Mammalian dihydroorotate dehydrogenase, the fourth enzyme of pyrimidine de novo synthesis is an integral protein of the inner mitochondrial membrane that faces the intermembrane space and is functionally connected to the respiratory chain via ubiquinone. Here, we describe the first cloning and analyzing of the complete cDNA of mouse dihydroorotate dehydrogenase. Based on our recent functional expression of the full-length rat and human dihydroorotate dehydrogenase, here we expressed N-terminal-truncated C-terminal-histidine-tagged constructs of the mouse, rat and human enzymes in Escherichia coli. These proteins were devoid of the N-terminal bipartite sequence consisting of the mitochondrial targeting sequence and adjacent hydrophobic domain necessary for import and proper location and fixation of the enzyme in the inner mitochondrial membrane. By employing metal-chelate affinity chromatography under native conditions, the enzymes were purified without detergents to a specific activity of more than 100 micromol x min(-1) x mg(-1) at pH optimum of 8.0--8.1. Flavin analyses by UV-visible spectrometry of the native enzymes gave fairly stoichiometric ratios of 0.6--1.2 mol flavin per mol protein. The kinetic constants of the truncated rat enzyme (K(m) = 11 microM dihydroorotate; K(m) = 7 microM ubiquinone) and human enzyme (K(m) = 10 microM dihydroorotate; K(m) = 14 microM ubiquinone) were very close to those recently reported for the full-size enzymes. The constants for the mouse enzyme, K(m) = 26 microM dihydroorotate and K(m) = 62 microM ubiquinone, were slightly elevated in comparison to those of the other species. The three truncated enzymes were tested for their efficacy with five inhibitors of topical clinical relevance against autoimmune disorders and tumors. Whereas the presence of the N-terminus of dihydroorotate dehydrogenase was essentially irrelevant for the efficacy of the malononitrilamides A77-1726, MNA715 and MNA279 with the rat and human enzyme, the N-termini were found to be important for the efficacy of the dianisidine derivative redoxal. Moreover, the complete N-terminal part of the human enzyme seemed to be of crucial importance for the 'slow-binding' features of the cinchoninic acid derivative brequinar, which was suggested to be one of the reasons for the narrow therapeutic window reported from clinical trials on its anti-proliferative and immunosuppressive action.

The mitochondrial membrane bound dihydroorotate dehydrogenase (DHODH; EC 1.3.99.11) catalyzes the fourth step of pyrimidine biosynthesis. By the present correction of a known cDNA sequence for Arabidopsis thaliana DHODH we revealed the importance of the very C-terminal part for its catalytic activity and the reason why^in contrast to mammalian and insect species^the recombinant plant £avoenzyme was unaccessible to date for in vitro characterization. Structure^activity relationship studies explained that potent inhibitors of animal DHODH do not signi¢cantly a¡ect the plant enzyme. These difference could be exploited for a novel approach to herb or pest growth control by limitation of pyrimidine nucleotide pools.

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Mammalian dihydroorotate dehydrogenase, the fourth enzyme of pyrimidine de novo synthesis is an integral protein of the inner mitochondrial membrane that faces the intermembrane space and is functionally connected to the respiratory chain via ubiquinone. Here, we describe the first cloning and analyzing of the complete cDNA of mouse dihydroorotate dehydrogenase. Based on our recent functional expression of the full-length rat and human dihydroorotate dehydrogenase, here we expressed N-terminal-truncated C-terminal-histidine-tagged constructs of the mouse, rat and human enzymes in Escherichia coli. These proteins were devoid of the N-terminal bipartite sequence consisting of the mitochondrial targeting sequence and adjacent hydrophobic domain necessary for import and proper location and fixation of the enzyme in the inner mitochondrial membrane. By employing metal-chelate affinity chromatography under native conditions, the enzymes were purified without detergents to a specific activity of more than 100 mmol´min 21´m g 21 at pH optimum of 8.0±8.1. Flavin analyses by UV-visible spectrometry of the native enzymes gave fairly stoichiometric ratios of 0.6±1.2 mol flavin per mol protein. The kinetic constants of the truncated rat enzyme (K m 11 mm dihydroorotate; K m 7 mm ubiquinone) and human enzyme (K m 10 mm dihydroorotate; K m 14 mm ubiquinone) were very close to those recently reported for the full-size enzymes. The constants for the mouse enzyme, K m 26 mm dihydroorotate and K m 62 mm ubiquinone, were slightly elevated in comparison to those of the other species. The three truncated enzymes were tested for their efficacy with five inhibitors of topical clinical relevance against autoimmune disorders and tumors. Whereas the presence of the N-terminus of dihydroorotate dehydrogenase was essentially irrelevant for the efficacy of the malononitrilamides A77-1726, MNA715 and MNA279 with the rat and human enzyme, the N-termini were found to be important for the efficacy of the dianisidine derivative redoxal. Moreover, the complete N-terminal part of the human enzyme seemed to be of crucial importance for the`slow-binding' features of the cinchoninic acid derivative brequinar, which was suggested to be one of the reasons for the narrow therapeutic window reported from clinical trials on its anti-proliferative and immunosuppressive action.

No abstract