Recombinant Dictyostelium discoideum Putative methylsterol monooxygenase DDB_G0270946 (DDB_G0270946)

What is DDB_G0270946 and what is its predicted function in Dictyostelium discoideum?

DDB_G0270946 is a protein encoded in the Dictyostelium discoideum genome that is annotated as a putative methylsterol monooxygenase. Based on sequence homology, it is predicted to function as an enzyme that catalyzes the oxidation of 4,4-dimethyl-5alpha-cholest-7-en-3beta-ol in sterol biosynthesis pathways. The protein consists of 267 amino acids and is believed to participate in sterol metabolism, which is critical for membrane integrity and signaling in eukaryotic cells . The "putative" designation indicates that while its function has been predicted through bioinformatic analyses, experimental validation of its specific enzymatic activity in D. discoideum may still be incomplete.

How does methylsterol monooxygenase activity contribute to cellular function?

• Conversion of 4,4-dimethyl-5alpha-cholest-7-en-3beta-ol to 4beta-hydroxymethyl-4alpha-methyl-5alpha-cholest-7-en-3beta-ol

• Further oxidation to 3beta-hydroxy-4beta-methyl-5alpha-cholest-7-ene-4alpha-carbaldehyde

• Final conversion to 3beta-hydroxy-4beta-methyl-5alpha-cholest-7-ene-4alpha-carboxylate

This enzymatic activity is essential for proper sterol composition in cellular membranes, which affects membrane fluidity, permeability, and the function of membrane-associated proteins in eukaryotic cells including D. discoideum.

What expression systems are available for producing recombinant proteins in Dictyostelium discoideum?

Dictyostelium discoideum has proven to be an efficient expression system for recombinant proteins, including both homologous and heterologous proteins. Several expression vectors have been developed specifically for D. discoideum that allow for:

• Constitutive expression using actin promoters

• Inducible expression systems

• Secretion of proteins using appropriate signal sequences

Studies have demonstrated that D. discoideum can efficiently secrete recombinant proteins such as soluble forms of normally cell-surface associated glycoproteins and heterologous proteins like glutathione-S-transferase (GST) from Schistosoma japonicum. Yields of up to 20 mg/L for some recombinant proteins have been achieved in standard peptone-based growth media . The expression is stable for at least one hundred generations in the absence of selection, making it a viable system for long-term protein production .

What are the advantages of using Dictyostelium discoideum as a host for recombinant protein expression?

Dictyostelium discoideum offers several advantages as a expression host for recombinant proteins:

• Eukaryotic post-translational modifications: As a eukaryotic organism, D. discoideum can perform complex post-translational modifications that bacterial systems cannot.

• Efficient secretion: It has been demonstrated that D. discoideum can efficiently secrete recombinant proteins into the culture medium, facilitating downstream purification processes .

• Correct processing: The secretion signal peptide is correctly cleaved from recombinant proteins, producing properly processed mature proteins .

• Stable expression: Expression of recombinant proteins in D. discoideum has been shown to be stable for extended periods (at least 100 generations) without selection pressure .

• Relatively high yields: Production levels of up to 20 mg/L for certain recombinant proteins have been reported, which is suitable for laboratory-scale investigations .

• Simplified purification: For secreted proteins, the relatively simple composition of the growth medium compared to mammalian cell culture systems can simplify purification protocols.

What are the methodological considerations when expressing DDB_G0270946 as a recombinant protein?

When expressing the putative methylsterol monooxygenase DDB_G0270946 as a recombinant protein, researchers should consider the following methodological aspects:

• Expression system selection: While expression in E. coli has been reported with His-tagging , researchers should evaluate whether a eukaryotic expression system might preserve enzymatic activity better due to proper folding and post-translational modifications. For membrane-associated proteins like methylsterol monooxygenase, proper folding is particularly critical.

• Codon optimization: Codon optimization for the expression host may improve translation efficiency and protein yield.

• Fusion tag considerations: The choice of fusion tag (e.g., His-tag as reported ) should be evaluated for its impact on protein folding and enzymatic activity. N-terminal vs. C-terminal tagging may have different effects on function.

