We characterized and determined the crystal structure of a putative glucokinase/hexokinase from Therm us thermophilus that belongs to the ROK (bacterial repressors, uncharacterized open reading frames, and sugar kinases) family. The protein possessed significant enzymatic activity against glucose and mannose, with V-max values of 260 and 68 mu mol.min(-1).mg(-1) protein, respectively. Therefore, we concluded that the enzyme is a hexokinase. However, the hexokinase showed little catalytic capacity for galactose and fructose. Circular dichroism measurements indicated that the enzyme was structurally stable at 90 degrees C. The crystal structure of the enzyme was determined at a resolution of 2.02 angstrom, with R-cryst and R-free values of 18.1% and 22.6%, respectively. The polypeptide structure was divided into large and small domains. The ROK consensus sequences 1 and 2 were included in the large domain. The cysteine-rich consensus sequence 2 folded into a zinc finger, and the bound zinc was confirmed by both electron density and X-ray absorption fine structure (XAFS) spectrum. The overall structure was a homotetramer that consisted of a dimer of dimers. The accessible surface area buried by the association of the dimers into the tetrameric structures was significantly higher in the T. thermophilus enzyme than in a homologous tetrameric ROK sugar kinase. (c) 2012, The Society for Biotechnology, Japan. All rights reserved.