Recombinant Debaryomyces hansenii Patatin-like phospholipase domain-containing protein DEHA2B04136g (DEHA2B04136g)

What is DEHA2B04136g and what is its basic structure?

DEHA2B04136g is a full-length patatin-like phospholipase domain-containing protein from the osmotolerant yeast Debaryomyces hansenii. The protein consists of 788 amino acids and belongs to the patatin-like phospholipase (PLP) family. When expressed recombinantly, it is typically fused to an N-terminal His tag for purification purposes. The full amino acid sequence includes multiple functional domains consistent with phospholipase activity .

Similar to other patatin-like phospholipases, the protein likely possesses a core structure with alpha/beta/alpha folding patterns, consisting of parallel β sheets flanked by α helices. This structural arrangement is characteristic of proteins in this family, as seen in the 3D modeling of related phospholipases .

How is DEHA2B04136g typically produced for research purposes?

For research applications, DEHA2B04136g is typically produced as a recombinant protein in E. coli expression systems. The full-length gene (encoding amino acids 1-788) is cloned into an appropriate expression vector that incorporates an N-terminal His tag for subsequent purification. Following expression, the protein is purified and provided as a lyophilized powder. For experimental use, reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL is recommended, with the addition of 5-50% glycerol for stability .

The production methodology must be carefully optimized as expression of eukaryotic proteins in prokaryotic systems can sometimes result in improper folding or reduced activity. Researchers should verify protein activity following reconstitution.

What are the recommended storage conditions for maintaining DEHA2B04136g stability?

Optimal storage conditions for DEHA2B04136g include:

• Long-term storage: Store at -20°C or -80°C upon receipt

• Working aliquots: Can be stored at 4°C for up to one week

• Storage buffer: Tris/PBS-based buffer containing 6% trehalose at pH 8.0

• Handling recommendations: Aliquot upon receipt to avoid repeated freeze-thaw cycles which can compromise protein integrity

• Pre-use preparation: Briefly centrifuge the vial before opening to bring contents to the bottom

For reconstituted protein, addition of 5-50% glycerol is recommended when preparing working solutions. Researchers should note that repeated freezing and thawing is not recommended as it may lead to protein denaturation and activity loss.

What enzymatic activities does DEHA2B04136g likely possess?

Based on studies of related patatin-like phospholipases, DEHA2B04136g likely possesses multiple enzymatic activities:

• Phospholipase activity: Ability to hydrolyze phospholipids at specific positions

• Potential lipase activity: Capability to hydrolyze triglycerides

• Potential phospholipase A1 and A2 activity: Cleaving fatty acid chains from phospholipids at specific positions

Comparable patatin-like phospholipases, such as Rv3091 from Mycobacterium tuberculosis, demonstrate phospholipase A1, phospholipase A2, and lipase activities . While direct enzymatic characterization of DEHA2B04136g is not fully detailed in the available literature, its classification suggests similar catalytic functions that would need experimental validation in specific research contexts.

How can researchers assay the enzymatic activity of DEHA2B04136g?

Researchers can employ several methodological approaches to assay DEHA2B04136g activity:

• Phospholipase activity assay: Using fluorescent or radiolabeled phospholipid substrates to monitor hydrolysis

• Lipase activity determination: Employing p-nitrophenyl esters (such as p-nitrophenyl butyrate) as substrates, where hydrolysis releases p-nitrophenol that can be measured spectrophotometrically

• In vitro phospholipid hydrolysis assays: Using thin-layer chromatography (TLC) or high-performance liquid chromatography (HPLC) to separate and quantify hydrolysis products

For reliable results, controls should include heat-inactivated enzyme and, when possible, site-directed mutants at putative active sites (comparable to the Ser214 and Asp407 mutations used to confirm activity in related patatin-like phospholipases) .

What methods are most effective for genetic manipulation of DEHA2B04136g in Debaryomyces hansenii?

Recent advances in genetic manipulation of D. hansenii provide efficient methodologies for studying DEHA2B04136g in its native context:

• PCR-based gene targeting: High-efficiency (>75%) homologous recombination can be achieved using PCR products with 50 bp flanks identical to the target site in the genome

• Heterologous selection markers: Complete heterologous selection markers have been developed for D. hansenii that avoid cross-reactivity with native genes

• Safe landing sites: Identified chromosomal harbor sites enable stable expression of heterologous proteins or modified versions of DEHA2B04136g

This targeted approach allows researchers to disrupt genes at high efficiency or express modified versions of DEHA2B04136g for functional studies. The methodology is particularly valuable as it works in wild-type isolates without requiring strains with pre-existing auxotrophic markers .

