Recombinant Dinoroseobacter shibae Phosphatidylserine decarboxylase proenzyme (psd)

What is Dinoroseobacter shibae phosphatidylserine decarboxylase proenzyme?

Dinoroseobacter shibae phosphatidylserine decarboxylase (PSD) proenzyme is an enzyme precursor found in the marine bacterium Dinoroseobacter shibae. This enzyme catalyzes the decarboxylation of phosphatidylserine (Ptd-Ser) to form phosphatidylethanolamine (PtdEtn), which is a critical phospholipid component of cellular membranes. The term "proenzyme" refers to its initial inactive form before autocatalytic cleavage occurs at a conserved Glycine-Serine-Threonine (GST) motif, resulting in the formation of α- and β-chains with a pyruvoyl group that becomes the active catalytic site . In D. shibae, this enzyme likely plays an important role in membrane phospholipid biosynthesis, potentially contributing to the organism's unique symbiotic-pathogenic lifecycle with dinoflagellates .

How does the PSD enzyme function in bacterial systems?

The PSD enzyme in bacterial systems like D. shibae functions through a pyruvoyl-dependent mechanism. Initially produced as a proenzyme, it undergoes autocatalytic cleavage at an internal Gly-Ser bond near the C-terminus, forming α- and β-chains . The serine residue at the cleavage site is converted to a pyruvoyl group, which serves as the catalytic center. Similar to other PSDs from eukaryotes like yeast, this activated enzyme then catalyzes the decarboxylation of phosphatidylserine to form phosphatidylethanolamine .

In bacterial systems such as D. shibae, the PSD enzyme likely plays crucial roles in:

• Maintaining proper membrane composition and fluidity

• Facilitating adaptation to environmental changes in marine habitats

• Contributing to the bacterium's complex relationship with dinoflagellates, where it transitions from a symbiotic partnership to a pathogenic interaction

The enzyme is typically targeted to specific membrane compartments, with bacterial PSDs generally associated with the cytoplasmic membrane, unlike their eukaryotic counterparts that can be found in multiple organellar membranes .

What is the significance of studying recombinant PSD from marine bacteria like D. shibae?

Studying recombinant PSD from marine bacteria like D. shibae offers several significant research advantages:

• Ecological insights: D. shibae exhibits a unique Jekyll-and-Hyde relationship with dinoflagellates, initially providing essential vitamins B12 and B1 but later becoming pathogenic and killing the dinoflagellate . Understanding its enzymes helps elucidate this complex ecological interaction.

• Membrane biology advancements: PSD enzymes are critical for phospholipid metabolism and membrane composition. D. shibae's marine adaptation may provide unique insights into membrane biochemistry under marine conditions.

• Enzymatic mechanism exploration: As a pyruvoyl-dependent enzyme, PSD represents an interesting catalytic mechanism different from many other enzymes, offering fundamental knowledge about enzyme evolution and function .

• Plasmid-encoded phenotypes: D. shibae's pathogenic capabilities are linked to its 191 kb plasmid . Understanding how plasmid-encoded functions interact with core metabolic enzymes like PSD may reveal novel mechanisms of bacterial pathogenicity.

• Marine microbiology advancement: Marine bacteria often possess unique adaptations, and studying their enzymes contributes to our understanding of microbial adaptation in ocean ecosystems.

Research on this enzyme informs both fundamental biochemistry and ecological studies of marine microorganisms with potential applications in biotechnology.

How is the D. shibae PSD gene typically expressed and purified for research purposes?

