Information on EC 3.6.3.39 - lipopolysaccharide-transporting ATPase

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

EC NUMBER
COMMENTARY hide
3.6.3.39
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RECOMMENDED NAME
GeneOntology No.
lipopolysaccharide-transporting ATPase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + H2O + lipopolysaccharide/in = ADP + phosphate + lipopolysaccharide/out
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of phosphoric ester
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transmembrane transport
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SYSTEMATIC NAME
IUBMB Comments
ATP phosphohydrolase (lipopolysaccharide-exporting)
ABC-type (ATP-binding cassette-type) ATPase, characterized by the presence of two similar ATP-binding domains. Does not undergo phosphorylation during the transport process. Enzymes of Gram-negative bacteria that export lipo-oligosaccharides and lipopolysaccharides.
CAS REGISTRY NUMBER
COMMENTARY hide
9000-83-3
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
MsbA protein, encoded by pA4997; serotype O5
SwissProt
Manually annotated by BRENDA team
biovar viciae
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Manually annotated by BRENDA team
gene msbA2 containing asparagine at position 33
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Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + H2O + (7-nitrobenz-2-oxa-1,3-diazole)-C12-sphingomyelin/in
ADP + phosphate + (7-nitrobenz-2-oxa-1,3-diazole)-C12-sphingomyelin/out
show the reaction diagram
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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
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-
?
ATP + H2O + (7-nitrobenz-2-oxa-1,3-diazole)-glucosylceramide/in
ADP + phosphate + (7-nitrobenz-2-oxa-1,3-diazole)-glucosylceramide/out
show the reaction diagram
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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
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?
ATP + H2O + (7-nitrobenz-2-oxa-1,3-diazole)-lactosylceramide/in
ADP + phosphate + (7-nitrobenz-2-oxa-1,3-diazole)-lactosylceramide/out
show the reaction diagram
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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
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?
ATP + H2O + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylcholine (16:0, 6:0)/in
ADP + phosphate + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylcholine (16:0, 6:0)/out
show the reaction diagram
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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
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?
ATP + H2O + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylethanolamine (16:0, 6:0)/in
ADP + phosphate + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylethanolamine (16:0, 6:0)/out
show the reaction diagram
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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
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?
ATP + H2O + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylethanolamine (18:1)/in
ADP + phosphate + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylethanolamine (18:1)/out
show the reaction diagram
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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
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?
ATP + H2O + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylglycerol (16:0, 6:0)/in
ADP + phosphate + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylglycerol (16:0, 6:0)/out
show the reaction diagram
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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
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-
?
ATP + H2O + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylserine (16:0, 6:0)/in
ADP + phosphate + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylserine (16:0, 6:0)/out
show the reaction diagram
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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
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-
?
ATP + H2O + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylserine (18:1)/in
ADP + phosphate + (7-nitrobenz-2-oxa-1,3-diazole)-phosphatidylserine (18:1)/out
show the reaction diagram
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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
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?
ATP + H2O + DMSO/in
ADP + phosphate + DMSO/out
show the reaction diagram
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?
ATP + H2O + ethidium/in
ADP + phosphate + ethidium/out
show the reaction diagram
ATP + H2O + Hoechst 33342
ADP + phosphate + Hoechst 33342/out
show the reaction diagram
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?
ATP + H2O + Hoechst 33342/in
ADP + phosphate + Hoechst 33342/out
show the reaction diagram
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?
ATP + H2O + Lipid A/in
ADP + phosphate + Lipid A/out
show the reaction diagram
ATP + H2O + lipo-oligosaccharide/in
ADP + phosphate + lipo-oligosaccharide/out
show the reaction diagram
ATP + H2O + lipopolysaccharide/in
ADP + phosphate + lipopolysaccharide/out
show the reaction diagram
ATP + H2O + lipopolysaccharide/in
ADP + phosphate + lipopolysacchride/out
show the reaction diagram
ATP + H2O + microcin J25/in
ADP + phosphate + microcin J25/out
show the reaction diagram
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?
ATP + H2O + verapamil/in
ADP + phosphate + verapamil/out
show the reaction diagram
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?
additional information
?
