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Human NAD(+)-dependent mitochondrial malic enzyme. cDNA cloning, primary structure, and expression in Escherichia coli.

1991; Elsevier BV; Volume: 266; Issue: 5 Linguagem: Inglês

10.1016/s0021-9258(18)49948-6

1083-351X

G Loeber, Anthony A. Infante, Ingrid Maurer-Fogy, E. Krystek, Mark B. Dworkin,

RNA modifications and cancer

Mitochondrial NAD+-dependent malic enzyme (EC 1.1.1.40)is expressed in rapidly proliferating cells and tumor cells, where it is probably linked to the conversion of amino acid carbon to pyruvate.In this paper, we report the cDNA cloning, amino acid sequence, and expression in Escherichia coli of functional human NAD+-dependent mitochondrial malic enzyme.The cDNA is 1,923 base pairs long and contains an open reading frame coding for a 584-amino acid protein.The molecular mass is 65.4 kDa for the unprocessed precursor protein.Comparison of the amino acid sequence of the human protein with the published NADP+-dependent mammalian cytosolic or plant chloroplast malic enzymes reveals highly conserved regions interrupted with long stretches of amino acids without significant homology.Expression of the processed protein in E. coli yielded an enzyme with the same kinetic and allosteric properties as malic enzyme purified from human cells.Malic enzyme (ME)' catalyzes the oxidative decarboxylation of malate to pyruvate, malate + NAD(P)+ --* pyruvate + CO, + NAD(P)H+, and can be found both in eukaryotic and prokaryotic cells.Three different isoforms of ME have been described in mammalian tissues: a strictly cytosolic NADP+dependent enzyme, an NADP+-dependent mitochondrial isoform, and a mitochondrial isoenzyme which can use both NAD' and NADP+ but is more effective with NAD' (Fraenkel, 1975).The mammalian isoforms are about 62-64 kDa in size (Moreadith and Lehninger, 1984b; Magnusson et al., 1986; Bagchi et al., 1987).A native size of 240,000 Da suggests a tetrameric structure for the active enzyme (Fraenkel, 1975; Moreadith and Lehninger, 1984b).The highest levels of the cytosolic ME activity are found in the liver and in adipose tissue, where this isoform is linked to the generation of cytosolic NADPH for de novo fatty acid synthesis.This isoenzyme is under dietary control and can be induced by a carbohydrate-rich diet or thyroid hormones (Fraenkel, 1975;Dozin et al., 1985).NADP+-dependent mitochondrial ME activity is found in many tissues, including brain, heart, and skeletal muscle and adrenals (Lin and Davis,