CONICET | Buscador de Institutos y Recursos Humanos

NADP-malic enzyme (NADP-ME 1.1.1.40) catalyses the oxidative decarboxylation of L-malate to yield pyruvate, CO2 and NADPH. In maize, two plastidic isoforms have been characterized, one of which is the specific C4-NADP-ME and the other a constitutively expressed NADP-ME not involved in photosynthesis. In this work, a cDNA for a novel NADP-ME isoform was isolated by RT-PCR using 1-2 day-old roots (AY864063). The recombinant NADP-ME was expressed in E.coli and the product obtained purified to homogeneity. The kinetic characterization of the recombinant NADP-ME (53 UI/mg, Km malate 1.4 mM, Km NADP 19 mM) significantly differs from the previous characterized maize NADP-MEs. From various metabolites analysed, succinate presented a strong activation effect, while fructose1,6-biP inhibited the enzyme. The analysis of the protein by native PAGE suggests that the enzyme exists in an hexameric u octameric form in vitro. RT-PCR studies using different maize tissues, indicate that the RNA from this isoform is only expressed in embryogenic tissues. In vitro import studies to chloroplasts using the complete cDNA isolated indicate that this NADP-ME protein is not sorted to this organelle, while prediction programmes detect a putative mitochondrial transit peptide in this sequence. The region encoding the putative transit peptide of this new NADP-ME was fused in frame to the GFP coding sequence. Transient transformation assays using N. tabacum BY-2 protoplasts and onion cells and stable Arabidopsis thaliana transformation, indicate that the transit peptide of this novel NADP-ME directs the product to mitochondria. In the present work a mitochondrial maize NADP-ME restricted to embryogenic maize tissue was characterized. This isoform resembles animal NADP-MEs (e.g. fumarate activation and subcellular localization) and its possible physiological function is discussed taking into account its pattern of expression and subcelullar localization