How might epigenetic factors influence DEHA2B04136g expression?

While specific data on DEHA2B04136g methylation is not available in the provided literature, research on related patatin-like phospholipase genes suggests important epigenetic considerations. Studies of PNPLA6 (another patatin-like phospholipase) demonstrate that DNA methylation significantly impacts gene expression. In particular:

• Increased methylation correlates with decreased mRNA expression

• Methylation inhibitors (such as 5-aza-2'-deoxycytidine) can significantly enhance transcription

• Age-related methylation changes may affect expression patterns

Researchers investigating DEHA2B04136g expression should consider examining methylation status using bisulfite pyrosequencing and correlating methylation levels with mRNA expression via RT-qPCR. Treatment with methylation inhibitors might serve as an experimental intervention to modulate expression levels.

How does DEHA2B04136g compare structurally to other patatin-like phospholipases?

Patatin-like phospholipases (PLPs) share several conserved structural features across species. Comparative analysis would likely reveal:

• Conserved catalytic residues: Similar to other PLPs, DEHA2B04136g likely contains conserved serine and aspartate residues in its catalytic site (comparable to Ser214 and Asp407 in Rv3091)

• Alpha/beta/alpha folding structure: The core structure likely consists of parallel β sheets flanked by α helices

• Conserved G-X-S-X-G motif: This sequence is typically found in the active site of phospholipases

• Species-specific variations: Unique insertions or deletions that may reflect adaptation to the osmotolerant lifestyle of D. hansenii

3D modeling using tools like SWISS MODEL with appropriate templates (such as PDB 5fya.1 used for Rv3091) would provide deeper insights into the structural conservation and divergence of DEHA2B04136g compared to other patatin-like phospholipases .

What functional differences might exist between DEHA2B04136g and patatin-like phospholipases from pathogenic organisms?

Several key functional differences may exist between DEHA2B04136g from the non-pathogenic D. hansenii and patatin-like phospholipases from pathogenic organisms:

• Cellular localization: Unlike the extracellular localization of some pathogen-derived PLPs (like Rv3091), DEHA2B04136g may have a different cellular distribution pattern

• Role in virulence: Pathogen-derived PLPs often serve as virulence factors enhancing intracellular survival and promoting phagosomal escape, functions likely absent in DEHA2B04136g

• Substrate specificity: DEHA2B04136g may have evolved substrate preferences optimized for D. hansenii's ecological niche rather than host-pathogen interactions

• Regulatory mechanisms: Expression and activity regulation likely differs between environmental and pathogenic contexts

These differences may manifest in distinct biochemical properties and physiological roles, which would require experimental validation through comparative functional assays.

How can DEHA2B04136g contribute to understanding D. hansenii's remarkable stress tolerance?

D. hansenii is known for its exceptional osmotolerance and stress resistance, making it valuable for biotechnological applications. DEHA2B04136g may play significant roles in these characteristics:

• Membrane phospholipid remodeling: As a phospholipase, DEHA2B04136g could modify membrane composition in response to osmotic stress

• Stress-induced signaling: Lipid metabolites generated by DEHA2B04136g activity might function as second messengers in stress response pathways

• Adaptation to marine environments: The enzymatic properties may be optimized for function in high-salt conditions typical of D. hansenii's natural habitats

• Biotechnological applications: Understanding DEHA2B04136g function could inform engineering of stress-resistant industrial strains

Research approaches might include analyzing DEHA2B04136g expression and activity under various stress conditions, creating knockout strains to assess stress sensitivity, and comparing wild-type and mutant strains for lipid composition changes during stress response.

What methodological approaches can be used to study DEHA2B04136g substrate specificity?

Determining substrate specificity of DEHA2B04136g requires sophisticated biochemical approaches:

• In vitro substrate screening:

  • Testing purified recombinant protein against a library of phospholipids with varying head groups and fatty acid compositions

  • Using mass spectrometry to identify reaction products and determine cleavage positions

• Structure-based predictions:

  • Molecular docking simulations with potential substrates

  • Site-directed mutagenesis of predicted substrate-binding residues

• Lipidomic analysis:

  • Comparing lipid profiles between wild-type and DEHA2B04136g-knockout D. hansenii strains

  • Stable isotope labeling to track phospholipid metabolism in vivo

• Physiological context assessment:

  • Evaluating activity under conditions mimicking D. hansenii's natural environment (high salt, varied pH)

  • Testing activity in the presence of potential regulatory molecules

Results should be validated across multiple methodological approaches, as substrate preferences in vitro may not fully reflect physiological function.

