BRENDA - Enzyme Database

The reconstituted Escherichia coli MsbA protein displays lipid flippase activity

Eckford, P.D.; Sharom, F.J.; Biochem. J. 429, 195-203 (2010)

Data extracted from this reference:

Activating Compound
EC Number
Activating Compound
Commentary
Organism
Structure
7.6.2.1
ATP
flippase activity is essentially supported only by ATP. If the ATP concentration in the assay falls below 5 mM, there is a significant decrease in flippase activity. Maximal translocation occurs at 10 mM ATP
Escherichia coli
7.6.2.1
additional information
GTP, AMP and AMP-PNP yield low levels of translocation compared to ATP
Escherichia coli
Cloned(Commentary)
EC Number
Commentary
Organism
7.5.2.6
expressed as a His-tagged fusion protein in Escherichia coli
Escherichia coli
Inhibitors
EC Number
Inhibitors
Commentary
Organism
Structure
7.6.2.1
AlFx
trapping of MsbA with 0.8 mM AlFx results in incomplete inhibition of activity
Escherichia coli
7.6.2.1
BeFx
trapping of MsbA with 0.8 mM BeFx results in incomplete inhibition of activity
Escherichia coli
7.6.2.1
lipid A
lipid A inhibits translocation by approx. 30% at 0.01-0.04 mg/ml
Escherichia coli
7.6.2.1
additional information
neither lipopolysaccharide from the Ra mutant of Escherichia coli nor deep rough chemotype lipopolysaccharide affect MsbA flippase activity significantly
Escherichia coli
7.6.2.1
phosphate analogue Vi
the presence of 0.1-0.2 m M Vi inhibits flippase activity of MsbA by approx. 50%
Escherichia coli
Localization
EC Number
Localization
Commentary
Organism
GeneOntology No.
Textmining
7.6.2.1
membrane
-
Escherichia coli
16020
-
Molecular Weight [Da]
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
7.6.2.1
65000
-
2 * 65000, SDS-PAGE
Escherichia coli
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
7.5.2.6
ATP + H2O + Lipid A/in
Escherichia coli
-
ADP + phosphate + Lipid A/out
-
-
?
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
7.5.2.6
Escherichia coli
-
-
-
7.6.2.1
Escherichia coli
P60752
-
-
Purification (Commentary)
EC Number
Commentary
Organism
7.6.2.1
Ni-NTA column chromatography
Escherichia coli
Specific Activity [micromol/min/mg]
EC Number
Specific Activity Minimum [Ámol/min/mg]
Specific Activity Maximum [Ámol/min/mg]
Commentary
Organism
7.5.2.6
additional information
-
purified MsbA is reconstituted into proteoliposomes of Escherichia coli lipid. In this system, the protein displays maximal lipid flippase activity of 7.7 nmol of lipid translocated per mg of protein over a 20 min period for (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylethanolamine (16:0, 6:0)
Escherichia coli
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
7.5.2.6
ATP + H2O + (7-nitrobenz-2-oxa-1,3-diazole)-C12-sphingomyelin/in
bacterial membrane vesicles isolated from Escherichia coli overexpressing MsbA display ATP-dependent translocation of several fluorescently NBD (7-nitrobenz-2-oxa-1,3-diazole)-labelled phospholipid species
711144
Escherichia coli
ADP + phosphate + (7-nitrobenz-2-oxa-1,3-diazole)-C12-sphingomyelin/out
-
-
-
?
7.5.2.6
ATP + H2O + (7-nitrobenz-2-oxa-1,3-diazole)-glucosylceramide/in
bacterial membrane vesicles isolated from Escherichia coli overexpressing MsbA display ATP-dependent translocation of several fluorescently NBD (7-nitrobenz-2-oxa-1,3-diazole)-labelled phospholipid species
711144
Escherichia coli
ADP + phosphate + (7-nitrobenz-2-oxa-1,3-diazole)-glucosylceramide/out
-
-
-
?
