Rat dihydroorotate dehydrogenase: isolation of the recombinant enzyme from mitochondria of insect cells.
Mammalian dihydroorotate dehydrogenase (EC 1.3.99.11), the fourth enzyme of pyrimidine de novo synthesis is located in the mitochondrial inner membrane with functional connection to the respiratory chain. From the cDNA of rat liver dihydroorotate dehydrogenase cloned in our laboratory the first complete sequence of a mammalian enzyme was deduced. Two hydrophobic stretches centered around residues 20 and 357, respectively, and a short N-terminal mitochondrial targeting sequence of 10 amino acids was proposed. A recombinant baculovirus containing the rat liver cDNA for dihydroorotate dehydrogenase was constructed and used for virus infection and protein expression in Trichoplusia ni cells. The targeting of the recombinant protein to mitochondria of the insect cells was monitored by activity determination of dihydroorotate dehydrogenase in subcellular compartments in comparison to succinate dehydrogenase activity (EC 1.3.5.1), which is a specific marker enzyme of the inner mitochondrial membrane. The results of subcellular distribution were verified by Western blotting with anti-dihydroorotate dehydrogenase immunoglobulins. The activity of the recombinant enzyme in the mitochondria of infected insect cells was found to be about 570-fold above the level of dihydroorotate dehydrogenase in rat liver mitochondria. By cation exchange chromatography of the Triton X-114 solubilisate of mitochondria, dihydroorotate dehydrogenase was purified to give a specific 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 resulted in a specific activity of 0.7 U/mg at pH 8.0. The characteristic flavin absorption spectrum obtained with the recombinant enzyme gave strong evidence that the rodent enzyme is a flavoprotein. By enzyme kinetic studies K(m) values for dihydroorotate and ubiquinone were 6.4 and 9.9 microM with the recombinant enzyme, and were 5.0 and 19.7 microM, respectively, with the rat liver enzyme. After expression of only truncated forms of human dihydroorotate dehydrogenase, the present successful 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 microbial enzyme.
Knecht
W
W
Institute for Physiological Chemistry, School of Medicine, Philipps-University, Frankfurt, Germany.
Altekruse
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D
Rotgeri
A
A
Gonski
S
S
Löffler
M
M
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D018076:DNA, Complementary / Q000235:genetics; D000090722:Dihydroorotate Dehydrogenase; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000737:chemistry; D008928:Mitochondria / Q000737:chemistry*; D008969:Molecular Sequence Data; D009036:Moths / Q000166:cytology / Q000378:metabolism*; D017924:Nucleopolyhedroviruses / Q000235:genetics; D009963:Orotic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D010088:Oxidoreductases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D044925:Oxidoreductases Acting on CH-CH Group Donors; D051381:Rats / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D018411:Spodoptera / Q000166:cytology / Q000378:metabolism; D013379:Substrate Specificity; D014451:Ubiquinone / Q000378:metabolism
D018076:DNA, Complementary; D000090722:Dihydroorotate Dehydrogenase; D011993:Recombinant Fusion Proteins; D014451:Ubiquinone; C004768:4,5-dihydroorotic acid; D009963:Orotic Acid; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors