Literature summary for 2.7.1.30 extracted from

Hokao, R.; Matsumura, H.; Katsumi, R.; Angkawidjaja, C.; Takano, K.; Kanaya, S.; Koga, Y.
Affinity shift of ATP upon glycerol binding to a glycerol kinase from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (2020), J. Biosci. Bioeng., 129, 657-663 .

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant overexpression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)	Thermococcus kodakarensis

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant wild-type enzyme in complex with glycerol and AMPPCP, and enzyme mutant K271E free or in complex with glycerol, sitting drop vapor diffusion method, 4-5 mg/ml protein in 5 mM Tris-HCl, pH 7.5, with or without 5 mM glycerol, and 5 mM AMPPCP, is mixed with 0.1 M HEPES, pH 8.0, containing 15% w/v PEG 1000 and 200 mM calcium acetate, 20°C, X-ray diffraction structure determination and analysis at 2.3-3.1 A resolution, molecular replacement using the structure of Tk-GK (PDB ID 2ZF5) as template, modelling	Thermococcus kodakarensis

Crystallization (Comment)

Organism

purified recombinant wild-type enzyme in complex with glycerol and AMPPCP, and enzyme mutant K271E free or in complex with glycerol, sitting drop vapor diffusion method, 4-5 mg/ml protein in 5 mM Tris-HCl, pH 7.5, with or without 5 mM glycerol, and 5 mM AMPPCP, is mixed with 0.1 M HEPES, pH 8.0, containing 15% w/v PEG 1000 and 200 mM calcium acetate, 20°C, X-ray diffraction structure determination and analysis at 2.3-3.1 A resolution, molecular replacement using the structure of Tk-GK (PDB ID 2ZF5) as template, modelling

Thermococcus kodakarensis

Protein Variants

Protein Variants	Comment	Organism
K271E	site-directed mutagenesis, the mutation disrupts the hexamer formation interface	Thermococcus kodakarensis

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
additional information	-	additional information	Michaelis-Menten kinetics, Tk-GK shows no negative cooperativity for ATP binding, the hexamer formation maintains a high ATP binding affinity	Thermococcus kodakarensis
0.0597	-	ATP	pH 7.5, 37°C, recombinant wild-type enzyme	Thermococcus kodakarensis
0.664	-	ATP	pH 7.5, 37°C, recombinant enzyme mutant K271E	Thermococcus kodakarensis

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Thermococcus kodakarensis

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
97400	101300	recombinant dimeric enzyme mutant K271E, gel filtration	Thermococcus kodakarensis
98500	-	recombinant dimeric wild-type enzyme, gel filtration	Thermococcus kodakarensis
316000	-	recombinant hexameric wild-type enzyme, gel filtration	Thermococcus kodakarensis

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
ATP + glycerol	Thermococcus kodakarensis	-	ADP + sn-glycerol 3-phosphate	-	?
ATP + glycerol	Thermococcus kodakarensis JCM 12380	-	ADP + sn-glycerol 3-phosphate	-	?
ATP + glycerol	Thermococcus kodakarensis ATCC BAA-918	-	ADP + sn-glycerol 3-phosphate	-	?

Organism

Organism	UniProt	Comment	Textmining
Thermococcus kodakarensis	O93623	-	-
Thermococcus kodakarensis ATCC BAA-918	O93623	-	-
Thermococcus kodakarensis JCM 12380	O93623	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by heat treatment at 90°C for 30 min, followed by ammonium sulfate fractionation, dialysis, and nickel affinity chromatography, then hydroxy apatite chromatography, gel ifltration, and again dialysis	Thermococcus kodakarensis

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
ATP + glycerol	-	Thermococcus kodakarensis	ADP + sn-glycerol 3-phosphate	-	?
ATP + glycerol	-	Thermococcus kodakarensis JCM 12380	ADP + sn-glycerol 3-phosphate	-	?
ATP + glycerol	-	Thermococcus kodakarensis ATCC BAA-918	ADP + sn-glycerol 3-phosphate	-	?
additional information	hexameric glycerol kinase (GK) from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (Tk-GK) is identified as the substrate-binding form of the enzyme	Thermococcus kodakarensis	?	-	-
additional information	hexameric glycerol kinase (GK) from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (Tk-GK) is identified as the substrate-binding form of the enzyme	Thermococcus kodakarensis JCM 12380	?	-	-
additional information	hexameric glycerol kinase (GK) from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (Tk-GK) is identified as the substrate-binding form of the enzyme	Thermococcus kodakarensis ATCC BAA-918	?	-	-

Subunits

Subunits	Comment	Organism
dimer	Tk-GK is a dimer in solution in absence of glycerol	Thermococcus kodakarensis
hexamer	Tk-GK has a hexameric form with a threefold axis in the crystal lattice	Thermococcus kodakarensis
More	in the absence of glycerol, Tk-GK is a dimer in solution. In the presence of its glycerol substrate, it becomes a hexamer consisting of three symmetrical dimers about the threefold axis. Through glycerol binding, all Tk-GK molecules in the hexamer are in closed form as a result of domain-motion. The closed form of Tk-GK has 10fold higher ATP affinity than the open form of Tk-GK. The hexamer structure stabilizes the closed conformation and enhances ATP binding affinity when the glycerol kinase is bound to glycerol	Thermococcus kodakarensis

Synonyms

Synonyms	Comment	Organism
Tk-GK	-	Thermococcus kodakarensis

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Thermococcus kodakarensis

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
108	-	the melting temperatures (Tm) of wild-type Tk-GK and enzyme mutant K271E are 107.8°C and 108.1°C, respectively, for the major transition	Thermococcus kodakarensis

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism	Structure
4.02	-	ATP	pH 7.5, 37°C, recombinant wild-type enzyme	Thermococcus kodakarensis
6.74	-	ATP	pH 7.5, 37°C, recombinant enzyme mutant K271E	Thermococcus kodakarensis

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Thermococcus kodakarensis

General Information

General Information	Comment	Organism
evolution	glycerol kinase is a member of the ATPase superfamily, which includes hexokinase, actin, and heat shock protein. These share a common betabetabetaalphabetaalphabetaalpha folding motif and markedly change conformation upon substrate binding because of interdomain motion	Thermococcus kodakarensis
metabolism	glycerol kinase (GK) is a key enzyme of glycerol metabolism. It participates in glycolysis and lipid membrane biosynthesis	Thermococcus kodakarensis
physiological function	glycerol kinase (GK) catalyzes the Mg/ATP-dependent phosphorylation of glycerol to produce glycerol-3-phosphate which is an important metabolic intermediate in glycolysis. In the absence of glycerol, Tk-GK was a dimer in solution. In the presence of its glycerol substrate, it becomes a hexamer consisting of three symmetrical dimers about the threefold axis. Through glycerol binding, all Tk-GK molecules in the hexamer are in closed form as a result of domain-motion. The closed form of Tk-GK has 10fold higher ATP affinity than the open form of enzyme Tk-GK. The hexamer structure stabilizes the closed conformation and enhances ATP binding affinity when the glycerol kinase is bound to glycerol	Thermococcus kodakarensis

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism	Structure
10.15	-	ATP	pH 7.5, 37°C, recombinant enzyme mutant K271E	Thermococcus kodakarensis
67.34	-	ATP	pH 7.5, 37°C, recombinant wild-type enzyme	Thermococcus kodakarensis