Recombinant Shewanella sediminis Phosphatidylserine decarboxylase proenzyme (psd)

What is Shewanella sediminis and what are its distinctive characteristics?

Shewanella sediminis is a psychrophilic rod-shaped marine bacterium originally isolated from Halifax Harbour sediment. It was first noted for its remarkable ability to degrade hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), making it potentially valuable for bioremediation applications .

This bacterium possesses several distinctive enzymatic features that differentiate it from other Shewanella species, including the presence of lysine decarboxylase (absent in other known Shewanella species), arginine dehydrolase, ornithine decarboxylase, and chitinase. Additionally, S. sediminis can oxidize and ferment N-acetyl-d-glucosamine and grows on several carbon sources including N-acetyl-d-glucosamine, Tween 40, Tween 80, acetate, succinate, butyrate, and serine .

Phylogenetically, S. sediminis belongs to the Na⁺-requiring group of Shewanella species, as demonstrated by both phenotypic analysis and 16S rRNA gene phylogenetic clustering. Its 16S rRNA gene sequence shows ≤97.4% similarity to all known Shewanella species, with closest relations to the bioluminescent species S. hanedai and S. woodyi .

What is phosphatidylserine decarboxylase and what is its biological significance?

Phosphatidylserine decarboxylase (PSD) is an enzyme that catalyzes the decarboxylation of phosphatidylserine (PS) to form phosphatidylethanolamine (PE), a crucial phospholipid in cellular membranes . This reaction represents a vital pathway in phospholipid metabolism across various organisms.

The enzyme exists initially as a proenzyme that must undergo proteolytic cleavage to become catalytically active. This maturation process involves self-catalyzed endoproteolytic processing, classifying PSD as a member of the serine protease family . The catalytic mechanism relies on a canonical D-H-S active site comprising conserved aspartic acid, histidine, and serine residues (specifically D139, H198, and S308 in Plasmodium knowlesi PSD) .

Why is recombinant Shewanella sediminis PSD of interest to researchers?

Recombinant S. sediminis PSD presents a unique research opportunity due to several factors. First, as a psychrophilic (cold-loving) bacterium, its enzymes including PSD may possess distinctive properties adapted to function efficiently at lower temperatures, potentially offering advantages for biotechnological applications requiring cold-active enzymes .

Second, S. sediminis demonstrates remarkable environmental adaptation capabilities, including RDX degradation and growth on various carbon sources, suggesting its enzymes may have evolved unique properties for survival in marine sediments . The study of its PSD can provide insights into how phospholipid metabolism contributes to these adaptive capabilities.

Finally, understanding the structure-function relationships and regulatory mechanisms of S. sediminis PSD can contribute to the broader understanding of phospholipid metabolism across bacterial species, potentially informing research on membrane biogenesis, bacterial adaptation, and even applications in synthetic biology.

How is the PSD gene structured and expressed in Shewanella sediminis?

The PSD gene in Shewanella sediminis, like in many bacteria, is likely part of an operon structure that regulates its expression in coordination with other genes involved in membrane phospholipid synthesis. While specific information about S. sediminis PSD gene organization is limited in the search results, insights can be drawn from related bacterial systems.

For example, in Escherichia coli, PSD is expressed as part of the psd-mscM operon, which is regulated by multiple transcription factors including sigma factor σE and CpxR . This regulatory mechanism suggests that PSD expression responds to membrane stress conditions. Similar regulatory elements might control PSD expression in S. sediminis.

To study PSD gene expression, researchers typically employ transcriptional fusions with reporter genes such as GFP. This approach allows for the monitoring of promoter activity by measuring fluorescence in living cells . Such methodologies can be adapted for studying S. sediminis PSD expression under various environmental conditions relevant to its marine sediment habitat.

What are the structural features of Shewanella sediminis PSD proenzyme?

The phosphatidylserine decarboxylase proenzyme typically consists of several key structural elements essential for its function. Based on homology with other bacterial PSDs and the information from search results, S. sediminis PSD proenzyme likely contains:

• A catalytic domain with a conserved D-H-S catalytic triad (aspartic acid, histidine, and serine residues) that forms the active site for both autoproteolytic processing and subsequent decarboxylase activity .

