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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Isolation of a cDNA encoding human holocarboxylase synthetase by functional complementation of a biotin auxotroph of Escherichia coli.
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Isolation of a cDNA encoding human holocarboxylase synthetase by functional complementation of a biotin auxotroph of Escherichia coli.

机译：通过大肠杆菌生物素营养缺陷型的功能互补分离编码人全羧化酶合成酶的cDNA。

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Holocarboxylase synthetase (HCS) catalyzes the biotinylation of the four biotin-dependent carboxylases in human cells. Patients with HCS deficiency lack activity of all four carboxylases, indicating that a single HCS is targeted to the mitochondria and cytoplasm. We isolated 21 human HCS cDNA clones, in four size classes of 2.0-4.0 kb, by complementation of an Escherichia coli birA mutant defective in biotin ligase. Expression of the cDNA clones promoted biotinylation of the bacterial biotinyl carboxyl carrier protein as well as a carboxyl-terminal fragment of the alpha subunit of human propionyl-CoA carboxylase expressed from a plasmid. The open reading frame encodes a predicted protein of 726 aa and M(r) 80,759. Northern blot analysis revealed the presence of a 5.8-kb major species and 4.0-, 4.5-, and 8.5-kb minor species of poly(A)+ RNA in human tissues. Human HCS shows specific regions of homology with the BirA protein of E. coli and the presumptive biotin ligase of Paracoccus denitrificans. Several forms of HCS mRNA are generated by alternative splicing, and as a result, two mRNA molecules bear different putative translation initiation sites. A sequence upstream of the first translation initiation site encodes a peptide structurally similar to mitochondrial presequences, but it lacks an in-frame ATG codon to direct its translation. We anticipate that alternative splicing most likely mediates the mitochondrial versus cytoplasmic expression, although the elements required for directing the enzyme to the mitochondria remain to be confirmed.
机译：全息羧化酶合成酶（HCS）催化人类细胞中四种生物素依赖性羧化酶的生物素化。具有HCS缺乏症的患者缺乏所有四种羧化酶的活性，这表明单个HCS靶向线粒体和细胞质。我们通过弥补生物素连接酶缺陷的大肠杆菌birA突变体的互补性，分离出21个人类HCS cDNA克隆，分为4个大小级别的2.0-4.0 kb。 cDNA克隆的表达促进了细菌生物素羧基载体蛋白的生物素化以及从质粒表达的人丙酰辅酶A羧化酶α亚基的羧基末端片段。开放阅读框编码预测的蛋白质为726aa和M（r）80,759。 Northern印迹分析显示在人组织中存在5.8（kb）主要种类和4.0、4.5和8.5kb次要种类的poly（A）+ RNA。人HCS显示与大肠杆菌的BirA蛋白和反硝化副球菌的生物素连接酶的特定同源性区域。 HCS mRNA的几种形式是通过交替剪接产生的，因此，两个mRNA分子带有不同的假定翻译起始位点。第一翻译起始位点上游的序列编码结构上类似于线粒体前序的肽，但是缺少指导其翻译的框内ATG密码子。我们预计，选择性剪接最有可能介导线粒体相对于细胞质的表达，尽管将酶引导至线粒体所需的元件仍有待确认。

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