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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
ATP + H2O + Lipid A/in
ADP + phosphate + Lipid A/out
show the reaction diagram
ATP + H2O + lipo-oligosaccharide/in
ADP + phosphate + lipo-oligosaccharide/out
show the reaction diagram
ATP + H2O + lipopolysaccharide/in
ADP + phosphate + lipopolysaccharide/out
show the reaction diagram
ATP + H2O + lipopolysaccharide/in
ADP + phosphate + lipopolysacchride/out
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mg2+
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required
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
adenosine 5'-(beta,gamma-imido)triphosphate
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AMP-PCP
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D-20133
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lipid-based drug, high affinity binding to MsbA
ethidium
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antimitotic drug, vinblastine, directly competes with ethidium for binding to MsbA
Hoechst 33342
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complete inhibition of MsbA-mediated Hoechst33342 transport by 0.025 mM taxol, noncompetitive kinetics with Ki of 0.0066 mM, overview
ilmofosine
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lipid-based drug, high affinity binding to MsbA
leupeptin
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lipopolysaccharide
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dependent on the origin
ortho-vanadate
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vanadate
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vinblastin
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vinblastine
additional information
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no inhibition by substrate lipid A also at high concentration, poor inhibition by verapamil, colchicine, and daunorubicine, poor inhiition by lipid-based drugs D-20133, and D-21266, along with LY335979
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Hoechst 33342
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Lipid
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from Escherichia coli, about 1.4fold stimulation
lipid A
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about 2fold stimulation of the ATPase activity of MsbA by its substrate
microcin J25
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the ATPase activity of the enzyme reconstituted in proteoliposomes is stimulated by microcin J25 in a concentration-dependent manner
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phosphocholine
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2fold stimulation of MsbA
taxol
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1.4fold stimulation of the wild-type enzyme, no stimulation of mutant S289A/S290A. Erythromycin resistance is conferred by wild-type or mutant MsbA, taxol is sufficient to reverse the erythromycin resistance of wild-type MsbA-expressing cells
additional information
activity shown to be selectively stimulated by different truncated versions of core oligosaccharides of lipopoysaccharides of Pseudomonas aeruginosa
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.002 - 4.5
ATP
0.0064
lipid A
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pH 7.5, 37C, recombinant His-tagged enzyme
0.048 - 0.05
lipopolysaccharide
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pH 7.5, 37C, recombinant His-tagged enzyme
additional information
additional information
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IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0395
adenosine 5'-(beta,gamma-imido)triphosphate
Escherichia coli
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pH and temperature not specified in the publication
1.25
AMP-PCP
Escherichia coli
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pH 7.5, 37C, recombinant His-tagged protein
0.48
leupeptin
Escherichia coli
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pH 7.5, 37C, recombinant His-tagged enzyme
0.0616
ortho-vanadate
Escherichia coli
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pH and temperature not specified in the publication
0.5
vinblastin
Escherichia coli
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above, pH 7.5, 37C, recombinant His-tagged enzyme
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.4
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ATPase activity, purified recombinant enzyme, labeled or unlabeled with 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid
0.624
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unstimulated recombinant His6-tagged wild-type enzyme activity
0.726
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recombinant His6-tagged mutant S289A/S290A in presence or absence of taxol
0.835
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taxol stimulated recombinant His6-tagged wild-type enzyme activity
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
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assay at
additional information
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ligand binding assays at 23C
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
27680
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NodJ, nucleotide sequence
28190
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NodJ, nucleotide sequence
34130
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NodI, nucleotide sequence
34300
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NodI, nucleotide sequence
56000
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2 * 56000, SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 65000, about, purified recombinant enzyme, mass spectrometry
homodimer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
