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Information on EC 7.5.2.6 - ABC-type lipid A-core oligosaccharide transporter and Organism(s) Escherichia coli and UniProt Accession P60752
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7.5.2.6 ABC-type lipid A-core oligosaccharide transporterIUBMB Comments
An ATP-binding cassette (ABC) type transporter, characterized by the presence of two similar ATP-binding domains/proteins and two integral membrane domains/proteins. The enzyme, best characterized from the bacterium Escherichia coli, is located in the inner membrane and mediates the movement of lipid A attached to the core oligosaccharide from the cytoplasm to the periplasmic side of the inner membrane, an important step in the lipopolysaccharide biosynthetic pathway. Not to be confused with EC 7.5.2.5, ABC-type lipopolysaccharide transporter (LptB), which is implicated in the translocation of LPS from the inner membrane to the outer membrane and acts later in the process.
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The taxonomic range for the selected organisms is: Escherichia coli
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
Reaction Schemes
+
+
lipopolysaccharide[side 1]
=
+
+
lipopolysaccharide[side 2]
+
+
lipid A-core oligosaccharide[side 1]
=
+
+
lipid A-core oligosaccharide[side 2]
Synonyms
msba2, msba protein, lipid a transporter, lps flippase, pa4997, ecmsba, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
MsbA
-
ATP-dependent lipid A-core flippase
-
-
-
-
lipid flippase
MsbA
additional information
-
the enzyme belongs to the ATP-binding cassette superfamily
lipid flippase
-
-
-
-
MsbA
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ATP + H2O + lipopolysaccharide[side 1] = ADP + phosphate + lipopolysaccharide[side 2]
spin labeling and electron paramagnetic resonance spectroscopy are used to systematically explore rearrangements in MsbA structure during the ATP hydrolysis cycle. Spin-label accessibility and local dynamics are determined in liposomes for the nucleotide-free intermediate and the transition state of ATP hydrolysis. The changes in the electron paramagnetic resonance parameters between these two intermediates fit a global pattern consistent with alternating access of the chamber. In the transition state of ATP hydrolysis, spin labels on the cytoplasmic side report increase dynamic restrictions and reduce water accessibility, while those on the extracellular side report increase water penetration
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SYSTEMATIC NAME
IUBMB Comments
ATP phosphohydrolase (ABC-type, lipid A-core oligosaccharide-translocating)
An ATP-binding cassette (ABC) type transporter, characterized by the presence of two similar ATP-binding domains/proteins and two integral membrane domains/proteins. The enzyme, best characterized from the bacterium Escherichia coli, is located in the inner membrane and mediates the movement of lipid A attached to the core oligosaccharide from the cytoplasm to the periplasmic side of the inner membrane, an important step in the lipopolysaccharide biosynthetic pathway. Not to be confused with EC 7.5.2.5, ABC-type lipopolysaccharide transporter (LptB), which is implicated in the translocation of LPS from the inner membrane to the outer membrane and acts later in the process.
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
ATP + H2O + Hoechst 33342/in
ADP + phosphate + Hoechst 33342/out
-
-
-
?
ATP + H2O + lipid A-core oligosaccharide[side 1]
ADP + phosphate + lipid A-core oligosaccharide[side 2]
ATP + H2O + lipopolysaccharide/in
ADP + phosphate + lipopolysaccharide/out
-
-
-
?
ATP + H2O + phosphoethanolamine[side 1]
ADP + phosphate + phosphoethanolamine[side 2]
fluorescently NBD (7-nitrobenz-2-oxa-1,3-diazole)-labeled
-
-
?
ATP + H2O + (7-nitrobenz-2-oxa-1,3-diazole)-C12-sphingomyelin/in
ADP + phosphate + (7-nitrobenz-2-oxa-1,3-diazole)-C12-sphingomyelin/out
-
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
-
-
?
ATP + H2O + (7-nitrobenz-2-oxa-1,3-diazole)-glucosylceramide/in
ADP + phosphate + (7-nitrobenz-2-oxa-1,3-diazole)-glucosylceramide/out
-
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
-
-
?
ATP + H2O + (7-nitrobenz-2-oxa-1,3-diazole)-lactosylceramide/in
ADP + phosphate + (7-nitrobenz-2-oxa-1,3-diazole)-lactosylceramide/out
-
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
-
-
?
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
-
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
-
-
?
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
-
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
-
-
?
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
-
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
-
-
?
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
-
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
-
-
?
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
-
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
-
-
?
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
-
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
-
-
?
ATP + H2O + lipopolysaccharide/in
ADP + phosphate + lipopolysaccharide/out
-
-
-
-
?
ATP + H2O + lipid A-core oligosaccharide[side 1]
ADP + phosphate + lipid A-core oligosaccharide[side 2]
-
-
-
?
ATP + H2O + lipid A-core oligosaccharide[side 1]
ADP + phosphate + lipid A-core oligosaccharide[side 2]
-
-
-
-
?
