Literature summary for 4.6.1.13 extracted from

Cheng, J.; Goldstein, R.; Stec, B.; Gershenson, A.; Roberts, M.F.
Competition between anion binding and dimerization modulates Staphylococcus aureus phosphatidylinositol-specific phospholipase C enzymatic activity (2012), J. Biol. Chem., 287, 40317-40327.

Cloned(Commentary)

Cloned (Comment)	Organism
expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21-Codonplus (DE3)-RIL	Staphylococcus aureus

Crystallization (Commentary)

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

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

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 [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	phospholipid binding kinetics of the recombinant enzyme, ooverview	Staphylococcus aureus

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
extracellular	the enzyme is secreted	Staphylococcus aureus	-	-

Organism

Organism	UniProt	Comment	Textmining
Staphylococcus aureus	P45723	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21-Codonplus (DE3)-RIL	Staphylococcus aureus

Substrates and Products (Substrate)

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

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

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 [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
28	-	assay at	Staphylococcus aureus

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
6.5	-	-	Staphylococcus aureus

General Information

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