Cloning, expression, and characterization of the hxk-1 Gene from the white truffle Tuber borchii vittad.: A first step toward understanding sugar metabolism.
Recent biochemical investigations of Tuber borchii Vittad. mycelium have demonstrated the presence of three distinct forms of hexokinase (HK(M1), HK(M2), and HKM3). In the investigation described here, a gene coding for hexokinase (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 recombinant protein was kinetically characterized. The K(cat) value for fructose is in agreement with the data reported for the hexokinase of Yarrowia lipolytica, the Km 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 represents the first characterization of the key enzyme of the glycolytic pathway and the related gene in a Tuber species.
Agostini
D
D
Istituto di Chimica Biologica Giorgio Fornaini, Università degli Studi di Urbino, Via A. Saffi 2, Urbino, (PU), 61029, Italy.
Polidori
E
E
Palma
F
F
Ceccaroli
P
P
Saltarelli
R
R
Tonelli
D
D
Stocchi
V
V
D000595:Amino Acid Sequence; D000818:Animals; D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D001483:Base Sequence; D050260:Carbohydrate Metabolism; D003001:Cloning, Molecular; D004271:DNA, Fungal; D004926:Escherichia coli; D005800:Genes, Fungal; D006593:Hexokinase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment
D004271:DNA, Fungal; D007527:Isoenzymes; D011994:Recombinant Proteins; D006593:Hexokinase