ATP + H2O + lipid A-core oligosaccharide[side 1]
ADP + phosphate + lipid A-core oligosaccharide[side 2]
-
-
-
?
ATP + H2O + lipid A-core oligosaccharide[side 1]
ADP + phosphate + lipid A-core oligosaccharide[side 2]
-
-
-
-
?
ATP + H2O + lipid A-core oligosaccharide[side 1]
ADP + phosphate + lipid A-core oligosaccharide[side 2]
-
-
-
?
ATP + H2O + Lipid A/in
ADP + phosphate + Lipid A/out
-
-
-
-
?
ATP + H2O + Lipid A/in
ADP + phosphate + Lipid A/out
-
substrate binding structure, overview
-
-
?
additional information
?
-
concerted conformational rearrangements occur during MsbA ATPase cycle
-
-
?
additional information
?
-
MsbA cannot efficiently transport a substrate lacking phosphorylation at the 4'-position of lipid A
-
-
?
additional information
?
-
purified MsbA from Escherichia coli displays high ATPase activity, and binds to lipids and lipid-like molecules, including lipid A, with affinity in the low micromolar range. Purified MsbA is reconstituted into proteoliposomes of Escherichia coli lipid and shows ability to translocate 7-nitrobenz-2-oxa-1,3-diazole (NBD)-labeled lipid derivatives. 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 an acyl chain-labeled phosphatidylethanolamine derivative. Lipid flippase activity requires ATP hydrolysis, and is dependent on the concentration of ATP and NBD-lipid. MsbA can accommodate lipids with bulky carbohydrate headgroups
-
-
?
additional information
?
-
-
purified MsbA from Escherichia coli displays high ATPase activity, and binds to lipids and lipid-like molecules, including lipid A, with affinity in the low micromolar range. Purified MsbA is reconstituted into proteoliposomes of Escherichia coli lipid and shows ability to translocate 7-nitrobenz-2-oxa-1,3-diazole (NBD)-labeled lipid derivatives. 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 an acyl chain-labeled phosphatidylethanolamine derivative. Lipid flippase activity requires ATP hydrolysis, and is dependent on the concentration of ATP and NBD-lipid. MsbA can accommodate lipids with bulky carbohydrate headgroups
-
-
?
additional information
?
-
-
MsbA functions as an ATP-dependent lipid translocase that transports lipid A from the inner to the outer leaflet of the cytoplasmic membrane. MsbA is able to hydrolyze TNP-ATP, albeit at a lower rate than its corresponding ATPase activity
-
-
?
additional information
?
-
-
MsbA is an essential ABC transporter in Gram-negative bacteria
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
ATP + H2O + lipid A-core oligosaccharide[side 1]
ADP + phosphate + lipid A-core oligosaccharide[side 2]
ATP + H2O + lipopolysaccharide/in
ADP + phosphate + lipopolysaccharide/out
-
-
-
?
ATP + H2O + Lipid A/in
ADP + phosphate + Lipid A/out
-
-
-
-
?
ATP + H2O + lipopolysaccharide/in
ADP + phosphate + lipopolysaccharide/out
-
-
-
-
?
ATP + H2O + lipid A-core oligosaccharide[side 1]
ADP + phosphate + lipid A-core oligosaccharide[side 2]
-
-
-
?
ATP + H2O + lipid A-core oligosaccharide[side 1]
ADP + phosphate + lipid A-core oligosaccharide[side 2]
-
-
-
-
?
ATP + H2O + lipid A-core oligosaccharide[side 1]
ADP + phosphate + lipid A-core oligosaccharide[side 2]
-
-
-
?
ATP + H2O + lipid A-core oligosaccharide[side 1]
ADP + phosphate + lipid A-core oligosaccharide[side 2]
-
-
-
-
?
ATP + H2O + lipid A-core oligosaccharide[side 1]
ADP + phosphate + lipid A-core oligosaccharide[side 2]
-
-
-
?
additional information
?
-
-
MsbA functions as an ATP-dependent lipid translocase that transports lipid A from the inner to the outer leaflet of the cytoplasmic membrane. MsbA is able to hydrolyze TNP-ATP, albeit at a lower rate than its corresponding ATPase activity
-
-
?
additional information
?
