Recombinant Desulfotalea psychrophila D-alanine--D-alanine ligase (ddl)

What is the function of D-alanine--D-alanine ligase in bacterial peptidoglycan synthesis?

D-alanine--D-alanine ligase (Ddl) catalyzes the ATP-dependent formation of the D-alanyl-D-alanine dipeptide, an essential building block in bacterial peptidoglycan biosynthesis . The reaction mechanism occurs in multiple steps:

• ATP and two Mg²⁺ ions bind to the enzyme, facilitating closure of the P-loop

• The first D-alanine (D-Ala 1) binds adjacent to ATP, inducing closure of the Ω-loop

• The carboxylate of D-Ala 1 attacks the γ-phosphate of ATP, forming an acylphosphate intermediate (D-Ala-phosphate)

• The second D-alanine (D-Ala 2) enters the active site in a deprotonated form

• The deprotonated D-Ala 2 attacks the phosphorylated carbonyl carbon of D-Ala 1

• A tetrahedral intermediate forms (D-Ala-D-Ala-phosphate), which resolves to the final dipeptide product

To study this enzyme function, researchers typically use phosphate detection assays that measure the inorganic phosphate generated during the D-Ala-D-Ala ligation reaction. The reaction can be monitored by light absorbance at 650 nm using 384-well or 96-well assay plates, or via cuvette measurements for lower-throughput applications .

How does the structure of Desulfotalea psychrophila Ddl enable its cold-adaptive properties?

Desulfotalea psychrophila is a marine sulfate-reducing delta-proteobacterium capable of growth at temperatures below 0°C . The cold-adaptive properties of its D-alanine--D-alanine ligase derive from structural modifications that enhance flexibility at low temperatures:

Researchers investigating these properties typically employ comparative structural analysis with mesophilic homologs, thermal stability assays, and molecular dynamics simulations to identify flexibility-enhancing features .

What assay methods can accurately measure D. psychrophila Ddl enzyme activity at low temperatures?

To accurately measure the activity of psychrophilic D-alanine--D-alanine ligase at low temperatures, researchers employ several methodological approaches:

• Phosphate detection assays - These measure the inorganic phosphate generated during the ATP-dependent ligation reaction, with detection by colorimetric methods at 650 nm

• Coupled enzyme assays - These link Ddl activity to NADH oxidation through auxiliary enzymes, allowing continuous monitoring via absorbance decrease at 340 nm

• Temperature-controlled kinetic studies - Comparing enzyme activity across temperature ranges (0-30°C) to determine:

  • Temperature optimum

  • Activation energy (Ea)

  • kcat/Km ratio at different temperatures

• ¹⁸O isotope exchange (PIX) experiments - These monitor enzyme activity through ³¹P NMR by tracking oxygen exchange between water and phosphoryl groups, as demonstrated with other Ddl enzymes

When designing these assays for D. psychrophila Ddl, researchers should:

• Pre-incubate all reagents at the target temperature

• Use temperature-controlled microplate readers or spectrophotometers

• Include appropriate controls for spontaneous ATP hydrolysis at each temperature

• Employ high-sensitivity detection methods for the lower activity typical at reduced temperatures

How does the genomic context of ddl in Desulfotalea psychrophila compare to other bacteria?

The D. psychrophila genome consists of a 3,523,383 bp circular chromosome with 3118 predicted genes and two plasmids of 121,586 bp and 14,663 bp . The genomic context of ddl in D. psychrophila reveals several features relevant to its adaptation to cold environments:

• D. psychrophila encodes more than 30 two-component regulatory systems, including a new Ntr subcluster of hybrid kinases, which likely help regulate metabolic adaptations to cold environments

• The genome contains nine putative cold shock proteins and nine potentially cold shock-inducible proteins, suggesting sophisticated cold-adaptive mechanisms

• Unlike typical mesophilic bacteria, D. psychrophila shows a distinct phylogenetic clustering of histidine kinases in the Ntr group, forming a new subcluster of phylogenetically distinct HKs not found in non-Desulfotalea members

• The ddl gene likely works in concert with genes encoding other cell wall biosynthesis enzymes, but with temperature-specific adaptations

• Comparative genomic analysis with Archaeoglobus fulgidus (a hyperthermophilic archaeon) revealed "many striking differences, but only a few shared features," highlighting divergent evolutionary adaptations for extreme temperature ranges

When studying the genomic context, researchers typically employ comparative genomics, transcriptomics at different temperatures, and gene neighborhood analysis to understand the cold-adaptive regulatory networks involving ddl.

