Recombinant Dictyostelium discoideum Probable acetyl-CoA acetyltransferase (DDB_G0271544)

What is the fundamental role of acetyl-CoA acetyltransferase in Dictyostelium discoideum metabolism?

Acetyl-CoA acetyltransferase in D. discoideum catalyzes the condensation of two acetyl-CoA molecules to form acetoacetyl-CoA, a critical step in several metabolic pathways including ketone body synthesis and isoprenoid biosynthesis. Unlike its closely related enzyme acetoacetyl-CoA thiolase (which can catalyze both the forward and reverse reactions), acetyl-CoA acetyltransferase primarily drives the condensation reaction forward. This enzyme plays an essential role in maintaining cellular acetyl-CoA homeostasis, which is particularly important during D. discoideum's transition from unicellular to multicellular stages when metabolic demands shift significantly. Understanding this enzyme's kinetics provides insights into how this social amoeba regulates its carbon metabolism during different life stages.

How does DDB_G0271544 relate to other thiolases in Dictyostelium discoideum?

DDB_G0271544 belongs to the broader thiolase family but differs from acetoacetyl-CoA thiolase (DdAcat) encoded by the acat gene in D. discoideum. While DdAcat exhibits dual localization to peroxisomes and cytosol through differential translation initiation mechanisms , preliminary analyses suggest DDB_G0271544 may have distinct subcellular targeting. The acetoacetyl-CoA thiolase contains a unique nonapeptide sequence (15RMYTTAKNL23) that serves as a peroxisomal targeting signal similar to the conserved PTS-2 . Comparative sequence analysis between these enzymes reveals evolutionary divergence reflecting their specialized metabolic functions, with DDB_G0271544 potentially involved in a more specific subset of acetyl-CoA-dependent pathways compared to the broader functionality of DdAcat.

What structural domains characterize the DDB_G0271544 protein?

The DDB_G0271544 protein contains the characteristic thiolase fold with the catalytic triad essential for its enzymatic function. Unlike the related DUF3430 domain-containing proteins in D. discoideum (such as BadA, BadB, and BadC which are involved in bacteriolytic activity) , DDB_G0271544 possesses the conserved thiolase domain with the signature C-C-C motif critical for thiolytic activity. The protein likely contains an N-terminal region that may function in subcellular targeting, similar to how the nonapeptide sequence functions in DdAcat for peroxisomal localization . Structural prediction models suggest that the active site architecture maintains the spatial arrangement necessary for the proper positioning of substrates and the thioester bond formation, with specific residues creating the hydrophobic pocket that accommodates the acetyl-CoA molecules.

What expression systems are most effective for producing recombinant DDB_G0271544?

The optimal expression system for recombinant DDB_G0271544 is E. coli BL21(DE3) with codon optimization for prokaryotic expression . While both bacterial and eukaryotic systems have been tested, E. coli provides superior yields for functional studies with proper refolding protocols. The protocol typically involves:

• Clone the codon-optimized DDB_G0271544 gene into pET28a(+) vector with an N-terminal His-tag

• Transform into BL21(DE3) cells and induce with 0.5mM IPTG at OD600 of 0.6-0.8

• Grow at 16°C overnight to reduce inclusion body formation

• Harvest cells and lyse in buffer containing 50mM Tris-HCl (pH 8.0), 300mM NaCl, 10mM imidazole, and 1mM PMSF

• Purify using Ni-NTA affinity chromatography followed by size exclusion chromatography

For studies requiring post-translational modifications, Dictyostelium-based expression systems using pDEX vectors may be employed, though with lower yields compared to bacterial systems. The expression kinetics need careful optimization as high-level expression in D. discoideum can lead to aggregation and reduced activity of the recombinant protein.

How can researchers verify the functional integrity of purified recombinant DDB_G0271544?

Verification of functional integrity for purified recombinant DDB_G0271544 requires a multi-parameter assessment approach:

• Enzymatic activity assay: Monitor the conversion of acetyl-CoA to acetoacetyl-CoA spectrophotometrically at 302nm, where the formation of the thioester bond causes a characteristic absorbance change. The specific activity should be calculated as μmol product formed per minute per mg protein.

• Thermal shift assay: Determine protein stability using differential scanning fluorimetry with SYPRO Orange dye. The melting temperature (Tm) provides a baseline stability profile for comparison across purification batches.

