Uncoupling de novo pyrimidine biosynthesis from mitochondrial electron transport by ectopic expression of cytosolic DHODH

Dihydroorotate dehydrogenase (DHODH) is an enzyme involved in the biosynthesis of pyrimidine nucleotides. In most eukaryotes, this enzyme is bound to the inner mitochondrial membrane, where it couples the synthesis of orotate with the reduction of ubiquinone. As ubiquinone must be regenerated by respiratory complex III, pyrimidine biosynthesis and cellular respiration are tightly coupled. Consequently, inhibition of respiration leads to cessation of DNA synthesis and impairs cell proliferation. We show that expression of Saccharomyces cerevisiae URA1 gene ( ScURA ) in mammalian cells uncouples biosynthesis of pyrimidines from mitochondrial electron transport. ScURA forms a homodimer in the cytosol that uses fumarate instead of ubiquinone as the electron acceptor, enabling oxygen-independent pyrimidine biosynthesis. Cells expressing ScURA are resistant to drugs that inhibit complex III and the mitochondrial ribosome. ScURA enables the growth of mtDNA-lacking ρ ⁰ cells in uridine-deficient medium and ameliorates the phenotype of cellular models of mitochondrial diseases. This genetic tool uncovers the contribution of pyrimidine biosynthesis to the phenotypes arising from electron transport chain defects.