Cloned (Comment) | Organism |
---|---|
wild-type and E267R protein are overexpressed in Escherichia coli | Haloarcula marismortui |
Crystallization (Comment) | Organism |
---|---|
the structure of the mutant E267R apoenzyme is determined to 2.6 A resolution and the structure of the wild-type apoenzyme is determined to 2.9 A resolution | Haloarcula marismortui |
Protein Variants | Comment | Organism |
---|---|---|
E267R | the numbering is not equivalent to the numbering of UniProt. The E267R mutation points into a central ordered water cavity, disrupting protein-solvent interactions. The mutant enzyme requires higher concentrations of the solvent salt for stability similar to that of the wild type | Haloarcula marismortui |
R207S/R292S | the numbering is not equivalent to the numbering of UniProt. The active tetrameric mutant enzyme R207S/R292S dissociates under certain conditions to active dimers and under other conditions to inactive dimers. These dimers further dissociate into folded monomers which eventually unfold. The mutant enzyme requires higher salt concentrations than the wild type for stability. Thermal inactivation starts at 35°C, whereas the wild type is stable up to 60°C. At 4 M NaCl (pH 8) the kinetics of unfolding of the mutant is measured by following the fluorescence emission during incubation at various temperatures. The process is biphasic between 35 and 48 °C, while the thermal deactivation kinetics of the wild type protein is first-order | Haloarcula marismortui |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Haloarcula marismortui | Q07841 | - |
- |
Haloarcula marismortui DSM 3752 | Q07841 | - |
- |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
oxaloacetate + NADH + H+ | - |
Haloarcula marismortui | (S)-malate + NAD+ | - |
? | |
oxaloacetate + NADH + H+ | - |
Haloarcula marismortui DSM 3752 | (S)-malate + NAD+ | - |
? |
Subunits | Comment | Organism |
---|---|---|
dimer | in the wild type the dimeric enzyme form is very unstable under low-salt conditions. The R207S/R292S mutation stabilizes the dimer sufficiently so that it can be observed and studied. The R207S/R292S mutant enzyme dissociates into dimers at 2 M NaCl, or at very low protein concentrations in 4 M NaCl above pH 7. This dimer is as active as the wild type tetramer at pH 8 but loses its activity at pH 7, without large changes in structure or association state | Haloarcula marismortui |
monomer | the monomer is in an inactive molten globule-like state, which can be reactivated through a structural change induced by NADH binding that allows it to associate into active dimers | Haloarcula marismortui |
tetramer | stabilized by ordered water molecule networks and intersubunit complex salt bridges locked in by bound solvent chloride and sodium ions | Haloarcula marismortui |
tetramer | the active tetrameric mutant enzyme R207S/R292S dissociates under certain conditions to active dimers and under other conditions to inactive dimers. These dimers further dissociate into folded monomers which eventually unfold. In 4 M NaCl (pH 8) and for the protein concentration range above 5 mg/ml, the R207S/R292S mutant enzyme is predominantly a tetramer | Haloarcula marismortui |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Haloarcula marismortui |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
45 | - |
30 min, 50% loss of activity, mutant enzyme R207S/R292S | Haloarcula marismortui |
75 | - |
30 min, 50% loss of activity, wild-type enzyme | Haloarcula marismortui |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Haloarcula marismortui |