Cloning, expression, and characterization of the hxk-1 gene from the whitetruffle Tuber borchii Vittad.: A first step toward understanding sugar metabolism

Recent biochemical investigations of Tuber borchii Vittad. mycelium have de monstrated the presence of three distinct forms of hexokinase (HKM1, HKM2, and HKM3). In the investigation described here, a gene coding for hexokinas e (hxk-1) from T. borchii was isolated and characterized. The hxk-1 gene is characterized by an ORF of 1494 nucleotides and codes for a polypeptide of 497 aa. The gene was overexpressed in Escherichia coli, and the recombinan t protein was kinetically characterized. The K-cat value for fructose is in agreement with the data reported for the hexokinase of Yarrowia lipolytica , the K-m for ATP is not dependent on the sugar used, and the enzyme is not inhibited by trehalose 6-phosphate or glucose 6-phosphate. The biochemical characteristics confirm that this enzyme is a hexokinase, as suggested by the Pileup results, and it corresponds to the HKM1 isoform. This work repre sents the first characterization of the key enzyme of the glycolytic pathwa y and the related gene in a Tuber species. (C) 2001 Academic Press.