-
-
MsbA is an essential ABC transporter in Gram-negative bacteria
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Mg2+
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
(2E)-3-[1-cyclopropyl-7-[(1S)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]naphthalen-2-yl]prop-2-enoic acid
-
(2E)-3-[6-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]quinolin-3-yl]prop-2-enoic acid
-
(2E)-3-[6-[(1S)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]quinolin-3-yl]prop-2-enoic acid
a quinoline compound with potent activity on purified Escherichia coli MsbA
(2E)-3-[6-[1-(2-chloro-6-cyclopropylphenyl)ethoxy]-4-cyclopropylquinolin-3-yl]prop-2-enoic acid
selective small-molecule antagonist with bactericidal activity and a dual-mode inhibitory mechanism. (2E)-3-[6-[1-(2-chloro-6-cyclopropylphenyl)ethoxy]-4-cyclopropylquinolin-3-yl]prop-2-enoic acid traps MsbA in an inward-facing, lipopolysaccharide-bound conformation by wedging into an architecturally conserved transmembrane pocket. (2E)-3-[6-[1-(2-chloro-6-cyclopropylphenyl)ethoxy]-4-cyclopropylquinolin-3-yl]prop-2-enoic acid gains access to MsbA through the bulk membrane. The 2-chloro-6 cyclopropylphenyl substituent of (2E)-3-[6-[1-(2-chloro-6-cyclopropylphenyl)ethoxy]-4-cyclopropylquinolin-3-yl]prop-2-enoic acid (A-ring) exhibits strong electron density and makes van der Waals interactions with side chains from transmembrane domains TM4 (L171, A175 and V178), TM5 (A259 and L263) and TM6 (M291 and L294). The quinoline core of (2E)-3-[6-[1-(2-chloro-6-cyclopropylphenyl)ethoxy]-4-cyclopropylquinolin-3-yl]prop-2-enoic acid (B-ring) is orthogonal to the plane of the phenyl substituent, where it is partially enclosed by residues from transmembrane domains TM4 (V178, S179 and I182), TM5 (A259) and TM6 (M295 and L298). A259 and M295 side chains contact the 4-cyclopropyl substitution of the quinoline core and delineate the inhibitor binding-pocket from the inner vestibule of MsbA. (2E)-3-[6-[1-(2-chloro-6-cyclopropylphenyl)ethoxy]-4-cyclopropylquinolin-3-yl]prop-2-enoic acid stabilizes a catalytically incompetent state of the transporter. The inhibitor may have broad relevance across the ABC transporter superfamily
lipid A
translocation of NBD-phosphatidylethanolamine is inhibited by the presence of the putative physiological substrate lipid A, probably due to competition for flipping of 7-nitrobenz-2-oxa-1,3-diazole-labeled phosphoethanolamine (NBD-PE)
vanadate
inhibitory effect of vanadate on the ATPase activity of MsbA
ethidium
-
antimitotic drug, vinblastine, directly competes with ethidium for binding to MsbA
Hoechst 33342
-
complete inhibition of MsbA-mediated Hoechst33342 transport by 0.025 mM taxol, noncompetitive kinetics with Ki of 0.0066 mM, overview
vinblastine
-
antimitotic drug, vinblastine, directly competes with ethidium for binding to MsbA
additional information
analysis of ABC transporter inhibition mechanism, small molecule inhibitor library screening, overview. (2E)-3-[6-[1-(2-chloro-6-cyclopropylphenyl)ethoxy]-4-cyclopropylquinolin-3-yl]prop-2-enoic acid traps MsbA in an inward-facing, lipopolysaccharide-bound conformation by wedging into an architecturally conserved transmembrane pocket. A second allosteric mechanism of antagonism occurs through structural and functional uncoupling of the nucleotide-binding domains
-
additional information
-
no inhibition by substrate lipid A also at high concentration, poor inhibition by verapamil, colchicine, and daunorubicine, poor inhibition by lipid-based drugs D-20133, and D-21266, along with LY335979
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
3-deoxy-D-mannooctulosonic acid (Kdo)2-lipid A
increases the ATPase activity 4-5fold, with half-maximal stimulation at 0.021 mM Kdo2-lipid A, addition of Kdo2-lipid A increases the Vmax and decreases the Km. The stimulation is only seen with hexaacylated lipid A species and not with precursors, such as diacylated lipid X or tetraacylated lipid IVA
-
lipid A
-
about 2fold stimulation of the ATPase activity of MsbA by its substrate
taxol
-
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
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.048 - 0.05
lipopolysaccharide
-
pH 7.5, 37°C, recombinant His-tagged enzyme
additional information
additional information
-
lipid binding kinetics, overview
-
0.379
ATP
reconstituted purified recombinant His-tagged enzyme in liposomes prepared from Escherichia coli phospholipids, pH 7.5, 37°C, ATP hydrolysis in presence of 10 mM Mg2+ and 0.021 mM Kdo2-lipid A
0.878
ATP
reconstituted purified recombinant His-tagged enzyme in liposomes prepared from Escherichia coli phospholipids, pH 7.5, 37°C, ATP hydrolysis in presence of 10 mM Mg2+
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.000005
(2E)-3-[1-cyclopropyl-7-[(1S)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]naphthalen-2-yl]prop-2-enoic acid
Escherichia coli
pH 7.5, 37°C, recombinant detagged enzyme
0.000018
(2E)-3-[6-[1-(2-chloro-6-cyclopropylphenyl)ethoxy]-4-cyclopropylquinolin-3-yl]prop-2-enoic acid
Escherichia coli
pH 7.5, 37°C, recombinant detagged enzyme
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
0.726
-
recombinant His6-tagged mutant S289A/S290A in presence or absence of taxol
