Arabidopsis thaliana Hexokinase cDNA Isolated by Complementation of Yeast Cells

The initial step in Glc and Fru metabolism in plants is the hexokinase reaction, which catalyzes their irreversible phosphorylation. Recently, the hexokinase reaction has been hypothesized to be involved in the sensing and signal transmission of sugar levels in sugar repression of photosynthetic genes in plants (Jang and Sheen, 1994). Hexose-phosphorylating enzymes are classified according to their hexose substrate specificities. In plants, hexosephosphorylating enzymes showing preferential affinity to Glc are generally classified as hexokinase (EC 2.7.1.1) and have also been referred to as glucokinases (Turner et al., 1977; see Renz and Stitt, 1993, for a survey of plant hexosephosphorylating enzymes). In addition, specific fructokinases (EC 2.7.1.4) with low or no activity toward Glc have also been studied (Renz and Stitt, 1993). Recently, a potato tuber fructokinase cDNA was cloned and characterized, with little sequence homology to the known hexokinase genes (Smith et al., 1993). Whereas severa1 hexokinase genes have been cloned from yeast and animals (GenBank survey), no hexokinase gene has been cloned from plants. To isolate a plant hexokinase gene, we used the strategy of functional complementation of a yeast triple mutant (hxkl hxk2 glkl), which lacks Glcand Fru-phosphorylating ability and hence is unable to grow on either Glc or Fru (Fraenkel, 1982). Transformants obtained after selection on Fru were able to grow on either Glc or Fru (Table I). Hexose-phosphorylating activity of the cloned gene was measured in re-transformed yeast cells to classify the gene product with respect to substrate specificity. Activity was measured by enzyme-linked assay, following Glc-6-P dehydrogenase-linked NAD reduction spectrophotometrically (Huber and Akazawa, 1986, with minor modifications), either in the absence of phosphoglucose isomerase (for Glc-phosphorylating activity) or in its presence (for Fru-phosphorylating activity). The enzyme showed K , values of 44 /.LM for Glc and 17 mM for Fru. These values are similar to those of hexokinases from plant sources (Renz and Stitt, 1993) and we therefore characterize the enzyme as hexokinase (EC 2.7.1.1), with preferential affinity to Glc.