• A self-cleavage site that undergoes endoproteolytic processing to generate the mature, active enzyme.

• Lipid-binding regions that facilitate interaction with membrane phospholipids, particularly anionic phospholipids like phosphatidylserine and phosphatidylglycerol, as demonstrated in other PSD enzymes .

The active PSD enzyme consists of two subunits (α and β) that remain associated after proteolytic processing. The larger α-subunit contains most of the enzyme structure, while the smaller β-subunit contains the pyruvate prosthetic group essential for catalytic activity.

How is the maturation of PSD proenzyme to active enzyme regulated?

The maturation of PSD proenzyme involves an autocatalytic cleavage event that converts the inactive proenzyme into the active enzyme. This process is tightly regulated and involves several factors:

• Self-catalyzed proteolysis: The PSD proenzyme functions as a serine protease that cleaves itself. This process relies on the conserved D-H-S catalytic triad. Site-directed mutagenesis studies in related PSDs have shown that altering any of these residues (Asp, His, or Ser) to alanine results in complete loss of endoproteolytic processing and subsequently, loss of PSD enzyme activity .

• Lipid-dependent regulation: The maturation process is influenced by the presence of specific phospholipids. In particular, anionic phospholipids like phosphatidylserine (PS) can facilitate the processing of the proenzyme . Research has shown that the PSD proenzyme physically interacts with PS and other anionic phospholipids like phosphatidylglycerol (PG), with binding affinities in the nanomolar range (Kd values of approximately 80.4 nM and 66.4 nM for PS and PG, respectively) .

• Inhibitory mechanisms: Some phospholipids may inhibit the maturation process. Studies have shown that phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylinositol (PI), and cardiolipin can inhibit the processing of PSD proenzymes in certain organisms .

The table below summarizes the effects of different phospholipids on PSD proenzyme maturation based on studies in related systems:

How does the PSD from Shewanella sediminis compare to PSD enzymes from other organisms?

Comparative analysis of PSD enzymes across different organisms reveals important evolutionary and functional insights. While specific comparative data for S. sediminis PSD is not explicitly provided in the search results, we can infer likely similarities and differences based on information about PSD enzymes from other organisms.

PSD enzymes can be categorized into two main types: prokaryotic (bacterial) PSDs and eukaryotic PSDs. S. sediminis PSD would belong to the prokaryotic category, which typically differs from eukaryotic counterparts in several aspects:

• Subcellular localization: Bacterial PSDs are typically membrane-associated, whereas eukaryotic PSDs can be found in various compartments including mitochondria (PSD1) and the Golgi/endosomal system (PSD2) .

• Regulatory mechanisms: The regulation of prokaryotic PSDs often involves transcriptional control through specific promoter elements. For instance, in E. coli, the psd-mscM operon is regulated by transcription factors like σE and CpxR . S. sediminis PSD might share similar regulatory elements or have evolved distinct mechanisms adapted to its marine environment.

• Substrate specificity and enzymatic properties: As a psychrophilic bacterium, S. sediminis PSD may exhibit cold-adapted properties compared to mesophilic counterparts, potentially including higher catalytic efficiency at lower temperatures, structural flexibility, or altered substrate affinities.

Further comparative analysis would require experimental determination of kinetic parameters, substrate preferences, and three-dimensional structures of S. sediminis PSD alongside other bacterial and eukaryotic PSDs.

What roles does PSD play in phospholipid homeostasis and membrane biogenesis in Shewanella sediminis?

PSD plays a critical role in phospholipid homeostasis and membrane biogenesis by catalyzing the production of phosphatidylethanolamine (PE), a major membrane phospholipid. In Shewanella sediminis, this function likely contributes to several aspects of cellular physiology:

• Membrane composition and physical properties: The conversion of phosphatidylserine (PS) to PE alters the charge distribution within the membrane, as PS is anionic while PE is zwitterionic. This transformation affects membrane curvature, fluidity, and protein-lipid interactions .

• Adaptation to environmental conditions: As a psychrophilic marine bacterium, S. sediminis encounters fluctuating temperatures, pressures, and salt concentrations. The activity of PSD may be regulated in response to these environmental factors to maintain appropriate membrane fluidity and integrity through modulation of PE content.

