Recombinant Citrobacter koseri Glycerol-3-phosphate acyltransferase (plsY)
Description
Biological Context and Significance
Glycerol-3-phosphate acyltransferase (plsY) is an essential enzyme in bacterial membrane phospholipid biosynthesis. In Citrobacter koseri, this enzyme catalyzes the first step in phospholipid formation, serving as the committed step in the pathway . Unlike conventional acyltransferases that utilize acyl-CoA or acyl-carrier protein as acyl donors, plsY uniquely employs acyl-phosphate for the acylation reaction . The enzyme is classified under EC 2.3.1.n3 and is also known by several alternative names including Acyl-PO4 G3P acyltransferase, Acyl-phosphate--glycerol-3-phosphate acyltransferase, and G3P acyltransferase (GPAT) .
Citrobacter koseri as a Source Organism
Citrobacter koseri is a facultative anaerobic Gram-negative bacillus belonging to the Enterobacteriaceae family. It is recognized as an opportunistic pathogen primarily associated with urinary tract infections, neonatal sepsis, and meningitis . C. koseri demonstrates remarkable genomic plasticity, exemplified by documented instances of horizontal gene transfer from other bacterial species. A notable example is the acquisition of a continuous 2 kb genomic fragment containing the virulence factor Pla from the Yersinia pestis pPCP1 plasmid . This mosaic gene structure reflects the adaptable lifestyle of C. koseri and its capacity for genomic exchange with different enteric bacteria.
Primary Structure and Sequence Information
The plsY protein from Citrobacter koseri (strain ATCC BAA-895 / CDC 4225-83 / SGSC4696) consists of 205 amino acids with the UniProt accession number A8APU2 . The complete amino acid sequence is:
MSAIAPGMILFAYLCGSISSAILVCRIAGLPDPRQSGSGNPGATNVLRIGGKGAAVAVLIFVDVLKGMLPVWGAYALGVTPFWLGLIAIAACLGHIWPVFFGFKGGKGVATAFGAIAPIGWDLTGVMAGTWLLTVLLSGYSSLGAIVSALIAPFYVWWFKPQFTFPVSMLSCLILLRHHDNIQRLWRRQETKIWTKLKKKREKDPQ
The gene encoding plsY in C. koseri is designated as plsY (gene name) with the ordered locus name CKO_04449 .
Three-Dimensional Structure
X-ray crystallography studies have determined the crystal structure of plsY at a high resolution of 1.48 Å, revealing a distinctive seven-transmembrane helix (7-TMH) fold . This structural data represents a significant advancement in understanding membrane-integral acyltransferases. In addition to the apo-protein structure, four substrate- and product-bound structures have been elucidated, providing atomic details of the enzyme's relatively inflexible active site . These structural insights are crucial for understanding the catalytic mechanism and for structure-based drug design targeting this enzyme.
Enzymatic Function
PlsY catalyzes the acylation of glycerol 3-phosphate to form lysophosphatidic acid, representing the committed and essential step in bacterial phospholipid biosynthesis . The reaction can be represented as:
Acyl-phosphate + Glycerol 3-phosphate → Lysophosphatidic acid + Phosphate
This reaction is fundamental to bacterial cell membrane formation and integrity, making plsY essential for bacterial survival.
Catalytic Mechanism
Unlike other acyltransferases, plsY employs a unique "substrate-assisted catalysis" mechanism that does not require a proteinaceous catalytic base to complete the acylation reaction . This distinctive mechanistic feature sets plsY apart from conventional acyltransferases and reflects its evolutionary adaptation to bacterial membrane phospholipid synthesis. Extensive mutagenesis studies, coupled with high-resolution structural data of substrate- and product-bound states, have provided evidence for this alternative catalytic pathway .
Enzymatic Assays
The enzymatic activity of plsY can be monitored using various techniques, including a coupled fluorescence-based assay that detects the release of inorganic phosphate. The progress curves from such assays typically show:
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An initial lag phase (approximately 2 minutes) reflecting the time required for glycerol 3-phosphate equilibration between the assay mix and lipid cubic phase
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A linear phase that can be used to calculate enzyme activity
The production of lysophosphatidic acid can be confirmed using thin layer chromatography (TLC), which can detect the product in samples containing active plsY but not in control samples without the enzyme .
