Molecular and biochemical characterization of a fructose-6-phosphate-forming and ATP-dependent fructokinase of the hyperthermophilic archaeon Thermococcus litoralis.
Close to an operon encoding an ABC transporter for maltose and trehalose, Thermococcus litoralis contains a gene whose encoded sequence showed similarity to sugar kinases. We cloned this gene, now called frk, and expressed it as a C-terminal His-tag version in Escherichia coli. We purified the recombinant protein, identified it as an ATP-dependent and fructose-6-phosphate-forming fructokinase (Frk) and determined its biochemical properties. At its optimal temperature of 80 degrees C, the apparent Km and Vmax values of Frk were 2.3 mM and 730 U/mg protein for fructose at saturating ATP concentration, and 0.81 mM and 920 U/mg protein for ATP at saturating fructose concentration. The enzyme did not lose activity at 80 degrees C for 4 h. Under denaturating conditions in SDS-PAGE, it exhibited a molecular mass of 35 kDa. Gel-filtration chromatography revealed a molecular mass of 58 kDa, indicating a dimer under nondenaturating, in vitro conditions.
Qu
Qiuhao
Q
Department of Biology, University of Konstanz, 78457, Konstanz, Germany.
Lee
Sung-Jae
SJ
Boos
Winfried
W
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D017124:Conserved Sequence; D017931:DNA Primers; D004795:Enzyme Stability; D005631:Fructokinases / Q000737:chemistry / Q000378:metabolism*; D019298:Glucose-6-Phosphate / Q000096:biosynthesis*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D019712:Thermococcus / Q000201:enzymology*; D013816:Thermodynamics
D017931:DNA Primers; D019298:Glucose-6-Phosphate; D000255:Adenosine Triphosphate; D005631:Fructokinases; C046173:fructokinase