Plant mitochondrial NAD+-dependent malic enzyme. cDNA cloning, deduced primary structure of the 59- and 62-kDa subunits, import, gene complexity and expression analysis.
The 59- and 62-kDa subunits of the mitochondrial NAD+-dependent malic enzyme (EC 1.1.1.39) were purified from Solanum tuberosum L. (potato). NH2-terminal and internal amino acid sequence information was used to identify cDNAs encoding the two subunits. Comparison of the nucleotide sequences revealed that the subunits have 60% identity at the DNA level and 65% identity at the deduced amino acid level, implying that they are derived from a common ancestral gene. The plant NAD+-dependent malic enzymes belong to a family of related enzymes, including cytosolic and chloroplastic NADP+-dependent malic enzymes (EC 1.1.1.40) and bacterial NAD+-dependent malic enzymes (EC 1.1.1.38). The cDNAs were transcribed and translated in vitro and the resultant polypeptides imported into isolated mitochondria and shown to be processed. Southern blot analysis of potato genomic DNA revealed a simple pattern of hybridization for both subunits, indicating a simple gene structure or small number of genes encoding the two subunits. Northern blot analysis of RNA from a range of potato tissues has shown that the steady state levels for the two subunits are equivalent, suggesting that they are coordinately expressed.
Winning
B M
BM
Department of Plant Sciences, University of Oxford, United Kingdom.
Bourguignon
J
J
Leaver
C J
CJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis / Q000302:isolation & purification; D018076:DNA, Complementary / Q000378:metabolism; D015870:Gene Expression; D017343:Genes, Plant; D006801:Humans; D046911:Macromolecular Substances; D008291:Malate Dehydrogenase / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D011198:Solanum tuberosum / Q000201:enzymology* / Q000235:genetics
D018076:DNA, Complementary; D046911:Macromolecular Substances; D009243:NAD; D004247:DNA; D008291:Malate Dehydrogenase; C030574:malate dehydrogenase (decarboxylating)