How can conformational changes in D. psychrophila Ddl be studied during substrate binding and catalysis?

Investigating conformational changes in D. psychrophila D-alanine--D-alanine ligase requires sophisticated biophysical techniques that can capture protein dynamics at low temperatures:

• Molecular Dynamics (MD) Simulations:

  • Simulate lid loop movements during substrate binding and catalysis

  • Model opening/closing transitions in response to substrate binding

  • Compare flexibility parameters with mesophilic homologs

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Monitor conformational flexibility and solvent accessibility changes at different temperatures

  • Compare regional dynamics between substrate-free and substrate-bound states

• Single-molecule FRET:

  • Introduce fluorophore pairs at strategic positions (e.g., lid loop and core domain)

  • Monitor real-time conformational changes during catalysis

  • Perform at different temperatures to assess cold-adaptive dynamics

• Cryo-EM or X-ray Crystallography:

  • Capture multiple conformational states by varying substrate combinations

  • Compare structures with mesophilic homologs like E. coli DdlB, which has been studied using these techniques

Research by Šink et al. on E. coli DdlB revealed that the lid loop undergoes significant conformational changes during catalysis, which are likely even more pronounced in psychrophilic variants to accommodate substrate binding at low temperatures .

What mechanisms explain D-cycloserine inhibition of D. psychrophila Ddl compared to mesophilic homologs?

D-cycloserine (DCS) is a known inhibitor of D-alanine--D-alanine ligase, but its inhibition mechanism may differ between psychrophilic and mesophilic enzymes. Recent research has revealed that DCS inhibition of E. coli DdlB involves formation of a phosphorylated DCS derivative:

• Novel inhibition mechanism:

  • DCS becomes phosphorylated by ATP in the enzyme active site

  • The phosphorylated DCS (DCSP) mimics the D-alanyl phosphate reaction intermediate

  • This phosphorylated form, not previously recognized, is the actual inhibitory species

• Comparative analysis for D. psychrophila Ddl:

  • Researchers should investigate whether the cold-adapted enzyme shows similar phosphorylation of DCS

  • Temperature-dependent kinetic analysis comparing inhibition constants (Ki) at various temperatures

  • Structural studies to determine if binding modes differ from mesophilic homologs

• Methodological approaches:

  • ³¹P NMR spectroscopy to detect phosphorylated inhibitor species in enzyme reactions

  • Crystal structures of enzyme-inhibitor complexes at different temperatures

  • Inhibition kinetics studies using phosphate-release assays

For D. psychrophila Ddl, the enhanced flexibility typical of psychrophilic enzymes might influence inhibitor binding and phosphorylation kinetics, potentially altering inhibition mechanisms compared to mesophilic homologs.

How can D. psychrophila Ddl be utilized as a model system for studying cold adaptation mechanisms?

D. psychrophila Ddl serves as an excellent model system for studying cold adaptation mechanisms due to its essential role in bacterial cell wall synthesis and well-characterized mesophilic counterparts:

• Comparative structural analysis:

  • Analyze amino acid composition showing adaptation patterns:

    • Lower proline content to reduce folding constraints at low temperatures

    • Potentially modified surface charges to maintain solubility in cold conditions

    • Reduced hydrophobic core packing to enhance flexibility

• Folding kinetics investigation:

  • Compare folding and unfolding rate constants at various temperatures

  • Assess the role of trigger factor (TF) and other cold-shock proteins in assisting proper folding