PlsY vs. PlsB: Distinct Bacterial GPATs
In bacteria, two distinct types of glycerol 3-phosphate acyltransferases (GPATs) catalyze the committed step in phospholipid biosynthesis: PlsB and PlsY . Table 1 summarizes the key differences between these acyltransferases:
| Feature | PlsY | PlsB |
|---|---|---|
| Distribution | Exclusively and ubiquitously in bacteria | Bacteria, with eukaryotic homologs |
| Acyl donor | Acyl-phosphate | Acyl-CoA or acyl-carrier protein |
| Structure | 7-transmembrane helix fold | Different structural organization |
| Catalytic mechanism | Substrate-assisted catalysis | Conventional catalytic mechanism requiring proteinaceous base |
| Role in Gram-positive bacteria | Sole and essential GPAT | Not typically present |
| Eukaryotic homologs | None | Present |
The absence of eukaryotic homologs and the exclusive presence in bacteria make PlsY an attractive target for antimicrobial development, particularly for addressing infections caused by Gram-positive pathogens such as Enterococcus faecium and Streptococcus pneumoniae .
Evolutionary and Functional Significance
PlsY represents a unique class of acyltransferase that lacks known acyltransferase motifs found in other enzymes of this category. Its unusual acyl donor preference and distinctive catalytic mechanism suggest a separate evolutionary path compared to conventional acyltransferases. In most Gram-positive bacteria, PlsY serves as the sole GPAT and is therefore essential for bacterial survival and proliferation .
Expression and Purification
Recombinant Citrobacter koseri plsY is typically produced in appropriate expression systems, considering its membrane-integral nature. Commercial preparations of the recombinant protein are available with various tags to facilitate purification and detection . Table 2 summarizes the typical characteristics of recombinant C. koseri plsY preparations:
| Parameter | Specification |
|---|---|
| Typical quantity | 50 μg (other quantities available) |
| Tag information | Tag type determined during production process |
| Storage buffer | Tris-based buffer, 50% glycerol, optimized for protein stability |
| Storage conditions | -20°C for regular storage; -20°C or -80°C for extended storage |
| Working storage | 4°C for up to one week |
| Expression region | 1-205 (full-length protein) |
| Special handling | Repeated freezing and thawing not recommended |
Research Applications
Recombinant plsY has valuable applications in biochemical and structural studies. The availability of high-resolution crystal structures, including those with bound substrates and products, provides a foundation for understanding its catalytic mechanism in detail . Additionally, high-throughput enzymatic assays developed for plsY can be utilized for virtual and experimental screening of potential inhibitors, facilitating the discovery of novel antimicrobial compounds .
Therapeutic Potential
The structural and biochemical uniqueness of PlsY positions it as a promising target for antimicrobial development. Several key factors contribute to its attractiveness as a therapeutic target:
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It is exclusively present in bacteria with no eukaryotic homologs, suggesting potential for selective toxicity
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It serves as the sole GPAT in most Gram-positive bacteria, including important pathogens like Enterococcus faecium and Streptococcus pneumoniae
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Its crystal structure at 1.48 Å resolution provides detailed insights for structure-based drug design
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Previous studies have identified several PlsY inhibitors as potential antimicrobials
Challenges and Future Directions
While PlsY represents a promising antimicrobial target, challenges remain in developing effective inhibitors that can penetrate the bacterial cell membrane and achieve sufficient in vivo efficacy. The high-resolution structural data and enzymatic assays developed for PlsY provide valuable tools for addressing these challenges. Future research directions may include:
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Structure-based design of novel PlsY inhibitors with improved pharmacokinetic properties
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Investigation of potential synergistic effects between PlsY inhibitors and existing antibiotics
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Development of delivery systems to enhance inhibitor penetration into bacterial cells
Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your preferred format in order notes for customized fulfillment.
Note: Standard shipping includes blue ice packs. Dry ice shipping requires prior arrangement and incurs additional charges.
The tag type is determined during the production process. Please specify your required tag type for preferential development.
Target Background
This enzyme catalyzes the transfer of an acyl group from acyl-phosphate (acyl-PO4) to glycerol-3-phosphate (G3P), resulting in the formation of lysophosphatidic acid (LPA). It utilizes acyl-phosphate as the fatty acyl donor, but not acyl-CoA or acyl-ACP.
KEGG: cko:CKO_04449
STRING: 290338.CKO_04449
Q&A
What is Citrobacter koseri Glycerol-3-phosphate acyltransferase (plsY) and what is its role in bacterial physiology?
