Recombinant Shewanella woodyi Phosphatidylserine decarboxylase proenzyme (psd)

What is phosphatidylserine decarboxylase and what reaction does it catalyze?

Phosphatidylserine decarboxylase (PSD) is an enzyme that catalyzes the conversion of phosphatidylserine (PS) to phosphatidylethanolamine (PE), a critical step in membrane biogenesis. This enzyme removes the carboxyl group from the serine head group of PS to form PE, which is an abundant component of most cellular membranes . The physical and chemical properties of PE are essential for multiple aspects of organelle membrane dynamics, making PSD activity crucial for maintaining proper membrane composition and function .

How is PSD structured and processed in cells?

PSD is synthesized as a proenzyme that undergoes post-translational processing to form the mature, active enzyme. The processing involves autocatalytic cleavage into α and β subunits, with the α subunit containing the catalytic site. In the mitochondrial form (PISD-M), the protein contains an N-terminal mitochondrial targeting sequence that directs it to the inner mitochondrial membrane. After import into mitochondria, the precursor is processed to remove the targeting sequence, followed by autocatalytic cleavage to form the mature enzyme .

What are the different isoforms of PSD and how are they distributed across species?

While many organisms encode multiple PSD enzymes that localize to different organelles, humans possess a single PSD locus (PISD) that undergoes alternative splicing to produce different isoforms. The two main human isoforms are:

• PISD-M: The conventional mitochondrial form

• PISD-LD: An alternatively spliced variant that can localize to both lipid droplets and mitochondria

In contrast, yeast (Saccharomyces cerevisiae) has two spatially segregated PSDs, and many fungi and plants express multiple PSD proteins encoded by distinct genes . Bacteria like E. coli rely exclusively on PSD for PE production, while mammalian systems can produce PE through multiple pathways including the Kennedy pathway, lysophospholipid-acylation pathway, and base-exchange pathways .

What expression systems are most effective for producing recombinant Shewanella woodyi PSD?

For expressing recombinant Shewanella woodyi PSD, E. coli-based expression systems have proven effective, similar to other PSD enzymes. The recommended approach involves using pET or pMAL vectors in BL21(DE3) strains with an N-terminal tag (such as MBP or His₆) to enhance solubility and facilitate purification. Expression should be induced with IPTG at lower temperatures (16-20°C) for 16-18 hours to maximize proper folding of this membrane-associated enzyme . This approach was successfully used for expressing recombinant PSD from Plasmodium knowlesi (MBP-His₆-Δ34PkPSD), suggesting similar strategies would be applicable for Shewanella woodyi PSD .

What purification strategy yields the highest activity for recombinant PSD?

A multi-step purification strategy is recommended to obtain high-activity recombinant PSD:

• Affinity chromatography (using the fusion tag) as the initial capture step

• Ion exchange chromatography to remove contaminants

• Size exclusion chromatography for final polishing

When purifying, it's crucial to include appropriate detergents (e.g., 0.05% Triton X-100) in all buffers to maintain enzyme stability and activity, as PSD is a membrane-associated enzyme. Purification should be conducted at 4°C to prevent denaturation. For Shewanella woodyi PSD specifically, maintaining reducing conditions with 1-2 mM DTT or β-mercaptoethanol throughout purification helps preserve enzymatic activity .

How can researchers verify the integrity and activity of purified recombinant PSD?

Verification of purified recombinant PSD should include:

• SDS-PAGE analysis to confirm proper processing into α and β subunits

• Western blotting using antibodies against the tag or PSD

• Activity assays using fluorescence-based methods such as the 1,2-diacetyl benzene/β-mercaptoethanol (1,2-DAB/β-ME) assay that forms a fluorescent iso-indole-mercaptide conjugate with PE

The 1,2-DAB/β-ME assay is particularly valuable as it has high sensitivity and specificity for PSD activity, with a signal/background ratio of 24, capable of detecting as little as 100 pmol of PE produced .

What are the most sensitive assays available for measuring PSD activity?

Several methods exist for measuring PSD activity, with varying degrees of sensitivity and specificity:

The 1,2-DAB/β-ME fluorescence assay is currently the recommended method for high-throughput applications due to its specificity and lack of background from PS . The assay involves a three-step process: first, conducting the enzyme reaction under optimal conditions; second, arresting the reaction with sodium tetraborate (pH 9.85) and adding Triton X-100 and 1,2-DAB/β-ME; and third, incubating for adduct formation and measuring fluorescence .

How can I optimize assay conditions for accurately measuring Shewanella woodyi PSD activity?