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)
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.5
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
37
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
malfunction
metabolism
physiological function
additional information
evolution
enzyme MsbA encoded is a member of the ABC transporter family
evolution
the msbA gene product belongs to the superfamily of ABC transporters, the ATP-binding cassette (ABC)-transporter superfamily, a universally conserved family of proteins characterized by a highly conserved ATP-binding domain
evolution
the MsbA protein is an essential ABC (ATP-binding-cassette) superfamily member in Gram-negative bacteria
malfunction
accumulation of lipid A species in the inner membranes of htrB- cells when msbA and orfE are not expressed. The tetra-acylated lipid A precursors that accumulate in htrB mutants may not be transported as efficiently by MsbA as are penta- or hexaacylated lipid A species. LPS accumulates in the inner membranes of htrB-deficient mutants. Introduction of msbA and orfE on low copy plasmids partially restores translocation of LPS to the outer membrane at 42°C in htrB mutants. The Escherichia coli msbA gene also functions as a multicopy suppressor of htrB mutations
malfunction
depletion or loss of function of MsbA results in the accumulation of lipopolysaccharide and phospholipids in the inner membrane of Escherichia coli
metabolism
the enzyme is involved in the biosynthetic pathways of lipid A
metabolism
the enzyme is involved in the lipid A biosynthesis. MsbA cannot efficiently transport a substrate lacking phosphorylation at the 4'-position of lipid A. This last essential step of lipid A biosynthesis is catalysed by LpxK, the two proteins are concatenated in some bacteria, suggesting tight coupling of their cellular activities
physiological function
Escherichia coli MsbA is a lipid-activated ATPase with a proposed role in phospholipid export
physiological function
genes msbA and orfE, forming an operon, are essential for bacterial viability under all growth conditions tested. Gene msbA is a multicopy suppressor of gene htrB, overview. When cloned in vectors maintained at two to four copies per cell, the msbA gene complements most of the HtrB phenotypes, including the morphological alterations, the overproduction of phospholipids, and the lethality exhibited at non-permissive temperatures. Neither HtrB nor MsbB can fully substitute for MsbA function
physiological function
MsbA is an essential ABC family transporter in lipid A and phospholipid biosynthesis, ATP-dependent transport of nascent core-lipid A molecules across the inner membrane by MsbA. The Escherichia coli msbA gene also functions as a multicopy suppressor of htrB mutations (temperature-sensitive growth of an Escherichia coli mutant lacking htrB and its suppression by extra copies of msbA). The HtrB-catalyzed transfer of laurate to lipid A may be necessary for efficient core-lipid A transport and MsbA and/or OrfE are components of the transport machinery
physiological function
the enzme is an LPS transporters. LPS transporters are ABC exporters that are known to export extremely hydrophobic compounds, such as lipids, drugs, and steroids. MsbA facilitates flipping of the lipid A-core structure across the inner membrane and exports antibiotics and chemotherapeutic drugs
physiological function
the enzyme is involved in the transport of lipid A-core, and not just lipid A, from the cytoplasmic side of the inner membrane to the periplasmic side. Expression of Escherichia coli MsbA but not Pseudomonas aeruginosa MsbA confers resistance to erythromycin in Pseudomonas aeruginosa, but gene msbA from Escherichia coli cannot cross complement the msbA merodiploid cells of Pseuomonas aeruginosa
physiological function
the homodimeric ATP-dependent lipid translocase or flippase transports lipid A from the inner to the outer leaflet of the cytoplasmic membrane
physiological function
the movement of core-lipopolysaccharide across the inner membrane of Gram-negative bacteria is catalysed by an essential ATP-binding cassette transporter, MsbA, structure of LPS in complex with EcMsbA, overview
physiological function
the movement of core-lipopolysaccharide across the inner membrane of Gram-negative bacteria is catalysed by an essential ATP-binding cassette transporter, MsbA. The ABC transporter MsbA flips the lipid to the outer leaflet of the inner membrane and the O-antigen is attached by WaaL to create LPS. LPS and the periplasmic protein, LptA (EC 2.7.8.43), are two essential components of Gram-negative bacteria. LPS (endotoxin) is asymmetrically distributed in the outer leaflet of the outer membrane of Gram-negative bacteria such as Escherichia coli and plays a role in the organism's natural defense in adverse environmental conditions. LptA is a member of the lipopolysaccharide transport protein (Lpt) family, which also includes LptC, LptDE, and LptBFG2, that functions to transport LPS through the periplasm to the outer leaflet of the outer membrane after MsbA flips LPS across the inner membrane
physiological function