Citrobacter koseri Glycerol-3-phosphate acyltransferase (plsY) is a critical enzyme in bacterial phospholipid biosynthesis, specifically catalyzing the transfer of an acyl group from acyl-phosphate to the 1-position of glycerol-3-phosphate. This reaction represents a key step in membrane phospholipid synthesis, which is essential for bacterial cell viability and membrane integrity. The enzyme belongs to the acyltransferase family and has several alternative names including Acyl-PO4 G3P acyltransferase, Acyl-phosphate--glycerol-3-phosphate acyltransferase, and G3P acyltransferase (GPAT) . In Citrobacter koseri, the plsY gene is identified by the locus name CKO_04449, and the protein plays a fundamental role in bacterial membrane biogenesis, which directly impacts cellular growth, division, and potentially pathogenicity.
How should Recombinant Citrobacter koseri plsY be stored and handled in laboratory settings?
For optimal stability and activity, Recombinant Citrobacter koseri plsY should be stored at -20°C in a Tris-based buffer containing 50% glycerol that has been optimized for this specific protein . For extended storage periods, conservation at -80°C is recommended to prevent protein degradation and maintain enzymatic activity. Working aliquots can be maintained at 4°C for up to one week to minimize freeze-thaw cycles, as repeated freezing and thawing is not recommended for this enzyme .
Handling procedures should include:
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Maintaining strict temperature control during all experimental procedures
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Using appropriate buffers that maintain optimal pH for enzyme stability
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Avoiding excessive exposure to proteases and oxidizing agents
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Implementing sterile technique to prevent microbial contamination
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Including protease inhibitors when working with crude preparations
These precautions help maintain the structural integrity and catalytic activity of the recombinant enzyme during experimental procedures.
What expression systems are typically used for producing Recombinant Citrobacter koseri plsY?
Recombinant Citrobacter koseri plsY is typically produced using bacterial expression systems, with Escherichia coli being the most common host due to its genetic similarity to Citrobacter species and established protocols for membrane protein expression. The expression region typically encompasses residues 1-205, representing the full-length protein . The process generally involves:
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Cloning the plsY gene (CKO_04449) into an appropriate expression vector
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Transformation into a compatible E. coli expression strain
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Induction of protein expression under optimized conditions
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Cell lysis and membrane fraction isolation
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Protein solubilization using detergents
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Purification via affinity chromatography (often using a fusion tag)
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Quality control testing for purity and activity
The tag type for purification is generally determined during the production process based on optimal expression and purification results . Common tags include His-tag, GST, or MBP to facilitate purification while maintaining protein functionality.
How does plsY contribute to Citrobacter koseri pathogenesis and virulence?
While the direct role of plsY in Citrobacter koseri pathogenesis hasn't been fully characterized, evidence suggests that bacterial membrane lipid composition significantly impacts virulence factors. Citrobacter koseri demonstrates a concerning preference for infecting the central nervous system (CNS) in the first two months of life, causing meningitis and brain abscesses that can lead to severe outcomes including intellectual disability and seizures .
The phospholipid biosynthesis pathway where plsY functions is essential for membrane biogenesis, which in turn affects:
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Membrane permeability and antibiotic resistance
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Cell surface properties that influence host-pathogen interactions
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Bacterial survival in diverse host environments
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Formation of biofilms that enhance virulence and persistence
Citrobacter koseri possesses several virulence factors associated with flagellar apparatus biosynthesis and iron uptake, including a High Pathogenicity Island (HPI) gene cluster similar to that found in pathogenic Yersinia strains . While plsY is not directly mentioned in relation to these specific virulence factors, the enzyme's role in membrane phospholipid synthesis makes it essential for proper membrane function and potentially influences the expression and functionality of membrane-associated virulence factors.
What experimental approaches can be used to study plsY inhibition as a potential antimicrobial strategy?