• Cofactor supplementation: Since methylsterol monooxygenase requires cytochrome b5 as a cofactor , expression systems should be evaluated for their ability to provide this cofactor, or supplementation strategies should be developed.

• Purification strategy: Development of a purification protocol should consider the potential membrane association of the protein and the need to maintain the native conformation for functional studies.

• Activity verification: Given the "putative" designation of DDB_G0270946, rigorous enzymatic activity assays should be designed to confirm its function as a methylsterol monooxygenase.

How can the enzymatic activity of recombinant DDB_G0270946 be assayed in vitro?

To assay the enzymatic activity of recombinant DDB_G0270946 putative methylsterol monooxygenase, researchers should consider the following comprehensive approach:

• Substrate preparation: Synthesize or obtain purified 4,4-dimethyl-5alpha-cholest-7-en-3beta-ol as the primary substrate.

• Cofactor requirements: Ensure the availability of:

  • NAD(P)H as electron donor

  • Cytochrome b5, which is required for methylsterol monooxygenase activity

  • Appropriate buffer conditions with controlled oxygenation

• Detection methods:

  • HPLC or LC-MS analysis to detect and quantify the conversion of substrate to intermediates and final product

  • Spectrophotometric monitoring of NAD(P)H oxidation at 340 nm

  • Oxygen consumption measurement using an oxygen electrode

• Reaction monitoring: Track all three steps of the reaction cascade independently:

  • Formation of 4beta-hydroxymethyl-4alpha-methyl-5alpha-cholest-7-en-3beta-ol

  • Conversion to 3beta-hydroxy-4beta-methyl-5alpha-cholest-7-ene-4alpha-carbaldehyde

  • Production of 3beta-hydroxy-4beta-methyl-5alpha-cholest-7-ene-4alpha-carboxylate

• Kinetic analysis: Determine enzyme kinetic parameters (Km, Vmax, kcat) under varying conditions of pH, temperature, and substrate/cofactor concentrations.

• Inhibition studies: Test known inhibitors of methylsterol monooxygenases to confirm the catalytic classification of DDB_G0270946.

What genome-editing approaches are most effective for studying DDB_G0270946 function in Dictyostelium discoideum?

For investigating the function of DDB_G0270946 in Dictyostelium discoideum, several genome-editing approaches can be employed:

• CRISPR-Cas9 gene knockout: Design guide RNAs targeting the DDB_G0270946 coding sequence to create complete gene knockouts. The efficiency of CRISPR-Cas9 in D. discoideum has improved in recent years, making this a preferred approach for generating null mutants.

• Homologous recombination: Traditional homologous recombination approaches remain effective in D. discoideum, allowing for:

  • Gene replacement with selectable markers

  • Introduction of point mutations to study specific amino acid contributions to enzyme function

  • Creation of conditional alleles

• RNAi-based knockdown: While less commonly used in D. discoideum than in some other model systems, RNAi approaches can be valuable for studying essential genes where complete knockout might be lethal.

• Inducible expression systems: Implementing tetracycline-inducible or similar systems to control gene expression levels, allowing for temporal regulation of DDB_G0270946 expression.

• Fluorescent protein tagging: Creating fusion proteins with GFP or other fluorescent tags to study subcellular localization and dynamics of DDB_G0270946.

When evaluating phenotypes, researchers should analyze growth rates, development patterns, membrane composition, and sterol profiles to comprehensively understand the role of this putative methylsterol monooxygenase in D. discoideum biology.

How does sterol metabolism in Dictyostelium discoideum compare to other model organisms, and what are the implications for studying DDB_G0270946?

Sterol metabolism in Dictyostelium discoideum exhibits several unique characteristics compared to other model organisms, with important implications for studying DDB_G0270946:

• Sterol auxotrophy: Unlike many fungi and plants, D. discoideum is a sterol auxotroph, meaning it cannot synthesize sterols de novo and must obtain them from its environment. This raises interesting questions about the role of a putative methylsterol monooxygenase like DDB_G0270946 in this organism.