The expression and purification of recombinant D. shibae PSD typically follows this methodological approach:

• Vector selection and cloning:

  • The PSD gene from D. shibae genomic DNA is amplified using PCR with specific primers

  • The gene is cloned into an expression vector (commonly pET-based) with an appropriate tag (His-tag, GST-tag)

  • The construct is verified by sequencing to ensure no mutations

• Expression in heterologous system:

  • Similar to approaches used for tomato PSD, expression in E. coli strains optimized for protein expression (BL21(DE3), Rosetta) is common

  • Expression is induced using IPTG when cultures reach appropriate density

  • Growth conditions are optimized for temperature (often lowered to 16-25°C after induction), media, and induction time

• Cell harvesting and lysis:

  • Cells are harvested by centrifugation and resuspended in buffer

  • Lysis is performed by sonication, French press, or chemical methods

  • Protease inhibitors are included to prevent degradation

• Purification workflow:

  • Initial purification by affinity chromatography (Ni-NTA for His-tagged protein)

  • Further purification by ion exchange and/or size exclusion chromatography

  • Protein purity is verified by SDS-PAGE and Western blotting

• Enzyme processing considerations:

  • Monitoring of autocatalytic processing from proenzyme to active form

  • Verification of α- and β-subunit formation by appropriate analytical methods

  • Activity assays using substrates like NBD-Ptd-Ser or Ptd-Ser with Triton X-100

The purification must carefully preserve the enzyme's native structure, as improper processing can affect the formation of the pyruvoyl group essential for activity.

What are the structural determinants of substrate specificity in D. shibae PSD compared to other bacterial PSDs?

The structural determinants of substrate specificity in D. shibae PSD involve several key features that distinguish it from other bacterial PSDs:

• Active site architecture:

  • The active site of D. shibae PSD likely contains specific residues that interact with the phosphatidylserine headgroup

  • Based on homology with other PSDs, conserved basic residues (Arg, Lys) probably form salt bridges with the phosphate group

  • The pyruvoyl group formed at the GST motif is essential for the decarboxylation reaction

• Membrane-binding domains:

  • D. shibae PSD likely contains hydrophobic regions that facilitate interaction with lipid bilayers

  • These regions may differ from those in PSDs from non-marine bacteria, reflecting adaptation to marine environments

  • The orientation of the enzyme relative to the membrane affects which phospholipids can access the active site

• Comparative analysis considerations:

  • D. shibae, as a marine organism, may have evolved unique structural adaptations compared to terrestrial bacteria

  • The substrate-binding pocket might accommodate phosphatidylserine molecules with specific fatty acid compositions typical of marine bacteria

  • Differences in the α/β subunit interaction surfaces could affect substrate channeling

• Regulatory elements:

  • Potential allosteric sites that respond to cellular conditions specific to the marine environment

  • Structural elements that might be involved in sensing the transition between symbiotic and pathogenic states

Elucidating these structural determinants would require detailed structural studies using X-ray crystallography or cryo-EM, combined with site-directed mutagenesis and enzymatic assays with various synthetic substrates to map the functional importance of specific residues.

How does the localization of PSD in D. shibae compare to its localization in other organisms?

The localization of PSD in D. shibae compared to other organisms reveals important distinctions with significant implications for membrane biogenesis:

• Comparative localization patterns:

  • In eukaryotes like yeast and mammals, PSDs are typically found in mitochondria (PSD1) and endoplasmic reticulum/Golgi (PSD2)

  • In plants, PSDs exist in both mitochondria and possibly chloroplasts, with the mitochondrial form being membrane-associated

  • In D. shibae, as a prokaryote, PSD is likely associated with the plasma membrane or possibly specialized membrane domains

• Targeting mechanisms:

  • Eukaryotic mitochondrial PSDs contain N-terminal targeting sequences and inner membrane sorting signals

  • Yeast PSD1 and Chinese hamster PSD have been shown to use these targeting sequences for proper localization

  • D. shibae PSD lacks these eukaryotic-specific signals but may contain bacterial membrane-targeting domains

• Implications for membrane organization:

  • Differential localization creates distinct pools of phosphatidylethanolamine in different membrane compartments

  • In D. shibae, the localization pattern may facilitate the rapid membrane remodeling needed during transitions between symbiotic and pathogenic states

  • Localization affects the enzyme's access to its substrate phosphatidylserine, which may have different concentrations in different membrane domains

• Methodological approaches to study localization:

  • Subcellular fractionation and Western blotting can confirm biochemical localization, as demonstrated with potato mitochondrial PSD

  • Membrane and matrix fraction separation can determine the precise submitochondrial localization in eukaryotes

  • Similar approaches adapted for bacterial systems could reveal D. shibae PSD localization

Understanding these localization patterns provides insights into how D. shibae organizes its lipid biosynthetic pathways and potentially how this organization contributes to its complex lifestyle as both a symbiont and pathogen.

