Dihydroorotate dehydrogenase arises from novel fused gene product with aspartate carbamoyltransferase in Bodo saliens

The ACT–DHOD gene in the kinetoplastid Bodo saliens encodes aspartate carbamoyltransferase and dihydroorotate dehydrogenase, the second and fourth enzymes of pyrimidine biosynthesis. Although the single mRNA species yielded a 70-kDa ACT–DHOD protein, Western blotting with anti-DHOD-peptide antibody showed a major band of 35-kDa and minor bands. In-gel digestion and liquid chromatography–tandem mass (MS/MS) spectrometry showed that the 35-kDa band contained DHOD-specific polypeptides and an ACT-specific polypeptide, suggesting the occurrence of independent DHOD and ACT. Immunoprecipitation and MS/MS analysis identified a 70-kDa ACT–DHOD and a 35-kDa DHOD independently, and the N-terminal amino acid of 35-kDa DHOD was blocked. In vitro processing assay showed that recombinant ACT–DHOD was decreased by the B. saliens lysate, accompanying the appearance of 35-kDa DHOD and 35-kDa ACT. These results indicate that fused ACT–DHOD is the precursor to mature DHOD. Large amount of 35-kDa DHOD in B. saliens is discussed from a viewpoint of its physiological roles.