  • Investigate the impact of prolyl isomerases on proper protein folding at low temperatures

• Enzyme kinetics across temperature gradients:

  • Determine temperature dependence of catalytic parameters (kcat, Km)

  • Calculate activation energy (Ea) and compare with mesophilic homologs

  • Analyze enthalpy-entropy compensation patterns unique to psychrophilic enzymes

• Protein engineering approaches:

  • Create chimeric enzymes with mesophilic counterparts to identify cold-adaptive regions

  • Perform site-directed mutagenesis to modify flexibility-enhancing residues

  • Test engineered variants across temperature ranges to validate cold-adaptive features

These methodological approaches together provide a comprehensive framework for understanding the molecular basis of enzyme cold adaptation using D. psychrophila Ddl as a model system.

What approaches can be used to engineer D. psychrophila Ddl for enhanced thermostability while maintaining activity at low temperatures?

Engineering D. psychrophila Ddl for improved thermostability while preserving cold activity represents a significant challenge requiring targeted molecular approaches:

• Computational design strategies:

  • Identify flexible regions using molecular dynamics simulations

  • Perform in silico mutation analysis to predict stabilizing modifications

  • Use approaches like FRESCO (Framework for Rapid Enzyme Stabilization by Computational libraries) to identify stabilizing mutations

• Targeted mutagenesis approaches:

  • Introduce disulfide bridges at strategic locations without affecting active site flexibility

  • Replace thermolabile residues (Asn, Gln) in non-essential positions

  • Optimize surface charge distribution to enhance stability without compromising activity

• Directed evolution methodologies:

  • Develop dual-selection systems that simultaneously screen for:

    • Activity at low temperatures (0-10°C)

    • Stability at moderate temperatures (30-40°C)

  • Use error-prone PCR or DNA shuffling to generate variant libraries

  • Employ high-throughput activity assays to identify promising candidates

• Evaluation protocol:

  • Measure kinetic parameters (kcat, Km) at multiple temperatures (0-40°C)

  • Determine thermal unfolding profiles using differential scanning calorimetry

  • Assess long-term storage stability at different temperatures

  • Verify catalytic efficiency at low temperatures is maintained

How can advanced mass spectrometry techniques be applied to study post-translational modifications in D. psychrophila Ddl?

Advanced mass spectrometry techniques offer powerful tools for characterizing post-translational modifications (PTMs) that may regulate D. psychrophila Ddl activity in cold environments:

• Bottom-up proteomics approach:

  • Enzymatic digestion of purified Ddl with multiple proteases (trypsin, chymotrypsin)

  • LC-MS/MS analysis with high-resolution mass spectrometers (Orbitrap or QTOF)

  • Database searching with variable modifications (phosphorylation, methylation, acetylation)

  • Validation of PTM sites through synthetic peptide standards

• Top-down proteomics strategy:

  • Direct analysis of intact Ddl protein

  • Electron transfer dissociation (ETD) or electron capture dissociation (ECD) fragmentation

  • Quantification of modification stoichiometry

  • Correlation of modification patterns with enzyme activity states

• Temperature-dependent modification analysis:

  • Compare PTM profiles at different temperatures (0°C, 10°C, 20°C)

  • Correlate modifications with temperature-specific activity regulation

  • Identify cold-adaptive PTM signatures

• Targeted phosphoproteomics:

  • Metal oxide affinity chromatography (MOAC) or immobilized metal affinity chromatography (IMAC) enrichment

  • Parallel reaction monitoring (PRM) or multiple reaction monitoring (MRM) for quantification

  • Site-specific phosphorylation kinetics during enzyme reaction cycle

These advanced mass spectrometry approaches can reveal how PTMs might contribute to cold adaptation mechanisms in D. psychrophila Ddl and potentially identify novel regulatory mechanisms specific to psychrophilic enzymes.

What comparative genomic and evolutionary approaches reveal the adaptation of D. psychrophila Ddl to cold environments?