the msbA gene expressed from a low-copy-number plasmid vector is able to suppress the temperature-sensitive growth phenotype of an Escherichia coli htrB null mutant as well as the accumulation of phospholipids. The msbA gene is essential for bacterial viability at all temperatures. Role for MsbA as a translocator of lipopolysaccharides or its precursors
additional information
inward facing structure of EcMsbA in complex with LPS, structure comparisons and molecular basis of active lipid transport, overview
additional information
nucleotide-free MsbA has an open structure where the two NBDs are separated by 50 A. The transmembrane (TM) helices are arranged in two wings that form a V-shaped chamber open to the cytoplasm and the inner leaflet of the bilayer. In the AMPPNP structure, the closed structure, the two NBDs form the canonical ATP dimer while the two TM wings of the transporter pack in the cytoplasm and split in an outward-facing conformation at the extracellular side. To create this opening, a twisting motion repacks the TM helices changing the identity of the swapped helices between the two monomers. Whereas TM1-TM2 share intersubunit contacts with TM3-TM6 in the closed structure, TM4-TM5 join the other subunit in the open conformation. As a result the inward and outward openings are mediated by different sets of helices. Nucleotide-free MsbA also crystallizes in an alternative conformation (closed-apo) that has the two NBDs closer than the open-apo and the chamber partially occluded. But it has the same helix packing in the TMD as the open-apo structure. Purified MsbA reconstitutes in liposomes. Structure-function analysis, detailed overview
additional information
reconstitution of purified MsbA into proteoliposomes, method, overview
additional information
-
reconstitution of purified MsbA into proteoliposomes, method, overview
additional information
structure comparisons and molecular basis of active lipid transport, overview
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
homodimer
homodimer
homodimer
the MsbA homodimer is comprised of two identical monomers, each with a cytosolic nucleotide binding domain (NBD) and a transmembrane domain (TMD)
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
apo,MgADP/Vi-bound and MgAMP-PNP-bound MsbA, X-ray diffraction structure determination and analysis
purified enzyme MsbA free and in complex with inhibitors (2E)-3-[6-[1-(2-chloro-6-cyclopropylphenyl)ethoxy]-4-cyclopropylquinolin-3-yl]prop-2-enoic acid or G902, sitting drop vapour diffusion method, mixing of 10 mg/ml protein in 50 mM Tris, pH 8.0, 500 mM NaCl, and 0.6% FA-3 detergent, with mother liquor containing 200 mM NaCl, 19% PEG 550 MME, 4% PEG 400, and 100 mM HEPES, pH 7.0, in a 1:1 v/v ratio, 19°C, 1 week, X-ray diffraction structure determination and analysis at 2.9 A resolution
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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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
A270T
A281C
the mutant displays a reduction in ATP-dependent Hoechst 33342 export activity
D510G
random mutagenesis, the mutant cannot support Escherichia coli growth, but it retains the ability to bind ATP in vitro
D512G
random mutagenesis, the mutant is able to hydrolyze ATP 3fold faster than the wild-type enzyme
E208A
the mutant shows greatly reduced activity compared to the wild type enzyme
E208A/A281C
the mutant displays a strong reduction in ATP-dependent Hoechst 33342 export activity
E208A/K212A
the mutant shows greatly reduced activity compared to the wild type enzyme
I385C/D512G
site-directed mutagenesis, the additional mutation of the spin-labeled reporter site causes slight changes upon MgATP/Vi binding
I385C/L511P
site-directed mutagenesis, the additional mutation of the spin-labeled reporter site causes changes upon MgATP/Vi binding
I517V
random mutagenesis, the MsbA mutant protein is still partly functional due to the fact that an Ile to Val change is a fairly conservative substitution, or because in the MDR proteins a Val residue is present at this position
K212A
the mutant shows greatly reduced activity compared to the wild type enzyme
L504C/D512G
site-directed mutagenesis, the additional mutation of the spin-labeled reporter site causes changes upon MgATP/Vi binding, the mutant exhibits a general broadening of the spectrum
L504C/L511P
site-directed mutagenesis, the additional mutation of the spin-labeled reporter site causes changes upon MgATP/Vi binding
L509P
random mutagenesis, the mutant cannot support Escherichia coli growth, but it retains the ability to bind ATP in vitro
L511P
random mutagenesis, the mutant is able to bind ATP at near-wild-type levels but is unable to maintain cell viability in an in vivo growth assay, it is dysfunctional at some point after ATP binding. The L511P mutation prevents effective ATP hydrolysis, only small amounts of ATP are hydrolyzed
Q485C/D512G
site-directed mutagenesis, the additional mutation of the spin-labeled reporter site causes changes upon MgATP/Vi binding
Q485C/L511P
site-directed mutagenesis, the additional mutation of the spin-labeled reporter site causes changes upon MgATP/Vi binding
S380C/D512G
site-directed mutagenesis, the additional mutation of the spin-labeled reporter site causes changes upon MgATP/Vi binding and exhibits an additional shift (approximately 30%) toward the immobilized population