7.5.2.6
ATP + H2O + (7-nitrobenz-2-oxa-1,3-diazole)-lactosylceramide/in
bacterial membrane vesicles isolated from Escherichia coli overexpressing MsbA display ATP-dependent translocation of several fluorescently NBD (7-nitrobenz-2-oxa-1,3-diazole)-labelled phospholipid species
711144
Escherichia coli
ADP + phosphate + (7-nitrobenz-2-oxa-1,3-diazole)-lactosylceramide/out
-
-
-
?
7.5.2.6
ATP + H2O + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylcholine (16:0, 6:0)/in
bacterial membrane vesicles isolated from Escherichia coli overexpressing MsbA display ATP-dependent translocation of several fluorescently NBD (7-nitrobenz-2-oxa-1,3-diazole)-labelled phospholipid species
711144
Escherichia coli
ADP + phosphate + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylcholine (16:0, 6:0)/out
-
-
-
?
7.5.2.6
ATP + H2O + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylethanolamine (16:0, 6:0)/in
bacterial membrane vesicles isolated from Escherichia coli overexpressing MsbA display ATP-dependent translocation of several fluorescently NBD (7-nitrobenz-2-oxa-1,3-diazole)-labelled phospholipid species. Translocation of (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylethanolamine is inhibited by the presence of the putative physiological substrate lipid A
711144
Escherichia coli
ADP + phosphate + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylethanolamine (16:0, 6:0)/out
-
-
-
?
7.5.2.6
ATP + H2O + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylethanolamine (18:1)/in
bacterial membrane vesicles isolated from Escherichia coli overexpressing MsbA display ATP-dependent translocation of several fluorescently NBD (7-nitrobenz-2-oxa-1,3-diazole)-labelled phospholipid species. Translocation of (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylethanolamine is inhibited by the presence of the putative physiological substrate lipid A
711144
Escherichia coli
ADP + phosphate + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylethanolamine (18:1)/out
-
-
-
?
7.5.2.6
ATP + H2O + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylglycerol (16:0, 6:0)/in
bacterial membrane vesicles isolated from Escherichia coli overexpressing MsbA display ATP-dependent translocation of several fluorescently NBD (7-nitrobenz-2-oxa-1,3-diazole)-labelled phospholipid species
711144
Escherichia coli
ADP + phosphate + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylglycerol (16:0, 6:0)/out
-
-
-
?
7.5.2.6
ATP + H2O + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylserine (16:0, 6:0)/in
bacterial membrane vesicles isolated from Escherichia coli overexpressing MsbA display ATP-dependent translocation of several fluorescently NBD (7-nitrobenz-2-oxa-1,3-diazole)-labelled phospholipid species
711144
Escherichia coli
ADP + phosphate + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylserine (16:0, 6:0)/out
-
-
-
?
7.5.2.6
ATP + H2O + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylserine (18:1)/in
bacterial membrane vesicles isolated from Escherichia coli overexpressing MsbA display ATP-dependent translocation of several fluorescently NBD (7-nitrobenz-2-oxa-1,3-diazole)-labelled phospholipid species
711144
Escherichia coli
ADP + phosphate + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylserine (18:1)/out
-
-
-
?
7.5.2.6
ATP + H2O + Lipid A/in
-
711144
Escherichia coli
ADP + phosphate + Lipid A/out
-
-
-
?
7.6.2.1
ATP + H2O + Lipid A/in
-
711144
Escherichia coli
ADP + phosphate + Lipid A/out
-
-
-
?
7.6.2.1
ATP + H2O + phosphatidylethanolamine/in
-
711144
Escherichia coli
ADP + phosphate + phosphatidylethanolamine/out
-
-
-
?
