Information on EC 4.6.1.13 - phosphatidylinositol diacylglycerol-lyase

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY hide
4.6.1.13
-
RECOMMENDED NAME
GeneOntology No.
phosphatidylinositol diacylglycerol-lyase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
1-phosphatidyl-1D-myo-inositol = 1D-myo-inositol 1,2-cyclic phosphate + 1,2-diacyl-sn-glycerol
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
P-O bond cleavage
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Inositol phosphate metabolism
-
-
SYSTEMATIC NAME
IUBMB Comments
1-phosphatidyl-1D-myo-inositol 1,2-diacyl-sn-glycerol-lyase (1D-myo-inositol-1,2-cyclic-phosphate-forming)
This enzyme is bacterial. Activity is also found in animals, but this activity is due to the presence of EC 3.1.4.11, phosphoinositide phospholipase C.
CAS REGISTRY NUMBER
COMMENTARY hide
37288-19-0
-
63551-76-8
-
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
metabolism
-
the enzyme is implicated in direct regulation of lipid biosynthesis
physiological function
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
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
1-phosphatidyl-1D-myo-inositol
1D-myo-inositol 1,2-cyclic phosphate + 1,2-diacyl-sn-glycerol
show the reaction diagram
1-phosphatidyl-1D-myo-inositol
1D-myo-inositol 1,2-cyclic phosphate + diacylglycerol
show the reaction diagram
1-phosphatidyl-1D-myo-inositol
1D-myo-inositol 1,2-cyclic phosphate + sn-1,2-diacylglycerol
show the reaction diagram
-
natural aggregate substrate, two-site enzyme model with interfacial cooperativity between the active site and a lipid-binding subsite, presumably adjacent to the active site
-
?
butyl-fluorescein myo-inositol phosphate
D-myo-inositol 1,2-cyclic phosphate + butyl-fluorescein
show the reaction diagram
-
two substrate molecules bind to enzyme, one at the active site and one at a subsite, causing an increase in activity, subsite interactions of PI-PLC
-
?
dibutyrylphosphatidylinositol
1D-myo-inositol 1,2-(cyclic)-phosphate + 1,2-dibutyryl-sn-glycerol
show the reaction diagram
-
is a poor substrate, necessitating long incubation times (2 to 5 h) if the same enzyme concentration is to be used in the absence and presence of salts and amphiphiles
-
-
?
dihexanoylphosphatidyl inositol
?
show the reaction diagram
-
-
-
-
?
dihexanoylphosphorothioyl-myo-inositol
myo-inositol cis(2-OH,S)-1,6-cyclic phosphorothioate + 1,2-dihexanoyl-sn-glycerol
show the reaction diagram
-
-
-
-
?
lysophosphatidylinositol + H2O
?
show the reaction diagram
methyl-fluorescein myo-inositol phosphate
D-myo-inositol 1,2-cyclic phosphate + methyl-fluorescein
show the reaction diagram
phosphatidylinositol
diacylglycerol + myo-inositol 1,2-cyclic phosphate
show the reaction diagram
phosphatidylinositol + H2O
diacylglycerol + myo-inositol 1,2-cyclic phosphate
show the reaction diagram
phosphatidylinositol + H2O
diacylglycerol + myo-inositol 1,2-cyclic phosphate + D-myo-inositol 1-phosphate
show the reaction diagram
-
at first the enzyme catalyzes phosphate transfer within the molecule of phosphatidylinositol from glycerol OH to 2-OH of myo-inositol, resulting in diacylglycerol and myo-inositol 1,2-cyclic phosphate. Next myo-inositol 1,2-cyclic phosphate is hydrolyzed by the enzyme to inositol 1-phosphate. Since the reaction rate of the first step (phosphotransferase) is 1000 times as much as that of the second step (cyclic phosphodiesterase) myo-inositol 1,2-cyclic phosphate accumulates as one of the major products during enzyme action
-
-
?
