EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF HISTIDINE-TAGGED RAT AND HUMAN FLAVOENZYME DIHYDROOROTATE DEHYDROGENASE

Mitochondrially bound dihydroorotate-ubiquinone oxidoreductase (dihydr oorotate dehydrogenase, EC 1.3.99.11) catalyzes the fourth sequential step in the de novo synthesis of uridine monophosphate. Based on the r ecent functional expression of the complete rat dihydroorotate dehydro genase by means of the baculovirus expression vector system in Trichop lusia ni cells, a procedure is described that allows the purification of baculovirus expressed enzyme protein fused to a carboxy-terminal ta g of eight histidines. Extracts from mitochondria of Spodoptera frugip erda cells infected with the recombinant virus using Triton X-100 were loaded onto Ni2+-nitrilotriacetic acid agarose and histidine-tagged r at protein was selectively eluted with imidazole-containing buffer. In view of our previously published work, the quality of the electrophor etic homogenous rat enzyme was markedly improved; specific activity wa s 130-150 mu mol dihydroorotate/min per milligram; and the stoichiomet ry of flavin content was 0.8-1.1 mol/mol protein. Efforts to generate mammalian dihydroorotate dehydrogenases with low production costs from bacteria resulted in successful overexpression of the carboxy-termina l-modified rat and human dihydroorotate dehydrogenase in XL-1 Blue cel ls. By employing the metal chelate affinity chromatography under nativ e conditions, the histidine-tagged human enzyme was purified with a sp ecific activity of 150 mu mol/min/mg and the rat enzyme with 83 mu mol /min/mg, respectively, at pH 8.0-8.1 optimum. Kinetic constants of the recombinant histidine-tagged rat enzyme from bacteria (dihydroorotate , K-m = 14.6 mu M; electron acceptor decylubiquinone, K-m = 9.5 mu M) were close to those reported for the enzyme from insect cells, with or without the affinity tag. HPLC analyses identified flavin mononucleot ide as cofactor of the rat enzyme; W-vis and fluorometric analyses ver ified a flavin/protein ratio of 0.8-1.1 mol/mol. By spectral analyses of the functional flavin with the native human enzyme, the interaction of the pharmacological inhibitors Leflunomide and Brequinar with thei r target could be clarified as interference with the transfer of elect rons from the flavin to the quinone. The combination of the bacterial expression system and metal chelate affinity chromatography offers an improved means to purify large quantities of mammalian membrane-bound dihydroorotate dehydrogenases which, by several criteria, possesses th e same functional activities as non-histidine-tagged recombinant enzym es. (C) 1998 Academic Press.