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 promoter in a pyrimidine auxotrophic strain of Escherichia coli lacking the endogenous 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 chromatographic steps. The purity of the resulting enzyme was judged to be > 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 kDa on native size-exclusion chromatography, suggesting that the native enzyme is multimeric. Recombinant DHODase displayed a specific activity and Km for dihydroorotate that were similar to those for the enzymes from bovine and human liver tissue. The pH dependence of the activity of the recombinant enzyme was likewise similar to that of the enzyme from human liver and may indicate the involvement of a critical histidine residue in catalytic turnover; only eight histidine residues remain in the truncated version of DHODase used here. The catalytic activity of the recombinant enzyme is inhibited in a dose-dependent fashion by the histidine-selective modifying agent diethylpyrocarbonate. These results further suggest a potential role for histidine in enzyme turnover. 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 Ki value similar to that for enzyme derived from human liver tissue. The recombinant DHODase thus displays enzymatic behavior similar to the 50-kDa full-length human liver enzyme, illustrating that the catalytically essential structural features of the enzyme, as well as the site of Brequinar binding, are contained within the 40-kDa truncated version of the enzyme that was expressed here.
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D000090722:Dihydroorotate Dehydrogenase; D004789:Enzyme Activation; D004794:Enzyme Repression; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D000090722:Dihydroorotate Dehydrogenase; D011994:Recombinant Proteins; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors