ALFALFA MALATE-DEHYDROGENASE (MDH) - MOLECULAR-CLONING AND CHARACTERIZATION OF 5 DIFFERENT FORMS REVEALS A UNIQUE NODULE-ENHANCED MDH

Malate dehydrogenase (MDH) catalyzes the readily reversible reaction o f oxaloacetate reversible arrow malate using either NADH or NADPH as a reductant. In plants, the enzyme is important in providing malate for C-4 metabolism, pH balance, stomatal and pulvinal movement, respirati on, beta-oxidation of fatty acids, and legume root nodule functioning. Due to its diverse roles the enzyme occurs as numerous isozymes in va rious organelles. While antibodies have been produced and cDNAs charac terized for plant mitochondrial, glyoxysomal, and chloroplast forms of MDH, little is known of other forms. Here we report the cloning and c haracterization of cDNAs encoding five different farms of alfalfa MDH, including a plant cytosolic MDH (cMDH) and a unique novel nodule-enha nced MDH (neMDH). Phylogenetic analyses show that neMDH is related to mitochondrial and glyoxysomal MDHs, but diverge from these forms early in land plant evolution. Four of the five forms could effectively com plement an E. coli Mdh(-) mutant. RNA and protein blots show that neMD H is most highly expressed in effective root nodules. Immunoprecipitat ion experiments show that antibodies produced to cMDH and neMDH are im munologically distinct and that the neMDH form comprises the major for m of total MDH activity and protein in root nodules. Kinetic analysis showed that neMDH has a turnover rate and specificity constant that ca n account for the extraordinarily high synthesis of malate in nodules.