Given the rising antibiotic resistance in Citrobacter species worldwide, with case-fatality rates reaching 30% and death rates of 33-48% in neonates , investigating plsY inhibition presents a promising antimicrobial strategy. The following experimental approaches can be employed:
| Experimental Approach | Methodology | Advantages | Challenges |
|---|---|---|---|
| High-throughput screening | Screen chemical libraries against purified recombinant plsY | Identifies novel inhibitor scaffolds | Requires robust activity assay |
| Structure-based drug design | Virtual screening based on enzyme structure | Rational design of specific inhibitors | Requires accurate protein structure |
| In vitro enzymatic assays | Measure plsY activity with substrate analogs | Direct measurement of inhibition | May not translate to whole-cell activity |
| Whole-cell antibacterial assays | Test compounds against Citrobacter strains | Confirms cellular penetration and effect | Cannot confirm specific plsY targeting |
| Genetic approaches | Gene knockdown/CRISPR interference | Validates plsY as essential target | Requires genetic manipulation tools |
| Membrane lipid analysis | LC-MS analysis of phospholipid changes | Direct measurement of pathway inhibition | Complex analytical requirements |
When developing plsY inhibitors, researchers should consider:
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The essentiality of plsY in bacterial survival
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Structural differences between bacterial and human homologs to ensure specificity
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The ability of compounds to penetrate the bacterial outer membrane
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Potential for resistance development
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Effects on membrane integrity and downstream virulence factors
How does the substrate specificity of Citrobacter koseri plsY compare with other bacterial acyltransferases?
The substrate specificity of Citrobacter koseri plsY is defined by its ability to recognize and utilize specific acyl-phosphate donors and glycerol-3-phosphate as acceptor. Comparative analysis with other bacterial acyltransferases reveals both conservation and specialization of function:
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Structural features: The Citrobacter koseri plsY contains transmembrane domains typical of acyltransferases, with specific amino acid residues in its active site that determine substrate preferences.
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Acyl chain preference: The enzyme typically shows preference for certain acyl chain lengths and saturation levels, which directly influences bacterial membrane composition.
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Evolutionary conservation: Comparative genomics indicates that the plsY gene is highly conserved across the Enterobacteriaceae family, reflecting its essential function.
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Differential regulation: Though structurally similar, regulatory mechanisms controlling plsY expression may vary between species, affecting adaptation to different environments.
Experimental approaches to study substrate specificity include:
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Enzyme kinetics with various acyl-phosphate donors
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Site-directed mutagenesis to identify specificity-determining residues
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Lipid profiling to assess in vivo substrate utilization
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Heterologous expression studies to compare functional conservation
What are the implications of antibiotic resistance on plsY functionality in Citrobacter koseri?
The emergence of multidrug-resistant (MDR) Citrobacter strains presents significant treatment challenges. While plsY itself is not typically a direct target of current antibiotics, membrane physiology and lipid composition can significantly impact bacterial susceptibility to antibiotics . Research considerations include:
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Membrane permeability changes: Alterations in phospholipid composition mediated by plsY can affect membrane permeability to antibiotics.
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Regulatory cross-talk: Stress responses induced by antibiotics may alter plsY expression and activity, potentially as part of adaptive resistance mechanisms.
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Metabolic compensation: Under antibiotic stress, bacteria may modulate membrane lipid synthesis pathways to maintain membrane integrity.
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Biofilm formation: Changes in membrane properties can influence biofilm formation, which contributes to antibiotic tolerance.
Citrobacter species have shown increasing resistance to β-lactam antibiotics, including amoxicillin, ampicillin, and cephalosporins . Additionally, concerning trends in carbapenem resistance and colistin resistance (mediated by mcr genes) have been reported in various Citrobacter species . While direct connections to plsY have not been established in the literature, the enzyme's fundamental role in membrane biogenesis suggests it may indirectly influence the bacterial response to antibiotic exposure.
What technological advances are enabling new insights into plsY structure-function relationships?
Recent technological advances have significantly enhanced our ability to study membrane-associated enzymes like plsY:
| Technology | Application to plsY Research | Potential Insights |
|---|---|---|
| Cryo-electron microscopy | High-resolution structural determination | Detailed active site configuration and membrane interaction |
| Molecular dynamics simulations | Modeling enzyme dynamics and substrate interactions | Catalytic mechanism and inhibitor binding modes |
| Native mass spectrometry | Analysis of protein-lipid interactions | Identification of specific lipid interactions that influence activity |
| CRISPR-Cas9 genome editing | Precise genetic manipulation | In vivo functional studies and essential residue identification |
| Lipidomics | Comprehensive lipid profiling | Impact of plsY variants on membrane composition |
| Single-molecule enzymology | Direct observation of catalytic events | Kinetic mechanisms and rate-limiting steps |
| Nanodiscs and lipid bilayer systems | Reconstitution in native-like environments | Effects of membrane composition on enzyme activity |
These technologies enable increasingly sophisticated investigations into how plsY structure influences its catalytic properties, membrane association, and potential as a drug target in Citrobacter koseri.