What role might PSD play in the Jekyll-and-Hyde behavior of D. shibae with dinoflagellates?

The potential role of PSD in the Jekyll-and-Hyde behavior of D. shibae with dinoflagellates involves several sophisticated mechanisms:

• Membrane remodeling during lifestyle transition:

  • PSD activity directly affects phospholipid composition, particularly phosphatidylethanolamine (PtdEtn) levels

  • Changes in membrane composition could trigger or facilitate the transition from symbiotic to pathogenic behavior

  • Specific membrane composition might influence the expression or function of virulence factors

• Interaction with the "killer plasmid":

  • The 191 kb plasmid (pDS191) of D. shibae is responsible for the killing phenotype of dinoflagellates

  • This plasmid contains 181 protein-encoding genes and is required for anaerobic growth

  • PSD activity might be necessary for proper functioning of proteins encoded by this plasmid, especially those involved in the type IV secretion system (T4SS)

  • The membrane composition could affect the assembly or secretion of toxins involved in algal killing

• Gene expression relationships:

  • Some genes on the killer plasmid show differential expression patterns during the transition from symbiotic to pathogenic phase

  • PSD activity might be regulated in coordination with these genes

  • The expression pattern of D. shibae PSD throughout the lifecycle with dinoflagellates could reveal its role in the transition

• Experimental approaches to test these hypotheses:

  • Creating D. shibae PSD mutants and testing their ability to transition to pathogenic behavior

  • Analyzing membrane composition changes during the symbiotic-to-pathogenic transition

  • Investigating potential regulatory interactions between PSD and killer plasmid-encoded proteins

Understanding this relationship would require sophisticated lipidomic analyses, genetic manipulation of D. shibae, and co-cultivation experiments with dinoflagellates under controlled conditions.

How can site-directed mutagenesis be used to investigate the catalytic mechanism of D. shibae PSD?

Site-directed mutagenesis is a powerful approach to investigate the catalytic mechanism of D. shibae PSD through the following methodological framework:

• Targeting the GST motif and autocatalytic processing:

  • Mutations of the conserved Glycine-Serine-Threonine motif to examine effects on autocatalytic cleavage

  • Substitution of Ser with Ala would prevent pyruvoyl group formation

  • Mutations of surrounding residues to determine factors influencing cleavage efficiency

• Investigating substrate binding residues:

  • Identification of putative substrate-binding residues through homology modeling

  • Systematic mutation of conserved basic residues (Arg, Lys) that likely interact with the phosphate group

  • Mutation of hydrophobic residues potentially involved in accommodating fatty acid chains

• Experimental design and workflow:

  • Construction of mutants using PCR-based mutagenesis

  • Expression and purification of mutant proteins using established protocols

  • Detailed kinetic analysis comparing wild-type and mutant enzymes using assays similar to those used for other PSDs

  • Structural analysis of mutants by techniques such as circular dichroism and thermal stability assays

• Assessment of catalytic parameters:

  • Determination of Km and kcat for each mutant using standardized PSD activity assays

  • Analysis of pH and temperature optima to detect alterations in catalytic properties

  • Evaluation of product profiles to identify potential alterations in specificity

• Data analysis and interpretation:

  Mutation Target	Expected Effect	Analytical Method
  GST motif	Disrupted processing	SDS-PAGE, Mass spectrometry
  Substrate binding	Altered Km	Enzyme kinetics
  Membrane interaction	Changed localization	Membrane binding assays
  Active site residues	Reduced kcat	Activity assays
  Allosteric sites	Changed regulation	Response to effectors

A systematic mutagenesis study would result in a comprehensive model of structure-function relationships in D. shibae PSD and could reveal unique features of the enzyme compared to other bacterial PSDs.

What analytical methods are most effective for assessing the activity and processing of recombinant D. shibae PSD?