What are the key considerations for designing knockout or knockdown experiments for DEHA2B04136g?

Researchers planning genetic manipulation of DEHA2B04136g should consider:

• Selection of appropriate targeting strategy:

  • Complete gene deletion vs. functional domain disruption

  • Conditional knockdown systems for essential genes

  • CRISPR-Cas9 approaches vs. homologous recombination

• Experimental design parameters:

  • Targeting efficiency: PCR-based gene targeting with 50 bp homology flanks provides >75% integration efficiency

  • Selection markers: Use heterologous markers to avoid cross-reactivity

  • Verification methods: PCR confirmation, Southern blotting, and RT-qPCR to confirm deletion/disruption

• Phenotypic analysis:

  • Growth rate under normal and stress conditions

  • Lipid profile analysis

  • Complementation studies to confirm phenotype specificity

• Controls:

  • Wild-type strains processed identically to mutants

  • Restoration of gene function through complementation

  • Off-target effect assessment

The recently developed PCR-based gene targeting system for D. hansenii provides an efficient method for DEHA2B04136g manipulation in wild-type isolates, eliminating the need for strains with pre-existing auxotrophic markers.

How should researchers approach expression and purification of DEHA2B04136g for structural studies?

Obtaining high-quality DEHA2B04136g for structural studies requires careful optimization:

• Expression system selection:

  • E. coli: Standard system but may have folding limitations for eukaryotic proteins

  • Yeast expression systems: Consider Pichia pastoris for proper folding and post-translational modifications

  • Cell-free systems: For problematic expressions

• Construct design:

  • Full-length vs. functional domains

  • Fusion tags: His-tag (N-terminal) for affinity purification

  • Protease cleavage sites for tag removal

• Purification strategy:

  • Multi-step purification: IMAC followed by size exclusion chromatography

  • Buffer optimization: Including stabilizers like trehalose (6%)

  • Protein quality control: SDS-PAGE, Western blot, mass spectrometry, and activity assays

• Sample preparation for structural studies:

  • Concentration optimization to prevent aggregation

  • Buffer screening for stability

  • Crystallization trials or NMR sample preparation

Researchers should verify protein activity following each purification step to ensure the structural studies reflect native conformations.

How can researchers resolve conflicting data regarding DEHA2B04136g function?

When faced with contradictory experimental results regarding DEHA2B04136g function:

• Methodological reconciliation:

  • Examine differences in experimental conditions (temperature, pH, salt concentration)

  • Compare protein preparations (recombinant vs. native, tags, purification methods)

  • Assess assay sensitivity and specificity

• Contextual analysis:

  • In vitro vs. in vivo discrepancies often reflect physiological regulation

  • Consider growth phase or environmental conditions of D. hansenii cultures

  • Evaluate potential cofactors or interacting partners

• Statistical approach:

  • Increase biological and technical replicates

  • Apply appropriate statistical tests with correction for multiple comparisons

  • Consider meta-analysis approaches when multiple datasets exist

• Validation through orthogonal methods:

  • Confirm key findings using alternative experimental approaches

  • Employ both gain-of-function and loss-of-function studies

  • Correlate biochemical data with physiological outcomes

When reporting results, researchers should transparently discuss limitations and potential sources of variability to advance collective understanding of DEHA2B04136g function.

What are the emerging research directions for patatin-like phospholipases in non-pathogenic organisms?

Emerging research directions for PLPs in non-pathogenic organisms like D. hansenii include:

• Biotechnological applications:

  • Exploitation of D. hansenii's osmotolerance for industrial processes

  • Engineering enhanced stress resistance through PLP modification

  • Development of biocatalysts for lipid modification

• Ecological and evolutionary studies:

  • Comparative genomics of PLPs across yeast species with varying stress tolerances

  • Adaptation mechanisms to extreme environments

  • Horizontal gene transfer and evolution of PLP functions

• Molecular mechanisms:

  • Roles in membrane remodeling during stress response

  • Involvement in lipid signaling networks

  • Interaction with other cellular pathways

• Structural biology:

  • Comparative structural analysis of PLPs from diverse ecological niches

  • Structure-function relationships determining substrate specificity

  • Rational design of modified PLPs with enhanced stability or altered specificity

These directions highlight the expanding significance of PLPs beyond pathogenicity studies, particularly in understanding fundamental aspects of cellular adaptation and biotechnological applications.