S380C/L511P
site-directed mutagenesis, the additional mutation of the spin-labeled reporter site causes changes upon MgATP/Vi binding
S423C/D512G
site-directed mutagenesis, the additional mutation of the spin-labeled reporter site causes slight changes upon MgATP/Vi binding
S423C/L511P
site-directed mutagenesis, the additional mutation of the spin-labeled reporter site causes changes upon MgATP/Vi binding
S482C/D512G
site-directed mutagenesis, the additional mutation of the spin-labeled reporter site causes changes upon MgATP/Vi binding
S482C/L511P
site-directed mutagenesis, the additional mutation of the spin-labeled reporter site causes changes upon MgATP/Vi binding
T541C/D512G
site-directed mutagenesis, the additional mutation of the spin-labeled reporter site causes changes upon MgATP/Vi binding
T541C/L511P
site-directed mutagenesis, the additional mutation of the spin-labeled reporter site causes changes upon MgATP/Vi binding
V426C/D512G
site-directed mutagenesis, the additional mutation of the spin-labeled reporter site causes changes upon MgATP/Vi binding
V426C/L511P
site-directed mutagenesis, the additional mutation of the spin-labeled reporter site causes changes upon MgATP/Vi binding, the mutant shows a more immobile spectrum in the presence of MgATP
V534C/D512G
site-directed mutagenesis, the additional mutation of the spin-labeled reporter site causes slight changes upon MgATP/Vi binding
V534C/L511P
site-directed mutagenesis, the additional mutation of the spin-labeled reporter site causes changes upon MgATP/Vi binding
E506Q
-
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
-
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
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/E506Q
-
significantly diminished rates of hydrolysis, about 4% of wild-type
S423C/H537A
-
significantly diminished rates of hydrolysis, about 4% of wild-type
additional information
A270T
site-directed mutagenesis, temperature-sensitive MsbA allele, the mutation renders cells temperature-sensitive for growth and lipid export, the mutant displays ATPase activity similar to that of the wild-type protein at 30°C but is significantly reduced at 42°C
A270T
site-directed mutagenesis, the mutation causes the protein to become inactive at high temperatures
additional information
amorphadiene, the precursor of antimalarial drug artemisinin from Artemisia annua, is secreted from Escherichia coli cells overexpressing the biosynthetic pathway. The overexpression of transporters in the lipopolysaccharide transport system (msbA, lptD, lptCABFG) improves amorphadiene (AD) production. AD production in both early stage (8 h) and final stage (24 h) is increased by more than twofold in the strains that overexpress lptCABFG or msbA. But co-overexpression of LptCABFG and LptD or LptD and TolC does not enhance AD-specific production synergistically, despite the fact that the AD titer is increased mainly due to the increased cell density, overview
additional information
generation of an Escherichia coli msbA insertion knockout mutant. The mutation has a deleterious effect on bacterial growth or viability. The insertion mutation affects the expression of both genes, msbA and orfE, overview
additional information
overexpression of msbA suppresses mutations in the htrB lipid A acyltransferase, as MsbA can transport the tetra-acylated LPS produced in htrB mutants, albeit inefficiently
additional information
random PCR mutagenesis of gene msbA resulting in six independent mutants, four of which result in single-amino-acid substitutions in non-conserved residues, the temperature-sensitive mutants are able to support cell growth at 30°C but not at 43°C. The remaining two mutants behave as recessive lethals, the mutations result in single-amino-acid substitutions in Walker motif B, one of the two highly conserved regions of the ATP-binding domain. The latter two mutants cannot support Escherichia coli growth, but they both retain the ability to bind ATP in vitro. N-acetyl [3H]-glucosamine, a precursor of Iipopolysaccharides, accumulates at the non-permissive temperature in the inner membrane of either htrB null or msbA conditional lethal strains. Translocation of the precursor to the outer membrane is restored by transformation with a plasmid containing the wild-type msbA gene. The Ts- phenotype exhibited at 43°C can be suppressed by supplementing the medium with 10 mM Mg2+ or Ca2+
additional information
-
random PCR mutagenesis of gene msbA resulting in six independent mutants, four of which result in single-amino-acid substitutions in non-conserved residues, the temperature-sensitive mutants are able to support cell growth at 30°C but not at 43°C. The remaining two mutants behave as recessive lethals, the mutations result in single-amino-acid substitutions in Walker motif B, one of the two highly conserved regions of the ATP-binding domain. The latter two mutants cannot support Escherichia coli growth, but they both retain the ability to bind ATP in vitro. N-acetyl [3H]-glucosamine, a precursor of Iipopolysaccharides, accumulates at the non-permissive temperature in the inner membrane of either htrB null or msbA conditional lethal strains. Translocation of the precursor to the outer membrane is restored by transformation with a plasmid containing the wild-type msbA gene. The Ts- phenotype exhibited at 43°C can be suppressed by supplementing the medium with 10 mM Mg2+ or Ca2+