The most effective analytical methods for assessing activity and processing of recombinant D. shibae PSD include a comprehensive suite of techniques:

• Enzyme activity assays:

  • Radiometric assays using 14C-labeled phosphatidylserine to directly measure decarboxylation, similar to methods used for other PSDs

  • Fluorescent assays using NBD-labeled phosphatidylserine, enabling real-time monitoring and providing a substrate that freely partitions into membranes

  • HPLC-based methods to separate and quantify substrate and product

• Processing and structural analysis:

  • SDS-PAGE to visualize the α and β subunits resulting from autocatalytic cleavage, as demonstrated with other PSDs

  • Western blotting with antibodies specific to different regions of the protein

  • Mass spectrometry to precisely identify the cleavage site and confirm pyruvoyl group formation

  • Protein fractionation to separate processed from unprocessed forms

• Subcellular localization studies:

  • Subcellular fractionation to isolate bacterial membranes

  • Immunoblotting of fractionated samples to detect PSD location, similar to methods used for potato mitochondrial PSD

  • Enzyme activity measurements in different cellular fractions to correlate with protein location

• Membrane interaction studies:

  • Liposome binding assays to quantify membrane association

  • Reconstitution experiments into lipid bilayers of defined composition

  • Analysis of enzyme activity in detergent-solubilized versus membrane-incorporated states

• Comparative analysis table:

  Analytical Method	Information Provided	Advantages	Limitations
  Radiometric assay	Direct activity measurement	High sensitivity	Requires radioactive materials
  NBD-fluorescent assay	Real-time monitoring	Non-radioactive, membrane partition	Less sensitive than radiometric
  SDS-PAGE/Western blot	Processing status	Visual confirmation of cleavage	Semi-quantitative
  Mass spectrometry	Precise cleavage site	Molecular detail	Requires specialized equipment
  Subcellular fractionation	Localization	In vivo relevance	Potential cross-contamination

A comprehensive analytical approach would combine multiple methods to provide a complete picture of enzyme processing, structure, and activity, enabling comparison with PSDs from other organisms and identification of unique features of the D. shibae enzyme.

How do environmental factors affect the expression and activity of D. shibae PSD in its natural habitat?

Environmental factors likely exert significant influence on D. shibae PSD expression and activity in marine environments, particularly during interactions with dinoflagellates:

• Nutrient availability effects:

  • Phosphate limitation may alter phospholipid metabolism, affecting PSD expression

  • Availability of B vitamins, which D. shibae provides to dinoflagellates during the symbiotic phase , may influence the transition to pathogenic behavior and associated PSD activity

  • Carbon source variations could affect membrane composition and consequently PSD function

• Salinity and osmotic pressure:

  • As a marine bacterium, D. shibae has adapted to high-salt environments

  • Salinity fluctuations may require membrane remodeling, potentially altering PSD expression

  • Osmotic stress response might include changes in membrane phospholipid composition requiring adjusted PSD activity

• Temperature fluctuations:

  • Marine environments experience temperature gradients that could affect enzyme kinetics

  • PSD expression may be thermally regulated to maintain appropriate membrane fluidity

  • Temperature changes could affect the autocatalytic processing efficiency of the proenzyme

• Interaction with host organisms:

  • The transition from symbiotic to pathogenic relationship with dinoflagellates may involve coordinated changes in PSD expression

  • Signaling molecules from the dinoflagellate host might regulate bacterial gene expression, including PSD

  • Different stages of interaction may require different membrane properties facilitated by altered PSD activity

• Oxygen availability:

  • The 191 kb plasmid containing genes potentially interacting with PSD is required for anaerobic growth

  • Oxygen levels may influence both plasmid function and PSD activity

  • Transitions between aerobic and anaerobic conditions in marine environments may trigger changes in PSD expression

Understanding these environmental influences would require field studies combined with laboratory simulations of natural conditions, as well as transcriptomic and proteomic analyses of D. shibae under various environmental scenarios.

What is the evolutionary relationship between D. shibae PSD and PSDs from other bacterial and eukaryotic species?

