Expression of rat liver ketohexokinase in yeast results in fructose intolerance

Abstract

Rat liver ketohexokinase (ATP:D-fructose 1-phosphotransferase; EC 2.7.1.3) 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 the total sequence, were determined. This sequence information was used to design oligonucleotide primers for a PCR using rat liver single-stranded cDNA as a template. The 224 bp PCR product was used as a probe to screen a rat liver cDNA library. A cDNA sequence of 1342 bp was obtained from three positive clones. This contained the entire coding region for ketohexokinase, and all seven peptides were identified in the predicted amino acid sequence. When ketohexokinase was expressed in Saccharomyces cerevisiae using the yeast expression vector pMA91, the cells became intolerant of the presence of fructose in their growth media. The growth of an exponential-phase culture was completely arrested within 90 min by the addition of fructose to a final concentration as low as 0.1% (w/v). This response is associated with an accumulation of fructose 1-phosphate. The cDNA for ketohexokinase encodes a protein composed of 299 amino acids with a combined molecular mass of 32,728 Da. This is in close agreement with the value for the isolated protein determined 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 prokaryotic phosphotransferases that have furanose substrates.

Molecular basis of essential fructosuria: molecular cloning and mutational analysis of human ketohexokinase (fructokinase)

“…The primary structure of rat liver KHK was, however, recendy described (5). This work demonstrated that KHK is structurally unlike other mammalian hexokinases, and does not show significant homology with other known mammalian protein families.…”

“…Val49De, on the other hand, does not produce fructosuria when present in the homozygous state, and is a common polymorphism in normal Europeans (data not shown). Finally, under the rather unlikely null hypothesis that essential fructosuria could result from a mutation unlinked to KHK, the concordant segregation between the mutations and the fructosuria in this family has a probability of only (U) 2 (K) 5 (excluding one proband) » 1 in 67.…”

“…This appears to result from alternative splicing to include either of two exons. The published rat cDNA sequence (5) corresponds in this region to the splice variant found in pHKHKl-2, referred to subsequently as form C. The other splice variant is referred to below as form A. Closer inspection ( Figure 1) shows that these two alternatively spliced exons can be aligned with each other to reveal conservation of several amino acid residues.…”

Structure and alternative splicing of the ketohexokinase gene

“…In its active form, it is a dimer [3] and requires K ϩ and ATP for activity. The physiological substrate for hepatic KHK is D-fructose, derived from dietary fructose, sucrose, or sorbitol, but it can also phosphorylate D-xylulose [1,3].Although most abundant in the liver, KHK is also found in kidney, small intestine and pancreas [2] and at much lower levels in heart, brain and muscle [3]. The distribution of highexpressing tissues is generally consistent with a major role in…”

“…This pathway metabolises fructose to the glycolytic intermediate glyceraldehyde-3-phosphate, in the process bypassing the important regulatory phosphofructokinase/fructose bisphosphatase step of glycolysis. KHK is most abundant in the liver, where it constitutes between 0.04% and 0.6 % of total protein [1,2]. The enzyme has been purified from human [3], bovine [1] and rat liver [2].…”