Recombinant expression of N-terminal truncated mutants of the membrane bound mouse, rat and human flavoenzyme dihydroorotate dehydrogenase - A versatile tool to rate inhibitor effects?

Mammalian dihydroorotate dehydrogenase, the fourth enzyme of pyrimidine de novo synthesis is an integral protein of the inner mitochondrial membrane t hat faces the intermembrane space and is functionally connected to the resp iratory chain via ubiquinone. Here, we describe the first cloning and analy zing of the complete cDNA of mouse dihydroorotate dehydrogenase. Based on o ur recent functional expression of the full-length rat and human dihydrooro tate dehydrogenase, here we expressed N-terminal-truncated C-terminal-histi dine-tagged constructs of the mouse, rat and human enzymes in Escherichia c oli. These proteins were devoid of the N-terminal bipartite sequence consis ting of the mitochondrial targeting sequence and adjacent hydrophobic domai n necessary for import and proper location and fixation of the enzyme in th e inner mitochondrial membrane. By employing metal-chelate affinity chromat ography under native conditions, the enzymes were purified without detergen ts to a specific activity of more than 100 mu mol.min(-1).mg(-1) at pH opti mum of 8.0-8.1. Flavin analyses by UV-visible spectrometry of the native en zymes gave fairly stoichiometric ratios of 0.6-1.2 mol flavin per mol prote in. The kinetic constants of the truncated rat enzyme (K-m = 11 mum dihydro orotate; K-m = 7 mum ubiquinone) and human enzyme (K-m = 10 mum dihydroorot ate; K-m = 14 mum ubiquinone) were very close to those recently reported fo r the full-size enzymes. The constants for the mouse enzyme, K-m = 26 mum d ihydroorotate and K-m = 62 mum ubiquinone, were slightly elevated in compar ison to those of the other species. The three truncated enzymes were tested for their efficacy with five inhibitors of topical clinical relevance agai nst autoimmune disorders and tumors. Whereas the presence of the N-terminus of dihydroorotate dehydrogenase was essentially irrelevant for the efficac y of the malononitrilamides A77-1726, MNA715 and MNA279 with the rat and hu man enzyme, the N-termini were found to be important for the efficacy of th e 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 c linical trials on its anti-proliferative and immunosuppressive action.