Glycerol kinase (GK; EC 184.108.40.206; ATP:glycerol 3-phosphotransferase) catalyzes the Mg/ATP-dependent phosphorylation of glycerol to produce glycerol-3-phosphate, an important metabolic intermediate in glycolysis. The enzyme is widely present in all three kingdoms of living organisms: bacteria, archaea and eukaryotes. Escherichia coli
GK has been the most extensively studied GK with respect to its function and structure. Since E. coli
GK is subject to several types of regulation involving protein–ligand and protein–protein interactions, the enzyme plays a key role in the regulation of glycerol metabolism in E. coli
(Zwaig & Lin, 1970
). Two allosteric inhibitors, fructose 1,6-bisphosphate (FBP) and IIAGlc
, a component of the glucose-specific phosphoenolpyruvate phosphotransferase system (PTS), have been studied with respect to their mechanisms of regulation of GK activity (de Riel & Paulus, 1978a
). The crystal structures of E. coli
GK in complex with FBP (Ormö et al.
) and with IIAGlc
(Hurley et al.
; Feese et al.
) have been determined. E. coli
GK exists in a dimer–tetramer equilibrium with an apparent dissociation constant of about 6.1 × 10−8
(Peng & Pettigrew, 2003
). FBP selectively binds to the tetramer and inhibits its activity (Liu et al.
). The crystal structure of the complex between E. coli
GK and the substrate analogue indicates that the FBP-bound tetrameric form of GK has an energetic barrier to the conformational change required to open the active site, which is necessary for efficient catalysis (Bystrom et al.
). In contrast, another allosteric inhibitor, IIAGlc
, binds to both the dimer and the tetramer and noncompetitively inhibits the GK activity for both substrates, glycerol and ATP (Novotony et al.
). The IIAGlc
-binding site, which is different from the FBP-binding site, is located far from the active site (Hurley et al.
GK is a member of the ATPase superfamily, which includes hexokinases, actins and heat-shock proteins (Hurley, 1996
). These proteins share a common βββαβαβα fold. The members of this superfamily are known to change conformation greatly upon substrate binding owing to interdomain motion. The crystal structure of the E. coli
GK–substrate complex indicates that GK forms an asymmetric dimer with one open-conformation subunit and one closed-conformation subunit. The conformational difference between these two subunits is brought about by interdomain motion, which is supposed to be associated with a catalytic step or allosteric inhibition by FBP or IIAGlc
(Bystrom et al.
The GK from the hyperthermophilic archaeon Thermococcus kodakaraensis
-GK) has been overproduced in E. coli
, purified and biochemically characterized (Koga et al.
). It shows high amino-acid sequence identity (57%) to E. coli
GK. Nevertheless, Tk
-GK is much more highly stable than E. coli
GK. For example, Tk
-GK is highly resistant to heat inactivation and fully retains enzymatic activity upon incubation at 353 K for 60 min, while E. coli
GK loses 90% of its enzymatic activity upon incubation at 323 K for 60 min. Comparison of a model of the Tk
-GK structure and the crystal structure of E. coli
GK, as well as mutational studies of these enzymes (Koga et al.
), have allowed us to propose that the increased number of ion pairs at the surface of the protein molecule and the stabilization of the subunit interface contribute to the thermal stability of Tk
-GK. However, the structural basis of the high stability of Tk
-GK remains to be analyzed. In addition, unlike E. coli
-GK exists only in a dimeric form, is not sensitive to FBP inhibition and does not show negative cooperativity for ATP binding (Koga et al.
). To understand the stabilization mechanism, subunit interaction and unique enzymatic properties of Tk
-GK at the atomic level, it is necessary to determine its crystal structure.
Here, we report the crystallization and preliminary X-ray crystallographic studies of Tk-GK and its complex with glycerol and ATP.