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
1-phosphatidyl-1D-myo-inositol
1D-myo-inositol 1,2-cyclic phosphate + 1,2-diacyl-sn-glycerol
show the reaction diagram
1-phosphatidyl-1D-myo-inositol
1D-myo-inositol 1,2-cyclic phosphate + diacylglycerol
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Cd2+
-
activates R69D mutant at low concentrations, no activation of wild-type PI-PLC
Cl-
-
1 M, 2-3fold activation of wild-type PI-PLC
Co2+
-
activates R69D mutant at low concentrations, no activation of wild-type PI-PLC
Li+
-
5fold activation of R69D mutant, slight activation of wild-type PI-PLC
Mn2+
-
activates R69D mutant at low concentrations, slight activation of wild-type PI-PLC
Sr2+
-
9fold activation of R69D mutant, slight activation of wild-type PI-PLC
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
AOT
-
bis(2-ethylhexyl)sulfosuccinate
-
Co2+
-
severe inhibition of wild-type PI-PLC
ET-18-OCH3
-
specific inhibitors of PI-PLC, 0.1 mM abolish stimulatory effect of 0.00005 mM heme in the PI-PLC/protein kinase C pathway
KCl
-
above 10 mM
Mn2+
-
2-5 mM, 50% inhibition
myo-inositol
Ni2+
-
severe inhibition of wild-type PI-PLC
p-chloromercuriphenylsulfonic-acid
-
-
U73122
-
specific inhibitors of PI-PLC, 0.05 mM abolish stimulatory effect of heme in the PI-PLC/protein kinase C pathway
vanadate
-
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
butyl-fluorescein myo-inositol phosphate
-
two molecules bind to enzyme, one at the active site and one at a subsite, causing an increase in activity, kinetics
deoxycholate
diacylglycerol
Diethyl ether
-
strongly activates
diheptanoyl phosphatidylcholine
-
activates
dihexanoyl phosphatidylcholine
-
activates, 4-5fold increase in catalytic efficiency, binds to a lipid-binding subsite, not to the active site, maximal activation at 0.4 mM
dihexanoylphosphatidylcholine
-
non-substrate activator lipid, maximum PI-PLC activity at 0.7-1 mM
dimethylformamide
-
water-miscible, enhances phosphotransferase activity
Dimethylsulfoxide
-
water-miscible, enhances phosphotransferase activity
ethanol
-
all PLC1-6 are activated in salt/ethanol extractions
heme
-
heme receptor mediates the stimulatory effect of heme on the (Na+ + K+)ATPase activity through a PIPLC/PKC signaling pathway
Isopropanol
KCl
-
ionic strength, and not the salt identity, is important for PI-PLC activation towards phosphatidylinositol in micelles. Added salt has a synergistic effect with zwitterionic phospholipids, leading to high specific activities for phosphatidylinositol cleavage with only moderate dilution of the anionic substrate in the interface. This kinetic activation correlates with weakening of strong PI-PLC hydrophobic interactions with the interface. PI-PLC cleavage of phosphatidylinositol presented in small unilamellar vesicles is activated by salt
phosphatidic acid
-
binding to nonsubstrate anionic interfaces enhances the catalytic activity of PI-PLC, interfacial binding studies, activation mechanism
phosphatidycholine
-
-
-
phosphatidylcholine
phosphatidylglycerol
-
binding to nonsubstrate anionic interfaces enhances the catalytic activity of PI-PLC, interfacial binding studies, activation mechanism
phosphatidylmethanol
-
binding to nonsubstrate anionic interfaces enhances the catalytic activity of PI-PLC, interfacial binding studies, activation mechanism
phosphatidylserine
-
binding to nonsubstrate anionic interfaces enhances the catalytic activity of PI-PLC, interfacial binding studies, activation mechanism
Salt
-
all PLC1-6 are activated in salt/ethanol extractions
SDS
-
strongly activates
Triton X-100
Tween 20
-
slight stimulation
additional information
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.061 - 0.81
methyl-fluorescein myo-inositol phosphate
1.4
phosphatidylinositol
-
-
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
66
methyl-fluorescein myo-inositol phosphate
Listeria monocytogenes
-
pH 7
additional information
additional information
Bacillus cereus
-
-
-
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2 - 5
Ca2+
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.2