Optimizing assay conditions for Shewanella woodyi PSD requires careful consideration of several parameters:

• pH optimization: Test activity across pH range 7.0-8.5, typically with optimal activity around pH 7.4-7.8

• Temperature: While 37°C is standard, Shewanella woodyi is a psychrophilic bacterium, so lower temperatures (20-25°C) may yield better activity

• Detergent concentration: Triton X-100 at 0.05-0.1% is typically required for activity

• Substrate concentration: Use PS concentrations near the Km (typically 50-100 μM) for inhibitor screening or saturation (>5x Km) for maximal activity

• Divalent cations: Test the effect of Mg²⁺ and Ca²⁺ (typically 1-5 mM)

For detection using the 1,2-DAB/β-ME method, ensure optimal adduct formation conditions: pH 9.85 with sodium tetraborate, 9.3 mM Triton X-100, and 1 mM/1.2 mM 1,2-DAB/β-ME respectively, followed by incubation at 22°C for 1 hour .

What controls are essential when measuring PSD activity in complex biological samples?

When measuring PSD activity in complex biological samples, the following controls are essential:

• Negative controls:

  • Heat-inactivated enzyme preparation (95°C for 10 minutes)

  • Reaction mixture without PS substrate

  • Reaction in the presence of EDTA (10 mM) to chelate metal ions

• Positive controls:

  • Commercial PE standards for calibrating the fluorescence assay

  • Well-characterized PSD enzyme preparation (e.g., from E. coli)

• Specificity controls:

  • Parallel assays with other phospholipids to confirm specificity

  • Assays in the presence of known inhibitors to confirm the signal represents true PSD activity

• Background subtraction:

  • Time-zero samples to account for any pre-existing PE or other fluorescent compounds

How is PSD localization regulated in different cellular conditions?

PSD localization is dynamically regulated by cellular nutritional state and metabolic conditions. In the case of PISD-LD (the lipid droplet-localizing isoform), subcellular targeting is controlled by a segment distinct from the catalytic domain and responds to nutritional conditions . Growth conditions that promote neutral lipid storage favor targeting to lipid droplets, while conditions promoting lipid droplet consumption favor mitochondrial targeting .

This conditional targeting has been observed across species. In yeast, phosphatidylserine decarboxylase targeting between mitochondria and endoplasmic reticulum depends on carbon source—growth on glucose favors mitochondrial targeting, while growth on fermentable carbon sources favors ER localization . This metabolic regulation of PSD localization appears to be an evolutionarily conserved mechanism to adapt phospholipid metabolism to cellular needs .

What structural elements determine the targeting of PSD to different organelles?

The targeting of PSD to different organelles is determined by specific structural elements within the protein sequence:

• Mitochondrial targeting: Contains typical N-terminal mitochondrial targeting sequences rich in positively charged residues. In PISD-LD, there appears to be at least two mitochondrial targeting signals, including one in the segment common to both PISD-LD and PISD-M (amino acids 74-375) .

• Lipid droplet targeting: Involves a modestly hydrophobic segment (amino acids 47-63) and a putative amphipathic alpha helix (amino acids 70-93) . The hydrophobic character of this region is crucial for proper targeting, as mutations disrupting hydrophobicity prevent localization to both lipid droplets and mitochondria .

• Proline residues: The hydrophobic segment of PISD-LD contains three proline residues resembling the "proline knot" motif that targets plant oleosin proteins to lipid droplets .

Mutation studies have shown that altering the hydrophobic character of targeting segments (e.g., changing Leu to Arg residues) causes failure of proper localization, emphasizing the importance of these structural features .

How does PSD contribute to lipid droplet biogenesis and neutral lipid storage?

PSD plays a previously unappreciated but crucial role in lipid droplet biogenesis and neutral lipid storage. Studies have shown that depletion of both forms of phosphatidylserine decarboxylase impairs triacylglycerol synthesis when cells are challenged with free fatty acids . This suggests that the PE produced by PSD, particularly the lipid droplet-associated PISD-LD isoform, is important for the proper formation and function of lipid droplets.

The mechanism likely involves:

• Local production of PE at lipid droplet surfaces to maintain appropriate phospholipid composition

• PE's conical shape influencing membrane curvature during lipid droplet formation

• PE potentially facilitating the recruitment of proteins involved in triacylglycerol synthesis

This connection between phospholipid metabolism and neutral lipid storage represents an important intersection of membrane biogenesis and energy homeostasis pathways .

What is the relationship between mitochondrial and lipid droplet-associated PSD activities?

The relationship between mitochondrial and lipid droplet-associated PSD activities appears to be coordinated and complementary. While PISD-M (mitochondrial form) produces most of the PE contained within mitochondrial membranes, PISD-LD serves a dual function by localizing to both mitochondria and lipid droplets depending on cellular metabolic state .