• Interorganellar communication: In eukaryotes, PSD activity has been implicated in nonvesicular lipid trafficking and membrane contact sites (MCS) . While bacterial cells lack the compartmentalization of eukaryotes, the principles of localized lipid metabolism may still apply to bacterial membrane domains or sites of cell division.

• Cell division and growth: PE is essential for proper cell division in many bacteria. PSD activity likely contributes to the coordination of membrane expansion with cell growth and division in S. sediminis.

Understanding these roles would require experimental approaches such as gene knockout studies, lipidomic profiling under various growth conditions, and fluorescent tagging to monitor PSD localization during different cellular processes.

How might the unique environmental adaptations of Shewanella sediminis influence its PSD function?

Shewanella sediminis has evolved several adaptations for its psychrophilic marine sediment lifestyle that may influence the function of its PSD enzyme:

• Cold adaptation: As a psychrophilic bacterium, S. sediminis grows optimally at lower temperatures. Its PSD enzyme likely exhibits cold-adapted features, potentially including higher catalytic efficiency at low temperatures, structural flexibility that prevents cold-induced rigidity, and possibly altered substrate specificities compared to mesophilic counterparts .

• Salt tolerance: Being a Na⁺-requiring marine bacterium, S. sediminis thrives in saline environments . Its PSD may have evolved structural features that maintain activity and stability under varying salt concentrations, potentially through modifications that affect protein-lipid interactions in high-salt conditions.

• Adaptation to sediment environment: S. sediminis was isolated from harbor sediment and has the ability to degrade compounds like RDX . These capabilities suggest adaptation to potentially contaminated environments, which might influence PSD function through altered regulation or substrate interactions related to stress responses.

• Metabolic versatility: The bacterium can utilize various carbon sources including N-acetyl-d-glucosamine, Tween compounds, and several organic acids . This metabolic flexibility may be reflected in adaptations of its phospholipid metabolism enzymes, including PSD, to accommodate shifts in carbon flux and membrane composition based on available nutrients.

Research investigating these adaptations would benefit from comparative studies of PSD activity across different Shewanella species, as well as experimental approaches examining enzyme kinetics under varying temperature, salt, and substrate conditions.

What are the optimal methods for cloning and expressing recombinant S. sediminis PSD?

Successful cloning and expression of recombinant S. sediminis PSD requires careful consideration of several factors to ensure proper folding, processing, and activity of the enzyme. Based on approaches used for related proteins, the following methodological strategy is recommended:

• Gene optimization and vector selection:

  • Optimize the S. sediminis psd gene sequence for expression in the chosen host (typically E. coli for initial studies)

  • Select an expression vector with an appropriate promoter (e.g., T7 for high expression or arabinose-inducible for titratable expression)

  • Consider including affinity tags (His6, FLAG, etc.) for purification, preferably with a cleavable linker to remove the tag if it interferes with activity

• Host strain selection:

  • For initial expression, standard E. coli strains like BL21(DE3) are suitable

  • For challenging expressions, consider specialized strains that provide additional chaperones or modified membrane compositions

  • Consider using PSD-deficient host strains for complementation studies to confirm functional expression

• Expression conditions:

  • As S. sediminis is psychrophilic, lower expression temperatures (16-25°C) may improve folding of its PSD

  • Use mild induction conditions to prevent formation of inclusion bodies

  • Consider co-expression with molecular chaperones if misfolding occurs

• Verification of expression and processing:

  • Monitor both proenzyme and processed forms using Western blot analysis with appropriate antibodies

  • Confirm self-processing ability by comparing wild-type PSD with catalytic site mutants (e.g., S308A) that should remain unprocessed

For functional studies, cell-free translation systems coupled with liposome incorporation have proven effective for studying PSD maturation and can be adapted for S. sediminis PSD research .

What assays can be used to measure S. sediminis PSD activity?

