RECOMBINANT HUMAN DIHYDROOROTATE DEHYDROGENASE - EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF A CATALYTICALLY FUNCTIONAL TRUNCATED ENZYME

An N-terminally truncated cDNA for human dihydroorotate dehydrogenase (DHODase) was placed under the control of the inducible T7 lac promote r in a pyrimidine auxotrophic strain of Escherichia coli lacking the e ndogenous enzyme, Induction of gene expression rescued growth in media lacking exogenous pyrimidines, The recombinant enzyme was purified to homogeneity from detergent extracts of bacterial membranes by two chr omatographic steps, The purity of the resulting enzyme was judged to b e > 95% based on SDS-PAGE with Coomassie staining, The enzyme displays an apparent molecular weight of ca. 40 kDa on SDS-PAGE and ca, 120 kD a on native size-exclusion chromatography, suggesting that the native enzyme is multimeric, Recombinant DHODase displayed a specific activit y and K-m for dihydroorotate that were similar to those for the enzyme s from bovine and human liver tissue, The pH dependence of the activit y of the recombinant enzyme was likewise similar to that of the enzyme hom human liver and may indicate the involvement of a critical histid ine residue in catalytic turnover; only eight histidine residues remai n in the truncated version of DHODase used here, The catalytic activit y of the recombinant enzyme is inhibited in a dose-dependent fashion b y the histidine-selective modifying agent diethylpyrocarbonate. These results further suggest a potential role for histidine in enzyme turno ver, Brequinar sodium, an experimental drug which has been shown to be a nanomolar noncompetitive inhibitor of mammalian DHODases, inhibited the activity of the purified recombinant enzyme with a K-i value simi lar to that for enzyme derived from human liver tissue, The recombinan t DHODase thus displays enzymatic behavior similar to the 50-kDa full- length human liver enzyme, illustrating that the catalytically essenti al structural features of the enzyme, as well as the site of Brequinar binding, are contained within the 40-kDa truncated version of the enz yme that was expressed here, (C) 1995 Academic Press, Inc.