Alfalfa malate dehydrogenase (MDH): molecular cloning and characterization of five different forms reveals a unique nodule‐enhanced MDH

• Published in: The Plant journal : for cell and molecular biology 1998-07, Vol.15 (2), p.173-184
• Main Authors: Miller, Susan S., Driscoll, Brian T., Gregerson, Robert G., Gantt, J. Stephen, Vance, Carroll P.
• Format: Article
• Language: English
• Subjects: Agronomy. Soil science and plant productions; Amino Acid Sequence; Biological and medical sciences; Cloning, Molecular; Cotyledon - enzymology; Economic plant physiology; Escherichia coli; Evolution, Molecular; Fundamental and applied biological sciences. Psychology; Genetic Complementation Test; Isoenzymes - chemistry; Isoenzymes - genetics; Isoenzymes - metabolism; Kinetics; Malate Dehydrogenase - chemistry; Malate Dehydrogenase - genetics; Malate Dehydrogenase - metabolism; Medicago sativa - enzymology; Medicago sativa - genetics; Meristem - enzymology; Molecular Sequence Data; Parasitism and symbiosis; Phylogeny; Plant Leaves - enzymology; Plant physiology and development; Plant Roots - enzymology; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Sequence Alignment; Sequence Homology, Amino Acid; Symbiosis; Symbiosis (nodules, symbiotic nitrogen fixation, mycorrhiza...)
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1046/j.1365-313X.1998.00192.x

Summary Malate dehydrogenase (MDH) catalyzes the readily reversible reaction of oxaloacetate ; 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, respiration, β‐oxidation of fatty acids, and legume root nodule functioning. Due to its diverse roles the enzyme occurs as numerous isozymes in various organelles. While antibodies have been produced and cDNAs characterized for plant mitochondrial, glyoxysomal, and chloroplast forms of MDH, little is known of other forms. Here we report the cloning and characterization of cDNAs encoding five different forms of alfalfa MDH, including a plant cytosolic MDH (cMDH) and a unique novel nodule‐enhanced 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 complement an E. coliMdh– mutant. RNA and protein blots show that neMDH is most highly expressed in effective root nodules. Immunoprecipitation experiments show that antibodies produced to cMDH and neMDH are immunologically distinct and that the neMDH form comprises the major form of total MDH activity and protein in root nodules. Kinetic analysis showed that neMDH has a turnover rate and specificity constant that can account for the extraordinarily high synthesis of malate in nodules.