This dual localization allows for integrated regulation of PE production according to cellular needs:

• Under conditions promoting lipid storage, PISD-LD preferentially localizes to lipid droplets

• Under conditions promoting lipid utilization, PISD-LD shifts to mitochondria

This metabolic regulation suggests a sophisticated mechanism for balancing phospholipid production between organelles, ensuring appropriate membrane composition for both mitochondrial function and lipid droplet dynamics . The shared catalytic mechanism between the two forms, with differences primarily in targeting sequences, provides an elegant solution for regulating PE production at different cellular locations.

What metabolic conditions regulate PSD activity and expression?

PSD activity and expression are regulated by several metabolic conditions:

• Nutritional state: Growth conditions that promote neutral lipid storage in lipid droplets favor targeting of PISD-LD to lipid droplets, while conditions promoting lipid droplet consumption favor mitochondrial targeting .

• Carbon source utilization: In yeast, carbon source availability affects PSD localization, with glucose favoring mitochondrial targeting and fermentable carbon sources favoring ER localization .

• Fatty acid availability: Challenge with free fatty acids requires functional PSD for proper triacylglycerol synthesis, suggesting upregulation of PSD activity under lipid-rich conditions .

• Membrane phospholipid composition: Likely provides feedback regulation, as PE is essential for multiple membrane functions including mitochondrial protein import, respiratory chain complex assembly, and mitochondrial fusion .

This metabolic regulation allows cells to adjust phospholipid metabolism in response to changing energy needs and membrane requirements .

How can recombinant PSD be used as a tool in membrane biology research?

Recombinant PSD serves as a valuable tool in membrane biology research through several applications:

• Artificial membrane modifications: Recombinant PSD can be used to convert PS to PE in artificial liposomes, allowing researchers to study the effects of lipid composition on membrane properties including curvature, fluidity, and protein interactions.

• Organelle membrane remodeling: By targeting recombinant PSD to specific organelles using appropriate targeting sequences, researchers can selectively modify the PE content of specific cellular compartments to study the role of PE in organelle function.

• Lipid droplet biology investigations: Recombinant PISD-LD can be employed to study the role of PE in lipid droplet formation, growth, and turnover, providing insights into neutral lipid storage mechanisms.

• Membrane asymmetry studies: Since PS is typically enriched in the cytoplasmic leaflet of the plasma membrane, controlled conversion to PE using recombinant PSD can help elucidate the importance of lipid asymmetry in membrane function.

• Phospholipid trafficking research: By monitoring the fate of newly synthesized PE produced by recombinant PSD, researchers can track phospholipid movement between organelles .

What makes PSD a potential target for antimicrobial and anti-cancer drug development?

PSD represents an attractive target for both antimicrobial and anti-cancer drug development for several reasons:

• Essential pathway in microorganisms: In many bacteria and fungi, PSD activity is the exclusive source of PE, making it essential for microbial viability .

• Structural differences: Bacterial and fungal PSDs differ structurally from mammalian counterparts, allowing for selective targeting.

• Cancer cell dependency: Many cancer cells show altered phospholipid metabolism and may have increased dependence on PSD-generated PE for rapid membrane biogenesis during proliferation.

• Mitochondrial function: Inhibiting mitochondrial PSD could selectively impact cancer cells that rely heavily on mitochondrial function.

• Assay availability: The development of fluorescence-based assays (particularly the 1,2-DAB/β-ME method) now enables high-throughput screening for PSD inhibitors that could be developed into novel therapeutics .

Recent application of fluorescence assays has already led to the discovery of five inhibitors of PSD enzyme, demonstrating the feasibility of this approach for drug discovery .

How can PSD inhibitors be identified and characterized for potential therapeutic applications?

The identification and characterization of PSD inhibitors follows a systematic process:

• Primary screening:

  • High-throughput screening using the 1,2-DAB/β-ME fluorescence assay in 96- or 384-well plates

  • Initial screening at substrate concentrations near Km (typically 50-100 μM PS)

  • Compounds showing >50% inhibition are considered hits

• Secondary validation:

  • Dose-response curves to determine IC50 values

  • Alternative assay methods (e.g., radioactive assay) to confirm activity

  • Enzyme kinetic studies to determine mechanism of inhibition (competitive, non-competitive, etc.)