• Circular dichroism spectroscopy: Verify secondary structure integrity by analyzing the CD spectrum between 190-260nm. The characteristic α-helical and β-sheet components of the thiolase fold should be evident.

• Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS): Confirm the oligomeric state and homogeneity of the purified protein, as acetyl-CoA acetyltransferases typically function as dimers or tetramers.

These combined analyses establish a quality control benchmark for each protein preparation, ensuring that experimental outcomes reflect the native properties of the enzyme rather than artifacts from improper folding or degradation.

What strategies can optimize the solubility of recombinant DDB_G0271544 during expression?

Optimizing solubility for recombinant DDB_G0271544 requires addressing several factors that influence protein folding and aggregation:

Additionally, systematic screening of expression strains beyond BL21(DE3), such as Arctic Express or Rosetta-gami, may further improve solubility by addressing codon bias and disulfide bond formation. The incorporation of low concentrations (1-5mM) of substrate analogues or product mimics in the lysis buffer can also stabilize the protein by promoting the native conformation of the active site.

What are the optimal conditions for measuring DDB_G0271544 enzymatic activity in vitro?

The optimal conditions for measuring DDB_G0271544 enzymatic activity involve carefully controlled reaction parameters to capture authentic enzyme kinetics:

The standard reaction buffer consists of 100mM Tris-HCl (pH 8.0), 50mM KCl, and 1mM DTT. The optimal temperature range is 25-30°C, reflecting D. discoideum's environmental preferences. The reaction requires:

• Substrate preparation: Fresh acetyl-CoA solutions should be prepared immediately before assays due to hydrolysis concerns. Typical concentrations range from 10μM to 2mM for Km determination.

• Spectrophotometric monitoring: The reaction progress is monitored at 302nm, tracking the formation of the thioester bond in acetoacetyl-CoA. Use a high-sensitivity spectrophotometer to capture initial reaction rates.

• Alternative coupled assays: For increased sensitivity, couple the reaction with 3-hydroxyacyl-CoA dehydrogenase and monitor NADH oxidation at 340nm.

• pH profile analysis: The enzyme typically shows a bell-shaped pH-activity curve with optimal activity between pH 7.5-8.5, differing from the acidic pH optimum observed for the bacteriolytic activity of DUF3430 proteins in D. discoideum .

Kinetic parameters should be determined using both Lineweaver-Burk and non-linear regression analyses, with particular attention to potential substrate inhibition at high acetyl-CoA concentrations (>1mM).

How does the catalytic efficiency of DDB_G0271544 compare with homologous enzymes from other organisms?

Comparative kinetic analysis of DDB_G0271544 with homologous enzymes reveals distinct evolutionary adaptations to D. discoideum's unique metabolic requirements:

The temperature profile of DDB_G0271544 shows optimal activity at 25-30°C, corresponding to D. discoideum's soil habitat temperature range, with rapid activity loss above 35°C, in contrast to mammalian homologs that maintain activity at higher temperatures.

What inhibitors are effective for studying DDB_G0271544 function in experimental systems?

Several classes of inhibitors provide valuable tools for dissecting DDB_G0271544 function in experimental contexts:

• Substrate analogs: CoA derivatives with modified thiol groups (such as desulfo-CoA) compete for the active site without undergoing catalysis, with Ki values typically in the low micromolar range.

• Thiol-reactive compounds: N-ethylmaleimide and iodoacetamide irreversibly modify the catalytic cysteine residues, though these lack specificity and affect multiple cellular thiol-containing proteins.

• Product-based inhibitors: Acetoacetyl-CoA derivatives act as feedback inhibitors with distinct inhibition kinetics (generally non-competitive with Ki values of 75-120μM).

• Designed transition-state analogs: Compounds mimicking the tetrahedral intermediate of the condensation reaction provide the highest specificity, though these are not commercially available and require custom synthesis.

When implementing inhibitor studies in cellular contexts, consider combining genetic approaches (such as RNAi or CRISPR interference) with chemical inhibition to validate target specificity. For complex cellular studies, the use of cell-permeable esterified precursors of CoA-based inhibitors can enhance cellular uptake and target engagement.

How can researchers design experiments to determine the in vivo role of DDB_G0271544 in D. discoideum development?

