RAT DIHYDROOROTATE DEHYDROGENASE - ISOLATION OF THE RECOMBINANT ENZYME FROM MITOCHONDRIA OF INSECT CELLS

Mammalian dihydroorotate dehydrogenase (EC 1.3.99.11), the fourth enzy me of pyrimidine de novo synthesis is located in the mitochondrial inn er membrane with functional connection to the respiratory chain. From the cDNA of rat liver dihydroorotate dehydrogenase cloned in our labor atory the first complete sequence of a mammalian enzyme was deduced. T wo hydrophobic stretches centered around residues 20 and 357, respecti vely, and a short N-terminal mitochondrial targeting sequence of 10 am ino acids was proposed. A recombinant baculovirus containing the rat l iver cDNA for dihydroorotate dehydrogenase was constructed and used fo r virus infection and protein expression in Trichoplusia ni cells. The targeting of the recombinant protein to mitochondria of the insect ce lls was monitored by activity determination of dihydroorotate dehydrog enase in subcellular compartments in comparison to succinate dehydroge nase activity (EC 1.3.5.1), which is a specific marker enzyme of the i nner mitochondrial membrane. The results of subcellular distribution w ere verified by Western blotting with anti-dihydroorotate dehydrogenas e immunoglobulins. The activity of the recombinant enzyme in the mitoc hondria of infected insect cells was found to be about 570-fold above the level of dihydroorotate dehydrogenase in rat liver mitochondria. B y cation exchange chromatography of the Triton X-114 solubilisate of m itochondria, dihydroorotate dehydrogenase was purified to give a speci fic activity of 15 U/mg at pH 8.0. This was a marked progress over the six-step purification procedure of the enzyme from rat liver which re sulted in a specific activity of 0.7 U/mg at pH 8.0. The characteristi c flavin absorption spectrum obtained with the recombinant enzyme gave strong evidence that the rodent enzyme is a flavoprotein. By enzyme k inetic studies K-m values for dihydroorotate and ubiquinone were 6.4 a nd 9.9 mu M with the recombinant enzyme, and were 5.0 and 19.7 mu M re spectively, with the rat liver enzyme. After expression of only trunca ted forms of human dihydroorotate dehydrogenase, the present successfu l generation of the complete rodent enzyme using insect cells and the efficient procedure will promote structure and function studies of the eukaryotic dihydroorotate dehydrogenases in comparison to the microbi al enzyme. (C) 1997 Academic Press.