Expression, purification and characterization of the mature photosynthetic NADP-malic enzyme from maize expressed in E. coli

DETARSIO, ENRIQUE; CAMPOS BERMUDEZ, VALERIA A.; ANDREO, CARLOS S. AND DRINCOVICH, MARÍA F.

Córdoba. Argentina

Congreso; XXXVII Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB); 2001

NADP-malic enzyme (NADP-ME, EC 1.1.1.40), which catalyses the oxidative decarboxylation of L-malate to yield pyruvate, CO2, and NADPH, is a widely distributed enzyme involved in different metabolic pathways. The photosynthetic isoform of the enzyme is located in bundle sheath chloroplasts (BSC) in certain C4 plants such as maize, and provides the CO2 to be fixed by RuBisCO. This specific isoform of the enzyme have evolved from non-photosynthetic forms in order to present high specific activity, low km values for the substrates and high expression in BSC. In order to express this enzyme in a bacterial system to study its particular properties, the entire coding region of the mature protein was obtained by PCR, cloned in pET32 and expressed in E. coli BL21. After removing the His-Tag that allowed rapid purification of the recombinant NADP-ME, the protein was analyzed by SDS-PAGE and native IEF. The purified NADP-ME thus obtained showed single bands, which corresponded, in all cases, to the enzyme present in maize crude extracts. The oligomerization state of the recombinant protein was analyzed by HPLC indicating that, as the purified NADP-ME from maize leaves, the enzyme was tetrameric at pH 8.0, but dissociates to a dimer at pH 7.0. Kinetic studies of the recombinant photosynthetic NADP-ME indicated that, although the km values for all the substrates were unaltered, the enzyme had higher Vmax than previously reported for the purified NADP-ME from maize. In vitro mutagenesis of this recombinant NADP-ME will now permit the detection of the relevant protein domains related to its particular kinetic properties.