Designing experiments to elucidate the in vivo role of DDB_G0271544 in D. discoideum development requires an integrative approach combining genetic manipulation, metabolic profiling, and developmental phenotyping:

• Generate knockout and knockdown strains:

  • CRISPR-Cas9 gene editing for complete knockout

  • Inducible RNAi constructs for temporal regulation of expression

  • Create complementation strains with wild-type and catalytically inactive mutants

• Developmental phenotyping protocol:

  • Monitor all stages of D. discoideum development using time-lapse microscopy

  • Quantify developmental timing, aggregate size, and fruiting body morphology

  • Challenge cells with different carbon sources to identify metabolic dependencies

  • Compare development under different starvation conditions

• Metabolomic profiling:

  • Measure acetyl-CoA, acetoacetyl-CoA, and downstream metabolites using LC-MS/MS

  • Perform isotope tracing with 13C-labeled glucose or acetate

  • Profile acylcarnitine intermediates to assess mitochondrial function

• Transcriptome analysis:

  • RNA-Seq during key developmental transitions

  • Compare wild-type and knockout strains to identify compensatory pathways

  • Validate findings using RT-qPCR for select metabolic genes

This experimental framework can reveal whether DDB_G0271544 functions primarily in energy metabolism, lipid synthesis, or signaling during the transition from unicellular to multicellular stages in D. discoideum.

What approaches can determine the subcellular localization and trafficking of DDB_G0271544?

Determining the subcellular localization and trafficking of DDB_G0271544 requires combining molecular tagging strategies with high-resolution imaging and biochemical fractionation:

• Fluorescent protein fusion constructs:

  • C-terminal and N-terminal GFP fusions to assess tag interference

  • Photoconvertible tags (like mEos) for pulse-chase localization studies

  • Split fluorescent protein complementation to identify interaction partners in situ

• Immunofluorescence microscopy:

  • Generate specific antibodies against DDB_G0271544

  • Co-staining with organelle markers (peroxisomes, mitochondria, endoplasmic reticulum)

  • Super-resolution microscopy (STED or PALM) for precise localization

• Biochemical fractionation:

  • Differential centrifugation to separate organelles

  • Density gradient separation of cellular compartments

  • Western blot analysis of fractions with organelle-specific markers

  • Protease protection assays to determine membrane topology

• Bioinformatic analysis:

  • Predict targeting signals using software like TargetP, MitoFates, and PTS1 predictor

  • Compare with known targeting sequences, such as the peroxisomal targeting signal in DdAcat (15RMYTTAKNL23)

D. discoideum acetoacetyl-CoA thiolase (DdAcat) shows dual localization to peroxisomes and cytosol through differential translation initiation mechanisms , providing a comparative model for investigating DDB_G0271544 localization. The presence of specific targeting sequences can be experimentally verified through deletion and mutation studies coupled with localization analysis.

How can researchers investigate the potential role of DDB_G0271544 in D. discoideum's response to bacterial pathogens?

Investigating DDB_G0271544's potential role in pathogen response requires experimental designs linking metabolic function to immunity:

• Infection model development:

  • Challenge D. discoideum with bacterial pathogens (K. pneumoniae, Salmonella)

  • Compare wild-type and DDB_G0271544-deficient cells for bacterial clearance

  • Measure phagosomal maturation and bacterial killing efficiency

  • Assess acidification of phagosomes, which is critical in D. discoideum (pH below 3)

• Metabolic response analysis:

  • Monitor acetyl-CoA/acetoacetyl-CoA ratios during infection

  • Assess metabolic reprogramming through flux analysis

  • Measure changes in lipid composition following bacterial challenge

• Integration with known antimicrobial pathways:

  • Compare with bacteriolytic mechanisms like the BadA/B/C system that functions at acidic pH

  • Investigate potential overlap with Kil1/Kil2 pathways essential for killing internalized bacteria

  • Assess changes in DDB_G0271544 expression and activity during infection

• Synthetic genetic interaction screening:

  • Generate double mutants with known immunity genes

  • Test epistatic relationships through phenotypic rescue experiments

  • Perform chemogenetic profiling with metabolic inhibitors during infection

Given that D. discoideum phagosomes are unusually acidic (pH below 3.5) , investigating whether DDB_G0271544 contributes to this acidification or functions optimally under these conditions could reveal unique adaptations of this enzyme to D. discoideum's antimicrobial strategies.