Several complementary assays can be employed to measure the activity of recombinant S. sediminis PSD:

• Radiometric assays:

  • Utilize radiolabeled substrates (e.g., [14C]-phosphatidylserine)

  • Measure the release of [14C]-CO2 or formation of [14C]-phosphatidylethanolamine

  • This provides direct quantification of decarboxylase activity

  • Requires appropriate radiation safety protocols

• Fluorescence-based assays:

  • Use fluorescently-labeled phosphatidylserine analogs

  • Monitor conversion to phosphatidylethanolamine via HPLC or TLC separation

  • Less hazardous than radiometric assays but may introduce artifacts due to the fluorescent label

• Mass spectrometry-based assays:

  • Quantify the conversion of PS to PE using LC-MS/MS

  • Allows for detailed analysis of substrate specificity using various PS species

  • Can be coupled with lipidomic approaches to assess broader effects on lipid composition

• Coupled enzyme assays:

  • Design systems where PSD activity is linked to other enzymatic reactions that produce measurable signals

  • Useful for high-throughput screening applications

• Proenzyme processing assays:

  • Monitor the self-cleavage of PSD proenzyme to its mature form using SDS-PAGE and Western blotting

  • This assay specifically examines the maturation process rather than catalytic activity

  • Can be combined with site-directed mutagenesis to identify residues critical for processing

The choice of assay should be guided by the specific research question, available equipment, and desired throughput.

How can protein-lipid interactions of S. sediminis PSD be studied experimentally?

Understanding the interactions between S. sediminis PSD and membrane phospholipids is crucial for elucidating its regulation and function. Several methodologies can be employed to study these interactions:

• Liposome cosedimentation assays:

  • Prepare multilamellar liposomes of defined composition (e.g., with varying PS, PG, PC content)

  • Incubate PSD proenzyme with liposomes, then centrifuge to separate bound and unbound protein

  • Analyze the distribution using SDS-PAGE and Western blotting

  • This approach has successfully demonstrated binding of PSD to PS and PG liposomes in related systems

• Surface plasmon resonance (SPR):

  • Immobilize liposomes of defined composition on sensor chips

  • Measure real-time association and dissociation of PSD

  • Determine binding kinetics and equilibrium dissociation constants (Kd)

  • SPR analysis has shown nanomolar affinity of PSD for PS and PG liposomes (Kd values of approximately 80.4 nM and 66.4 nM, respectively)

• Solid-phase binding assays:

  • Immobilize lipids on solid supports such as nitrocellulose membranes

  • Probe with purified PSD followed by immunodetection

  • Useful for screening multiple lipid types simultaneously

• Fluorescence-based techniques:

  • Förster resonance energy transfer (FRET) between fluorescently labeled PSD and lipids

  • Fluorescence anisotropy to monitor changes in protein mobility upon lipid binding

  • These approaches can provide insights into the dynamics of interactions

• Computational methods:

  • Molecular docking simulations to predict lipid binding sites

  • Molecular dynamics simulations to study the dynamics of protein-lipid interactions

  • These in silico approaches can guide experimental designs and interpretation

When studying S. sediminis PSD specifically, it would be valuable to compare its lipid-binding properties with those of PSDs from mesophilic bacteria to identify any adaptations related to its psychrophilic lifestyle.

How can researchers address the challenges in distinguishing between effects on PSD processing versus catalytic activity?

• Sequential analysis of processing and activity:

  • First, monitor proenzyme processing using SDS-PAGE and Western blotting to track the conversion of proenzyme to mature enzyme

  • Subsequently, measure the catalytic activity of the processed enzyme using substrate conversion assays

  • This sequential approach allows temporal separation of the two processes

• Catalytic site mutants as controls:

  • Generate mutants affecting either:

    • The catalytic residues for decarboxylase activity but not self-processing

    • The self-processing catalytic triad (e.g., D139A, H198A, S308A mutations)

  • Compare these mutants with wild-type enzyme to isolate specific effects

• In vitro reconstitution:

  • Separately study:

    • Proenzyme processing in cell-free systems supplemented with various lipids

    • Activity of pre-processed enzyme with different substrates and conditions

  • This approach artificially separates the two processes for individual analysis

• Kinetic modeling:

  • Develop mathematical models that incorporate both processing and catalytic steps

  • Fit experimental data to these models to extract parameters specific to each process

  • This quantitative approach can help disambiguate complex experimental results

• Structural studies:

  • Use techniques like X-ray crystallography or cryo-EM to capture different conformational states

  • Compare structures of proenzyme, processing intermediates, and mature enzyme

  • Identify structural elements specifically involved in processing versus catalysis

By employing these strategies, researchers can more accurately interpret experimental results and identify factors that specifically affect proenzyme processing, catalytic activity, or both.

What controls and validation steps are essential when studying recombinant S. sediminis PSD?

When studying recombinant S. sediminis PSD, several controls and validation steps are essential to ensure reliable and interpretable results:

• Expression and purification controls:

  • Include negative control (empty vector) to confirm band specificity in protein expression

  • Verify protein identity via mass spectrometry or N-terminal sequencing

  • Analyze protein purity using multiple methods (SDS-PAGE, size exclusion chromatography)

  • Confirm appropriate oligomeric state through native PAGE or analytical ultracentrifugation

• Activity validation:

  • Compare activity of recombinant enzyme with that of native enzyme (if available)

  • Generate catalytic site mutants (e.g., S308A) as negative controls for activity assays

  • Perform substrate specificity tests with various phosphatidylserine species

  • Confirm product identity (phosphatidylethanolamine) using mass spectrometry

• Processing verification:

  • Monitor both proenzyme and processed forms via Western blotting

  • Verify processing kinetics under different conditions

  • Confirm that processing inhibitors (e.g., serine protease inhibitors like PMSF) block maturation

• Lipid interaction controls:

  • Include zwitterionic phospholipids (e.g., PC) as negative or baseline controls in binding assays

  • Verify specificity of binding using competition assays

  • Include appropriate controls for non-specific binding in all interaction studies

• Functional complementation:

  • Test if recombinant S. sediminis PSD can functionally complement PSD-deficient bacterial strains

  • This validates in vivo activity and proper folding/processing

The table below summarizes key validation experiments and their corresponding controls:

How should researchers interpret discrepancies between in vitro and in vivo studies of S. sediminis PSD?

Discrepancies between in vitro and in vivo studies of S. sediminis PSD are common and can provide valuable insights if properly interpreted. Researchers should consider several factors when faced with such inconsistencies:

• Membrane environment differences:

  • In vitro systems often use simplified membrane models (liposomes with defined composition)

  • In vivo membranes contain complex mixtures of lipids, proteins, and have defined curvature and domains

  • Solution: Gradually increase complexity of in vitro systems to better mimic cellular environments; use lipidomic analysis to characterize native membrane composition

• Post-translational modifications:

  • Recombinant systems may lack cellular machinery for proper protein modification

  • Solution: Compare post-translational modification status between recombinant and native enzyme; consider expression in more similar host systems

• Protein partners and regulators:

  • In vivo, PSD likely interacts with protein partners absent in purified systems

  • Solution: Perform protein-protein interaction studies (e.g., pull-downs, crosslinking) to identify potential interaction partners; include candidate partners in in vitro studies

• Substrate accessibility:

  • In cells, substrate channeling and compartmentalization affect enzyme access to substrates

  • In vitro, substrates are often presented in non-physiological conformations

  • Solution: Develop more sophisticated in vitro systems that account for substrate presentation in membranes

• Environmental parameters:

  • Temperature, ionic strength, pH, and crowding effects differ between test tube and cellular environments

  • Solution: Systematically vary these parameters in vitro to identify conditions that better recapitulate in vivo observations

• Transcriptional regulation:

  • Expression levels and timing differ between native and recombinant systems

  • Solution: Use inducible expression systems that allow titration of expression levels; monitor native expression patterns

What are the most promising applications of recombinant S. sediminis PSD research?

