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

摘要

A full-length cDNA clone encoding fructose-6-phosphate, 2-kinase/fructose-2,6-bisphosphatase from Arabidopsis thaliana (AtF2KP) was isolated. The encoded protein is composed of two different regions: (i) a 400 amino acid COOH-terminal region, covering the catalytic region of the protein which is homologous to enzymes from other eukaryotes. This region is highly conserved among plant species (88% identity to spinach F2KP). (ii) A 345 amino acid plant-specific NH_2-terminal region, with 59% identity to spinach F2KP, which is composed of homologous motifs and intermittent variable sequences. Western blots show that F2KP from several plant species migrates in sodium dodecyl sulphate-polyacrylamide gel electrophoresis as a similar sized (93 kDa) protein. AtF2KP was expressed in Escherichia coli as a full length and a truncated (without the NH_2-terminal region) fusion protein. Both forms had kinase as well as phosphatase activity, but presence of the NH_2-terminal region influenced the ratio between the two activities. It is suggested that the NH_2-terminal region represents a regulatory region, which defines specific properties of the plant enzymes. A genomic clone for the corresponding gene, AtF2KP, was isolated. The clone (9519 bp) included 23 exons, 22 introns and the promoter sequence. Southern blot analysis showed only one copy of the gene in the A. thaliana genome.