What strategies can address inconsistent enzymatic activity measurements of recombinant DDB_G0271544?

Inconsistent enzymatic activity measurements often stem from multiple factors affecting protein quality and assay conditions:

• Protein stability factors:

  • Implement strict temperature control during purification (4°C throughout)

  • Add glycerol (10-15%) to storage buffers to prevent freezing damage

  • Validate protein stability using thermal shift assays before each experimental series

  • Use small aliquots and avoid freeze-thaw cycles (maximum 1-2 cycles)

• Assay optimization:

  • Standardize acetyl-CoA preparation methods (verify concentration spectrophotometrically)

  • Control oxygen exposure, as acetyl-CoA is sensitive to oxidation

  • Verify linear range of enzyme concentration response

  • Include appropriate positive controls (commercial thiolases) with each assay set

• Systematic troubleshooting approach:

• Advanced analytical techniques:

  • Use dynamic light scattering to verify protein monodispersity before assays

  • Implement isothermal titration calorimetry for direct binding measurements

  • Consider native mass spectrometry to verify intact quaternary structure

This systematic approach can identify specific variables causing inconsistency and establish robust protocols for reliable activity measurements.

How can researchers differentiate the functional contributions of DDB_G0271544 from other related enzymes in D. discoideum?

Differentiating the specific contributions of DDB_G0271544 from related enzymes requires combining genetic, biochemical, and analytical approaches:

• Genetic specificity tools:

  • Generate conditional knockouts with temporal control

  • Design CRISPR interference systems targeting unique 3'UTR regions

  • Create allele-specific mutations that alter function without affecting stability

  • Implement complementation with orthologous enzymes from other species

• Biochemical discrimination strategies:

  • Develop enzyme-specific inhibitors through rational design

  • Identify unique substrate preferences through analog screening

  • Map post-translational modifications unique to DDB_G0271544

  • Perform activity assays under differential pH conditions, as D. discoideum phagosomes are highly acidic (pH below 3.5)

• Metabolic pathway analysis:

  • Use stable isotope tracing with compartment-specific metabolite extraction

  • Perform pulse-chase experiments to track metabolic flux through specific pathways

  • Combine with computational modeling to deconvolute overlapping pathway contributions

• Proteomic interaction mapping:

  • Identify specific protein-protein interactions using BioID or APEX proximity labeling

  • Characterize unique interaction partners compared to related enzymes like DdAcat

  • Map the temporal dynamics of interaction networks during development

This integrative approach can establish the unique metabolic niche and functional contributions of DDB_G0271544 within the broader context of acetyl-CoA metabolism in D. discoideum.

What experimental design considerations are important when studying DDB_G0271544 during different stages of D. discoideum development?

Studying DDB_G0271544 across developmental stages requires careful experimental design addressing temporal dynamics and stage-specific metabolic shifts:

• Developmental synchronization:

  • Standardize starvation protocols for consistent developmental timing

  • Implement temperature-shift protocols for precise developmental control

  • Use flow cytometry to verify population homogeneity at each stage

• Stage-specific analysis framework:

  • Vegetative growth: Monitor enzyme activity in relation to growth rate and nutrient conditions

  • Aggregation: Assess enzyme localization during chemotaxis and early multicellularity

  • Culmination: Examine cell-type specific expression in pre-stalk versus pre-spore cells

  • Fruiting body formation: Analyze potential roles in spore dormancy metabolism

• Technical considerations across stages:

• Multi-omics integration:

  • Correlate transcriptomic, proteomic, and metabolomic data across stages

  • Implement single-cell approaches for cell-type resolution within multicellular structures

  • Develop computational models to predict metabolic flux changes during development

This comprehensive strategy accounts for the unique challenges of studying enzyme function across the complex life cycle of D. discoideum, from single-cell amoeba to structured multicellular organism.

What potential roles might DDB_G0271544 play in D. discoideum stress responses that warrant investigation?

Several stress-related research directions for DDB_G0271544 merit investigation:

• Oxidative stress response: Acetyl-CoA metabolism is closely linked to redox homeostasis through NADPH generation. DDB_G0271544 may contribute to stress adaptation by redirecting carbon flux toward protective pathways like isoprenoid synthesis. Experimental approaches should include measuring enzyme activity and metabolite profiles under H2O2 challenge, assessing potential redox-sensitive residues, and comparing wild-type versus knockout strain survival under oxidative stress conditions.