Research on recombinant Shewanella sediminis PSD offers several promising applications in various scientific and biotechnological domains:

• Bioremediation technology:

  • S. sediminis is known for its ability to degrade environmental contaminants like RDX

  • Understanding how PSD contributes to membrane adaptations during contaminant exposure could enhance bioremediation strategies

  • Engineered PSD variants might improve the stability and efficiency of S. sediminis in bioremediation applications

• Cold-adapted enzyme biotechnology:

  • As a psychrophilic bacterium, S. sediminis PSD likely possesses cold-adaptation features

  • Characterization of these features could inform the design of enzymes for low-temperature industrial processes

  • Applications include biocatalysis in cold environments, detergent additives, and food processing enzymes

• Phospholipid biosynthesis engineering:

  • Recombinant PSD could be employed in in vitro systems for the enzymatic production of phosphatidylethanolamine

  • Such systems might be valuable for producing defined phospholipids for research, pharmaceutical, or nutritional applications

• Membrane biology insights:

  • Comparative studies between S. sediminis PSD and homologs from other organisms can reveal fundamental principles of enzyme-membrane interactions

  • These insights could advance understanding of membrane biogenesis across diverse organisms

• Antimicrobial target exploration:

  • While not directly applicable to S. sediminis, research on bacterial PSDs can inform antimicrobial strategies targeting phospholipid metabolism in pathogenic bacteria

  • Structural and functional insights from S. sediminis PSD could guide such efforts

Each of these applications benefits from the fundamental research on S. sediminis PSD structure, function, and regulation, demonstrating the value of basic research for addressing applied challenges.

What methodological advances would most benefit S. sediminis PSD research?

Several methodological advances would significantly accelerate research on S. sediminis PSD:

• Structural biology techniques:

  • High-resolution structures of S. sediminis PSD in both proenzyme and mature forms

  • Cryo-electron microscopy to visualize the enzyme in membrane environments

  • Time-resolved structural methods to capture the dynamics of proenzyme processing

• Advanced membrane mimetic systems:

  • Development of more sophisticated membrane models that better recapitulate the native environment

  • Nanodiscs or supported bilayers with controlled composition to study lipid-specific effects

  • Methods to study PSD activity in asymmetric membranes mimicking bacterial membrane architecture

• Single-molecule techniques:

  • Single-molecule FRET to monitor conformational changes during processing and catalysis

  • Single-particle tracking to study the dynamics of PSD in native membranes

  • Force spectroscopy to investigate protein-lipid interactions at the molecular level

• Genetic tools for Shewanella sediminis:

  • Improved transformation protocols and expression systems for S. sediminis

  • CRISPR-Cas9 based genome editing for precise manipulation of the native psd gene

  • Reporter systems optimized for psychrophilic bacteria to monitor gene expression in situ

• Computational methods:

  • Enhanced molecular dynamics simulations of membrane-protein interactions

  • Machine learning approaches to predict effects of mutations on PSD function

  • Systems biology models integrating PSD activity with broader cellular phospholipid metabolism

These methodological advances would enable researchers to address more sophisticated questions about S. sediminis PSD, particularly regarding its adaptation to psychrophilic marine environments and its integration with cellular metabolism.

What are the critical unresolved questions in S. sediminis PSD research?

Despite advances in understanding phosphatidylserine decarboxylase enzymes, several critical questions specific to Shewanella sediminis PSD remain unresolved:

• Evolutionary adaptation mechanisms:

  • How has S. sediminis PSD evolved to function in psychrophilic marine environments?

  • What structural adaptations enable activity at low temperatures while maintaining stability?

  • How do these adaptations compare to PSDs from other extremophiles?

• Regulatory networks:

  • How is S. sediminis PSD expression regulated in response to environmental stressors?

  • What transcription factors control psd gene expression in this species?

  • Are there post-translational regulatory mechanisms beyond proenzyme processing?

• Membrane integration and dynamics:

  • How does S. sediminis PSD localize within the bacterial membrane?

  • Does it form complexes with other enzymes involved in phospholipid synthesis?

  • How does membrane lipid composition feedback to regulate PSD activity?

• Substrate specificity determinants:

  • What structural features determine the specificity of S. sediminis PSD for different PS species?

  • How does this specificity compare to PSD enzymes from mesophilic bacteria?

  • Can the enzyme utilize alternative substrates beyond canonical PS?

• Connection to environmental adaptation:

  • How does PSD activity contribute to S. sediminis' ability to degrade environmental contaminants?

  • Does membrane phospholipid composition change during growth on different carbon sources?

  • What role does PSD play in adapting to fluctuating temperatures and salinities in marine sediments?