-
mutant Y246S/Y247S/Y248S, with 8 mM cIP as substrate
0.4
-
mutant Y246S/Y247S/Y248S/Y251S, with 8 mM cIP as substrate
0.6
-
mutant Y247S/Y251S, with 8 mM cIP as substrate
0.8
-
mutant Y246S/Y247S/Y248S/Y251S, with 8 mM cIP as substrate, in the presence of 5 mM diheptanoylphosphatidylcholine
1.6
-
mutant Y246S/Y247S/Y248S/Y251S, with phosphatidylinositol as substrate, in the presence of 2 mM POPC (for the small unilamellar vesicles)
2.1
-
mutant Y246S/Y247S/Y248S, with phosphatidylinositol as substrate, in the presence of 2 mM POPC (for the small unilamellar vesicles)
2.3
-
wild-type, with 8 mM cIP as substrate
2.9
-
mutant Y246S/Y247S/Y248S, with 8 mM cIP as substrate, in the presence of 5 mM diheptanoylphosphatidylcholine
6.7
-
mutant Y247S/Y251S, with phosphatidylinositol as substrate, in the presence of 2 mM POPC (for the small unilamellar vesicles)
9.5
-
wild-type, with phosphatidylinositol as substrate, in the presence of 2 mM POPC (for the small unilamellar vesicles)
30
-
pH 7.2, phosphatidylinositol as substrate, in absence of MgCl2
41
-
mutant Y247S/Y251S, with 8 mM cIP as substrate, in the presence of 5 mM diheptanoylphosphatidylcholine
62
-
mutant Y246S/Y247S/Y248S/Y251S, with phosphatidylinositol as substrate, in the presence of 16 mM Triton X-100
73
-
wild-type, with 8 mM cIP as substrate, in the presence of 5 mM diheptanoylphosphatidylcholine
98
-
mutant Y246S/Y247S/Y248S, with phosphatidylinositol as substrate, in the presence of 16 mM Triton X-100
112
-
mutant Y246S/Y247S/Y248S/Y251S, with phosphatidylinositol as substrate, in the presence of 32 mM diheptanoylphosphatidylcholine
239
-
mutant Y247S/Y251S, with phosphatidylinositol as substrate, in the presence of 16 mM Triton X-100
301
-
mutant Y246S/Y247S/Y248S, with phosphatidylinositol as substrate, in the presence of 32 mM diheptanoylphosphatidylcholine
375
-
wild-type, with phosphatidylinositol as substrate, in the presence of 16 mM Triton X-100
401
-
W47I mutant PI-PLC, phosphotransferase activity
556
-
wild-type PI-PLC, phosphotransferase activity
558
-
W47F mutant PI-PLC, phosphotransferase activity
560
-
wild-type, with phosphatidylinositol as substrate, in the presence of 32 mM diheptanoylphosphatidylcholine
658
-
W242F mutant PI-PLC, phosphotransferase activity
670
-
mutant Y247S/Y251S, with phosphatidylinositol as substrate, in the presence of 32 mM diheptanoylphosphatidylcholine
684
-
W242I mutant PI-PLC, phosphotransferase activity
700 - 1300
-
pH 7.2, phosphatidylinositol as substrate, in presence of MgCl2
1630
-
wild-type, for phosphatidylinositol/diC7-phosphatidylcholine, in 50 mM HEPES buffer, pH 7.5, with 1 mM EDTA, 5 mM dithiothreitol, and 0.1 mg/ml bovine serum albumin, at 28C
3560
-
pH 7.5, 37C, wild-type PI-PLC, in absence of Ca2+
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 9
-
wild-type PI-PLC, in the absence of 1 M Cl-
7 - 8
-
wild-type PI-PLC, in the presence of 1 M Cl-
7 - 7.2
-
assay at
7.1
-
cleavage of phosphatidylinositol solubilized in diheptanoyl phosphatidylcholine
7.2 - 7.5
-
-
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8
-
cleavage of phosphatidylinositol solubilized in diheptanoyl phosphatidylcholine, drop in activity around pH 8, consistent with the drop in binding affinity for activating surfaces
additional information
-
pH-dependence study of wild-type and mutant PI-PLC
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
-
assay at
39
-
assay at
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 70
-
20C: about 80% of maximal activity, 70C: about 35% of maximal activity
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.4
-
theoretical pI
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
-
recombinant PI-PLC, expressed in Escherichia coli BL21
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Staphylococcus aureus (strain Newman)
Staphylococcus aureus (strain Newman)
Staphylococcus aureus (strain Newman)
Staphylococcus aureus (strain Newman)
Staphylococcus aureus (strain Newman)
Staphylococcus aureus (strain Newman)
Staphylococcus aureus (strain Newman)