Subunits
EC Number
Subunits
Commentary
Organism
7.6.2.1
homodimer
2 * 65000, SDS-PAGE
Escherichia coli
Temperature Optimum [░C]
EC Number
Temperature Optimum [░C]
Temperature Optimum Maximum [░C]
Commentary
Organism
7.5.2.6
37
-
assay at
Escherichia coli
Activating Compound (protein specific)
EC Number
Activating Compound
Commentary
Organism
Structure
7.6.2.1
ATP
flippase activity is essentially supported only by ATP. If the ATP concentration in the assay falls below 5 mM, there is a significant decrease in flippase activity. Maximal translocation occurs at 10 mM ATP
Escherichia coli
7.6.2.1
additional information
GTP, AMP and AMP-PNP yield low levels of translocation compared to ATP
Escherichia coli
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
7.5.2.6
expressed as a His-tagged fusion protein in Escherichia coli
Escherichia coli
Inhibitors (protein specific)
EC Number
Inhibitors
Commentary
Organism
Structure
7.6.2.1
AlFx
trapping of MsbA with 0.8 mM AlFx results in incomplete inhibition of activity
Escherichia coli
7.6.2.1
BeFx
trapping of MsbA with 0.8 mM BeFx results in incomplete inhibition of activity
Escherichia coli
7.6.2.1
lipid A
lipid A inhibits translocation by approx. 30% at 0.01-0.04 mg/ml
Escherichia coli
7.6.2.1
additional information
neither lipopolysaccharide from the Ra mutant of Escherichia coli nor deep rough chemotype lipopolysaccharide affect MsbA flippase activity significantly
Escherichia coli
7.6.2.1
phosphate analogue Vi
the presence of 0.1-0.2 m M Vi inhibits flippase activity of MsbA by approx. 50%
Escherichia coli
Localization (protein specific)
EC Number
Localization
Commentary
Organism
GeneOntology No.
Textmining
7.6.2.1
membrane
-
Escherichia coli
16020
-
Molecular Weight [Da] (protein specific)
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
7.6.2.1
65000
-
2 * 65000, SDS-PAGE
Escherichia coli
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
7.5.2.6
ATP + H2O + Lipid A/in
Escherichia coli
-
ADP + phosphate + Lipid A/out
-
-
?
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
7.6.2.1
Ni-NTA column chromatography
Escherichia coli
Specific Activity [micromol/min/mg] (protein specific)
EC Number
Specific Activity Minimum [Ámol/min/mg]
Specific Activity Maximum [Ámol/min/mg]
Commentary
Organism
7.5.2.6
additional information
-
purified MsbA is reconstituted into proteoliposomes of Escherichia coli lipid. In this system, the protein displays maximal lipid flippase activity of 7.7 nmol of lipid translocated per mg of protein over a 20 min period for (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylethanolamine (16:0, 6:0)
Escherichia coli
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
7.5.2.6
ATP + H2O + (7-nitrobenz-2-oxa-1,3-diazole)-C12-sphingomyelin/in
bacterial membrane vesicles isolated from Escherichia coli overexpressing MsbA display ATP-dependent translocation of several fluorescently NBD (7-nitrobenz-2-oxa-1,3-diazole)-labelled phospholipid species
711144
Escherichia coli
ADP + phosphate + (7-nitrobenz-2-oxa-1,3-diazole)-C12-sphingomyelin/out
-
-
-
?
7.5.2.6
ATP + H2O + (7-nitrobenz-2-oxa-1,3-diazole)-glucosylceramide/in
bacterial membrane vesicles isolated from Escherichia coli overexpressing MsbA display ATP-dependent translocation of several fluorescently NBD (7-nitrobenz-2-oxa-1,3-diazole)-labelled phospholipid species
711144
Escherichia coli
ADP + phosphate + (7-nitrobenz-2-oxa-1,3-diazole)-glucosylceramide/out
-
-
-
?
