Dihydroorotate dehydrogenase arises from novel fused gene product with aspartate carbamoyltransferase in Bodo saliens
Dihydroorotate dehydrogenase arises from novel fused gene product with aspartate carbamoyltransferase in Bodo saliens
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DOI：
10.1016/j.bbrc.2007.04.102
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发表时间：
2007-06-22
影响因子：
3.1
通讯作者：
Aoki, Takashi
中科院分区：
文献类型：
--
作者：
Annoura, Takeshi;Sariego, Idalia;Aoki, Takashi
动叶绿体中的ACT-DHOD基因编码天冬氨酸氨基甲酰基转移酶和二氢甲酸脱氢酶，它们是嘧啶生物合成的第二和第四酶。虽然单一的mRNA物种产生了70 kDa的ACT-DHOD蛋白，但用抗DHOD多肽抗体进行的Western blotting显示了一条35 kDa的主带和次要条带。凝胶内消化和液相色谱串联质谱(MS/MS)分析表明，35 kDa条带中含有DHOD特异多肽和ACT特异多肽，提示DHOD和ACT独立存在。免疫沉淀和MS/MS分析分别鉴定出一个70 kDa的ACT-DHOD和一个35 kDa的DHOD，35 kDa DHOD的N端氨基酸被封闭。体外加工实验表明，重组ACT-DHOD可被裂解液抑制，同时出现35-kDa的DHOD和35-kDa的ACT。这些结果表明融合的ACT-DHOD是成熟DHOD的前体。从35 kDa脱氢表雄酮的生理功能出发，探讨了脱氢表观代谢产物35 kDa脱氢表雄酮的生理功能。(C)2007 Elsevier Inc.保留所有权利。
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 R 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. (c) 2007 Elsevier Inc. All rights reserved.