Molecular and biochemical characterizations of a plastidic glycerol-3-phosphate dehydrogenase from Arabidopsis

Glycerol-3-phosphate dehydrogenase (NAD⁺-G-3-P oxidoreductase, EC 1.1.1.8) (GPDH) catalyses the reduction of dihydroxyacetone phosphate (DHAP) to form glycerol-3-phosphate (G-3-P), which is essential for phospholipid synthesis through both the prokaryotic and the eukaryotic glycerolipid pathway. A cDNA (AtGPDHp) encoding for the plastidic GPDH from Arabidopsis thaliana was cloned. The predicted AtGPDHp has in its amino-terminal region a chloroplast targeting signal peptide-like sequence. Transgenic tobacco plants with a chimeric green fluorescent protein (GFP) construct showed that the N-terminal peptide of AtGPDHp targeted GFP into chloroplasts. The functional identity of AtGPDHp was experimentally confirmed by expression of AtGPDHp in an Escherichia coli (gpsA) mutant auxotrophic of G-3-P. The K_m value and V_max of a purified recombinant AtGPDHp for DHAP were 12.8 μM and 3.2 μkat·min^-1·mg^-1 protein, respectively. Northern blot experiments indicated that AtGPDHp gene is expressed in all tissues examined, but its transcript is particularly abundant in developing siliques and flowers. This study represents the first molecular cloning and expression analysis of a plastidic GPDH in plants.