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Literature summary for 7.5.2.6 extracted from
- Biochemical characterization of MsbA from Pseudomonas aeruginosa (2007), J. Biol. Chem., 282, 26939-26947 .
Activating Compound
| Activating Compound | Comment | Organism | Structure |
|---|---|---|---|
| additional information | activity of MsbA can be selectively stimulated by different truncated versions of core oligosaccharides of Pseudomonas aeruginosa lipopolysaccharide (LPS). Phosphate substituents in the lipid A-core are important for stimulating ATPase activity of MsbA | Pseudomonas aeruginosa |
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| gene msbA, recombinant expression in Pseudomonas aeruginosa wild-type cells and msbA merodiploid cells | Escherichia coli |
| gene msbA, recombinant overexpression of N-terminal His6-tagged enzyme in Escherichia coli strain BL21 (DE3) from low-copy-number plasmid pRK404 | Pseudomonas aeruginosa |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| additional information | disruption of the chromosomal msbA is achieved only when a functional copy of the gene is provided in trans, mutation in the gene is lethal to the bacterium. Gene msbA from Escherichia coli K-12 (msbAEc) cannot cross complement the msbA merodiploid cells of Pseuomonas aeruginosa | Pseudomonas aeruginosa |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | Michaelis-Menten kinetics of MsbA ATPase activity | Pseudomonas aeruginosa | |
| 0.573 | - |
ATP | pH 7.5, 37°C, detergent-solubilized recombinant His6-tagged enzyme, 8 mM ATP, ATPase activity | Pseudomonas aeruginosa |  |
| 4.5 | - |
ATP | pH 7.5, 37°C, reconstituted recombinant His6-tagged enzyme, 8 mM ATP, ATPase activity | Pseudomonas aeruginosa | |
Localization
| Localization | Comment | Organism | GeneOntology No. | Textmining |
|---|---|---|---|---|
| inner membrane | - |
Pseudomonas aeruginosa | - |
- |
| inner membrane | - |
Escherichia coli | - |
- |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Mg2+ | required | Pseudomonas aeruginosa | |
| Mg2+ | required | Escherichia coli | |
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] | Pseudomonas aeruginosa | - |
ADP + phosphate + lipid A-core oligosaccharide[side 2] | - |
? | |
| ATP + H2O + lipid A-core oligosaccharide[side 1] | Escherichia coli | - |
ADP + phosphate + lipid A-core oligosaccharide[side 2] | - |
? | |
| ATP + H2O + lipid A-core oligosaccharide[side 1] | Pseudomonas aeruginosa ATCC 15692 | - |
ADP + phosphate + lipid A-core oligosaccharide[side 2] | - |
? | |
| ATP + H2O + lipid A-core oligosaccharide[side 1] | Pseudomonas aeruginosa 1C | - |
ADP + phosphate + lipid A-core oligosaccharide[side 2] | - |
? | |
| ATP + H2O + lipid A-core oligosaccharide[side 1] | Pseudomonas aeruginosa PRS 101 | - |
ADP + phosphate + lipid A-core oligosaccharide[side 2] | - |
? | |
| ATP + H2O + lipid A-core oligosaccharide[side 1] | Pseudomonas aeruginosa DSM 22644 | - |
ADP + phosphate + lipid A-core oligosaccharide[side 2] | - |
? | |
| ATP + H2O + lipid A-core oligosaccharide[side 1] | Pseudomonas aeruginosa CIP 104116 | - |
ADP + phosphate + lipid A-core oligosaccharide[side 2] | - |
? | |
| ATP + H2O + lipid A-core oligosaccharide[side 1] | Pseudomonas aeruginosa LMG 12228 | - |
ADP + phosphate + lipid A-core oligosaccharide[side 2] | - |
? | |
| ATP + H2O + lipid A-core oligosaccharide[side 1] | Pseudomonas aeruginosa JCM 14847 | - |
ADP + phosphate + lipid A-core oligosaccharide[side 2] | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Escherichia coli | P60752 | - |
- |
| Pseudomonas aeruginosa | Q9HUG8 | - |
- |
| Pseudomonas aeruginosa 1C | Q9HUG8 | - |
- |
| Pseudomonas aeruginosa ATCC 15692 | Q9HUG8 | - |
- |
| Pseudomonas aeruginosa CIP 104116 | Q9HUG8 | - |
- |
| Pseudomonas aeruginosa DSM 22644 | Q9HUG8 | - |
- |
| Pseudomonas aeruginosa JCM 14847 | Q9HUG8 | - |
- |
| Pseudomonas aeruginosa LMG 12228 | Q9HUG8 | - |
- |
| Pseudomonas aeruginosa PRS 101 | Q9HUG8 | - |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant N-terminal His6-tagged enzyme from Escherichia coli strain BL21 (DE3) by ultracentrifugation delivering inside-out membrane vesicles, solubilization by detergent, again ultracentrifugation, metal affinity chromatography, and desalting gel filtration. The suitable detergent for solubilization of MsbA is examined among a number of detergents including octylglucoside at 2% w/v, lauryldimethylamine oxide at 2% w/v, dodecyl beta-D-maltoside at 1% w/v and sarcosyl at 1% w/v. Samples are then incubated with gentle stirring for 1 h. No difference in the amount of solubilized proteins is observed when the membranes are incubated with detergents for longer than 1 h. Sarcosyl is most effective for solubilizing MsbA from the membranes resulting in the highest yield of solubilized protein compared with the other three detergents | Pseudomonas aeruginosa |
