MICROSEQUENCING AND CDNA CLONING OF THE CALVIN CYCLE OPPP ENZYME RIBOSE-5-PHOSPHATE ISOMERASE (EC-5.3.1.6) FROM SPINACH-CHLOROPLASTS

Ribose-5-phosphate isomerase (RPI) catalyses the interconversion of ri bose-5-phosphate and ribulose-5-phosphate in the reductive and oxidati ve pentose phosphate pathways in plants. RPI from spinach chloroplasts was purified and microsequenced. Via PCR with degenerate primers desi gned against microsequenced peptides, a hybridisation probe was obtain ed and used to isolate several cDNA clones which encode RPI. The nucle ar-encoded 239 amino acid mature RPI subunit has a predicted size of 2 5.3 kDa and is translated as a cytosolic precursor possessing a 50 ami no acid transit peptide. The processing site of the transit peptide wa s identified from protein sequence data. Spinach leaves possess only o ne type of homodimeric RPI enzyme which is localized in chloroplasts a nd is encoded by a single nuclear gene. Molecular characterization of RPI supports the view that a single amphibolic RPI enzyme functions in the oxidative and reductive pentose phosphate pathways of spinach pla stids.