybrid molecules linking ddlB inhibition with other mechanisms

  • Design sequential treatment protocols to prevent resistance development

• Species-selective approaches:

  • Engineer inhibitors targeting unique features of specific bacterial ddlB variants

  • Develop narrow-spectrum agents for precision antimicrobial therapy

  • Design inhibitors exploiting pathogen-specific metabolic dependencies

  • Create prodrugs activated by pathogen-specific enzymes

• Formulation and delivery considerations:

  • Develop nanoparticle formulations for targeted delivery

  • Create biofilm-penetrating formulations

  • Design controlled-release systems for sustained inhibitor activity

  • Explore topical applications for localized infections