additional information
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construction of point and deletion muitants, e.g. DELTAK382, change-in-specificity mutations colocalize in a major groove in each of the two wings of transmembrane helices, that point away from one another to ward the periplasm. Near the apex of the groove, the periplasmic side of transmembrane helice 6, TMH6, in both monomers contains a hot spot of change-in-specificity mutations and residues which, when replaced with cysteines in ABCB1, covalently interact with thiol-reactive drug analogues, drug-protein interaction analysis, overview
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant FLAG-tagged enzyme from Escherichia coli strain Rosetta 2 (DE3) by affinity chromatography, the Flag tag is removed using TEV protease, followed by gel filtration and ultrafiltration
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli by ultraccentrifugation, detergent solubilization, again ultraccentrifugation, and nickel affinity chromatography, to over 95% purity
recombinant N-terminally His6-tagged enzyme from isolated Escherichia coli inside-out membrane vesicles by nickel affinity chromatography
recombinant N-terminally His6-tagged wild-type and mutant enzymes from Escherichia coli NovaBlue cells by cobalt affinity chromatography and ultrafiltration
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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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene msbA, DNA and amino acid sequence determination and analysis, sequence comparisons, gene msbA may form an operon with a downstream gene orfE, complementation of the Escherichia coli htrB- phenotypes by expression of gene msbA from plasmid pK-Bst
gene msbA, overexpression of gene msbA in Escherichia coli strain K12 MG1655 DELTArecADELTAendA DE3, quantitative real-time PCR expression analysis
gene msbA, overexpression of N-terminally His6-tagged enzyme in Escherichia coli
gene msbA, recombinant expression in Escherichia coli strain BL21 inner membranes. At 42°C and in the absence of transducer arabinose, the msbA gene is transcribed poorly, if at all
gene msbA, recombinant expression in Pseudomonas aeruginosa wild-type cells and msbA merodiploid cells
gene msbA, recombinant expression of N-terminally His6-tagged wild-type and mutant enzymes in Escherichia coli NovaBlue cells
gene msbA, recombinant overexpression of His6-tagged wild-type and mutant enzymes in Escherichia coli
gene msbA, recombinant overexpresssion of FLAG-tagged enzyme in Escherichia coli strain Rosetta 2 (DE3)
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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RENATURED/Commentary
ORGANISM
UNIPROT
LITERATURE
MsbA is reconstituted into 65:25:10 phosphatidylethanolamine: phosphatidylglycerol: cardiolipin inner membrane liposomes at a 250:1 lipid:protein molar ratio
reconstitution of purified MsbA into proteoliposomes, method, overview
the enzyme is reconstituted into liposomes prepared from Escherichia coli phospholipids
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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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Woebking, B.; Velamakanni, S.; Federici, L.; Seeger, M.A.; Murakami, S.; van Veen, H.W.
Functional role of transmembrane helix 6 in drug binding and transport by the ABC transporter MsbA
Biochemistry
47
10904-10914
2008
Escherichia coli, Salmonella enterica subsp. enterica serovar Typhimurium (P63359)
Eckford, P.D.; Sharom, F.J.
Functional characterization of Escherichia coli MsbA: interaction with nucleotides and substrates
J. Biol. Chem.
283
12840-12850
2008
Escherichia coli
Eckford, P.D.; Sharom, F.J.
The reconstituted Escherichia coli MsbA protein displays lipid flippase activity
Biochem. J.
429
195-203
2010
Escherichia coli
Zou, P.; McHaourab, H.S.
Alternating access of the putative substrate-binding chamber in the ABC transporter MsbA
J. Mol. Biol.
393
574-585
2009
Escherichia coli
Schultz, K.; Merten, J.; Klug, C.
Characterization of the E506Q and H537A dysfunctional mutants in the E. coli abc transporter MsbA
Biochemistry
50
3599-3608
2011
Escherichia coli
Kawai, T.; Caaveiro, J.; Abe, R.; Katagiri, T.; Tsumoto, K.
Catalytic activity of MsbA reconstituted in nanodisc particles is modulated by remote interactions with the bilayer
FEBS Lett.
585
3533-3537
2011
Escherichia coli
Weng, J.; Fan, K.; Wang, W.
The conformational transition pathway of ATP binding cassette transporter MsbA revealed by atomistic simulations
J. Biol. Chem.
285
3053-3063
2010
Escherichia coli
Syberg, F.; Suveyzdis, Y.; Koetting, C.; Gerwert, K.; Hofmann, E.
Time-resolved Fourier transform infrared spectroscopy of the nucleotide-binding domain from the ATP-binding cassette transporter MsbA: ATP hydrolysis is the rate-limiting step in the catalytic cycle
J. Biol. Chem.
287
23923-23931
2012
Escherichia coli (P60752)
Doshi, R.; Van Veen, H.