• Selectivity profiling:

  • Testing against mammalian PSD to determine selectivity for microbial targets

  • Assessing activity against related enzymes to establish specificity

• Cellular activity evaluation:

  • Growth inhibition assays in target organisms (bacteria, fungi, cancer cells)

  • Lipidomic analysis to confirm on-target activity (reduction in PE levels)

  • Cytotoxicity testing in mammalian cells to establish therapeutic window

• Lead optimization:

  • Structure-activity relationship studies

  • Improvement of pharmacokinetic properties

  • In vivo efficacy testing in appropriate disease models

The 1,2-DAB/β-ME fluorescence assay is particularly well-suited for inhibitor screening as it provides a high signal/background ratio (24) and can detect inhibition of PE formation in various biological preparations including bacterial extracts, fungal mitochondria, and cancer cell mitochondria .

How do post-translational modifications affect PSD activity and localization?

Post-translational modifications significantly influence PSD activity and localization through several mechanisms:

• Autocatalytic processing: All PSDs undergo self-catalyzed cleavage to generate α and β subunits, which is essential for catalytic activity. This autocatalytic process involves formation of a pyruvoyl group at the N-terminus of the α subunit, which serves as the prosthetic group for catalysis .

• Proteolytic processing: The mitochondrial targeting sequence of PISD-M is removed by mitochondrial processing peptidase after import, which is necessary for proper folding and subsequent autocatalytic processing.

• Phosphorylation: Although not specifically documented in the search results, phosphorylation sites have been identified in PSD from various organisms and may regulate enzyme activity or localization in response to cellular signaling.

• Lipid modifications: The hydrophobic nature of the lipid droplet targeting sequence in PISD-LD suggests potential interactions with lipids that could influence localization patterns between organelles .

The precise regulation of these modifications in response to cellular conditions remains an important area for future research, particularly regarding how they coordinate the balance between mitochondrial and lipid droplet targeting of PSD .

What are the challenges in expressing and studying membrane-associated enzymes like PSD?

Expressing and studying membrane-associated enzymes like PSD presents several significant challenges:

• Solubility issues: Membrane proteins often form inclusion bodies during heterologous expression due to hydrophobic domains. Solutions include:

  • Using solubility-enhancing tags (MBP, SUMO)

  • Expressing truncated forms lacking transmembrane segments

  • Optimizing expression conditions (lower temperature, reduced inducer concentration)

• Maintaining native conformation: Detergent selection is critical as inappropriate detergents can disrupt enzyme structure or activity. A detergent screen is often necessary to identify optimal conditions.

• Reconstituting activity: PSD requires a lipid environment for optimal activity. Options include:

  • Detergent-solubilized enzyme with added lipids

  • Reconstitution into liposomes or nanodiscs

  • Cell-free expression systems with supplied lipids

• Assay development: As demonstrated by the evolution from radioactive to fluorescent assays for PSD, developing appropriate activity assays for membrane enzymes is challenging but essential .

• Structural studies: Obtaining structural information requires specialized approaches:

  • Crystallization in lipidic cubic phases

  • Cryo-electron microscopy of protein-detergent complexes

  • NMR studies of reconstituted systems

The development of the 1,2-DAB/β-ME fluorescence assay represents an important advance in addressing the analytical challenges associated with studying PSD .

How can alternative splicing of PSD be studied to understand differential targeting mechanisms?

Studying alternative splicing of PSD to understand differential targeting mechanisms requires a multi-faceted approach:

• Transcriptomic analysis:

  • RNA-seq to identify alternative splice variants across different tissues and conditions

  • qRT-PCR with isoform-specific primers to quantify relative expression levels of PISD-M versus PISD-LD

  • 5' RACE to identify potential alternative transcription start sites

• Fluorescent protein fusion studies:

  • Construction of GFP fusions with different PSD isoforms

  • Live-cell imaging to track localization under different metabolic conditions

  • FRAP (Fluorescence Recovery After Photobleaching) to assess dynamics of organelle targeting

• Mutation analysis:

  • Site-directed mutagenesis of key residues in targeting sequences

  • Deletion analysis to map minimal regions required for specific organelle targeting

  • Domain swapping between PISD-M and PISD-LD to identify critical targeting determinants

• Metabolic manipulation:

  • Treatment with different carbon sources or lipid supplements

  • Nutrient deprivation to induce lipid droplet consumption

  • Time-course studies to track re-localization following metabolic shifts

• Protein-protein interaction studies:

  • Proximity labeling approaches (BioID, APEX) to identify organelle-specific interaction partners

  • Co-immunoprecipitation to identify factors that regulate targeting

  • Yeast two-hybrid or mammalian two-hybrid screens using targeting domains as bait

These approaches have already revealed important insights, such as the identification of hydrophobic segments and proline residues in PISD-LD that resemble the "proline knot" motif targeting plant oleosin proteins to lipid droplets .