Human mitochondrial
dihydroorotate dehydrogenase (the fourth
enzyme of
pyrimidine de novo synthesis) has been overproduced by means of a recombinant baculovirus that contained the human
cDNA fragment for this
protein. After
virus infection and
protein expression in Trichoplusia ni cells (BTI-Tn-5B1-4), the subcellular distribution of the recombinant
dihydroorotate dehydrogenase was determined by two distinct
enzyme-activity assays and by Western blot analysis with anti-(
dihydroorotate dehydrogenase) Ig. The targeting of the
recombinant protein to the mitochondria of the insect cells was verified. The activity of the recombinant
enzyme in the mitochondria of infected cells was about 740-fold above the level of
dihydroorotate dehydrogenase in human liver mitochondria. In a three-step procedure,
dihydroorotate dehydrogenase was purified to a specific activity of greater than 50 U/mg. Size-exclusion chromatography showed a molecular mass of 42 kDa and confirmed the existence of the fully active
enzyme as a monomeric species. Fluorimetric cofactor analysis revealed the presence of
FMN in recombinant
dihydroorotate dehydrogenase. By kinetics analysis, Km values for
dihydroorotate and ubiquinone-50 were found to be 4 microM and 9.9 microM, respectively, while Km values for
dihydroorotate and
decylubiquinone were 9.4 microM and 13.7 microM, respectively. The applied expression system will allow preparation of large quantities of the
enzyme for structure and function studies. Purified recombinant human dihytdroorotate
dehydrogenase was tested for its sensitivity to a reported inhibitor
A77 1726 (2-hydroxyethyliden-cyanoacetic acid 4-trifluoromethyl anilide), which is the active metabolite of the
isoxazole derivative
leflunomide [5-methyl-N-(4-trifluoromethyl-phenyl)-4-
isoxazole carboximide]. An IC50 value of 1 microM was determined for
A77 1726. Detailed kinetics experiments revealed uncompetitive inhibition with respect to
dihydroorotate (Kiu = 0.94 microM) and non-competitive inhibition with respect to
decylubiquinone (Kic = 1.09 microM, Kiu = 1.05 microM). These results suggest that the immunomodulating agent
A77 1726 (currently in clinical phase III studies for the treatment of
rheumatoid arthritis) is a very good inhibitor of human
dihydroorotate dehydrogenase.