Recombinant Klebsiella pneumoniae subsp. pneumoniae Bifunctional protein aas (aas)
Product Specs
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Target Background
This bifunctional protein plays a crucial role in lysophospholipid acylation. It catalyzes the transfer of fatty acids to the 1-position of lysophospholipids via an enzyme-bound acyl-ACP intermediate, requiring ATP and magnesium. Its primary physiological function is the regeneration of phosphatidylethanolamine from 2-acyl-glycero-3-phosphoethanolamine (2-acyl-GPE), a byproduct of transacylation reactions or phospholipase A1 degradation.
KEGG: kpn:KPN_03245
STRING: 272620.KPN_03245
Q&A
What is Klebsiella pneumoniae Bifunctional protein aas?
Klebsiella pneumoniae Bifunctional protein aas is a multifunctional enzyme encoded by the aas gene in Klebsiella pneumoniae bacteria. The protein is classified as "bifunctional" due to its dual enzymatic activities, which include 2-acylglycerophosphoethanolamine acyltransferase (EC= 2.3.1.40) functionality . This enzyme plays a critical role in bacterial membrane phospholipid metabolism and maintenance. The full-length protein consists of 719 amino acids and has been successfully expressed as a recombinant protein in E. coli expression systems with various tags (such as His-tag) to facilitate purification and downstream research applications .
What are the optimal storage and handling conditions for recombinant aas protein?
For maintaining optimal stability and activity of recombinant Klebsiella pneumoniae Bifunctional protein aas, the following storage and handling protocols are recommended:
| Storage Parameter | Recommended Condition |
|---|---|
| Long-term storage | -20°C/-80°C |
| Working storage | 4°C (up to one week) |
| Physical form | Lyophilized powder |
| Storage buffer | Tris/PBS-based buffer with 6% Trehalose, pH 8.0 |
| Reconstitution | Deionized sterile water to 0.1-1.0 mg/mL |
| Stability additive | 5-50% glycerol (final concentration) |
| Special considerations | Aliquoting necessary for multiple use; avoid repeated freeze-thaw cycles |
When handling the reconstituted protein, it is recommended to briefly centrifuge the vial prior to opening to bring contents to the bottom . For long-term storage after reconstitution, adding glycerol (typically to a final concentration of 50%) and aliquoting the sample can help preserve protein integrity and enzymatic activity .
What functional domains and mechanisms characterize the Bifunctional protein aas?
The Klebsiella pneumoniae Bifunctional protein aas contains at least two distinct functional domains that contribute to its enzymatic versatility:
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2-acylglycerophosphoethanolamine acyltransferase domain (EC= 2.3.1.40): This domain catalyzes the transfer of acyl groups to glycerophosphoethanolamine substrates, playing a crucial role in phospholipid remodeling .
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Acyl-[acyl-carrier-protein]--phospholipid domain: This functionality suggests the protein's involvement in transferring acyl groups from acyl-carrier proteins to phospholipids, contributing to membrane lipid biosynthesis and turnover .
The bifunctional nature of this protein enables it to coordinate multiple aspects of bacterial membrane phospholipid metabolism within a single polypeptide chain. This arrangement likely facilitates efficient coupling between different steps in phospholipid biosynthesis and remodeling pathways, providing Klebsiella pneumoniae with adaptive mechanisms for maintaining membrane integrity under varying environmental conditions.
Sequence analysis indicates the presence of motifs characteristic of acyltransferases, including potential substrate binding pockets and catalytic residues. The protein's sequence suggests it may have membrane-associated regions, consistent with its role in phospholipid metabolism.
What methodologies can be employed to measure aas protein enzymatic activity in vitro?
To effectively measure the enzymatic activities of Klebsiella pneumoniae Bifunctional protein aas in vitro, researchers can implement several complementary approaches:
| Assay Type | Methodology | Detection | Advantages |
|---|---|---|---|
| Spectrophotometric | Using DTNB (Ellman's reagent) to detect free CoA release | Absorbance at 412 nm | Real-time monitoring, quantitative |
| Radiometric | 14C-labeled acyl-CoA incorporation into phospholipids | Scintillation counting after TLC separation | High sensitivity, direct measurement of product formation |
| Chromatographic | HPLC or LC-MS analysis of substrate consumption and product formation | UV, MS, or ELSD detection | High resolution, identification of multiple products |
| Fluorescence-based | Fluorescently labeled substrates or coupled enzyme reactions | Fluorescence emission | High sensitivity, potential for high-throughput screening |
A typical reaction buffer composition for acyltransferase activity measurement would include:
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50 mM Tris-HCl or HEPES buffer, pH 7.5-8.0
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100-150 mM NaCl
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5-10 mM MgCl2 (as a cofactor)
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0.1-1 mM DTT (to maintain reducing conditions)
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0.05-0.1% detergent (for membrane-associated enzyme)
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Appropriate substrates: acyl-CoA donors and phospholipid acceptors
When establishing an activity assay, researchers should:
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Determine the linear range of the assay with respect to enzyme concentration and time
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Generate a calibration curve with known product concentrations
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Include appropriate controls (heat-inactivated enzyme, no-substrate controls)
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Calculate specific activity in terms of μmol product formed per minute per mg of protein
This methodological framework provides a robust foundation for characterizing the enzymatic properties of the aas protein, including substrate specificity, kinetic parameters, and the effects of potential inhibitors.
