Cloned (Comment) | Organism |
---|---|
expressed in Escherichia coli BL21(DE3)pLysS cells | Escherichia coli |
Crystallization (Comment) | Organism |
---|---|
the crystal structure of glycerol kinase mutant G230D is determined to 2.0 A resolution using a microfluidics based crystallization platform | Escherichia coli |
using modified microfluidic scale-up diffraction device, crystals form after one week at ambient temperature unter crystallization conditions 0.3 M magnesium chloride, 0.1 M TrisHCl (pH 8.5), and 20% PEG 1500. Using vapor diffusion crystallization, crystals appear after one week at ambient temperature using the crystallization conditions 0.1 M magnesium chloride, 0.1 M TrisHCl (pH 8.5), and 10% PEG 1500. Glycerol kinase of the mutant G230D crystallied in space group P21 with two tetramers of 222 point symmetry in the asu. The average B factor for the overall structure of the G230D mutant is 21.2 A2. | Escherichia coli |
Protein Variants | Comment | Organism |
---|---|---|
G230D | structural analysis reveal that the decreased allosteric regulation in the G230D mutant is a result of the altered fructose 1,6-bisphosphate binding loop conformations in the mutant that interfere with the wild-type fructose 1,6-bisphosphate binding site. The altered fructose 1,6-bisphosphate binding loop conformation in the G230D mutant of glycerol kinase are supported through a series of intramolecular loop interactions. The appearence of Asp230 in the fructose 1,6-bisphosphate binding loops also repositions the wildtype fructose 1,6-bisphosphate binding residues away from the fructose 1,6-bisphosphate binging site. | Escherichia coli |
G230D | hyperactive mutant enzyme | Escherichia coli |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
113000 | - |
light scattering, dimer peak in the absence of fructose 1,6-bisphosphate, G230D mutant | Escherichia coli |
127000 | - |
light scattering, dimer peak in the presence of fructose 1,6-bisphosphate, G230D mutant | Escherichia coli |
177000 | - |
light scattering, dimer peak in the absence of fructose 1,6-bisphosphate, wild type enzyme | Escherichia coli |
227000 | - |
light scattering, tetramer peak (about 2% of the principal peak) in the absence of fructose 1,6-bisphosphate, G230D mutant | Escherichia coli |
391000 | - |
light scattering, tetramer peak (about 9% of the principal peak) in the absence of fructose 1,6-bisphosphate, wild type enzyme | Escherichia coli |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | P0A6F3 | - |
- |
Purification (Comment) | Organism |
---|---|
metal-chelate affinity chromatography using a Ni-NTA column, anion exchange chromatography using a Mono Q 5/50 GL column and gel filtration chromatography using s Superdex 200 10/30 GL column,all purification steps are performed in standard buffer (20 mM TrisHCl (pH 7.5), 10 mM glycerol, 1 mM beta-mercaptoethanol) at 4°C excluding the affinity chromatography purification | Escherichia coli |
mutant enzyme G230D | Escherichia coli |
Subunits | Comment | Organism |
---|---|---|
dimer | light scattering analysis confirmed G230D is a dimer and is resistant to tetramer formation in the presence of fructose 1,6-bisphosphate, whereas the wild type enzyme dimers are converted into putatively inactive tetramers in the presence of fructose 1,6-bisphosphate. | Escherichia coli |
Synonyms | Comment | Organism |
---|---|---|
GK | glycerol kinase | Escherichia coli |