• Sterol modification capacity: Despite being unable to synthesize sterols completely, D. discoideum possesses enzymes for modifying dietary sterols, suggesting DDB_G0270946 might be involved in sterol modification rather than de novo synthesis.

• Developmental regulation: Sterol composition changes during D. discoideum development from unicellular to multicellular forms, potentially implicating DDB_G0270946 in developmental processes.

• Evolutionary position: As a member of Amoebozoa, D. discoideum represents an evolutionary position distinct from fungi, plants, and animals, making comparative studies of sterol metabolism enzymes valuable for understanding the evolution of these pathways.

Implications for studying DDB_G0270946:

• The function of DDB_G0270946 might differ from its homologs in sterol-synthesizing organisms, potentially involving dietary sterol modification.

• Phenotypic studies should examine both vegetative growth and developmental stages to capture potential stage-specific functions.

• Comparative genomics approaches comparing DDB_G0270946 with methylsterol monooxygenases from diverse organisms may reveal specialized adaptations.

• Metabolomic profiling of sterol intermediates in wild-type versus DDB_G0270946 mutants under various growth conditions would be particularly informative.

What is the relationship between DDB_G0270946 and epigenetic regulation in Dictyostelium discoideum?

The relationship between the putative methylsterol monooxygenase DDB_G0270946 and epigenetic regulation in Dictyostelium discoideum represents an intriguing area for investigation, particularly considering D. discoideum's unique epigenetic landscape:

• Limited DNA methylation: Genome-wide studies have revealed that D. discoideum has extremely low levels of DNA methylation. Deep sequencing of bisulfite-converted genomic DNA found only a few hundred sites with any detectable methylation out of approximately 7.5 million cytosines in the genome . This contrasts with the extensive DNA methylation observed in many other eukaryotes.

• Potential sterol-epigenetic connections: In other organisms, sterols and their derivatives can influence gene expression through interactions with nuclear receptors. Although classical sterol-responsive nuclear receptors haven't been characterized in D. discoideum, sterol-dependent signaling pathways might influence chromatin organization or other epigenetic mechanisms.

• Developmental regulation: Both sterol metabolism and epigenetic mechanisms change during D. discoideum development. Studies could investigate whether:

  • DDB_G0270946 expression correlates with specific developmental stages

  • Alterations in DDB_G0270946 function affect the expression of developmentally regulated genes

  • Sterol modifications catalyzed by DDB_G0270946 influence chromatin accessibility

• Experimental approaches: To explore these connections, researchers might:

  • Perform RNA-seq on DDB_G0270946 mutants to identify gene expression changes

  • Combine DDB_G0270946 mutations with epigenetic modifications (e.g., in histone modifying enzymes)

  • Use chromatin immunoprecipitation (ChIP) to examine changes in histone modifications or chromatin accessibility in DDB_G0270946 mutants

  • Investigate the localization of DDB_G0270946 protein during different developmental stages

While direct evidence linking DDB_G0270946 to epigenetic regulation is not apparent from the current literature, the intersection of sterol metabolism and gene regulation presents an open area for investigation in D. discoideum biology.

What purification strategies are most effective for recombinant DDB_G0270946 protein?

Based on available information and general principles for purifying membrane-associated enzymes, the following purification strategies are recommended for recombinant DDB_G0270946:

• Affinity chromatography: The reported His-tagged version of recombinant DDB_G0270946 expressed in E. coli facilitates purification using immobilized metal affinity chromatography (IMAC). Consider the following:

  • Ni-NTA, Co-TALON, or Cu-IMAC resins with optimization of imidazole concentrations for binding and elution

  • Addition of glycerol (10-15%) and reducing agents (1-5 mM DTT or β-mercaptoethanol) to maintain protein stability

  • Evaluation of detergent requirements if membrane association poses purification challenges

• Detergent selection: If DDB_G0270946 shows membrane association, evaluate multiple detergents:

  • Mild detergents (DDM, CHAPS, OG) for initial solubilization

  • Detergent screening panel to identify optimal conditions that maintain enzymatic activity

  • Consider detergent exchange during purification if initial solubilization detergent affects activity

• Additional purification steps:

  • Ion exchange chromatography (typically anion exchange as a secondary step)

  • Size exclusion chromatography for final polishing and to assess oligomeric state

  • Hydroxyapatite chromatography which often works well for sterol-metabolizing enzymes

• Quality assessment:

  • SDS-PAGE and western blotting to confirm purity and identity

  • Enzymatic activity assays at each purification step to track specific activity

  • Mass spectrometry to confirm protein identity and potential post-translational modifications

  • Dynamic light scattering to assess homogeneity and aggregation state

The purification protocol should be optimized with specific attention to maintaining the native conformation and enzymatic activity of DDB_G0270946.

How can researchers effectively analyze the impact of DDB_G0270946 on sterol profiles in Dictyostelium discoideum?

To comprehensively analyze the impact of DDB_G0270946 on sterol profiles in Dictyostelium discoideum, researchers should employ a multi-faceted approach:

• Sterol extraction and analytical methods:

  • Develop optimized extraction protocols using chloroform/methanol or similar solvent systems

  • Employ gas chromatography-mass spectrometry (GC-MS) for comprehensive sterol analysis

  • Utilize liquid chromatography-mass spectrometry (LC-MS/MS) for targeted analysis of specific sterols and potential novel intermediates

  • Consider supercritical fluid chromatography (SFC) for enhanced separation of structurally similar sterols

• Experimental design:

  • Compare wild-type, DDB_G0270946 knockout, and DDB_G0270946 overexpression strains

  • Analyze sterol profiles across different developmental stages (vegetative cells, aggregation, slug formation, culmination)

  • Perform feeding experiments with different sterol precursors to trace metabolic fate

  • Conduct time-course experiments following sterol supplementation

• Data analysis approaches:

  • Targeted analysis of known sterols and their derivatives

  • Untargeted metabolomics to identify novel sterol compounds

  • Stable isotope labeling to track sterol metabolism dynamics

  • Multivariate statistical analysis to identify pattern changes across experimental conditions

• Complementary approaches:

  • Analyze membrane properties (fluidity, rafts) using techniques like laurdan fluorescence or detergent resistance

  • Examine gene expression changes in sterol metabolism pathways in response to DDB_G0270946 manipulation

  • Investigate phenotypic effects of altered sterol profiles on development, phagocytosis, and stress resistance

A representative data table from such analysis might look like this:

*p < 0.01 compared to wild-type (n=5 biological replicates)

What are the most informative functional assays to determine if DDB_G0270946 truly functions as a methylsterol monooxygenase?

To definitively establish whether DDB_G0270946 functions as a true methylsterol monooxygenase, the following comprehensive functional assays should be conducted:

• In vitro enzyme activity assays:

  • Direct enzyme activity assay using purified recombinant DDB_G0270946 with 4,4-dimethyl-5α-cholest-7-en-3β-ol substrate

  • Monitor formation of all three expected reaction intermediates and products using LC-MS/MS

  • Measure NAD(P)H consumption spectrophotometrically

  • Determine oxygen consumption using oxygen electrodes

  • Evaluate cytochrome b5 dependency by comparing activity with and without this cofactor

• Substrate specificity profiling:

  • Test activity against a panel of structurally related sterols

  • Measure kinetic parameters (Km, kcat, Vmax) for each potential substrate

  • Compare substrate preference profile with characterized methylsterol monooxygenases from other organisms

• Genetic complementation studies:

  • Express DDB_G0270946 in yeast strains with mutations in the corresponding ERG25 methylsterol monooxygenase

  • Assess rescue of growth defects or sterol profile abnormalities

  • Perform reciprocal complementation by expressing yeast ERG25 in DDB_G0270946 knockout D. discoideum

• Structure-function analysis:

  • Generate site-directed mutants altering predicted catalytic residues

  • Assess the impact of mutations on enzymatic activity and sterol profiles

  • Perform homology modeling to predict structural features and validate through mutagenesis

• Cellular localization studies:

  • Determine subcellular localization using fluorescently tagged DDB_G0270946

  • Confirm endoplasmic reticulum localization (typical for sterol-metabolizing enzymes)

  • Analyze co-localization with other sterol biosynthesis enzymes

• Metabolomic footprinting:

  • Compare sterol profiles in wild-type, knockout, and overexpression strains

  • Conduct stable isotope tracing experiments to follow carbon flow through sterol pathways

  • Identify accumulation of substrate or depletion of product in knockout strains

Results from these assays would provide convergent evidence to establish whether DDB_G0270946 functions as predicted or has an alternative or additional enzymatic activity.

How can DDB_G0270946 serve as a model for studying evolutionary conservation of sterol metabolism enzymes?

DDB_G0270946 offers a unique opportunity to study the evolutionary conservation and divergence of sterol metabolism enzymes for several compelling reasons:

This research direction could yield fundamental insights into principles of enzyme evolution and adaptation while placing DDB_G0270946 in a broader evolutionary context.

What potential applications exist for using recombinant DDB_G0270946 in biotechnology and synthetic biology?

Recombinant DDB_G0270946, if confirmed as a functional methylsterol monooxygenase, holds several promising applications in biotechnology and synthetic biology:

• Sterol modification for pharmaceutical development:

  • Engineering sterol structures to create novel compounds with pharmaceutical potential

  • Producing modified sterols with enhanced bioavailability or biological activity

  • Generating intermediates for semi-synthetic production of steroid hormones or drugs

• Biocatalysis applications:

  • Using DDB_G0270946 for regioselective and stereoselective oxidation of sterols and related compounds

  • Incorporating the enzyme into multi-enzyme cascades for complex transformations

  • Engineering the enzyme for broader substrate scope or enhanced stability for industrial applications

• Metabolic engineering platforms:

  • Integrating DDB_G0270946 into yeast or bacterial systems engineered for sterol production

  • Creating synthetic pathways for novel sterol-derived compounds

  • Optimizing production of valuable sterol derivatives through pathway engineering

• Biosensor development:

  • Using DDB_G0270946 as a component in biosensors for specific sterols or related compounds

  • Developing high-throughput screening systems for compounds that interact with sterol pathways

  • Creating reporter systems to monitor sterol metabolism in living cells

• Educational and research tools:

  • Utilizing DDB_G0270946 as a model system for teaching enzyme kinetics and sterol biochemistry

  • Developing standardized assays for studying oxidoreductase mechanisms

  • Creating research tools for investigating membrane-protein interactions

The potential for these applications would be enhanced through protein engineering approaches such as:

• Directed evolution to enhance stability, activity, or substrate scope

• Structure-guided mutagenesis to modify catalytic properties

• Computational design to optimize performance under specific conditions

How does the study of DDB_G0270946 contribute to our understanding of Dictyostelium discoideum as a model organism?

The study of DDB_G0270946 contributes significantly to our understanding of Dictyostelium discoideum as a model organism in several key dimensions:

• Metabolic adaptations and evolution:

  • Investigating DDB_G0270946 provides insights into how D. discoideum has adapted its metabolism despite being a sterol auxotroph

  • Comparison with other organisms helps place D. discoideum's metabolic capabilities in evolutionary context

  • Understanding the role of sterol modification in an organism that cannot synthesize sterols de novo but can modify exogenous sterols

• Developmental biology insights:

  • Exploring how sterol metabolism through DDB_G0270946 might regulate the transition from unicellular to multicellular states

  • Investigating potential roles of modified sterols in signaling during development

  • Understanding how membrane composition changes mediate developmental processes

• Host-pathogen interactions:

  • D. discoideum serves as a model for phagocytosis and interactions with microorganisms

  • Sterols play critical roles in membrane organization and phagocytosis

  • Understanding how DDB_G0270946 and sterol metabolism influence D. discoideum's interactions with bacteria and fungi