7.5.2.6
ATP + H2O + (7-nitrobenz-2-oxa-1,3-diazole)-lactosylceramide/in
bacterial membrane vesicles isolated from Escherichia coli overexpressing MsbA display ATP-dependent translocation of several fluorescently NBD (7-nitrobenz-2-oxa-1,3-diazole)-labelled phospholipid species
711144
Escherichia coli
ADP + phosphate + (7-nitrobenz-2-oxa-1,3-diazole)-lactosylceramide/out
-
-
-
?
7.5.2.6
ATP + H2O + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylcholine (16:0, 6:0)/in
bacterial membrane vesicles isolated from Escherichia coli overexpressing MsbA display ATP-dependent translocation of several fluorescently NBD (7-nitrobenz-2-oxa-1,3-diazole)-labelled phospholipid species
711144
Escherichia coli
ADP + phosphate + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylcholine (16:0, 6:0)/out
-
-
-
?
7.5.2.6
ATP + H2O + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylethanolamine (16:0, 6:0)/in
bacterial membrane vesicles isolated from Escherichia coli overexpressing MsbA display ATP-dependent translocation of several fluorescently NBD (7-nitrobenz-2-oxa-1,3-diazole)-labelled phospholipid species. Translocation of (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylethanolamine is inhibited by the presence of the putative physiological substrate lipid A
711144
Escherichia coli
ADP + phosphate + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylethanolamine (16:0, 6:0)/out
-
-
-
?
7.5.2.6
ATP + H2O + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylethanolamine (18:1)/in
bacterial membrane vesicles isolated from Escherichia coli overexpressing MsbA display ATP-dependent translocation of several fluorescently NBD (7-nitrobenz-2-oxa-1,3-diazole)-labelled phospholipid species. Translocation of (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylethanolamine is inhibited by the presence of the putative physiological substrate lipid A
711144
Escherichia coli
ADP + phosphate + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylethanolamine (18:1)/out
-
-
-
?
7.5.2.6
ATP + H2O + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylglycerol (16:0, 6:0)/in
bacterial membrane vesicles isolated from Escherichia coli overexpressing MsbA display ATP-dependent translocation of several fluorescently NBD (7-nitrobenz-2-oxa-1,3-diazole)-labelled phospholipid species
711144
Escherichia coli
ADP + phosphate + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylglycerol (16:0, 6:0)/out
-
-
-
?
7.5.2.6
ATP + H2O + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylserine (16:0, 6:0)/in
bacterial membrane vesicles isolated from Escherichia coli overexpressing MsbA display ATP-dependent translocation of several fluorescently NBD (7-nitrobenz-2-oxa-1,3-diazole)-labelled phospholipid species
711144
Escherichia coli
ADP + phosphate + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylserine (16:0, 6:0)/out
-
-
-
?
7.5.2.6
ATP + H2O + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylserine (18:1)/in
bacterial membrane vesicles isolated from Escherichia coli overexpressing MsbA display ATP-dependent translocation of several fluorescently NBD (7-nitrobenz-2-oxa-1,3-diazole)-labelled phospholipid species
711144
Escherichia coli
ADP + phosphate + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylserine (18:1)/out
-
-
-
?
7.5.2.6
ATP + H2O + Lipid A/in
-
711144
Escherichia coli
ADP + phosphate + Lipid A/out
-
-
-
?
7.6.2.1
ATP + H2O + Lipid A/in
-
711144
Escherichia coli
ADP + phosphate + Lipid A/out
-
-
-
?
7.6.2.1
ATP + H2O + phosphatidylethanolamine/in
-
711144
Escherichia coli
ADP + phosphate + phosphatidylethanolamine/out
-
-
-
?
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
7.6.2.1
homodimer
2 * 65000, SDS-PAGE
Escherichia coli
Temperature Optimum [░C] (protein specific)
EC Number
Temperature Optimum [░C]
Temperature Optimum Maximum [░C]
Commentary
Organism
7.5.2.6
37
-
assay at
Escherichia coli