4.6.1.13: phosphatidylinositol diacylglycerol-lyase

PI-PLC double mutant, 50fold activation by 1 mM Ca2+

R69A

R69C

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+

R69G

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

K44A

K44E

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

729401

P42G

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

650231

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

W242A

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

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

709019

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

709019

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

709019

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-

DP-L1936

genotype, deltaplcA/deltaplcB. Phenotype PI-PLC-/PC-PLC-

DP-L2161

genotype, deltahyl. Phenotype LLO- (listeriolysin O)

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 5°C 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