EXPRESSION OF RAT-LIVER KETOHEXOKINASE IN YEAST RESULTS IN FRUCTOSE INTOLERANCE

Rat liver ketohexokinase (ATP:D-fructose 1-phosphotransferase; EC2.7.1 .3) was purified to homogeneity and the molecular mass of the protein was found by mass spectrometry to be 32 800 Da. The enzyme was cleaved and the amino acid sequences of seven peptides, comprising 24 % of th e total sequence, were determined. This sequence information was used to design oligonucleotide primers for a PCR using rat liver single-str anded cDNA as a template. The 224 bp PCR product was used as a probe t o screen a rat liver cDNA library. A cDNA sequence of 1342 bp was obta ined from three positive clones. This contained the entire coding regi on for ketohexokinase, and all seven peptides were identified in the p redicted amino acid sequence. When ketohexokinase was expressed in Sac charomyces cerevisiae using the yeast expression vector pMA91, the cel ls became intolerant of the presence of fructose in their growth media . The growth of an exponential-phase culture was completely arrested w ithin 90 min by the addition of fructose to a final concentration as l ow as 0.1 % (w/v). This response is associated with an accumulation of fructose I-phosphate. The cDNA for ketohexokinase encodes a protein c omposed of 299 amino acids with a combined molecular mass of 32728 Da. This is in close agreement with the value for the isolated protein de termined by mass spectrometry. The primary structure does not show any significant homology with those of other eukaryotic hexokinases, but it contains a highly conserved region that is present in three prokary otic phosphotransferases that have furanose substrates.