Cloning, characterization and expression of a bifunctional fructose-6-phosphate, 2-kinase/fructose-2,6-bisphosphatase...

We have isolated cDNA clones encoding the regulatory enzyme fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase from a potato (Solanum tuberosum) leaf cDNA library. All clones represented transcripts of the same gene (F2KP1). Functionality of the encoded protein was verified by expression of the active enzyme in Escherichia coli. The expressed enzyme had both kinase activity which forms fructose-2,6-bisphosphate from fructose-6-phosphate and ATP, and phosphatase activity which degrade fructose-2,6-bisphosphate. The recombinant potato enzyme was radiolabelled by [2-32P]fructose-2,6-bisphosphate verifying conservation of the phosphatase catalytic mechanism which involves a phospho-protein intermediate. The deduced amino acid sequence corresponding to the catalytic core for F2KPI is homologous to the fructose-6-phosphate, 2-kinase/fructose-2,6-bisphosphatase isolated from animals and yeast, with conservation of amino acids involved in substrate binding and catalytic mechanisms. The sequence for F2KP1 also includes a 102 amino acids long NH2-terminal with no homology to any previously identified enzymes. This NH2 terminal may be even longer since an upstream stop codon has not yet been identified. Northern blot analysis of potato showed that the F2KP1 transcript is present in several tissues including source leaves, sink leaves and flowers, whereas the transcripts were not detectable in developing tubers. Southern blot analysis of Solanum phureja suggest there to be only one copy of the gene.