• Technical advances in the model system:

  • Development of new tools for protein expression and analysis in D. discoideum

  • Optimization of methods for studying membrane-associated enzymes

  • Creation of reporter systems and biosensors applicable to other D. discoideum proteins

• Fundamental cell biology:

  • Insights into membrane organization and dynamics in amoeboid cells

  • Understanding of sterol trafficking and homeostasis mechanisms

  • Elucidation of connections between metabolism and other cellular processes

The comprehensive study of DDB_G0270946 thus enriches multiple aspects of D. discoideum biology, strengthening its position as a valuable model organism that bridges gaps between unicellular and multicellular systems.

What are the current knowledge gaps regarding DDB_G0270946 and how might they be addressed?

Several significant knowledge gaps exist regarding DDB_G0270946, presenting opportunities for future research:

• Enzymatic confirmation: The designation of DDB_G0270946 as a "putative" methylsterol monooxygenase indicates that its precise enzymatic activity remains to be definitively confirmed. This could be addressed through:

  • Comprehensive biochemical characterization of the purified enzyme

  • Detailed substrate specificity profiling

  • Structural studies to confirm the presence of catalytic motifs characteristic of methylsterol monooxygenases

• Physiological role: The specific biological function of DDB_G0270946 in D. discoideum remains unclear, particularly given that this organism is a sterol auxotroph. Research priorities include:

  • Phenotypic characterization of knockout mutants under various growth conditions

  • Analysis of sterol profiles in wild-type versus mutant strains

  • Investigation of the enzyme's role during different developmental stages

• Regulation and interactions: Little is known about how DDB_G0270946 is regulated or its protein-protein interactions. Future studies should examine:

  • Transcriptional and post-translational regulation mechanisms

  • Interaction partners, particularly cytochrome b5 and other sterol metabolism enzymes

  • Subcellular localization and potential relocation during different cellular states

• Comparative biology: How DDB_G0270946 differs from methylsterol monooxygenases in sterol-synthesizing organisms remains to be fully explored through:

  • Detailed comparative genomics and protein structure studies

  • Heterologous expression experiments in different host systems

  • Evolutionary rate analysis to identify potential adaptive changes

Addressing these knowledge gaps will require integrated approaches combining molecular biology, biochemistry, structural biology, and systems biology methodologies.

How might emerging technologies advance our understanding of DDB_G0270946 function and applications?

Emerging technologies offer exciting opportunities to deepen our understanding of DDB_G0270946 function and expand its potential applications:

• Advanced structural biology techniques:

  • Cryo-electron microscopy for determining the structure of DDB_G0270946, particularly in the context of membrane association

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

  • Hydrogen-deuterium exchange mass spectrometry to probe protein dynamics and substrate interactions

• Single-cell technologies:

  • Single-cell RNA-seq to examine expression heterogeneity during development

  • Single-cell metabolomics to identify cell-to-cell variations in sterol profiles

  • Spatial transcriptomics to map DDB_G0270946 expression patterns in multicellular structures

• CRISPR technologies:

  • CRISPR interference (CRISPRi) for tunable gene repression

  • CRISPR activation (CRISPRa) for enhanced expression

  • Base editing for introducing precise mutations without double-strand breaks

  • Prime editing for targeted insertions, deletions, and all possible base-to-base conversions

• Advanced imaging:

  • Super-resolution microscopy to visualize DDB_G0270946 localization with nanometer precision

  • Live-cell imaging with genetically encoded biosensors to track enzyme activity in real time

  • Correlative light and electron microscopy to connect protein localization with ultrastructural features

• Synthetic biology approaches:

  • Cell-free expression systems for rapid protein engineering

  • Engineered protein scaffolds to enhance enzymatic efficiency

  • Minimal cell systems to study enzyme function in simplified contexts

• Computational advances:

  • Molecular dynamics simulations to study enzyme-substrate interactions

  • Machine learning approaches for predicting functional impacts of mutations

  • Network biology to place DDB_G0270946 in broader cellular contexts