The evolutionary relationship between D. shibae PSD and PSDs from other species reveals important insights about enzyme adaptation and function:

• Phylogenetic relationships:

  • PSDs are widely distributed across bacteria, archaea, and eukaryotes, suggesting ancient origins

  • Marine bacterial PSDs like that from D. shibae likely form distinct clades reflecting adaptation to marine environments

  • Eukaryotic PSDs show evidence of having originated from bacterial ancestors through endosymbiotic events

  • The catalytic GST motif shows high conservation across all domains of life, indicating its fundamental importance

• Functional evolution:

  • While the core catalytic mechanism is conserved, targeting sequences have diverged significantly

  • Eukaryotic PSDs have acquired mitochondrial targeting sequences and inner membrane sorting signals

  • Plants have developed both mitochondrial and chloroplastic PSDs, representing further functional specialization

  • D. shibae PSD may contain unique adaptations related to its marine lifestyle and symbiotic-pathogenic behavior

• Comparative enzyme properties:

  • Yeast has two distinct PSDs (PSD1 in mitochondria, PSD2 in Golgi/vacuole) with specialized functions

  • Plants have both mitochondrial and extramitochondrial PSDs, with evidence suggesting the latter predominates

  • D. shibae PSD likely represents a bacterial form that may share features with ancestral PSDs that gave rise to eukaryotic forms

• Domain architecture conservation:

  • The catalytic domain containing the GST motif is highly conserved

  • Membrane interaction domains show greater variability between species

  • Regulatory domains have evolved differently across lineages to respond to different cellular signals

A comprehensive evolutionary analysis would provide context for understanding the unique features of D. shibae PSD and might reveal adaptation mechanisms relevant to its marine lifestyle and complex ecological interactions with dinoflagellates.

How might knowledge of D. shibae PSD contribute to understanding marine microbial ecology?

Understanding D. shibae PSD can significantly enhance our knowledge of marine microbial ecology through several interconnected pathways:

• Symbiotic-pathogenic transitions:

  • D. shibae's Jekyll-and-Hyde behavior with dinoflagellates represents a model system for studying bacterial-algal interactions

  • PSD's role in membrane remodeling may be crucial for transitions between mutualistic and antagonistic relationships

  • This could provide insights into the molecular mechanisms underlying the stability and breakdown of marine symbioses

• Nutrient cycling implications:

  • Phospholipid metabolism, including PSD activity, affects phosphorus cycling in marine ecosystems

  • The interaction between D. shibae and dinoflagellates influences vitamin B12 and B1 cycling in marine environments

  • Understanding how membrane phospholipid metabolism is regulated provides insights into bacterial adaptation to nutrient limitation

• Plasmid-mediated traits:

  • The 191 kb "killer plasmid" in D. shibae is responsible for its pathogenic capabilities

  • Studying how this plasmid interacts with core metabolic functions like PSD could reveal mechanisms of horizontal gene transfer and integration of acquired functions

  • This knowledge may help explain how marine bacteria rapidly adapt to changing ecological conditions

• Membrane adaptations in marine environments:

  • Marine bacteria face unique challenges including high salinity, pressure variations, and temperature fluctuations

  • PSD's role in maintaining appropriate membrane composition may represent key adaptations to these challenges

  • Comparative studies with terrestrial bacteria could highlight marine-specific adaptations

• Methodological approaches for ecological studies:

  • Development of molecular markers based on PSD genes for tracking D. shibae populations

  • Creation of reporter systems to monitor PSD expression as an indicator of physiological state

  • Design of inhibitors targeting PSD to investigate its role in symbiotic-pathogenic transitions

This research connects molecular biochemistry with ecosystem-level processes, potentially revealing fundamental principles governing microbial interactions in marine environments and their impact on global biogeochemical cycles.

What challenges exist in expressing and characterizing membrane-associated enzymes like D. shibae PSD?

Expressing and characterizing membrane-associated enzymes like D. shibae PSD presents several significant challenges with specific methodological solutions:

• Expression hurdles:

  • Membrane proteins often cause toxicity when overexpressed in heterologous hosts

  • Improper folding and aggregation in non-native membrane environments

  • Inefficient processing of the proenzyme to the active form

  Solutions include:

  • Using tightly controlled induction systems

  • Expressing fusion proteins with solubility-enhancing tags

  • Co-expressing with chaperones to assist folding

  • Testing multiple expression hosts including marine bacteria

• Purification difficulties:

  • Requirement for detergents that may affect enzyme structure and function

  • Low yields compared to soluble proteins

  • Maintaining native lipid interactions during purification

  Solutions include:

  • Screening multiple detergents for optimal extraction

  • Using nanodisc technology to provide a more native-like environment

  • Developing purification protocols that preserve essential lipid interactions

• Activity assay complexities:

  • Need for appropriate membrane-like environments for activity assays

  • Substrate accessibility issues in reconstituted systems

  • Distinguishing between effects on substrate binding versus catalysis

  Solutions include:

  • Using fluorescent substrates like NBD-Ptd-Ser that partition into membranes

  • Developing assays in various membrane mimetics (liposomes, nanodiscs)

  • Comparing detergent-solubilized versus membrane-reconstituted activity

• Structural characterization limitations:

  • Difficulty in obtaining crystals for X-ray crystallography

  • Size limitations for solution NMR studies

  • Conformational heterogeneity complicating structural analyses

  Solutions include:

  • Cryo-electron microscopy approaches

  • Limited proteolysis coupled with mass spectrometry

  • Computational modeling based on homologous structures

• Comparative challenges across different systems:

  Challenge	Impact on Research	Potential Solutions
  Proenzyme processing	Heterogeneous protein population	Optimize conditions for complete processing
  Membrane requirement	Difficult to study in solution	Reconstitution into defined lipid systems
  Detergent effects	May alter enzyme properties	Screen multiple detergents, use lipid nanodiscs
  Expression toxicity	Low yields	Inducible systems, specialized expression hosts
  Conformational dynamics	Structural heterogeneity	Time-resolved methods, stabilizing mutations

Addressing these challenges requires interdisciplinary approaches combining biochemistry, structural biology, and biophysics to fully characterize membrane-associated enzymes like D. shibae PSD.

How can complementation studies in model organisms advance our understanding of D. shibae PSD function?

Complementation studies in model organisms provide powerful tools for understanding D. shibae PSD function through several methodological approaches:

• Yeast complementation systems:

  • Using yeast psd1 psd2 double mutants that require ethanolamine for growth

  • Testing whether D. shibae PSD can complement this growth defect, similar to successful tomato PSD complementation

  • Analyzing subcellular localization of the recombinant protein in yeast

  • Measuring PSD activity in the complemented strains using standardized assays

• Experimental design considerations:

  • Construction of different versions of D. shibae PSD:

    • Full-length protein with native targeting sequences

    • Truncated versions removing potential targeting sequences

    • Chimeric constructs with yeast targeting sequences

  • Analysis of growth rates under various conditions

  • Measurement of phospholipid composition changes in complemented strains

• E. coli complementation systems:

  • Using E. coli PSD mutants

  • Testing complementation with various D. shibae PSD constructs

  • Analyzing effects on membrane composition

  • Measuring growth under various environmental stresses

• Complementation in D. shibae itself:

  • Creating PSD knockout mutants in D. shibae

  • Complementing with wild-type and mutated versions of the PSD gene

  • Testing effects on interaction with dinoflagellates

  • Analyzing changes in membrane composition and plasmid maintenance

• Data interpretation framework:

  Complementation System	Information Gained	Limitations	Controls Needed
  Yeast psd1 psd2	Basic enzyme function	Different cellular context	Yeast PSD1 positive control
  E. coli PSD mutant	Prokaryotic context	Different from marine environment	E. coli PSD positive control
  D. shibae knockout	Native context	More challenging genetics	Empty vector negative control
  Plant knockout	Different eukaryotic context	Complex phospholipid pathways	Plant PSD positive control

Complementation studies have already proven valuable for understanding plant PSDs and would likely yield similar insights for D. shibae PSD, especially regarding targeting, processing, and functional conservation across diverse organisms.