Specific Activity [micromol/min/mg]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| 0.1445 | - |
pH 7.5, 37°C, reconstituted recombinant His6-tagged enzyme, substrate 8 mM ATP, ATPase activity | Pseudomonas aeruginosa |
Storage Stability
| Storage Stability | Organism |
|---|---|
| -80°C, recombinant His6-tagged enzyme in purified membrane vesicles, the ATPase activity of MsbA is stable upon storage for up to three months | Pseudomonas aeruginosa |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| ATP + H2O + lipid A-core oligosaccharide[side 1] | - |
Pseudomonas aeruginosa | ADP + phosphate + lipid A-core oligosaccharide[side 2] | - |
? | |
| ATP + H2O + lipid A-core oligosaccharide[side 1] | - |
Escherichia coli | ADP + phosphate + lipid A-core oligosaccharide[side 2] | - |
? | |
| ATP + H2O + lipid A-core oligosaccharide[side 1] | - |
Pseudomonas aeruginosa ATCC 15692 | ADP + phosphate + lipid A-core oligosaccharide[side 2] | - |
? | |
| ATP + H2O + lipid A-core oligosaccharide[side 1] | - |
Pseudomonas aeruginosa 1C | ADP + phosphate + lipid A-core oligosaccharide[side 2] | - |
? | |
| ATP + H2O + lipid A-core oligosaccharide[side 1] | - |
Pseudomonas aeruginosa PRS 101 | ADP + phosphate + lipid A-core oligosaccharide[side 2] | - |
? | |
| ATP + H2O + lipid A-core oligosaccharide[side 1] | - |
Pseudomonas aeruginosa DSM 22644 | ADP + phosphate + lipid A-core oligosaccharide[side 2] | - |
? | |
| ATP + H2O + lipid A-core oligosaccharide[side 1] | - |
Pseudomonas aeruginosa CIP 104116 | ADP + phosphate + lipid A-core oligosaccharide[side 2] | - |
? | |
| ATP + H2O + lipid A-core oligosaccharide[side 1] | - |
Pseudomonas aeruginosa LMG 12228 | ADP + phosphate + lipid A-core oligosaccharide[side 2] | - |
? | |
| ATP + H2O + lipid A-core oligosaccharide[side 1] | - |
Pseudomonas aeruginosa JCM 14847 | ADP + phosphate + lipid A-core oligosaccharide[side 2] | - |
? | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| lipid A export ATP-binding/permease protein | UniProt | Pseudomonas aeruginosa |
| lipid A export ATP-binding/permease protein | UniProt | Escherichia coli |
| MsbA | - |
Pseudomonas aeruginosa |
| MsbA | - |
Escherichia coli |
| pA4997 | - |
Pseudomonas aeruginosa |
| pA4997 | locus name | Pseudomonas aeruginosa |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 37 | - |
assay at | Pseudomonas aeruginosa |
| 37 | - |
assay at | Escherichia coli |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 7.5 | - |
assay at | Pseudomonas aeruginosa |
| 7.5 | - |
assay at | Escherichia coli |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| ATP | - |
Pseudomonas aeruginosa | |
| ATP | - |
Escherichia coli | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| evolution | enzyme MsbA encoded is a member of the ABC transporter family | Escherichia coli |
| evolution | Pseudomonas aeruginosa MsbA encoded is a member of the ABC transporter family, but this protein has distinctive features when compared with other MsbA proteins | Pseudomonas aeruginosa |
| malfunction | msbA is an essential gene in this organism and mutation in this gene is lethal to the bacterium. Disruption of the chromosomal msbA is achieved only when a functional copy of the gene is provided in trans. Gene msbA from Escherichia coli (msbAEc) cannot cross complement the msbA merodiploid cells of Pseuomonas aeruginosa | Pseudomonas aeruginosa |
| metabolism | the enzyme is involved in the biosynthetic pathways of lipid A | Pseudomonas aeruginosa |
| metabolism | the enzyme is involved in the biosynthetic pathways of lipid A | Escherichia coli |
| physiological function | lipopolysaccharide of Pseudomonas aeruginosa is a major constituent of the outer membrane, and it is composed of three distinct regions: lipid A, core oligosaccharide, and O antigen. Lipid A and core oligosaccharides (OS) are synthesized and assembled at the cytoplasmic side of the inner membrane and then translocated to the periplasmic side of the membrane where lipid A-core becomes the acceptor of the O antigens. The translocation through the inner membrane is catalyzed by ABC transporter MsbA. 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. Gene msbA is essential in the organism. Expression of Escherichia coli MsbA but not Pseudomonas aeruginosa MsbA confers resistance to erythromycin in Pseudomonas aeruginosa. Neither MsbA nor MsbAEc have an impact on the susceptibility of Pseudomonas aeruginosa to ciprofloxacin, tobramycin, or ofloxacin used in treatment of Pseudomonas aeruginosa infections | Pseudomonas aeruginosa |
| 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 | Escherichia coli |
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