Comparative genomic and evolutionary analyses provide critical insights into the adaptation mechanisms of D. psychrophila Ddl to cold environments:

• Phylogenetic analysis:

  • Construct phylogenetic trees using Ddl sequences from psychrophilic, mesophilic, and thermophilic bacteria

  • Identify evolutionary lineages associated with temperature adaptation

  • Map key adaptive mutations on phylogenetic branches

  • Calculate evolutionary rates in different protein regions

• Sequence conservation analysis:

  • Calculate amino acid conservation scores across Ddl homologs

  • Identify cold-specific amino acid substitution patterns

  • Map conservation onto structural models to identify adaptive hotspots

  • Compare with other D. psychrophila cold-adapted enzymes

• Codon usage analysis:

  • Examine codon bias patterns in psychrophilic ddl genes

  • Correlate with tRNA abundance and optimal expression at low temperatures

  • Compare with housekeeping genes to identify cold-specific signatures

• Horizontal gene transfer assessment:

  • Analyze genomic context and GC content

  • Evaluate potential acquisition of cold-adaptive features through HGT

  • Compare with closely related Desulfotalea species

The comparative genomic analysis between D. psychrophila and the hyperthermophilic Archaeoglobus fulgidus showed "many striking differences, but only a few shared features" , highlighting the divergent evolutionary pathways these organisms have taken to adapt to extreme temperature environments. The unique clustering of histidine kinases in D. psychrophila further suggests specialized regulatory adaptations to cold environments that may include regulation of cell wall synthesis enzymes like Ddl.

What are the optimal storage conditions for maintaining the stability and activity of recombinant D. psychrophila Ddl?

Proper storage of recombinant D. psychrophila Ddl is critical for maintaining its structural integrity and enzymatic activity:

• Storage temperature recommendations:

  • Liquid form: 6 months at -20°C/-80°C

  • Lyophilized form: 12 months at -20°C/-80°C

  • Working aliquots: Up to one week at 4°C

• Buffer formulation guidelines:

  • Include 5-50% glycerol as a cryoprotectant (50% is recommended as default)

  • Use deionized sterile water for reconstitution

  • Reconstitute to 0.1-1.0 mg/mL concentration

• Stability enhancement strategies:

  • Aliquot in small volumes to avoid repeated freeze-thaw cycles

  • Add protease inhibitors to prevent degradation

  • Consider adding reducing agents (DTT or β-mercaptoethanol) to prevent oxidation of cysteine residues

• Quality control monitoring:

  • Periodically check enzymatic activity using phosphate release assays

  • Monitor protein integrity by SDS-PAGE

  • Assess aggregation state by dynamic light scattering

These storage recommendations balance the inherent cold-adapted nature of the enzyme with practical laboratory considerations to maintain optimal activity for experimental use.

How can researchers validate the functional integrity of recombinant D. psychrophila Ddl?

To ensure the recombinant D. psychrophila D-alanine--D-alanine ligase maintains its functional integrity after expression and purification, researchers should employ multiple validation techniques:

• Enzymatic activity assays:

  • Measure ATP-dependent D-Ala-D-Ala formation using phosphate detection methods

  • Determine specific activity (μmol/min/mg) under standard conditions

  • Compare activity values to reference standards or literature values

• Thermal sensitivity profile:

  • Measure activity across a temperature range (0-30°C)

  • Verify expected psychrophilic behavior (higher activity at low temperatures compared to mesophilic homologs)

  • Determine temperature optimum and stability limits

• Circular dichroism (CD) spectroscopy:

  • Assess secondary structure integrity

  • Monitor thermal unfolding profiles

  • Compare with theoretical predictions based on sequence

• Inhibition studies:

  • Verify susceptibility to D-cycloserine or other Ddl inhibitors

  • Determine inhibition constants (Ki)

  • Compare inhibition profile with characterized Ddl enzymes

• Mass spectrometry confirmation:

  • Verify intact mass matches theoretical prediction

  • Perform peptide mapping to confirm sequence coverage

  • Check for unexpected modifications or truncations