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Literature summary for 7.5.2.6 extracted from
- Association/dissociation of the nucleotide-binding domains of the ATP-binding cassette protein MsbA measured during continuous hydrolysis (2013), J. Biol. Chem., 288, 20785-20796 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| gene msbA, recombinant expression of His10-tagged wild-type and mutant enzymes in Escherichia coli strain BL21-Codon plus(DE3)-RIL | Salmonella enterica subsp. enterica serovar Typhimurium |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| C315S | site-directed mutagenesis of the active site Cys | Salmonella enterica subsp. enterica serovar Typhimurium |
| C88A/C315A/E506Q/T561C | site-directed mutagenesis, replacement of the catalytic carboxylate E506, the catalytically deficient mutant E506Q/T561C has a very low activity (1% of the T561C activity) | Salmonella enterica subsp. enterica serovar Typhimurium |
| C88A/C315A/T561C | site-directed mutagenesis, inactive cysteine mutant, the ATPase activity of T561C is highly dependent on temperature, addition of Mg2x01 decreased the LRET signal intensity of the ATP-bound T561C, and subsequent addition of Vi increased the signal back toward the ATP-bound state | Salmonella enterica subsp. enterica serovar Typhimurium |
| C88S | site-directed mutagenesis of the active site Cys | Salmonella enterica subsp. enterica serovar Typhimurium |
| additional information | the mutant enzymes are T561C and E506Q/T561C are labeled with thiol-reactive LRET probes, time course of nucleotide-binding domains (NBDs) association/dissociation monitored by LRET, kinetics of the conformational changes | Salmonella enterica subsp. enterica serovar Typhimurium |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | kinetics of the conformational change from ATP-bound T561C to the dynamic equilibrium during continuous ATP hydrolysis (in MgATP) by stopped flow measurements, kinetics of the conformational changes of mutant enzymes | Salmonella enterica subsp. enterica serovar Typhimurium |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Mg2+ | required, residue Glu506, located at the end of motif B, is the catalytic carboxylate involved in Mg2+ coordination | Salmonella enterica subsp. enterica serovar Typhimurium |  |
Molecular Weight [Da]
| Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
|---|---|---|---|
| 134000 | - |
gel filtration | 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 His10-tagged wild-type and mutant enzymes from Escherichia coli strain BL21-Codon plus(DE3)-RIL by solubilization from membranes with 2% n-dodecyl-beta-D-maltopyranoside and 0.4% cholate, cobalt affinity chromatography, and gel filtration in presence of detergents | Salmonella enterica subsp. enterica serovar Typhimurium |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| ATP + H2O + lipid A-core oligosaccharide[side 1] | - |
Salmonella enterica subsp. enterica serovar Typhimurium | ADP + phosphate + lipid A-core oligosaccharide[side 2] | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| homodimer | - |
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 | induces conformational changes of the enzyme during catalysis, kinetics of the conformational changes of mutant enzymes, overview | Salmonella enterica subsp. enterica serovar Typhimurium | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| additional information | structure of the AMPPNP-bound MsbA, structure-activity model, overview | Salmonella enterica subsp. enterica serovar Typhimurium |
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