Staphylococcus aureus (strain Newman)
Staphylococcus aureus (strain Newman)
Staphylococcus aureus (strain Newman)
Staphylococcus aureus (strain Newman)
Staphylococcus aureus (strain Newman)
Staphylococcus aureus (strain Newman)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
28200
-
laser light scattering
29000
-
gel filtration
33000
-
sequence analysis
34460
-
R69C mutant PI-PLC, electrospray ionization mass spectrometry
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
monomer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
in complex with myo-inositol
-
X-ray crystal structure
-
mutant Y247S/Y251S in the absence and presence of myo-inositol as well as mutant Y246S/Y247S/Y248S/Y251S, both mutant proteins crystallize as monomers, are very similar to one another, and have no change in the active site region
-
wild-type and D274N mutant PI-PLC
-
recombinant PI-PLC
-
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, 20C, 2 weeks, X-ray diffraction structure determination and analysis at 2.16-2.3 A resolution
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
-
some thermal unfolding of PI-PLC occurs
30
-
Tm-value in presence of 30% isopropanol, myo-inositol enhances thermostability in isopropanol
41.5
-
Tm-value, pH 8, W178A mutant PI-PLC
47
-
Tm-value, pH 8, W280A mutant PI-PLC
50
-
pH 8.0, 30 min, stable below
54.6
-
Tm-value, pH 8, W47A/W242A double mutant PI-PLC
56.2
-
Tm-value, pH 8, W242A mutant PI-PLC
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
myo-inositol stabilizes
-
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
isopropanol
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by sonication and centrifugation, on chitin column and by gel filtration, more than 85% pure
-
PI-PLC mutants
-
Q-Sepharose column chromatography and phenyl Sepharose column chromatography
-
Q-sepharose fast-flow column and phenyl-Sepharose column, monitored by SDS-PAGE
-
R69C mutant PI-PLC
-
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21-Codonplus (DE3)-RIL
recombinant PI-PLC
recombinant wild-type and mutant PI-PLC
wild-type and mutant PI-PLC
-
wild-type and mutants, by gel filtration
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloned into the L4440 vector, transformation of plasmids into Escherichia coli HT115DE3
-
expressed in Escherichia coli BL21-CodonPlus (DE3)-RIL cells
-
expressed in Saccharomyces cerevisiae strain BY4741
-
expression in Escherichia coli MM294
-
expression in Escherichia coli, construction of four vectors for high-level expression
expression in Listeria monocytogenes. Listeria monocytogenes expressing Bacillus anthracis phosphatidylinositol-specific phospholipase C shows significantly decreased efficiencies of ascape from a phagosome and in cell-to-cell spread
-
expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21-Codonplus (DE3)-RIL
inserted into the pTYB11 N-terminal fusion vector between Sap I and EcoR I restriction sites. Recombinant plasmid, IMPACT-Y, transformed into Escherichia coli ER2566 cells for overexpression
-
overexpressed in Escherichia coli
-
overexpression in Escherichia coli BL21
-
PI-PLC gene is part of the virulence gene cluster, overexpression in Escherichia coli MM294
-
transformed into Escherichia coli BL21-Codonplus (DE3)-RIL cells
-
wild-type and mutant PI-PLC, expression in Escherichia coli BL21
-
wild-type and mutants overexpressed in Escherichia coli
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
PLC2, PLC4 and PLC6 show the highest mRNA levels
PLC5 reveals a drastic lower mRNA level compared to all other PLCs, this gene shows characteristics of a non-functional pseudogene. Adequate knockdown levels for almost all PLCs in the RNAi lines, except for PLC5 with only 53.85%
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D274A
-
mutation of an catalytic diad residue, mutant with abolished activity, NMR study
D274N
-
mutation of an catalytic diad residue, 4.2% of wild-type activity
H32A
-
mutation of an catalytic diad residue, mutant with abolished activity, NMR study
D33N/R69D
-
PI-PLC double mutant, 50fold activation by 1 mM Ca2+