targeted molecular dynamics simulation methods reveal a clear spatiotemporal order of the conformational movements from the outward-facing to the inward-facing states. The disruption of the nucleotide binding sites at the nucleotide-binding dimer interface is the very first event that initiates the following conformational changes. The conserved x-loops of the nucleotide binding sites participate in the interaction network that stabilizes the cytoplasmic tetrahelix bundle of the transmembrane domains and play an important role in mediating the cross-talk between the nucleotide-binding domains and transmembrane domains. The movement of the nucleotide-binding domain dimer is transmitted through x-loops to break the tetrahelix bundle, inducing the packing rearrangements of the transmembrane helices at the cytoplasmic side and the periplasmic side sequentially
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X-ray diffraction structure determination and analysis at 3.7 A resolution and analysis of structure PDB ID 3b60
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Ni-NTA column chromatography and DEAE column chromatography
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Ni2+ affinity resin column chromatography
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Ni2+-nitrilotriacetic acid resin column chromatography
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recombinant His6-tagged wild-type and mutant enzyme by nickel affinity chromatography
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recombinant His6-tagged wild-type and mutant enzymes from Lactobacillus lactis by soluibilization from membranes with detergent n-dodecyl beta-D-maltoside, and nickel affinity chromatography, formation of inside-out membrane vesicles
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recombinant protein, gel filtration
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed as a His-tagged fusion protein in Escherichia coli
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expressed in Escherichia coli ER2566 cells
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expressed in Escherichia coli XL1 Blue cells
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expressed in Escherichia coli, host strains BL21-DE3, JM109, SM10, different plasmids used and listed
expression of His6-tagged wild-type and mutant enzyme in Escherichia coli
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gene msbA, DNA and amino acid sequence determination and analysis of wild-type and mutant enzymes, expression of His6-tagged proteins in Lactococcus lactis strain NZ9000 DELTAlmrA DELTAlmrCD
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gene msbA, overexpression of the N-terminally His-tagged enzyme in Escherichia coli strain BL21(DE3)
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nodI and nodJ gene
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nodJ gene cloned, expression in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A281C
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the mutant displays a reduction in ATP-dependent Hoechst 33342 export activity
E208A
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the mutant shows greatly reduced activity compared to the wild type enzyme
E208A/A281C
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the mutant displays a strong reduction in ATP-dependent Hoechst 33342 export activity
E208A/K212A
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the mutant shows greatly reduced activity compared to the wild type enzyme
E208Q
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the mutant shows 44.4% of wild type activity
E506Q
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mutation leads to dysfunctional protein, loss of cell viability. Mutant protein maintains its ability to bind ATP, but hydrolysis is severely inhibited. Hydrolysis does occur over time. Protein adopts a closed dimer conformation, indicating that events within the cell can induce a stable, closed conformation of the MsbA homodimer that does not reopen even in the absence of nucleotide
H537A
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mutation leads to dysfunctional protein, loss of cell viability. Mutant protein maintains its ability to bind ATP, but hydrolysis is severely inhibited. Hydrolysis does occur over time. Protein adopts a closed dimer conformation, indicating that events within the cell can induce a stable, closed conformation of the MsbA homodimer that does not reopen even in the absence of nucleotide
K212A
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the mutant shows greatly reduced activity compared to the wild type enzyme
S289A/S290A
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site-directed mutagenesis, the mutant enzyme is not stimulated by taxol in contrast to the wild-type enzyme. The mutation does not alter the interaction of MsbA with Hoechst33342 but reduces the level of inhibition of MsbA-mediated Hoechst33342 transport by taxol. The mutant MsbA is affected in the binding and transport of ethidium
S423C
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mutant shows a Vmax similar to WT
S423C/E506Q
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significantly diminished rates of hydrolysis, about 4% of wild-type
S423C/H537A
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significantly diminished rates of hydrolysis, about 4% of wild-type
C315S
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site-idrected mutagenesis, structure analysis in comparison to the wild-type enzyme
C88S
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site-directed mutagenesis, structure analysis in comparison to the wild-type enzyme
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant His6-tagged MsbA is reconstituted into proteoliposomes, that contain phosphocholine, using detergent destabilization
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reconstitution of ABC transporter MsbA in nanodiscs of various sizes and lipid compositions. ATP hydrolysis rates, Michaelis-Menten parameters, and dissociation constants of substrate analog ATP-gamma-S demonstrate that physicochemical properties of the bilayer modulate binding and ATPase activity. ATPase rates of MsbA reconstituted in nanodiscs are 6-15 times faster than those of protein solubilized in micelles of n-dodecyl-beta-D-maltoside. Enzymatic activity is modulated by a complex interplay between at least the bilayer size, and lipid composition
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