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Literature summary for 7.5.2.6 extracted from
- The ABC transporter MsbA interacts with lipid A and amphipathic drugs at different sites (2009), Biochem. J., 419, 317-328 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| gene msbA, recombinant expression of His-tagged wild-type and mutant C88S/C135S enzymes in Escherichia coli strain BL21-Codonplus(DE3)-RIL in cytoplasmic membrane vesicles | Salmonella enterica subsp. enterica serovar Typhimurium |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| C315S | site-directed mutagenesis, the mutant ATPase activity of the C88S and C315S mutants does not differ substantially from that of wild-type MsbA | Salmonella enterica subsp. enterica serovar Typhimurium |
| C88S | site-directed mutagenesis, the mutant ATPase activity of the C88S and C315S mutants does not differ substantially from that of wild-type MsbA | Salmonella enterica subsp. enterica serovar Typhimurium |
| C88S/C315S | site-directed mutagenesis | Salmonella enterica subsp. enterica serovar Typhimurium |
| additional information | the mutant enzymes are T561C and E506Q/T561C are labeled with MIANS fluorescent probes | Salmonella enterica subsp. enterica serovar Typhimurium |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.52 | - |
ATP | recombinant His-tagged wild-type enzyme, ATPase activity, pH 7.5, 37°C | Salmonella enterica subsp. enterica serovar Typhimurium |  |
Localization
| Localization | Comment | Organism | GeneOntology No. | Textmining |
|---|---|---|---|---|
| inner membrane | topological model of the MsbA protein, the six transmembrane helices in each MsbA monomer extend into the cytosol, forming an intracellular domain that may play a role in coupling ATP hydrolysis to substrate transport, overview | Salmonella enterica subsp. enterica serovar Typhimurium | - |
- |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Mg2+ | required | Salmonella enterica subsp. enterica serovar Typhimurium | |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| ATP + H2O + lipid A-core oligosaccharide[side 1] | Salmonella enterica subsp. enterica serovar Typhimurium | - |
ADP + phosphate + lipid A-core oligosaccharide[side 2] | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Salmonella enterica subsp. enterica serovar Typhimurium | P63359 | - |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant N-terminally His6-tagged wild-type and mutant C88S/C135S enzymes from Escherichia coli strain BL21-Codonplus(DE3)-RIL by n-dodecyl-beta-D-maltopyranoside solubilization from membranes, nickel affinity chromatography, and ultrafiltration | Salmonella enterica subsp. enterica serovar Typhimurium |
Specific Activity [micromol/min/mg]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| 0.4 | - |
purified recombinant His-tagged wild-type enzyme, ATPase activity, pH 7.5, 37°C | Salmonella enterica subsp. enterica serovar Typhimurium |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| ATP + H2O + daunorubicin[side 1] | - |
Salmonella enterica subsp. enterica serovar Typhimurium | ADP + phosphate + daunorubicin[side 2] | - |
? | |
| ATP + H2O + lipid A-core oligosaccharide[side 1] | - |
Salmonella enterica subsp. enterica serovar Typhimurium | ADP + phosphate + lipid A-core oligosaccharide[side 2] | - |
? | |
| additional information | simultaneous high affinity binding to MsbA of lipid A (the putative physiological substrate) and daunorubicin, which suggests that the protein has separate binding sites for these two compounds. The effects of nucleotide and lipid A/daunorubicin binding to MsbA are additive, and binding could occur in any order. The two substrate-binding sites appear to communicate with each other, and also with the nucleotide-binding site in the nucleotide-binding domains, NBDs. The two monomers function independently, and do not interact co-operatively with each other, analysis using MIANS-labeled enzyme, MIANS, i.e. 2-(4-maleimidylanilino)naphthalene-6-sulfonic acid, is a cysteine-reactive fluorescent probe for soluble and membrane-bound proteins, fluorescence quenching studies. The percentage quenching values observed for lipid A and daunorubicin are also similar regardless of the order of titration. The binding affinity for lipid A is reduced about 5fold at 23°C and about 7fold at 10°C when the daunorubicin-binding site is occupied first | Salmonella enterica subsp. enterica serovar Typhimurium | ? | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| homodimer | the two monomers function independently, and do not interact co-operatively with each other | Salmonella enterica subsp. enterica serovar Typhimurium |
| More | secondary structure analysis of MsbA | Salmonella enterica subsp. enterica serovar Typhimurium |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| MsbA | - |
Salmonella enterica subsp. enterica serovar Typhimurium |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 37 | - |
assay at | Salmonella enterica subsp. enterica serovar Typhimurium |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 7.5 | - |
assay at | Salmonella enterica subsp. enterica serovar Typhimurium |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| ATP | - |
Salmonella enterica subsp. enterica serovar Typhimurium | |
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