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.
Annoura
Takeshi
T
Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan.
Sariego
Idalia
I
Nara
Takeshi
T
Makiuchi
Takashi
T
Fujimura
Tsutomu
T
Taka
Hikari
H
Mineki
Reiko
R
Murayama
Kimie
K
Aoki
Takashi
T
D000595:Amino Acid Sequence; D000818:Animals; D001221:Aspartate Carbamoyltransferase / Q000235:genetics / Q000378:metabolism*; D000090722:Dihydroorotate Dehydrogenase; D050939:Gene Fusion; D017125:Genes, Protozoan; D016830:Kinetoplastida / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D005810:Multigene Family; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000032:analysis; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry
D000090722:Dihydroorotate Dehydrogenase; D010455:Peptides; D011994:Recombinant Proteins; D044925:Oxidoreductases Acting on CH-CH Group Donors; D001221:Aspartate Carbamoyltransferase