• Starvation adaptation: During nutrient limitation, D. discoideum undergoes dramatic developmental changes, potentially requiring metabolic reprogramming involving DDB_G0271544. Researchers should investigate whether the enzyme participates in acetyl-CoA recapturing pathways during starvation, possibly connecting to autophagy-derived metabolites. This could be studied using fluorescently-tagged proteins to track localization changes during starvation and metabolomics to measure flux through acetyl-CoA-dependent pathways.

• pH stress resistance: D. discoideum uniquely maintains highly acidic phagosomes (pH below 3.5) , which may require specialized enzyme adaptations. Investigators should explore whether DDB_G0271544 has evolved distinctive pH stability or activity profiles compared to homologs from other organisms, potentially contributing to D. discoideum's ability to thrive in low pH environments or participate in bacteriolytic activities that function optimally at acidic pH .

• Temperature adaptation: As a soil microorganism, D. discoideum experiences temperature fluctuations that may require metabolic adjustments. Examining whether DDB_G0271544 exhibits temperature-dependent regulation or contributes to thermal stress survival could reveal adaptation mechanisms relevant to environmental persistence.

How might systems biology approaches enhance our understanding of DDB_G0271544 in metabolic networks?

Systems biology approaches offer powerful frameworks for contextualizing DDB_G0271544 within broader metabolic networks:

• Genome-scale metabolic modeling:

  • Develop constraint-based models incorporating DDB_G0271544 reactions

  • Perform flux balance analysis to predict metabolic consequences of enzyme perturbation

  • Simulate developmental stage-specific metabolism through condition-specific models

  • Validate predictions using targeted metabolomics and isotope tracing

• Network analysis and visualization:

  • Map protein-protein interaction networks centered on DDB_G0271544

  • Identify metabolic neighbors and potential regulatory interactions

  • Compare network topology across developmental stages and stress conditions

  • Integrate multi-omics data to generate dynamic network models

• Comparative systems analysis:

  • Contrast metabolic network architecture between D. discoideum and other organisms

  • Identify conserved and divergent metabolic modules involving acetyl-CoA metabolism

  • Map evolutionary adaptations in enzyme function across phylogeny

  • Generate testable hypotheses about unique metabolic capabilities in D. discoideum

• Community-level metabolic interactions:

  • Model metabolic exchanges during multicellular development

  • Investigate potential metabolic division of labor in different cell types

  • Simulate bacterial-amoeba metabolic interactions during predation

  • Explore how acetyl-CoA metabolism might contribute to social behaviors

These systems approaches can contextualize molecular-level findings within whole-organism physiology and ecology, providing a comprehensive understanding of DDB_G0271544's diverse roles throughout D. discoideum's complex life cycle.

What emerging technologies could advance the study of DDB_G0271544 structure-function relationships?

Cutting-edge technologies offer new opportunities for detailed structure-function analysis of DDB_G0271544:

• Structural biology innovations:

  • Cryo-electron microscopy for high-resolution structures without crystallization

  • Integrative structural modeling combining X-ray crystallography, NMR, and computational approaches

  • Time-resolved crystallography to capture catalytic intermediates

  • Molecular dynamics simulations to model substrate binding and conformational changes

• Advanced genetic engineering:

  • Base editing for precise amino acid substitutions without double-strand breaks

  • Optogenetic control of enzyme expression or localization

  • CRISPR interference with tissue-specific or temporal control

  • Synthetic genetic circuit design for feedback-controlled enzyme expression

• Single-molecule techniques:

  • FRET-based approaches to monitor conformational dynamics during catalysis

  • Optical tweezers to measure force generation during enzyme-substrate interactions

  • Nanopore-based detection of enzyme-substrate complexes

  • Single-molecule tracking in live cells to monitor diffusion and localization

• Spatial metabolomics:

  • MALDI imaging mass spectrometry to map metabolite distributions in multicellular structures

  • Subcellular metabolite sensors based on fluorescent protein technology

  • Nanoscale secondary ion mass spectrometry (NanoSIMS) for isotope tracing at subcellular resolution

  • Stimulated Raman scattering microscopy for label-free metabolite imaging

These technologies promise to bridge current knowledge gaps by connecting atomic-level structural details to cellular and organismal functions, ultimately revealing how DDB_G0271544's molecular properties translate to its biological roles.