Substrate binding stabilizes a pre-translocation intermediate in the ATP-binding cassette transport protein MsbA
J. Biol. Chem.
288
21638-21647
2013
Escherichia coli
Doshi, R.; Ali, A.; Shi, W.; Freeman, E.V.; Fagg, L.A.; van Veen, H.W.
Molecular disruption of the power stroke in the ATP-binding cassette transport protein MsbA
J. Biol. Chem.
288
6801-6813
2013
Escherichia coli (P60752)
Xie, X.; Li, C.; Yang, Y.; Jin, L.; Tan, J.; Zhang, X.; Su, J.; Wang, C.
Allosteric transitions of ATP-binding cassette transporter MsbA studied by the adaptive anisotropic network model
Proteins Struct. Funct. Bioinform.
83
1643-1653
2015
Escherichia coli
Eckford, P.D.; Sharom, F.J.
The reconstituted Escherichia coli MsbA protein displays lipid flippase activity
Biochem. J.
429
195-203
2010
Escherichia coli (P60752), Escherichia coli
Schultz, K.; Merten, J.; Klug, C.
Effects of the L511P and D512G mutations on the Escherichia coli ABC transporter MsbA
Biochemistry
50
2594-2602
2011
Escherichia coli (P60752), Escherichia coli K12 (P60752)
Zhang, C.; Chen, X.; Stephanopoulos, G.; Too, H.P.
Efflux transporter engineering markedly improves amorphadiene production in Escherichia coli
Biotechnol. Bioeng.
113
1755-1763
2016
Escherichia coli (P60752)
Zhou, Z.; White, K.; Polissi, A.; Georgopoulos, C.; Raetz, C.
Function of Escherichia coli MsbA, an essential ABC family transporter, in lipid A and phospholipid biosynthesis
J. Biol. Chem.
273
12466-12475
1998
Escherichia coli (P60752), Escherichia coli W3110 (P60752)
Doerrler, W.T.; Raetz, C.R.
ATPase activity of the MsbA lipid flippase of Escherichia coli
J. Biol. Chem.
277
36697-36705
2002
Escherichia coli (P60752), Escherichia coli K12 (P60752)
Ghanei, H.; Abeyrathne, P.; Lam, J.
Biochemical characterization of MsbA from Pseudomonas aeruginosa
J. Biol. Chem.
282
26939-26947
2007
Escherichia coli (P60752), Escherichia coli, Pseudomonas aeruginosa (Q9HUG8), Pseudomonas aeruginosa, Pseudomonas aeruginosa ATCC 15692 (Q9HUG8), Pseudomonas aeruginosa 1C (Q9HUG8), Pseudomonas aeruginosa PRS 101 (Q9HUG8), Pseudomonas aeruginosa DSM 22644 (Q9HUG8), Pseudomonas aeruginosa CIP 104116 (Q9HUG8), Pseudomonas aeruginosa LMG 12228 (Q9HUG8), Pseudomonas aeruginosa JCM 14847 (Q9HUG8)
Zou, P.; Bortolus, M.; McHaourab, H.S.
Conformational cycle of the ABC transporter MsbA in liposomes detailed analysis using double electron-electron resonance spectroscopy
J. Mol. Biol.
393
586-597
2009
Escherichia coli (P60752)
Polissi, A.; Georgopoulos, C.
Mutational analysis and properties of the msbA gene of Escherichia coli, coding for an essential ABC family transporter
Mol. Microbiol.
20
1221-1233
1996
Escherichia coli (P60752), Escherichia coli
Karow, M.; Georgopoulos, C.
The essential Escherichia coli msbA gene, a multicopy suppressor of null mutations in the htrB gene, is related to the universally conserved family of ATP-dependent translocators
Mol. Microbiol.
7
69-79
1993
Escherichia coli (P60752)
Ho, H.; Miu, A.; Alexander, M.K.; Garcia, N.K.; Oh, A.; Zilberleyb, I.; Reichelt, M.; Austin, C.D.; Tam, C.; Shriver, S.; Hu, H.; Labadie, S.S.; Liang, J.; Wang, L.; Wang, J.; Lu, Y.; Purkey, H.E.; Quinn, J.; Franke, Y.; Clark, K.; Beresini, M.H.; Tan, M.W.; Sellers, B.D.; Maurer, T.; Koehler, M.F.T.; Wecksler, A.T.
Structural basis for dual-mode inhibition of the ABC transporter MsbA
Nature
557
196-201
2018
Escherichia coli (P60752), Salmonella enterica subsp. enterica serovar Typhimurium (P63359)
EXTERNAL LINKS (specific for EC-Number 7.5.2.6)
Transporter Classification Database (TCDB): 3.A.1.106.20, 3.A.1.106.1
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