Isolation and characterization of a cDNA encoding cytosolic fructose-1,6-bisphosphatase from spinach.

Abstract

Photosynthetic cells require fructose-1,6-bisphosphatase (FBPase) (EC 3.1.3.11) activity in the chloroplast as well as in the cytoplasm [ 1, 11 ]. In both cases the enzyme catalyzes the hydrolysis of fructose-l,6-bisphosphate (F1,6P2) to fructose6-phosphate (F6P) and inorganic phosphate (Pi). The chloroplast FBPase is an essential enzyme in the photosynthetic pathway of CO2 fixation into sugars. The cytosolic FBPase is required for the synthesis of sucrose from triosephosphate, the major form of reduced carbon exported from the chloroplast during photosynthesis. Due to the importance of the cytosolic FBPase in sucrose biosynthesis in photosynthetic tissues and in gluconeogenesis in non-photosynthetic tissues, a great effort has been focused on understanding the regulatory mechanism of the enzyme Ireviewed in 20, 21]. Nevertheless, most biochemical information, like reactive sites, came from animal systems [7-10, 14, 24]. In this paper, we report the first DNA sequence of a plant cytosolic FBPase. We have isolated and characterized a cDNA encoding a cytosolic FBPase from spinach, compared the amino acid sequence derived from the nucleotide sequence with other published FBPases, and analyzed the possible functional amino acid residues on the basis of structural studies [7-10]. These data have been presented previously in abstract form [6]. A gene encoding yeast FBPase was obtained from Dr D.G. Fraenkel [19]. A 600 bp Barn HI-Xba I fragment from the center of the 2.5 kb yeast genomic clone was used as a hybridization probe to screen 300 000 clones in a spinach leaf cDNA library constructed in the expression vector 2gtl l [22]. The cDNA inserts were subcloned into the Eco RI site of the plasmid vector pGEM3 (Promega, Madison, WI). To sequence both strands as well as to obtain overlapped fragments, a series of unidirectional deletions were generated [5] from two recombinant plasmids, pFBPY and pFPBI, carrying the cDNA insert in the forward and inverted orientation, respectively. The inserts were sequenced by the dideoxy chain termination method of Sanger etal. [18], using the Sequenase system (USB, Cleveland, OH) as described by the manufacturer. Figure 1 shows the complete nucleotide sequence of 1585 bp of a cDNA clone encoding a cytosolic FBPase and the deduced amino acid