Structure and heterologous expression of a gene encoding fructose-6-phosphate, 2-kinase/fructose-2,6-bisphosphatase from Arabidopsis thaliana

A full-length cDNA clone encoding fructose-6-phosphate,2-kinase/fructose-2, 6-bisphosphatase from Arabidopsis thaliana (AtF2KP) was isolated. The encod ed protein is composed of two different regions: (i) a 400 amino acid COOH- terminal region, covering the catalytic region of the protein which is homo logous to enzymes from other eukaryotes. This region is highly conserved am ong plant species (88% identity to spinach F2KP). (ii) A 345 amino acid pla nt-specific NH2-terminal region, with 59% identity to spinach F2KP, which i s composed of homologous motifs and intermittent variable sequences. Wester n blots show that F2KP from several plant species migrates in sodium dodecy l sulphate-polyacrylamide gel electrophoresis as a similar sized (93 kDa) p rotein. AtF2KP was expressed in Escherichia coli as a full length and a tru ncated (without the NH2-terminal region) fusion protein. Both forms had kin ase as well as phosphatase activity, but presence of the NH2-terminal regio n influenced the ratio between the two activities. It is suggested that the NH2-terminal region represents a regulatory region, which defines specific properties of the plant enzymes. A genomic clone for the corresponding gen e, AtF2KP, was isolated. The clone (9519 bp) included 23 exons, 22 introns and the promoter sequence, Southern blot analysis showed only one copy of t he gene in the A. thaliana genome. (C) 2000 Elsevier Science B.V. All right s reserved.