Cloned (Comment) | Organism |
---|---|
expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21-Codonplus (DE3)-RIL | Staphylococcus aureus |
Crystallization (Comment) | Organism |
---|---|
purified recombinant wild-type enzyme and mutant Y253S complexed with 1,2-dibutyroyl-sn-glycero-3-phosphocholine, hanging drop vapour diffusion method, mixing of 0.0015 ml of 10 mg/ml protein, 50 mM myo-inositol and 2.5 mM 1,2-dibutyroyl-sn-glycero-3-phosphocholine, with 0.0015 ml of reservoir solution containing 150 mM ammonium acetate, 100 mM sodium acetate, pH 4.6, with 1 mM Mg(NO3)2 (100 mM for the mutant enzymes), and 16-20% PEG 4000, 20°C, 2 weeks, X-ray diffraction structure determination and analysis at 2.16-2.3 A resolution | Staphylococcus aureus |
Protein Variants | Comment | Organism |
---|---|---|
F249W | site-directed mutagenesis, the mutant shows no significant changes in structure and thermal stability, but altered enzyme activity compared to the wild-type enzyme | Staphylococcus aureus |
H86E | site-directed mutagenesis, the mutant shows no significant changes in structure and thermal stability, but altered enzyme activity compared to the wild-type enzyme | Staphylococcus aureus |
H86Y | site-directed mutagenesis, the mutant shows no significant changes in structure and thermal stability, but altered enzyme activity compared to the wild-type enzyme | Staphylococcus aureus |
V44C | site-directed mutagenesis, the mutant shows no significant changes in structure and thermal stability, but altered enzyme activity compared to the wild-type enzyme | Staphylococcus aureus |
V44W | site-directed mutagenesis, the mutant shows no significant changes in structure and thermal stability, but altered enzyme activity compared to the wild-type enzyme | Staphylococcus aureus |
Y253K | site-directed mutagenesis, the mutant shows no significant changes in structure and thermal stability, but altered enzyme activity compared to the wild-type enzyme | Staphylococcus aureus |
Y253S | site-directed mutagenesis, the mutant shows no significant changes in structure and thermal stability, but altered enzyme activity compared to the wild-type enzyme | Staphylococcus aureus |
Y253S/Y255S | site-directed mutagenesis, the mutant has the same secondary structure content but a 5°C lower thermal denaturation temperature than the wild-type and an altered enzyme activity | Staphylococcus aureus |
Y253W | site-directed mutagenesis, the mutant shows no significant changes in structure and thermal stability, but altered enzyme activity compared to the wild-type enzyme | Staphylococcus aureus |
Y290A | site-directed mutagenesis, the mutant shows no significant changes in structure and thermal stability, but altered enzyme activity compared to the wild-type enzyme | Staphylococcus aureus |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | phospholipid binding kinetics of the recombinant enzyme, ooverview | Staphylococcus aureus |
Localization | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
extracellular | the enzyme is secreted | Staphylococcus aureus | - |
- |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Staphylococcus aureus | P45723 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21-Codonplus (DE3)-RIL | Staphylococcus aureus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | formation of inositol 1,2-(cyclic)phosphate from L-alpha-phoshatidylinositol, NMR spectroscopy analysis | Staphylococcus aureus | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
dimer | the enzyme can form a dimer via helix B, a structural feature present in all secreted, bacterial phosphatidylinositol-specific phospholipases C that is important for membrane binding. The small interface is critical for optimal enzyme activity. The enzyme dimerization is enhanced in membranes containing phosphatidylcholine, fluorescence correlation spectroscopy binding analysis | Staphylococcus aureus |
Synonyms | Comment | Organism |
---|---|---|
More | cf. EC 3.1.4.11 | Staphylococcus aureus |
phosphatidylinositol-specific phospholipase C | - |
Staphylococcus aureus |
PI-PLC | - |
Staphylococcus aureus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
28 | - |
assay at | Staphylococcus aureus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
6.5 | - |
- |
Staphylococcus aureus |
General Information | Comment | Organism |
---|---|---|
additional information | the enzyme can form a dimer via helix B, a structural feature present in all secreted, bacterial phosphatidylinositol-specific phospholipases C that is important for membrane binding. The small interface is critical for optimal enzyme activity. The enzyme dimerization is enhanced in membranes containing phosphatidylcholine, the zwitterionic phospholipid acts not by specific binding to the protein, but rather by reducing anionic lipid interactions with a cationic pocket on the surface of the enzyme that stabilizes monomeric protein. Staphylococcus aureus phosphatidylinositol-specific phospholipase C appears to have a unique mechanism where enzyme activity is modulated by competition between binding of soluble anions or anionic lipids to the cationic sensor and transient dimerization on the membrane | Staphylococcus aureus |
physiological function | the enzyme is a virulence factor in the pathogenicity of Staphylococcus aureus | Staphylococcus aureus |