Expression, purification and characterization of a plastidic non-photosynthetic NADP-malic enzyme from maize in E. coli

MARIANA SAIGO; FEDERICO BOLOGNA; CARLOS ANDREO; MARÍA F. DRINCOVICH

Villa Carlos Paz, Argentina

Congreso; Sociedad Argentina de Investigación en Bioquímica y Biología Molecular XXXVIII Reunión Anual; 2002

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

NADP-malic enzyme (NADP-ME, EC 1.1.1.40) catalyzes the oxidative decarboxylation of L-malate to produce pyruvate, CO2 and NADPH in a wide variety of organisms. In plants, the isoform involved in C4 photosynthesis is the best characterized, since it has a central role providing CO2 to be fixed by RuBisCO. Comparative analysis of many NADP-ME cDNA sequences suggests that the C4 specific forms could have evolved from others non related to photosynthesis. In maize, a typical C4-type plant, two isoforms of NADP-ME have been purified and characterized, one is involved in the C4 cycle and the other plays a constitutive role in the provision of reducing power and pyruvate for anaplerotic reactions. After obtaining a cDNA fragment from maize roots, the sequence corresponding to the plastidic transit peptide was removed by PCR and the sequence coding for a mature NADP-ME was inserted in the vector pET-32c. Several conditions for protein expression, purification and cleavage of the poli-Histidine tag were tested. Once obtained the pure protein, different properties of the enzyme were studied. The molecular weight and isoelectric point, determined by electrophoretic techniques, and kinetic parameters as substrates binding affinities, specific activity and the optimum pH for activity were measured. All the results obtained indicate that this is a novel uncharacterized isoform, clearly different from the 72 kDa form previously purified from non-photosynthetic tissues of maize. We are now planning a set of experiments to establish the expression pattern of this enzyme to find out its function in vivo.