Purification and characterization of an NAD-dependent malate dehydrogenase from leaves of the crassulacean acid metabolism plant Aptenia cordifolia

An NAD-malate dehydrogenase (NAD-MDH, EC 1.1.1.37) was purified and characterized from leaves of Aptenia cordifolia L. f. (Schwant). This plant performs crassulacean acid metabolism (CAM), as indicated by: (a) elevated levels of phosphoenolpyruvate carboxylase (PEPC) and NAD(P) malic enzyme; (b) regulation of PEPC compatible with its function during the night; (c) characteristic day/night changes in titratable acidity; and (d) gas exchange profile consistent with that shown by CAM plants. These features remained unchanged by water availability or salt stress, suggesting constitutive CAM. The purified MDH showed a subunit molecular mass of 39.4 kDa, a native mass of 83 kDa (dimer) and a pI of 5.8. It cross-reacted with antibodies against cytosolic malate dehydrogenase (cMDH) from pineapple. Maximum activities for oxaloacetate (OAA) reduction or malate oxidation were observed at pH 7.0 and between pH 7.2 and 8.4, respectively. The enzyme was inhibited by excess OAA, in a pH-dependent manner. A discontinuity was observed in Arrhenius plots at 33 °C, with an activation energy twice as high below this temperature. Although immunologically related, some physical and kinetic dissimilarities between the A. cordifolia and pineapple enzymes suggest that diverse CAM metabolic subtypes may require different MDH isozymes to carry out OAA reduction.