What challenges exist in expressing and purifying recombinant Klebsiella pneumoniae aas protein?
Expression and purification of recombinant Klebsiella pneumoniae Bifunctional protein aas presents several technical challenges that researchers should consider when designing experimental protocols:
Expression Challenges:
| Challenge | Description | Potential Solutions |
|---|---|---|
| Membrane association | Protein sequence suggests membrane-interacting regions | Use specialized expression strains; include detergents in lysis buffer |
| Size complexity | 719 amino acid length increases susceptibility to premature termination | Optimize codon usage; lower expression temperature (16-20°C) |
| Potential toxicity | Membrane protein overexpression may disrupt host cell membranes | Use tightly regulated expression systems; reduce induction levels |
| Proper folding | Multi-domain protein requires coordinated folding | Co-express with chaperones; include folding enhancers |
| Solubility limitations | Hydrophobic regions may cause aggregation | Fusion tags (SUMO, MBP, GST); solubility-enhancing additives |
Purification Challenges:
The recombinant protein is typically expressed with a His-tag to facilitate purification , but additional considerations include:
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Optimization of lysis conditions: Inclusion of appropriate detergents to solubilize membrane-associated protein while maintaining activity
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Multi-step purification strategy: Combining affinity chromatography (IMAC) with ion exchange and/or size exclusion chromatography
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Activity preservation: Identifying buffer components that maintain structural integrity and prevent aggregation
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Purity assessment: Achieving >90% purity as determined by SDS-PAGE
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Removal of contaminating bacterial lipids: Additional wash steps or specialized chromatography
Current successful expression strategies utilize E. coli as the expression host with His-tagged constructs , which provides a starting point for further optimization based on specific research requirements.
How can site-directed mutagenesis be utilized to investigate the active sites of aas protein?
Site-directed mutagenesis represents a powerful approach for dissecting the structure-function relationships of the Klebsiella pneumoniae Bifunctional protein aas, particularly for elucidating the roles of specific residues in catalysis and substrate binding:
Systematic Mutagenesis Strategy:
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Target residue identification:
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Computational prediction of catalytic residues through sequence alignment with characterized homologs
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Identification of conserved motifs typical for acyltransferases
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Structure-based prediction of substrate-binding residues
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Mutagenesis approaches:
| Approach | Description | Application |
|---|---|---|
| Alanine scanning | Systematic replacement with alanine | Identify essential residues |
| Conservative substitutions | Replace with similar amino acids (e.g., Asp→Glu) | Probe specific chemical requirements |
| Non-conservative substitutions | Dramatic changes in residue properties | Test hypotheses about residue function |
| Domain swapping | Create chimeric proteins with homologous domains | Investigate domain-specific functions |
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Experimental workflow:
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Design of PCR-based mutagenesis protocols
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Expression and purification of mutant proteins using established protocols
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Parallel characterization of wild-type and mutant proteins
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Functional analysis of mutants:
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Enzyme activity assays to determine effects on catalytic parameters (kcat, Km)
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Substrate binding studies to assess changes in binding affinity
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Thermal stability analysis to detect structural perturbations
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Structural studies (if feasible) to observe conformational changes
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Data interpretation and modeling:
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Structure-function correlation analysis
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Development of mechanistic models for enzymatic action
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Identification of potential targets for inhibitor design
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This systematic approach can provide detailed insights into the catalytic mechanism of aas protein and potentially reveal strategies for targeted inhibition, which may have relevance for antimicrobial development against Klebsiella pneumoniae.
How can research literature contradiction detection be applied to resolve conflicting findings about aas protein?
In the evolving field of research on Klebsiella pneumoniae Bifunctional protein aas, contradictory findings may emerge due to methodological differences, strain variations, or interpretation discrepancies. Modern contradiction detection approaches can help researchers navigate and resolve these conflicts:
Systematic Contradiction Analysis Framework:
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Literature mining and contradiction identification:
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Categorization of contradictions:
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Implementation of contradiction detection systems:
Recent advances in clinical contradiction detection using distant supervision over clinical ontologies have demonstrated statistically significant improvements in identifying contradictions in medical literature . These approaches can be adapted to the biochemical research domain, particularly for bacterial proteins like aas.
A systematic approach to contradiction detection can help researchers:
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Identify knowledge gaps requiring further investigation
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Prioritize research questions with the greatest uncertainty
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Design definitive experiments to resolve contradictory findings
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Build consensus models that integrate diverse experimental data