R69D
-
reduced activity compared with wild-type enzyme, mutant is activated by Ca2+, mutation engineers a catalytic metal binding site into the calcium-independent PI-PLC leading to enhanced stereoselectivity
R69E
-
mutation of the catalytic Arg-69, inactive mutant, not activated by Ca2+
R69N
-
mutation of the catalytic Arg-69, not activated by Ca2+
D274A
-
catalytic aspartate mutation, 0.005% of wild-type activity, no activation by exogenous anions
D274E
-
catalytic aspartate mutation, 50% of wild-type activity, no activation by chloride ions
D274G
-
catalytic aspartate mutation, activation of mutant PI-PLC by exogenous anions, e.g. Cl-
D274N
-
catalytic aspartate mutation, 40fold decreased activity compared with wild-type enzyme, no activation by chloride ions
G238W
-
study of the kinetic activation by diheptanoyl phosphatidylcholine and water-miscible isopropanol
G238W/W242A
-
double mutant with enhanced activation and affinity for phosphatidylcholine interfaces above that of wild-type PI-PLC
G48A
-
single mutant
G48W/W47A
-
double mutant, study of the kinetic activation by diheptanoyl phosphatidylcholine and water-miscible isopropanol
H32A
-
active site mutant, but binds to pure phosphatidylglycerol and pure phosphatidylcholine small unilamellar vesicles with essentially the same affinities as mutant N168C
I43A
-
single mutant
I43W
-
single mutant
I43W/W47A
-
double mutant with recovered kinetic interfacial activation, lower specific activity than wild-type PI-PLC
I43W/W47I
-
are made by introducing the second mutation in the gene coding for a single mutant
L39A
-
single mutant
L39A/V46A
-
are made by introducing the second mutation in the gene coding for a single mutant
M49W/W47A
-
double mutant, study of the kinetic activation by diheptanoyl phosphatidylcholine and water-miscible isopropanol
N168C
-
1% relative activity
N243W/W242A
-
double mutant, study of the kinetic activation by diheptanoyl phosphatidylcholine and water-miscible isopropanol
P245Y
-
the mutant shows reduced activity and membrane affinity
Q45A
-
single mutant
Q45W/W47A
-
double mutant, study of the kinetic activation by diheptanoyl phosphatidylcholine and water-miscible isopropanol
R69D
-
active site mutant with low specific activity towards phosphatidylinositol, interfacial binding study
S236W/W242A
-
double mutant, study of the kinetic activation by diheptanoyl phosphatidylcholine and water-miscible isopropanol
V46A
-
single mutant
W178A
-
mutant with reduced stability and specific activity, study of kinetic activation by micellar phosphatidylcholine
W242F
-
kinetic analysis, binding studies to phosphatidylcholine vesicles
W242I
-
kinetic analysis, binding studies to phosphatidylcholine vesicles
W280A
-
mutant with reduced stability, study of kinetic activation by micellar phosphatidylcholine
W47A/W242A
W47F
-
kinetic analysis, binding studies to phosphatidylcholine vesicles
W47I
-
kinetic analysis, binding studies to phosphatidylcholine vesicles
Y246A
-
the variant shows significant membrane binding defects
Y246A/Y247A
-
the variant shows significant membrane binding defects
Y246S/Y247S/Y248S
-
less active toward phosphatidylinositol solubilized in diheptanoylphosphatidylcholine and when changing the detergent matrix to Triton X-100, as the wild-type
Y246S/Y247S/Y248S/N168C
-
impaired phosphatidylcholine binding, but still binds most tightly to mixed lipid vesicles. Has similar affinities for pure phosphatidylglycerol vesicles than mutant N168C, while the apparent Kd of for pure phosphatidylcholine vesicles is ca. 3 orders of magnitude higher than that of mutant N168C. Apparent Kd toward small unilamellar vesicles is about 1000fold higher than that of mutant N168C
Y246S/Y247S/Y248S/Y251S
-
less active toward phosphatidylinositol solubilized in diheptanoylphosphatidylcholine and when changing the detergent matrix to Triton X-100, as the wild-type
Y247A
-
the variant shows significant membrane binding defects
Y247S/Y251S
-
exhibits specific activity toward phosphatidylinositol solubilized in diheptanoylphosphatidylcholine comparable to wild-type. Reduced specific activity, when changing the detergent matrix to Triton X-100
