Literature summary for 2.7.1.30 extracted from

Anderson, M.J.; DeLabarre, B.; Raghunathan, A.; Palsson, B.O.; Brunger, A.T.; Quake, S.R.
Crystal structure of a hyperactive Escherichia coli glycerol kinase mutant Gly230 --> Asp obtained using microfluidic crystallization devices (2007), Biochemistry, 46, 5722-5731.

Cloned(Commentary)

Cloned (Comment)	Organism
expressed in Escherichia coli BL21(DE3)pLysS cells	Escherichia coli

Crystallization (Commentary)

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

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

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 [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

Organism	UniProt	Comment	Textmining
Escherichia coli	P0A6F3	-	-

Purification (Commentary)

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

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

Synonyms	Comment	Organism
GK	glycerol kinase	Escherichia coli