FUNCTIONAL EXPRESSION OF A FRAGMENT OF HUMAN DIHYDROOROTATE DEHYDROGENASE BY MEANS OF THE BACULOVIRUS EXPRESSION VECTOR SYSTEM, AND KINETICINVESTIGATION OF THE PURIFIED RECOMBINANT ENZYME

Human mitochondrial dihydroorotate dehydrogenase (the fourth enzyme of pyrimidine de novo synthesis) has been overproduced by means of a rec ombinant baculovirus that contained the human cDNA fragment for this p rotein, After virus infection and protein expression in Trichoplusia n i cells (BTI-Tn-5B1-4), the subcellular distribution of the recombinan t dihydroorotate dehydrogenase was determined by two distinct enzyme-a ctivity assays and by Western blot analysis with anti-(dihydroorotate dehydrogenase) Ig. The targeting of the recombinant protein to the mit ochondria of the insect cells was verified. The activity of the recomb inant enzyme in tile mitochondria of infected cells was about 740-fold above the level of dihydroorotate dehydrogenase in human liver mitoch ondria, In a three-step procedure, dihydroorotate dehydrogenase was pu rified to a specific activity of greater than 50 U/mg. Size-exclusion chromatography showed a molecular mass of 42 kDa and confirmed the exi stence of the fully active enzyme as a monomeric species. Fluorimetric cofactor analysis revealed the presence of FMN in recombinant dihydro orotate dehydrogenase. By kinetics analysis K-m values for dihydroorot ate and ubiquinone-50 were found to be 4 mu M and 9.9 mu M, respective ly, while K-m values for dihydroorotate and decylubiquinone were 9.4 m u M and 13.7 mu M, respectively. The applied expression system will al low preparation of large quantities of the enzyme for structure and fu nction studies. Purified recombinant human dihydroorotate dehydrogenas e was tested for its sensitivity to a reported inhibitor A77 1726 (2-h ydroxyethylidene-cyanoacetic acid 4-trifluoromethyl anilide). which is the active metabolite of the isoxazol derivative leflunomide [5-methy l-N-(4-trifluoromethyl-phenyl)-4-isoxazole carboximide]. An IC50 value of 1 mu M was determined for A77 1726, Detailed kinetics experiments revealed uncompetitive inhibition with respect to dihydroorotate (K-in = 0.94 mu M) and non-competitive inhibition with respect to decylubiq uinone (K-ic = 1.09 mu M, K-in = 1.05 mu M). These results suggest tha t the immunomodulating agent A77 1726 (currently in clinical phase III studies for the treatment of rheumatoid arthritis is a very good inhi bitor of human dihydroorotate dehydrogenase.