Isolation and Characterization of Plastidic Glucose-6-Phosphate Dehydrogenase (G6PDH) from Castor (Ricinus communis L.)

Abstract

Abstract

Plant cells contain plastids, organelles dedicated to performing specific biochemical processes including photosynthesis, starch and oil biosynthesis. Fatty acid biosynthesis in oil seeds occurs in one type of plastid termed the leucoplast. Anabolic metabolism in leucoplasts includes the production of fatty acids and amino acids that depend on the availability of reductants such as NADPH. NADPH can be generated in plastid by glucose 6-phosphate dehydrogenase (G6PDH) which is the chief control enzyme and first step in the Oxidative Pentose Phosphate Pathway (OPPP). G6PDH catalyses the reaction of NADP+ and glucose 6-phosphate to NADPH and 6-phosphogluconate. At least two compartment-specific isoforms of G6PDH exist in plants, a cytosolic and a plastidic form. In this study, castor oil seed (COS) (Ricinus communis L.) was used as a model enzyme system for the ongoing study of oil biosynthesis in plants. This is the first ever report of the full-length clone of the plastidic isoform of G6PDH being isolated from a castor cDNA library using polyclonal potato plastidic G6PDH antiserum. The full-length cDNA was sequenced and compared to other G6PDH genes from higher plants, the castor sequence reveals conserved regions and conserved cysteine residues similar to other higher plant G6PDH. Over expression of the recombinant cleaved fusion protein in an E. coli expression system from the isolation of the cDNA clone shows it is enzymatically active, stable and unlike other plastid G6PDH’s dithiothreitol insensitive. In fact this G6PDH shows increased activation in the presence of dithiothreitol. Initial kinetic characteristics shows that it behaves in a similar fashion enzymatically when compared to other higher plant chloroplast G6PDH. The gene sequence and initial kinetic findings for castor G6PDH concur with other higher plant, non-photosynthetic, plastidic isoforms.