Y86A/Y88A
-
the mutations decrease membrane affinity for the enzyme
Y88A
-
2.92% relative activity, mutation near the lipid binding region. Is an extremely active enzyme whose specific activity is 3fold higher than recombinant PI-PLC, binds more weakly to small unilamellar vesicles than wild-type
P245Y
-
the mutant shows reduced activity and membrane affinity
-
Y246A
-
the variant shows significant membrane binding defects
-
Y246A/Y247A
-
the variant shows significant membrane binding defects
-
Y247A
-
the variant shows significant membrane binding defects
-
Y86A/Y88A
-
the mutations decrease membrane affinity for the enzyme
-
DP-L1552
genotype, deltaplcA. Phenotype PI-PLC-
DP-L1935
genotype, deltaplcb. Phenotype PC-PLC-
DP-L1936
genotype, deltaplcA/deltaplcB. Phenotype PI-PLC-/PC-PLC-
DP-L2161
genotype, deltahyl. Phenotype LLO- (listeriolysin O)
F237A
-
most approaches wild-type PI-PLC in its dependence on enzyme concentration
F237W
-
even at high concentrations, has high specific activity comparable to dilute unaltered recombinant PI-PLC, does not form the aggregates with anionic lipid-rich vesicles that are disrupted by excess detergent and salt, although it is still activated to about the same extent as wild-type by salt
L151A
-
added KCl (0.15 M) still enhances PI-PLC cleavage of phosphatidylinositol in TX-100 micelles, although KCl effects are much more modest (1.6fold increase) compared to wild-type, F237A or F237W
L235A
-
added KCl (0.15 M) still enhances PI-PLC cleavage of phosphatidylinositol in TX-100 micelles, although KCl effects are much more modest (1.4fold increase) compared to wild-type, F237A or F237W
W49A
-
added KCl (0.15 M) still enhances PI-PLC cleavage of phosphatidylinositol in TX-100 micelles, although KCl effects are much more modest (1.8fold increase) compared to wild-type, F237A or F237W
DP-L1552
-
genotype, deltaplcA. Phenotype PI-PLC-
-
DP-L1935
-
genotype, deltaplcb. Phenotype PC-PLC-
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DP-L1936
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genotype, deltaplcA/deltaplcB. Phenotype PI-PLC-/PC-PLC-
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DP-L2161
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genotype, deltahyl. Phenotype LLO- (listeriolysin O)
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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
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
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
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
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
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
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
Y253S/Y255S
site-directed mutagenesis, the mutant has the same secondary structure content but a 5C lower thermal denaturation temperature than the wild-type and an altered enzyme activity
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
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
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
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use of the enzyme and specific antibodies for the enzyme for the examination of the growth inhibition, morphological change and ectoenzyme release of the LLC-PK1 cells from pig, effective for the investigation of the function of the glycosyl-phosphatidylinositol-anchor protein
medicine
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PI-PLC is a virulence factor of the animal and human pathogen causing listeriosis
molecular biology
additional information
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optimization of PI-PLC binding to substrate-containing vesicles is a balancing act between anchoring the protein in the correct conformation and orientation while also allowing it to dissociate in order to find substrate phospholipids or GPI-anchored proteins by scooting and/or hopping
Show AA Sequence (181 entries)
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